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Sample records for hemolysins

  1. POSSIBLE ROLE OF FUNGAL HEMOLYSINS IN SICK BUILDING SYNDROME

    EPA Science Inventory

    Many fungi produce proteinaceous hemolytic agents. Like bacterial hemolysins, fungal hemolysins create pores or holes in membranes. Depending on which membranes are damaged, fungal hemolysins can produce a variety of effects. Fungal hemolysins can cause histamine release from ...

  2. Hemolysin patterns of Actinobacillus pleuropneumoniae.

    PubMed Central

    Frey, J; Nicolet, J

    1990-01-01

    The secreted hemolytic activities produced by the reference strains and field isolates of the 12 serotypes and 2 subtypes of Actinobacillus pleuropneumoniae were analyzed. Serotype 1 produced a Ca2(+)-inducible hemolysin, which was previously characterized as a 105-kilodalton protein and was named hemolysin I (HlyI). Serotypes 2, 4, 6, 7, and 8 produced a different hemolytic activity that was not inducible by Ca2+ but required this ion for its activity. The hemolytic activity produced by these serotypes was much weaker than that found in serotype 1 and was not neutralized by rabbit antibodies against HlyI. It was, however, neutralized by serum from pigs that were experimentally infected with a serotype 2 strain and was called hemolysin II (HlyII). Serotypes 5a, 5b, 9, 10, and 11 produced both HlyI and HlyII. In these strains, HlyI was the major contributor to the hemolytic activity. The remaining serotypes, 3 and 12, produced a very weak hemolytic activity, which was not further analyzed. Immunoblot analysis of the culture supernatants from all 12 serotypes with rabbit polyclonal antibodies directed against HlyI revealed reactions with a protein in the 105-kilodalton size range for all serotypes, indicating that HlyI and HlyII might be serologically related. Strains producing active HlyI seem to belong to serotypes that are generally considered to be virulent types and that are frequently isolated from pigs in severe pleuropneumonia outbreaks. Images PMID:2312671

  3. Binding properties of Paracentrotus lividus (Echinoidea) hemolysin.

    PubMed

    Canicatti, C

    1991-01-01

    1. Paracentrotus lividus hemolysin binds erythrocytes, zymosan particles, lipopolysaccharide and laminarin surfaces but not auto and allogeneic cell membranes. 2. The binding could, at least for erythrocytes, involve phospholipids and cholesterol. 3. The protease activity of the coelomic fluid is not related to hemolysis. 4. The finding that very low concentrations of Zn2+ inactivate the hemolysin suggests a possible regulative function of the ion in the hemolytic reaction. 5. Ultrastructural observations on rabbit erythrocyte membranes indicate that most likely the transmembrane pores are induced by the lytic molecules. PMID:1674457

  4. CHARACTERIZATION OF THE HEMOLYSIN, FROM STACHYBOTRYS CHARTARUM

    EPA Science Inventory

    Stachybotrys chartarum is a toxigenic fungus that has been associated with human health concerns, including pulmonary hemorrhage and hemosiderosis. This fungus produces a hemolysin, stachylysin, which in its apparent monomeric form has a molecular mass of 11,920
    Da as determ...

  5. Hemolysin of uropathogenic Escherichia coli: A cloak or a dagger?

    PubMed

    Ristow, Laura C; Welch, Rodney A

    2016-03-01

    Hemolysin from uropathogenic Escherichia coli (UPEC) is a hemolytic and cytotoxic protein active against a broad range of species and cell types. Expression of hemolysin correlates with severity of infection, as up to 78% of UPEC isolates from pyelonephritis cases express hemolysin. Despite decades of research on hemolysin activity, the mechanism of intoxication and the function of hemolysin in UPEC infection remain elusive. Early in vitro research established the role of hemolysin as a lytic protein at high doses. It is hypothesized that hemolysin is secreted at sublytic doses in vivo and recent research has focused on understanding the more subtle effects of hemolysin both in vitro and in elegant infection models in vivo, including inoculation by micropuncture of individual kidney nephrons. As the field continues to evolve, comparisons of hemolysin function in isolates from a range of UTI infections will be important for delineating the role of this toxin. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.

  6. Characteristics of heat-stable extracellular hemolysin from Pseudomonas aeruginosa.

    PubMed Central

    Fujita, K; Akino, T; Yoshioka, H

    1988-01-01

    Heat-stable hemolysin produced by the cellophane-agar plate method consisted of two major acidic glycolipids. One was composed of 2 mol of rhamnose and 2 mol of beta-hydroxydecanoic acid, and the other was composed of 1 mol of rhamnose and 2 mol of beta-hydroxydecanoic acid. The hemolysis-producing moiety of the hemolysin was shown to be the dimer of beta-hydroxydecanoic acid contained in the glycolipid molecule. Images PMID:3128485

  7. Characterization of a Vibrio alginolyticus strain, isolated from Alaskan oysters, carrying a hemolysin gene similar to the thermostable direct hemolysin-related hemolysin gene (trh) of Vibrio parahaemolyticus.

    PubMed

    González-Escalona, Narjol; Blackstone, George M; DePaola, Angelo

    2006-12-01

    A Vibrio strain isolated from Alaskan oysters and classified by its biochemical characteristics as Vibrio alginolyticus possessed a thermostable direct hemolysin-related hemolysin (trh) gene previously reported only in Vibrio parahaemolyticus. This trh-like gene was cloned and sequenced and was 98% identical to the trh2 gene of V. parahaemolyticus. This gene seems to be functional since it was transcriptionally active in early-stationary-phase growing cells. To our knowledge, this is the first report of V. alginolyticus possessing a trh gene.

  8. [Effect of Bacillus cereus hemolysin II on hepatocyte cells].

    PubMed

    Kholodkov, O A; Budarina, Zh; Kovalevskaya, J I; Si'unov, A V; Solonin, A

    2015-01-01

    We investigated the efficiency of increasing the permeability (permeabilization) of cell membranes in primary liver cells by Bacillus cereus hemolysin II. An assessment of the degree of permeabilization was car ried out by measuring the fluorescence intensity of various low molecular weight dyes, which enter through pores into hepatocyte cells cultivated with hemolysin. We uncovered a high efficacy of hemolysin HlyII action on hepatocyte cell walls, which exceeded the effect of nonionic detergent, digitonin, which is commonly employed for pore formation in various cell membranes. Our results also point to the reversibility of membrane permeabilization in primary hepatocytes. The data obtained in this study can be utilized for assessments of pore-forming activity, in studies of hepatic mechanisms of action, and also the determination of the liver toxicity for different low molecular weight drugs. PMID:26027363

  9. [Effect of Bacillus cereus hemolysin II on hepatocyte cells].

    PubMed

    Kholodkov, O A; Budarina, Zh; Kovalevskaya, J I; Si'unov, A V; Solonin, A

    2015-01-01

    We investigated the efficiency of increasing the permeability (permeabilization) of cell membranes in primary liver cells by Bacillus cereus hemolysin II. An assessment of the degree of permeabilization was car ried out by measuring the fluorescence intensity of various low molecular weight dyes, which enter through pores into hepatocyte cells cultivated with hemolysin. We uncovered a high efficacy of hemolysin HlyII action on hepatocyte cell walls, which exceeded the effect of nonionic detergent, digitonin, which is commonly employed for pore formation in various cell membranes. Our results also point to the reversibility of membrane permeabilization in primary hepatocytes. The data obtained in this study can be utilized for assessments of pore-forming activity, in studies of hepatic mechanisms of action, and also the determination of the liver toxicity for different low molecular weight drugs.

  10. Virulence of Bordetella bronchiseptica: role of adenylate cyclase-hemolysin.

    PubMed Central

    Gueirard, P; Guiso, N

    1993-01-01

    Bordetella bronchiseptica is a pathogen of laboratory, domestic, and wild animals and sometimes of humans. In the present study some characteristics of the virulence of B. bronchiseptica isolates of different origin were studied. All isolates had similar phenotypes, similar bacteriological characters, and synthesized adenylate cyclase-hemolysin, filamentous hemagglutinin and pertactin but not pertussis toxin. These isolates, however, differed in their ability to express dermonecrotic toxin and to cause a lethal infection, but no correlation was found with the human or animal origin of the isolates. The fact that the most virulent isolate did not express dermonecrotic toxin suggests that this toxin does not play an important role in the virulence of the bacteria in the murine model. After infection with virulent B. bronchiseptica a very early synthesis and a persistence of anti-adenylate cyclase-hemolysin and anti-filamentous hemagglutinin antibodies were observed in the sera of infected mice, suggesting a persistence of the bacteria or of its antigens. B. bronchiseptica adenylate cyclase-hemolysin was purified and was shown to be a major protective antigen against B. bronchiseptica infection. Furthermore, we showed that its immunological and protective properties were different from that of B. pertussis adenylate cyclase-hemolysin, confirming that Bordetella species are immunologically different. Images PMID:8406794

  11. CHARACTERIZATION OF THE HEMOLYSIN, STACHYLYSIN, FROM STACHYBOTRYS CHARTARUM

    EPA Science Inventory

    Stachybotrys chartarum is a toxigenic fungus that has been associated with human health concerns, including pulmonary hemorrhage/hemosiderosis. This fungus produces a hemolysin, stachylysin, which in its monomeric form, has a molecular wieght of 11,920 daltons as determined by m...

  12. HEMOLYSIN, CHRYSOLYSIN FROM PENICILLIUM CHRYSOGENUM PROMOTES INFLAMMATORY RESPONSE

    EPA Science Inventory

    Some strains of Penicillium chrysogenum produce a proteinaceous hemolysin, chrysolysin, when incubated on sheep's blood agar at 37 �C but not at 23 �C. Chrysolysin is an aggregating protein composed of approximately 2 kDa monomers, contains one cysteine amino acid, and has an is...

  13. Water transport by the bacterial channel alpha-hemolysin

    NASA Technical Reports Server (NTRS)

    Paula, S.; Akeson, M.; Deamer, D.

    1999-01-01

    This study is an investigation of the ability of the bacterial channel alpha-hemolysin to facilitate water permeation across biological membranes. alpha-Hemolysin channels were incorporated into rabbit erythrocyte ghosts at varying concentrations, and water permeation was induced by mixing the ghosts with hypertonic sucrose solutions. The resulting volume decrease of the ghosts was followed by time-resolved optical absorption at pH 5, 6, and 7. The average single-channel permeability coefficient of alpha-hemolysin for water ranged between 1.3x10-12 cm/s and 1.5x10-12 cm/s, depending on pH. The slightly increased single-channel permeability coefficient at lower pH-values was attributed to an increase in the effective pore size. The activation energy of water transport through the channel was low (Ea=5.4 kcal/mol), suggesting that the properties of water inside the alpha-hemolysin channel resemble those of bulk water. This conclusion was supported by calculations based on macroscopic hydrodynamic laws of laminar water flow. Using the known three-dimensional structure of the channel, the calculations accurately predicted the rate of water flow through the channel. The latter finding also indicated that water permeation data can provide a good estimate of the pore size for large channels.

  14. UPEC hemolysin: more than just for making holes.

    PubMed

    Justice, Sheryl S; Hunstad, David A

    2012-01-19

    During acute cystitis, uropathogenic Escherichia coli (UPEC) induce bladder epithelial cell exfoliation, which eliminates infected cells and promotes UPEC dissemination. Dhakal and Mulvey (2012) uncover the mechanism that induces this exfoliation and reintroduce the pore-forming toxin, hemolysin, as an effector that surprisingly targets multiple host pathways to facilitate infection.

  15. Molecular characterization of the hemolysin determinant of Serratia marcescens.

    PubMed Central

    Poole, K; Schiebel, E; Braun, V

    1988-01-01

    The nucleotide sequence of a 7.3-kilobase-pair fragment of DNA encoding a hemolytic activity from Serratia marcescens was determined. Two large open reading frames were identified, designated shlA (Serratia hemolysin) and shlB, capable of encoding polypeptides of 165, 056 and 61,897 molecular weight, respectively. Both reading frames were expressed in vivo. The shlB gene product was localized to the outer membrane of Escherichia coli cells harboring the S. marcescens hemolysin determinant. Consistent with this location, a signallike sequence was identified at the N terminus of the polypeptide predicted from the nucleotide sequence of the shlB gene. Hyperexpression of the shlB locus permitted the identification of two shlB-encoded polypeptides of 65,000 and 62,000 molecular weight, respectively. Determination of the N-terminal amino acid sequence of the purified 62,000-molecular-weight protein confirmed that it was the mature form of the ShlB protein initially synthesized as a precursor (65,000-molecular-weight protein). By using polyclonal antisera raised against the purified proteins, ShlA and ShlB were identified in the outer membrane of S. marcescens. The shlA gene product was shown to interact with erythrocyte membranes, confirming it as the hemolysin proper. Both hemolysis and the interaction of ShlA with erythrocyte membranes did, however, require the ShlB function. Progressive deletion of the C terminus of the ShlA protein gradually reduced hemolytic activity until 37% of the amino acids had been removed. Elimination of 54% of the amino acids produced a nonhemolytic protein which, however, was still capable of associating with erythrocyte membranes. Images PMID:3290200

  16. Erythrocyte Lysis and Xenopus laevis Oocyte Rupture by Recombinant Plasmodium falciparum Hemolysin III

    PubMed Central

    Moonah, Shannon; Sanders, Natalie G.; Persichetti, Jason K.

    2014-01-01

    Malaria kills more than 1 million people per year worldwide, with severe malaria anemia accounting for the majority of the deaths. Malaria anemia is multifactorial in etiology, including infected erythrocyte destruction and decrease in erythrocyte production, as well as destruction or clearance of noninfected erythrocytes. We identified a panspecies Plasmodium hemolysin type III related to bacterial hemolysins. The identification of a hemolysin III homologue in Plasmodium suggests a potential role in host erythrocyte lysis. Here, we report the first characterization of Plasmodium falciparum hemolysin III, showing that the soluble recombinant P. falciparum hemolysin III is a pore-forming protein capable of lysing human erythrocytes in a dose-, time-, and temperature-dependent fashion. The recombinant P. falciparum hemolysin III-induced hemolysis was partially inhibited by glibenclamide, a known channel antagonist. Studies with polyethylene glycol molecules of different molecular weights indicated a pore size of approximately 3.2 nm. Heterologous expression of recombinant P. falciparum hemolysin III in Xenopus oocytes demonstrated early hypotonic lysis similar to that of the pore-forming aquaporin control. Live fluorescence microscopy localized transfected recombinant green fluorescent protein (GFP)-tagged P. falciparum hemolysin III to the essential digestive vacuole of the P. falciparum parasite. These transfected trophozoites also possessed a swollen digestive vacuole phenotype. Native Plasmodium hemolysin III in the digestive vacuole may contribute to lysis of the parasitophorous vacuole membrane derived from the host erythrocyte. After merozoite egress from infected erythrocytes, remnant P. falciparum hemolysin III released from digestive vacuoles could potentially contribute to lysis of uninfected erythrocytes to contribute to severe life-threatening anemia. PMID:25148832

  17. Erythrocyte lysis and Xenopus laevis oocyte rupture by recombinant Plasmodium falciparum hemolysin III.

    PubMed

    Moonah, Shannon; Sanders, Natalie G; Persichetti, Jason K; Sullivan, David J

    2014-10-01

    Malaria kills more than 1 million people per year worldwide, with severe malaria anemia accounting for the majority of the deaths. Malaria anemia is multifactorial in etiology, including infected erythrocyte destruction and decrease in erythrocyte production, as well as destruction or clearance of noninfected erythrocytes. We identified a panspecies Plasmodium hemolysin type III related to bacterial hemolysins. The identification of a hemolysin III homologue in Plasmodium suggests a potential role in host erythrocyte lysis. Here, we report the first characterization of Plasmodium falciparum hemolysin III, showing that the soluble recombinant P. falciparum hemolysin III is a pore-forming protein capable of lysing human erythrocytes in a dose-, time-, and temperature-dependent fashion. The recombinant P. falciparum hemolysin III-induced hemolysis was partially inhibited by glibenclamide, a known channel antagonist. Studies with polyethylene glycol molecules of different molecular weights indicated a pore size of approximately 3.2 nm. Heterologous expression of recombinant P. falciparum hemolysin III in Xenopus oocytes demonstrated early hypotonic lysis similar to that of the pore-forming aquaporin control. Live fluorescence microscopy localized transfected recombinant green fluorescent protein (GFP)-tagged P. falciparum hemolysin III to the essential digestive vacuole of the P. falciparum parasite. These transfected trophozoites also possessed a swollen digestive vacuole phenotype. Native Plasmodium hemolysin III in the digestive vacuole may contribute to lysis of the parasitophorous vacuole membrane derived from the host erythrocyte. After merozoite egress from infected erythrocytes, remnant P. falciparum hemolysin III released from digestive vacuoles could potentially contribute to lysis of uninfected erythrocytes to contribute to severe life-threatening anemia.

  18. Structure of Staphylococcal α-Hemolysin, a Heptameric Transmembrane Pore

    NASA Astrophysics Data System (ADS)

    Song, Langzhou; Hobaugh, Michael R.; Shustak, Christopher; Cheley, Stephen; Bayley, Hagan; Gouaux, J. Eric

    1996-12-01

    The structure of the Staphylococcus aureus α-hemolysin pore has been determined to 1.9 overset{circ}{mathrm A} resolution. Contained within the mushroom-shaped homo-oligomeric heptamer is a solvent-filled channel, 100 overset{circ}{mathrm A} in length, that runs along the sevenfold axis and ranges from 14 overset{circ}{mathrm A} to 46 overset{circ}{mathrm A} in diameter. The lytic, transmembrane domain comprises the lower half of a 14-strand antiparallel β barrel, to which each protomer contributes two β strands, each 65 overset{circ}{mathrm A} long. The interior of the β barrel is primarily hydrophilic, and the exterior has a hydrophobic belt 28 overset{circ}{mathrm A} wide. The structure proves the heptameric subunit stoichiometry of the α-hemolysin oligomer, shows that a glycine-rich and solvent-exposed region of a water-soluble protein can self-assemble to form a transmembrane pore of defined structure, and provides insight into the principles of membrane interaction and transport activity of β barrel pore-forming toxins.

  19. The thermostable direct hemolysin-related hemolysin (trh) gene of Vibrio parahaemolyticus: Sequence variation and implications for detection and function.

    PubMed

    Nilsson, William B; Turner, Jeffrey W

    2016-07-01

    Vibrio parahaemolyticus is a leading cause of bacterial food-related illness associated with the consumption of undercooked seafood. Only a small subset of strains is pathogenic. Most clinical strains encode for the thermostable direct hemolysin (TDH) and/or the TDH-related hemolysin (TRH). In this work, we amplify and sequence the trh gene from over 80 trh+strains of this bacterium and identify thirteen genetically distinct alleles, most of which have not been deposited in GenBank previously. Sequence data was used to design new primers for more reliable detection of trh by endpoint PCR. We also designed a new quantitative PCR assay to target a more conserved gene that is genetically-linked to trh. This gene, ureR, encodes the transcriptional regulator for the urease gene cluster immediately upstream of trh. We propose that this ureR assay can be a useful screening tool as a surrogate for direct detection of trh that circumvents challenges associated with trh sequence variation.

  20. Occurrence of genes for P and S fimbriae and hemolysin in urinary Escherichia coli.

    PubMed

    Jusková, E; Ciznár, I

    1994-01-01

    Escherichia coli is the common causative agent of urinary tract infections. Sixty-one strains of E. coli isolated from children with urinary tract infections were tested by colony hybridization for the presence of genes determining P and S fimbriae and hemolysin. Of these strains, 46 possess a gene for hemolysin, 44 for P fimbriae and 28 for S fimbriae. Only 30 strains formed lytic zones around the colonies on plates with sheep erythrocytes. The results indicated that simultaneous occurrence of genes in urinary E. coli was highest for P fimbriae and hemolysin and lower for other combinations of the tested genes.

  1. COMPARATIVE KINETICS OF HEMOLYSIS INDUCED BY BACTERIAL AND OTHER HEMOLYSINS

    PubMed Central

    Bernheimer, Alan W.

    1947-01-01

    A study has been made of the kinetics of lysis induced by various hemolytic agents. The course of bemolysis was followed by mixing lysin with washed human erythrocytes, removing samples from the mixture, and estimating colorimetrically the hemoglobin in the supernatant fluid of the centrifuged samples. Most of the curves (but not all of them, e.g. tyrocidine) obtained by plotting degree of hemolysis against time, were S-shaped. The initiation of lysis by streptolysin S' was delayed, and in this property, streptolysin S' was similar to Cl. septicum hemolysin. None of the other lysins studied exhibited a long latent period preceding lysis. The maximum rate of hemoglobin liberation was found, in the range of lysin concentrations studied, to be a linear function of concentration when theta toxin of Cl. welchii, pneumolysin, tetanolysin, or streptolysin S' was the lytic agent. With comparable concentrations of saponin, sodium taurocholate, cetyl pyridinium chloride, tyrocidine, duponol C, lecithin-atrox venom mixture, or streptolysin O, the relation between rate and concentration was non-linear. The critical thermal increment associated with hemolysis was determined for systems containing pneumolysin, theta toxin, streptolysin S', streptolysin O, tetanolysin, and saponin. The findings concerning the effect of concentration and temperature on the rate of hemolysis provide a basis for classifying hemolytic agents (Tables I and II). Hemolysis induced by Cl. septicum hemolysin was found to be preceded by two phases: a phase of alteration of the erythrocytes and a phase involving swelling. Antihemolytic serum inhibited the first but not the second phase while sucrose inhibited the second but not the first phase. PMID:19873499

  2. Characterization of hemolysins of Staphylococcus strains isolated from human and bovine, southern Iran.

    PubMed

    Moraveji, Z; Tabatabaei, M; Shirzad Aski, H; Khoshbakht, R

    2014-01-01

    The staphylococci are important pathogenic bacteria causing various infections in animals and human. Hemolysin is one of the virulence factors of coagulase-positive (CPS) and coagulase-negative staphylococci (CNS). The aims of the study were to characterize hemolysins of Staphylococcus spp. isolated from human and bovine origin, phenotypic- and genotypically. Characterization of hemolysin phenotypically based on hemolysis pattern of Staphylococcus spp. was done on the sheep, horse and rabbit blood agar plates. Genes encoding hemolysin were amplified with specific primers by using polymerase chain reaction (PCR) technique. Hemolytic activities phenotypically were determined in 60 and 90% of the total bovine and human isolates, respectively. All non hemolytic isolates were CNS (P≤0.05). In all isolates, hla and hld genes were determined by PCR amplification. None of the bovine and human isolates showed phenotypically and genotypically gamma hemolysin. The results from this study suggest that, in accordance with what is generally believed, some differences are apparent in hemolysin types among Staphylococcus strains of bovine and human origin. Furthermore, this study showed that CNS can be important as new pathogens.

  3. Characterization of hemolysins of Staphylococcus strains isolated from human and bovine, southern Iran

    PubMed Central

    Moraveji, Z; Tabatabaei, M; Shirzad Aski, H; Khoshbakht, R

    2014-01-01

    The staphylococci are important pathogenic bacteria causing various infections in animals and human. Hemolysin is one of the virulence factors of coagulase-positive (CPS) and coagulase-negative staphylococci (CNS). The aims of the study were to characterize hemolysins of Staphylococcus spp. isolated from human and bovine origin, phenotypic- and genotypically. Characterization of hemolysin phenotypically based on hemolysis pattern of Staphylococcus spp. was done on the sheep, horse and rabbit blood agar plates. Genes encoding hemolysin were amplified with specific primers by using polymerase chain reaction (PCR) technique. Hemolytic activities phenotypically were determined in 60 and 90% of the total bovine and human isolates, respectively. All non hemolytic isolates were CNS (P≤0.05). In all isolates, hla and hld genes were determined by PCR amplification. None of the bovine and human isolates showed phenotypically and genotypically gamma hemolysin. The results from this study suggest that, in accordance with what is generally believed, some differences are apparent in hemolysin types among Staphylococcus strains of bovine and human origin. Furthermore, this study showed that CNS can be important as new pathogens. PMID:27175125

  4. Inhibition of Colony-spreading Activity of Staphylococcus aureus by Secretion of δ-Hemolysin*

    PubMed Central

    Omae, Yosuke; Sekimizu, Kazuhisa; Kaito, Chikara

    2012-01-01

    Staphylococcus aureus spreads on the surface of soft agar, a phenomenon we termed “colony spreading.” Here, we found that S. aureus culture supernatant inhibited colony spreading. We purified δ-hemolysin (Hld, δ-toxin), a major protein secreted from S. aureus, as a compound that inhibits colony spreading. The culture supernatants of hld-disrupted mutants had 30-fold lower colony-spreading inhibitory activity than those of the parent strain. Furthermore, hld-disrupted mutants had higher colony-spreading ability than the parent strain. These results suggest that S. aureus negatively regulates colony spreading by secreting δ-hemolysin. PMID:22411996

  5. Genetic and biochemical properties of a hemolysin (pyolysin) produced by a swine isolate of Arcanobacterium (Actinomyces) pyogenes.

    PubMed

    Ikegami, M; Hashimoto, N; Kaidoh, T; Sekizaki, T; Takeuchi, S

    2000-01-01

    Arcanobacterium (Actinomyces) pyogenes, a causative agent of various pyogenic diseases in domestic animals, produces a hemolysin which is thought to be an important virulence factor. This hemolysin was purified from the culture supernatant of A. pyogenes swine isolate. The purified hemolysin showed a single band with a molecular mass of 56 kDa on SDS-polyacrylamide gel electrophoresis, and its isoelectric point was 9.2. The activity of this hemolysin was not enhanced by the addition of L-cysteine or sodium thioglycolate, but it was inhibited by cholesterol. The gene encoding the hemolysin was cloned, sequenced and expressed in Escherichia coli by means of ZAP Express vector. Analysis by SDS-polyacrylamide gel electrophoresis with immunoblotting showed that the molecular weight of the hemolysin expressed in E. coli is the same as that of the hemolysin purified from A. pyogenes. Nucleotide sequence analysis revealed an open reading frame of 1,605 bp encoding a 534 amino acid protein of 57,989 Da. The nucleotide sequence of the hemolysin gene from A. pyogenes swine isolate differed only slightly (97.6% identity) from the sequence of plo gene from A. pyogenes strain BBR1 reported by Billington et al (J. Bacteriol. 179: 6100-6106, 1997). The cysteine residue existed in the undecapeptide region of the hemolysin, which is highly conserved in thiol-activated cytolysins (cholesterol-binding cytolysins), and is replaced with alanine. Therefore, the hemolysin of A. pyogenes seems to be a novel member of the thiol-activated cytolysin family. PMID:10711593

  6. NIGERLYSINTM, HEMOLYSIN PRODUCED BY ASPERGILLUS NIGER, CAUSES LETHALITY OF PRIMARY RAT CORTICAL NEURONAL CELLS IN VITRO

    EPA Science Inventory

    Aspergillus niger produced a proteinaceous hemolysin, nigerlysinTM when incubated on sheep's blood agar at both 23° C and 37°C. Nigerlysin was purified from tryptic soy broth culture filtrate. Purified nigerlysin has a molecular weight of approximately 72 kDa, with an...

  7. A molecular modeling based screening for potential inhibitors to alpha hemolysin from Staphylococcus aureus

    PubMed Central

    Rashidieh, Behnam; Etemadiafshar, Sarah; Memari, Golnaz; Mirzaeichegeni, Masoumeh; Yazdi, Shahrzad; Farsimadan, Fatemeh; Alizadeh, Soodabeh

    2015-01-01

    Staphylococcus aureus, a Gram-positive bacterium is pathogenic in nature. It is known that secreted toxins remain active after antibiotic treatment. The alpha hemolysin or alpha toxin damages cell membrane and induces apoptosis and degradation of DNA. The titer of alphahemolysin increases and causes hemostasis disturbances, thrombocytopenia, and pulmonary lesions during staphylococcal infection. Therefore, it is of interest to inhibit alpha hemolysin using novel compounds. We used the structure of alpha hemolysin(PDB: 7AHL) to screen structures for 100,000 compounds from the ZINC database using molecular docking with AutoDock VINA. Nine (9) successive hits were then subjected for pharmacokinetic and toxicity properties by PROTOX (a webserver for the prediction of oral toxicities of small molecules) and FAFDrugs (a tool for prediction of ADME and Toxicity). This exercise further identified hit #1 ({[3a-(Dihydroxymethyl)-6-hydroxy-2,2-dimethyl-1,3,4-trioxatetrahydro-2H-pentalen-5- yl]methyl}amino(9H-fluoren-9-yl)acetate with binding affinity: -10.3 kcal/mol) and hit #2 (6-(Dihydroxymethyl)-2-{2-[3- (methylamino)propyl]-2-azatricyclo[9.4.0.03,8]pentadeca-1(11),3,5,7,12,14-hexaen-6-yloxy}tetrahydro-2H-pyran-3,4,5-triol with binding affinity: -9.6 kcal/mol) with acceptable toxicity and ADME properties for potential predicted hemolysin inhibition. These compounds should then be evaluated in vitro using inhibitory studies. PMID:26420917

  8. X-ray Crystal Structure of the B Component of Hemolysin BL from Bacillus cereus

    SciTech Connect

    Madegowda,M.; Eswaramoorthy, S.; Burley, S.; Swaminathan, S.

    2008-01-01

    Bacillus cereus Hemolysin BL enterotoxin, a ternary complex of three proteins, is the causative agent of food poisoning and requires all three components for virulence. The X-ray structure of the binding domain of HBL suggests that it may form a pore similar to other soluble channel forming proteins. A putative pathway of pore formation is discussed.

  9. INITIAL CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST THE FUNGAL HEMOLYSIN STACHYLYSIN FROM STACHYBOTRYS CHARTARUM

    EPA Science Inventory

    Stachybotrys chartarum is known to produce the hemolysin stachylysin and its detection in human serum has been proposed as a biomarker for exposure to the fungus. In this study we report the initial characterization of monoclonal antibodies (mAbs) against stachylysin and the dev...

  10. Expression of a cloned Staphylococcus aureus alpha-hemolysin determinant in Bacillus subtilis and Staphylococcus aureus.

    PubMed Central

    Fairweather, N; Kennedy, S; Foster, T J; Kehoe, M; Dougan, G

    1983-01-01

    A DNA sequence encoding Staphylococcus aureus alpha-hemolysin, which had been previously cloned and mapped in Escherichia coli K-12, was introduced into Bacillus subtilis BD170 and several strains of S. aureus by using plasmid vectors, some of which could replicate in all three organisms. The determinant was cloned on a 3.3-kilobase pair DNA fragment into B. subtilis by using the vector plasmid pXZ105 to form the hybrid plasmid pXZ111. B. subtilis cells harboring pXZ111 produced large zones of alpha-hemolysis after 18 h of growth at 37 degrees C on rabbit blood agar plates, and alpha-hemolysin activity was detected in supernatants prepared from growing cultures of this strain. The alpha-hemolysin was apparently secreted across the B. subtilis cell envelope. Polypeptides of molecular weights 34,000 and 33,000 were precipitated with anti-alpha-hemolysin serum from lysates prepared from BD170 cells harboring pXZ111. A hybrid replicon which could replicate in both E. coli and S. aureus was constructed in E. coli by ligating a HindIII fragment encoding the replication functions and chloramphenicol resistance genes of S. aureus plasmid pCW59 to the pBR322 alpha-hemolysin hybrid plasmid pDU1150. The DNA of this plasmid, pDU1212, was prepared in E. coli and used to transform protoplasts prepared from a non-alpha-hemolytic, nonrestricting strain of S. aureus RN4220. Some of the transformants contained plasmids which had suffered extensive deletions. Some plasmids, however, were transformed intact into RN4220. Such plasmids were subsequently maintained in a stable manner. pDU1212 DNA was prepared from RN4220 and transformed into alpha-hemolytic S. aureus 8325-4 and two mutant derivatives defective in alpha-hemolysin synthesis. All three strains expressed alpha-hemolysin when harboring pDU1212. Images PMID:6411618

  11. Secretion of Alpha-Hemolysin by Escherichia coli Disrupts Tight Junctions in Ulcerative Colitis Patients

    PubMed Central

    Mirsepasi-Lauridsen, Hengameh Chloé; Du, Zhengyu; Struve, Carsten; Charbon, Godefroid; Karczewski, Jurgen; Krogfelt, Karen Angeliki; Petersen, Andreas Munk; Wells, Jerry M

    2016-01-01

    Objectives: The potential of Escherichia coli (E. coli) isolated from inflammatory bowel disease (IBD) patients to damage the integrity of the intestinal epithelium was investigated. Methods: E. coli strains isolated from patients with ulcerative colitis (UC) and healthy controls were tested for virulence capacity by molecular techniques and cytotoxic assays and transepithelial electric resistance (TER). E. coli isolate p19A was selected, and deletion mutants were created for alpha-hemolysin (α-hemolysin) (hly) clusters and cytotoxic necrotizing factor type 1 (cnf1). Probiotic E. coli Nissle and pathogenic E. coli LF82 were used as controls. Results: E. coli strains from patients with active UC completely disrupted epithelial cell tight junctions shortly after inoculation. These strains belong to phylogenetic group B2 and are all α-hemolysin positive. In contrast, probiotic E. coli Nissle, pathogenic E. coli LF82, four E. coli from patients with inactive UC and three E. coli strains from healthy controls did not disrupt tight junctions. E. coli p19A WT as well as cnf1, and single loci of hly mutants from cluster I and II were all able to damage Caco-2 (Heterogeneous human epithelial colorectal adenocarcinoma) cell tight junctions. However, this phenotype was lost in a mutant with knockout (Δ) of both hly loci (P<0.001). Conclusions: UC-associated E. coli producing α-hemolysin can cause rapid loss of tight junction integrity in differentiated Caco-2 cell monolayers. This effect was abolished in a mutant unable to express α-hemolysin. These results suggest that high Hly expression may be a mechanism by which specific strains of E. coli pathobionts can contribute to epithelial barrier dysfunction and pathophysiology of disease in IBD. PMID:26938480

  12. Prevalence of seroreactors to the 104-kilodalton hemolysin of Actinobacillus pleuropneumoniae in swine herds.

    PubMed Central

    Devenish, J; Rosendal, S; Bossé, J T; Wilkie, B N; Johnson, R

    1990-01-01

    A rabbit homologous polyclonal antiserum to the 104-kilodalton hemolysin of Actinobacillus pleuropneumoniae serotype 1 strain CM-5 was specifically produced and used in an antigen capture enzyme-linked immunosorbent assay (ELISA) to detect swine serum antibodies to this potentially important virulence factor. Sera from pigs experimentally infected with the most common disease-producing serotypes (serotypes 1, 2, 5, and 7) of A. pleuropneumoniae produced positive results in this ELISA. Of 144 serum samples collected from 10 herds free of pleuropneumonia and 155 serum samples from 11 herds with a history of the disease, 68 (47%) and 148 (95%), respectively, were found positive by the ELISA. In addition, pigs naturally infected with Actinobacillus suis produced antibodies which seroreacted in this ELISA. The results indicated that a high proportion of swine have antibodies seroreactive with the 104-kilodalton hemolysin produced by A. pleuropneumoniae. PMID:2332472

  13. Prelytic and lytic conformations of erythrocyte-associated Escherichia coli hemolysin.

    PubMed Central

    Moayeri, M; Welch, R A

    1997-01-01

    Flow cytometry was developed as a method to assess the conformation of erythrocyte-bound Escherichia coli hemolysin polypeptide (HlyA). Topology of membrane-associated hemolysin (HlyA(E)) was investigated by testing surface accessibility of HlyA regions in lytic and nonlytic bound states, using a panel of 12 anti-HlyA monoclonal antibodies (MAbs). Hemolysin associates nonlytically with erythrocytes at 0 to 2 degrees C. To test the hypothesis that the nonlytic HlyA(E) conformation at 0 to 2 degrees C differs from the lytic conformation at 23 degrees C, MAb epitope reactivity profiles at the two temperatures were compared by flow cytometry. Four MAbs have distinctly increased reactivity at 0 to 2 degrees C compared to 23 degrees C. HlyA requires HlyC-dependent acylation at lysine residues 563 and 689 for lytic function. Toxin with cysteine substitution mutations at each lysine (HlyA(K563C) and HlyA(K689C)) as well as the nonacylated form of hemolysin made in a HlyC-deficient strain were examined by flow cytometry at 0 to 2 and 23 degrees C. The three mutants bind erythrocytes at wild-type toxin levels, but there are conformational changes reflected by altered MAb epitope accessibility for six of the MAbs. To test further the surface accessibility of regions in the vicinity of MAb-reactive epitopes, HlyA(E) was proteolytically treated prior to testing for MAb reactivity. Differences in protease susceptibility at 0 to 2 degrees and 23 degrees C for the reactivities of three of the MAbs further support the model of two distinct conformations of cell-associated toxin. PMID:9169756

  14. Gal-Gal binding and hemolysin phenotypes and genotypes associated with uropathogenic Escherichia coli.

    PubMed

    O'Hanley, P; Low, D; Romero, I; Lark, D; Vosti, K; Falkow, S; Schoolnik, G

    1985-08-15

    To determine whether uropathogenic strains of Escherichia coli exhibit a distinctive constellation of phenotypes, we examined 44 urinary isolates from women with radiologically normal urinary tracts and pyelonephritis, cystitis, or asymptomatic bacteriuria and 73 fecal isolates from healthy control subjects. The strains were characterized by their O serogroup, by their binding specificity (as determined by adhesins), and by their production of hemolysin and colicin V. In addition, the strains were assessed for homologous gene sequences by means of DNA-hybridization probes prepared from cistrons that encode hemolysin and the Gal-Gal binding adhesin--two determinants of virulence, which cause tissue injury and promote bacterial colonization of uroepithelia, respectively. In contrast to most isolates from normal feces and from the urine of patients with asymptomatic bacteriuria, pyelonephritis strains belong to a small number of O serogroups; all express the Gal--Gal binding adhesin and 75 per cent are hemolytic. A gene probe for the Gal--Gal binding adhesin, derived from the chromosome of one strain from a patient with pyelonephritis, hybridized with the DNA of all other pyelonephritis strains. The probe for the hemolysin gene hybridized with DNA from all other hemolytic strains. These data indicate that most cases of pyelonephritis are due to a small number of pathogenic clones that express critical determinants of virulence, and that the nucleotide sequences for hemolysin and the Gal--Gal binding adhesin in heterologous strains share homology. We are tempted to speculate that the gene products of these shared regions of the genome might form the basis for a vaccine against pyelonephritis.

  15. Nucleobase recognition in ssDNA at the central constriction of the αhemolysin pore

    PubMed Central

    Stoddart, David; Heron, Andrew J.; Klingelhoefer, Jochen; Mikhailova, Ellina; Maglia, Giovanni; Bayley, Hagan

    2010-01-01

    Nanopores are under investigation for single-molecule DNA sequencing. The α-hemolysin (αHL) protein nanopore contains three recognition points capable of nucleobase discrimination in individual immobilized ssDNA molecules. We have modified the recognition point R1 by extensive mutagenesis of residue 113. Amino acids that provide an energy barrier to ion flow (e.g. bulky or hydrophobic residues) strengthen base identification, while amino acids that lower the barrier weaken it. Amino acids with related side chains produce similar patterns of nucleobase recognition providing a rationale for the redesign of recognition points. PMID:20704324

  16. Hemagglutinin, urease, and hemolysin production by Proteus mirabilis from clinical sources.

    PubMed

    Mobley, H L; Chippendale, G R

    1990-03-01

    Proteus mirabilis, a common cause of urinary tract infection, can lead to serious complications including pyelonephritis. Adherence factors, urease, and hemolysin may be virulence determinants. These factors were compared for bacteria cultured from 16 patients with acute pyelonephritis and 35 with catheter-associated bacteriuria and for 20 fecal isolates. Pyelonephritis isolates were more likely (P less than .05) to express the mannose-resistant/Proteus-like (MR/P) hemagglutinin in the absence of mannose-resistant/Klebsiella-like (MR/K) hemagglutinin than were catheter-associated or fecal isolates. Pyelonephritis isolates produced urease activity of 63 +/- 27 (mean +/- SD) mumol of NH3/min/mg of protein, not significantly different from catheter-associated or fecal isolates. Hybridization of Southern blots of P. mirabilis chromosomal DNA with two urease gene probes demonstrated that urease gene sequences were conserved in all isolates. Geometric mean of reciprocal hemolytic titers for pyelonephritis isolates was 27.9; for urinary catheter isolates, 18.0; and for fecal isolates, 55.7 (not significantly different, P greater than .1). Although in vivo expression of urease and hemolysin may not be reliable indexes of virulence, MR/P hemagglutination in the absence of MR/K hemagglutination may be necessary for development of pyelonephritis.

  17. Hemolysin-producing Listeria monocytogenes affects the immune response to T-cell-dependent and T-cell-independent antigens.

    PubMed Central

    Hage-Chahine, C M; Del Giudice, G; Lambert, P H; Pechere, J C

    1992-01-01

    A murine experimental infection with a hemolysin-producing (Hly+) strain of Listeria monocytogenes and a non-hemolysin-producing (Hly-) mutant was used as an in vivo model to evaluate the role of hemolysin production in the immune response. No antilisterial antibodies were detectable following sublethal infection with Hly+ bacteria, but consistent antilisterial immunoglobulin G (IgG) and IgM antibody production was observed following sublethal infection with the Hly- mutant. Hly+ but not Hly- L. monocytogenes induced transient inhibition of antibody response to Hly- bacteria and to unrelated T-cell-dependent (tetanus toxoid) and T-cell-independent (pneumococcal polysaccharide 3) antigens. Transient inhibition of the activation of an antigen-specific T-cell clone was also observed following Hly+ infection of antigen-presenting cells but not following Hly- infection. These results suggest that hemolysin production by L. monocytogenes is an important factor in modulating the immune response to T-cell-dependent and T-cell-independent antigens in infected individuals. Images PMID:1548067

  18. ADAM10 Cell Surface Expression but Not Activity Is Critical for Staphylococcus aureus α-Hemolysin-Mediated Activation of the NLRP3 Inflammasome in Human Monocytes

    PubMed Central

    Ezekwe, Ejiofor A.D.; Weng, Chengyu; Duncan, Joseph A.

    2016-01-01

    The Staphylococcus aureus toxin, α-hemolysin, is an important and well-studied virulence factor in staphylococcal infection. It is a soluble monomeric protein that, once secreted by the bacterium, forms a heptameric pore in the membrane of a broad range of host cell types. Hemolysin was recently discovered to bind and activate a disintegrin and metalloprotease 10 (ADAM10). In epithelial and endothelial cells, ADAM10 activation is required for the toxin’s activity against these cells. In host monocytic cells, α-hemolysin activates the nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing 3 (NLRP3) inflammasome leading to production of pro-inflammatory cytokines and cell death. We now show that ADAM10 is critical for α-hemolysin-mediated activation of the NLRP3 inflammasome in human monocytes as siRNA knockdown or chemical blockade of ADAM10-α-hemolysin interaction leads to diminished inflammasome activation and cell death by reducing the available ADAM10 on the cell surface. Unlike epithelial cell and endothelial cell damage, which requires α-hemolysin induced ADAM10 activation, ADAM10 protease activity was not required for NLRP3 inflammasome activation. This work confirms the importance of ADAM10 in immune activation by α-hemolysin, but indicates that host cell signal induction by the toxin is different between host cell types. PMID:27043625

  19. Impact of brief exposure to antifungal agents on the post-antifungal effect and hemolysin activity of oral Candida albicans

    PubMed Central

    ELLEPOLA, Arjuna Nishantha; KHAJAH, Rana; JAYATILAKE, Sumedha; SAMARANAYAKE, Lakshman; SHARMA, Prem; KHAN, Zia

    2015-01-01

    Post-antifungal effect (PAFE) of Candida and its production of hemolysin are determinants of candidal pathogenicity. Candida albicans is the foremost aetiological agent of oral candidosis, which can be treated with polyene, azole, and echinocandin antifungals. However, once administered, the intraoral concentrations of these drugs tend to be subtherapeutic and transient due to the diluent effect of saliva and cleansing effect of the oral musculature. Hence, intra-orally, Candida may undergo a brief exposure to antifungal drugs. Objective Therefore, the PAFE and hemolysin production of oral C. albicans isolates following brief exposure to sublethal concentrations of the foregoing antifungals were evaluated. Material and Methods A total of 50 C. albicans oral isolates obtained from smokers, diabetics, asthmatics using steroid inhalers, partial denture wearers and healthy individuals were exposed to sublethal concentrations of nystatin, amphotericin B, caspofungin, ketoconazole and fluconazole for 60 min. Thereafter, the drugs were removed and the PAFE and hemolysin production were determined by previously described turbidometric and plate assays, respectively. Results Nystatin, amphotericin B, caspofungin and ketoconazole induced mean PAFE (hours) of 2.2, 2.18, 2.2 and 0.62, respectively. Fluconazole failed to produce a PAFE. Hemolysin production of these isolates was suppressed with a percentage reduction of 12.27, 13.47, 13.33, 8.53 and 4.93 following exposure to nystatin, amphotericin B, caspofungin, ketoconazole and fluconazole, respectively. Conclusions Brief exposure to sublethal concentrations of antifungal drugs appears to exert an antifungal effect by interfering with the growth as well as hemolysin production of C. albicans. PMID:26398514

  20. Structure and Function of Thermostable Direct Hemolysin (TDH) from Vibrio Parahaemolyticus

    NASA Astrophysics Data System (ADS)

    Hashimoto, Hiroshi; Yamane, Tsutomu; Ikeguchi, Mitsunori; Nakahira, Kumiko; Yanagihara, Itaru

    Thermostable direct hemolysin (TDH) is a major virulence factor of Vibrio parahaemolyticus that causes pandemic food-borne enterocolitis mediated by seafood. TDH exists as a tetramer in solution, and it possesses extreme hemolytic activity. Here, we present the crystal structure of the TDH tetramer at 1.5 Å resolution. The TDH tetramer forms a central pore with dimensions of 23 Å in diameter and ∼50 Å in depth. π-cation interactions between protomers comprising the tetramer were indispensable for hemolytic activity of TDH. The N-terminal region was intrinsically disordered outside the pore. Molecular dynamics (MD) simulations suggested that water molecules permeate freely through the central and side channel pores. These findings imply a novel membrane attachment mechanism by a soluble tetrameric pore-forming toxin.

  1. Size-dependent forced PEG partitioning into channels: VDAC, OmpC, and α-hemolysin

    PubMed Central

    Aksoyoglu, M. Alphan; Podgornik, Rudolf; Bezrukov, Sergey M.; Gurnev, Philip A.; Muthukumar, Murugappan; Parsegian, V. Adrian

    2016-01-01

    Nonideal polymer mixtures of PEGs of different molecular weights partition differently into nanosize protein channels. Here, we assess the validity of the recently proposed theoretical approach of forced partitioning for three structurally different β-barrel channels: voltage-dependent anion channel from outer mitochondrial membrane VDAC, bacterial porin OmpC (outer membrane protein C), and bacterial channel-forming toxin α-hemolysin. Our interpretation is based on the idea that relatively less-penetrating polymers push the more easily penetrating ones into nanosize channels in excess of their bath concentration. Comparison of the theory with experiments is excellent for VDAC. Polymer partitioning data for the other two channels are consistent with theory if additional assumptions regarding the energy penalty of pore penetration are included. The obtained results demonstrate that the general concept of “polymers pushing polymers” is helpful in understanding and quantification of concrete examples of size-dependent forced partitioning of polymers into protein nanopores. PMID:27466408

  2. Itraconazole-resistant Candida auris with phospholipase, proteinase and hemolysin activity from a case of vulvovaginitis.

    PubMed

    Kumar, Dharmendra; Banerjee, Tuhina; Pratap, Chandra Bhan; Tilak, Ragini

    2015-04-15

    Since the emergence of pathogenic non-albicans Candida species, a number of new isolates have been added to the list. One such unusual species is Candida auris (C. auris), recently isolated and studied in few reports. In this study, a case of vulvovaginitis caused by Candida auris incidentally identified by molecular methods using internal transcribed spacer polymerase chain reaction (ITS PCR) is described. Antifungal susceptibility testing revealed the isolate to be resistant to itraconazole (MIC ≥ 2 µg/ml) and expressed important virulence factors including phospholipase, proteinase and hemolysin activity. The patient was successfully treated with oral fluconazole and did not have any invasive fungemia. Very few cases of this emerging pathogen have been reported. However, its isolation from clinical specimens reveals the significance of non-albicans candida species over C. albicans and the diversity of Candida spp causing infections.

  3. Isorhamnetin Attenuates Staphylococcus aureus-Induced Lung Cell Injury by Inhibiting Alpha-Hemolysin Expression.

    PubMed

    Jiang, Lanxiang; Li, Hongen; Wang, Laiying; Song, Zexin; Shi, Lei; Li, Wenhua; Deng, Xuming; Wang, Jianfeng

    2016-03-01

    Staphylococcus aureus, like other gram-positive pathogens, has evolved a large repertoire of virulence factors as a powerful weapon to subvert the host immune system, among which alpha-hemolysin (Hla), a secreted pore-forming cytotoxin, plays a preeminent role. We observed a concentration-dependent reduction in Hla production by S. aureus in the presence of sub-inhibitory concentrations of isorhamnetin, a flavonoid from the fruits of Hippophae rhamnoides L., which has little antibacterial activity. We further evaluate the effect of isorhamnetin on the transcription of the Hla-encoding gene hla and RNAIII, an effector molecule in the agr system. Isorhamnetin significantly down-regulated RNAIII expression and subsequently inhibited hla transcription. In a co-culture of S. aureus and lung cells, topical isorhamnetin treatment protected against S. aureus-induced cell injury. Isorhamnetin may represent a leading compound for the development of anti-virulence drugs against S. aureus infections. PMID:26643966

  4. Size-dependent forced PEG partitioning into channels: VDAC, OmpC, and α-hemolysin.

    PubMed

    Aksoyoglu, M Alphan; Podgornik, Rudolf; Bezrukov, Sergey M; Gurnev, Philip A; Muthukumar, Murugappan; Parsegian, V Adrian

    2016-08-01

    Nonideal polymer mixtures of PEGs of different molecular weights partition differently into nanosize protein channels. Here, we assess the validity of the recently proposed theoretical approach of forced partitioning for three structurally different β-barrel channels: voltage-dependent anion channel from outer mitochondrial membrane VDAC, bacterial porin OmpC (outer membrane protein C), and bacterial channel-forming toxin α-hemolysin. Our interpretation is based on the idea that relatively less-penetrating polymers push the more easily penetrating ones into nanosize channels in excess of their bath concentration. Comparison of the theory with experiments is excellent for VDAC. Polymer partitioning data for the other two channels are consistent with theory if additional assumptions regarding the energy penalty of pore penetration are included. The obtained results demonstrate that the general concept of "polymers pushing polymers" is helpful in understanding and quantification of concrete examples of size-dependent forced partitioning of polymers into protein nanopores. PMID:27466408

  5. Characterization of Interstrand DNA-DNA Cross-Links Using the α-Hemolysin Protein Nanopore.

    PubMed

    Zhang, Xinyue; Price, Nathan E; Fang, Xi; Yang, Zhiyu; Gu, Li-Qun; Gates, Kent S

    2015-12-22

    Nanopore-based sensors have been studied extensively as potential tools for DNA sequencing, characterization of epigenetic modifications such as 5-methylcytosine, and detection of microRNA biomarkers. In the studies described here, the α-hemolysin protein nanopore embedded in a lipid bilayer was used for the detection and characterization of interstrand cross-links in duplex DNA. Interstrand cross-links are important lesions in medicinal chemistry and toxicology because they prevent the strand separation that is required for read-out of genetic information from DNA in cells. In addition, interstrand cross-links are used for the stabilization of duplex DNA in structural biology and materials science. Cross-linked DNA fragments produced unmistakable current signatures in the nanopore experiment. Some cross-linked substrates gave irreversible current blocks of >10 min, while others produced long current blocks (10-100 s) before the double-stranded DNA cross-link translocated through the α-hemolysin channel in a voltage-driven manner. The duration of the current block for the different cross-linked substrates examined here may be dictated by the stability of the duplex region left in the vestibule of the nanopore following partial unzipping of the cross-linked DNA. Construction of calibration curves measuring the frequency of cross-link blocking events (1/τon) as a function of cross-link concentration enabled quantitative determination of the amounts of cross-linked DNA present in samples. The unique current signatures generated by cross-linked DNA in the α-HL nanopore may enable the detection and characterization of DNA cross-links that are important in toxicology, medicine, and materials science.

  6. Hybrid MD-Nernst Planck Model of Alpha-hemolysin Conductance Properties

    NASA Technical Reports Server (NTRS)

    Cozmuta, Ioana; O'Keefer, James T.; Bose, Deepak; Stolc, Viktor

    2006-01-01

    Motivated by experiments in which an applied electric field translocates polynucleotides through an alpha-hemolysin protein channel causing ionic current transient blockade, a hybrid simulation model is proposed to predict the conductance properties of the open channel. Time scales corresponding to ion permeation processes are reached using the Poisson-Nemst-Planck (PNP) electro-diffusion model in which both solvent and local ion concentrations are represented as a continuum. The diffusion coefficients of the ions (K(+) and Cl(-)) input in the PNP model are, however, calculated from all-atom molecular dynamics (MD). In the MD simulations, a reduced representation of the channel is used. The channel is solvated in a 1 M KCI solution, and an external electric field is applied. The pore specific diffusion coefficients for both ionic species are reduced 5-7 times in comparison to bulk values. Significant statistical variations (17-45%) of the pore-ions diffusivities are observed. Within the statistics, the ionic diffusivities remain invariable for a range of external applied voltages between 30 and 240mV. In the 2D-PNP calculations, the pore stem is approximated by a smooth cylinder of radius approx. 9A with two constriction blocks where the radius is reduced to approx. 6A. The electrostatic potential includes the contribution from the atomistic charges. The MD-PNP model shows that the atomic charges are responsible for the rectifying behaviour and for the slight anion selectivity of the a-hemolysin pore. Independent of the hierarchy between the anion and cation diffusivities, the anionic contribution to the total ionic current will dominate. The predictions of the MD-PNP model are in good agreement with experimental data and give confidence in the present approach of bridging time scales by combining a microscopic and macroscopic model.

  7. Molecular epidemiology of adhesin and hemolysin virulence factors among uropathogenic Escherichia coli.

    PubMed

    Arthur, M; Johnson, C E; Rubin, R H; Arbeit, R D; Campanelli, C; Kim, C; Steinbach, S; Agarwal, M; Wilkinson, R; Goldstein, R

    1989-02-01

    The pap, prs, pil, and hly operons of the pyelonephritic Escherichia coli isolate J96 code for the expression of P, F, and type 1 adhesins and the production of hemolysin, respectively; the afaI operon of the pyelonephritic E. coli KS52 encodes an X adhesin. Using different segments of these operons as probes, colony hybridizations were performed on 97 E. coli urinary tract and 40 fecal clinical isolates to determine (i) the presence in the infecting bacteria of nucleotide sequences related to virulence operons, and (ii) the phenotypic properties associated with such sequences. Coexpression of P and F adhesins encoded by pap-related sequences was detected more frequently among isolates from patients with pyelonephritis (32 of 49, 65%) than among those with cystitis (11 of 48, 23%; P less than 0.0001) or from fecal specimens (6 of 40, 15%; P less than 0.0001). Therefore, the expression of both adhesins appears to be critical in the colonization of the upper urinary tract. In contrast, afaI-related sequences were detected significantly more frequently among isolates from patients with cystitis, suggesting that this class of X adhesin may have a role in lower urinary tract infections. Urinary tract isolates differed from fecal isolates by a low incidence of type 1 adhesin expression among pil probe-positive isolates. hly-related sequences were only detected in pap probe-positive isolates. The frequency of hemolysin production among pap probe-positive isolates was not associated with a particular pattern of infection. The distribution of these virulence factors was similar in the presence or absence of reflux, indicating that structural abnormalities of the urinary tract did not facilitate colonization by adhesin-negative isolates.

  8. Molecular detection of HpmA and HlyA hemolysin of uropathogenic Proteus mirabilis.

    PubMed

    Cestari, Silvia Emanoele; Ludovico, Marilucia Santos; Martins, Fernando Henrique; da Rocha, Sérgio Paulo Dejato; Elias, Waldir Pereira; Pelayo, Jacinta Sanchez

    2013-12-01

    Urinary tract infection (UTI) is one of the bacterial infections frequently documented in humans. Proteus mirabilis is associated with UTI mainly in individuals with urinary tract abnormality or related with vesicular catheterism and it can be difficult to treat because of the formation of stones in the bladder and kidneys. These stones are formed due to the presence of urease synthesized by the bacteria. Another important factor is that P. mirabilis produces hemolysin HpmA, used by the bacteria to damage the kidney tissues. Proteus spp. samples can also express HlyA hemolysin, similar to that found in Escherichia coli. A total of 211 uropathogenic P. mirabilis isolates were analyzed to detect the presence of the hpmA and hpmB genes by the techniques of polymerase chain reaction (PCR) and dot blot and hlyA by PCR. The hpmA and hpmB genes were expressed by the RT-PCR technique and two P. mirabilis isolates were sequenced for the hpmA and hpmB genes. The presence of the hpmA and hpmB genes was confirmed by PCR in 205 (97.15 %) of the 211 isolates. The dot blot confirmed the presence of the hpmA and hpmB genes in the isolates that did not amplify in the PCR. None of the isolates studied presented the hlyA gene. The hpmA and hpmB genes that were sequenced presented 98 % identity with the same genes of the HI4320 P. mirabilis sample. This study showed that the PCR technique has good sensitivity for detecting the hpmA and hpmB genes of P. mirabilis. PMID:23884594

  9. Antibody-forming cells and serum hemolysin responses of pastel and sapphire mink inoculated with Aleutian disease virus.

    PubMed

    Lodmell, D L; Bergman, R K; Hadlow, W J

    1973-11-01

    The effect of Aleutian disease virus (ADV) on serum hemolysin titers and antibody-forming cells in lymph nodes and spleens of sapphire and pastel mink inoculated with goat erythrocytes (G-RBC) was investigated. ADV injected 1 day after primary antigenic stimulation with G-RBC did not depress the immune responses of either color phase for a period of 26 days. However, when G-RBC were injected 47 days after ADV, both the number of antibody-forming cells and hemolysin titers were more markedly depressed in sapphire than in pastel mink. The results are discussed in relation to the greater susceptibility of sapphire mink and the variable susceptibility of pastel mink to the Pullman isolate of ADV.

  10. Antibody-Forming Cells and Serum Hemolysin Responses of Pastel and Sapphire Mink Inoculated with Aleutian Disease Virus

    PubMed Central

    Lodmell, Donald L.; Bergman, R. Kaye; Hadlow, William J.

    1973-01-01

    The effect of Aleutian disease virus (ADV) on serum hemolysin titers and antibody-forming cells in lymph nodes and spleens of sapphire and pastel mink inoculated with goat erythrocytes (G-RBC) was investigated. ADV injected 1 day after primary antigenic stimulation with G-RBC did not depress the immune responses of either color phase for a period of 26 days. However, when G-RBC were injected 47 days after ADV, both the number of antibody-forming cells and hemolysin titers were more markedly depressed in sapphire than in pastel mink. The results are discussed in relation to the greater susceptibility of sapphire mink and the variable susceptibility of pastel mink to the Pullman isolate of ADV. PMID:4584051

  11. Single-molecule study of thymidine glycol and i-motif through the alpha-hemolysin ion channel

    NASA Astrophysics Data System (ADS)

    He, Lidong

    Nanopore-based devices have emerged as a single-molecule detection and analysis tool for a wide range of applications. Through electrophoretically driving DNA molecules across a nanosized pore, a lot of information can be received, including unfolding kinetics and DNA-protein interactions. This single-molecule method has the potential to sequence kilobase length DNA polymers without amplification or labeling, approaching "the third generation" genome sequencing for around $1000 within 24 hours. alpha-Hemolysin biological nanopores have the advantages of excellent stability, low-noise level, and precise site-directed mutagenesis for engineering this protein nanopore. The first work presented in this thesis established the current signal of the thymidine glycol lesion in DNA oligomers through an immobilization experiment. The thymidine glycol enantiomers were differentiated from each other by different current blockage levels. Also, the effect of bulky hydrophobic adducts to the current blockage was investigated. Secondly, the alpha-hemolysin nanopore was used to study the human telomere i-motif and RET oncogene i-motif at a single-molecule level. In Chapter 3, it was demonstrated that the alpha-hemolysin nanopore can differentiate an i-motif form and single-strand DNA form at different pH values based on the same sequence. In addition, it shows potential to differentiate the folding topologies generated from the same DNA sequence.

  12. Indirect enzyme-linked immunosorbent assay for detection of antibody to a 110,000-molecular-weight hemolysin of Actinobacillus pleuropneumoniae.

    PubMed Central

    Ma, J N; Inzana, T J

    1990-01-01

    An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect swine antibody to a 110,000-molecular-weight hemolysin (110K hemolysin) of Actinobacillus pleuropneumoniae. Affinity-purified rabbit polyclonal or mouse monoclonal immunoglobulin G to the hemolysin of A. pleuropneumoniae serotype 5 strain J45, followed by hemolysin-rich concentrated culture supernatant, was used to bind swine antibody to hemolysin to microdilution plates. Sixty-nine serum samples from swine that were clinically normal, presented with clinical evidence of pleuropneumonia, were experimentally immunized or challenged, or were free of pleuropneumonia were tested, and their ELISA titers were compared with complement fixation (CF) titers. On the basis of serum samples from swine that were clinically normal and negative by CF, an ELISA titer of 1:320 or greater was considered positive. In comparison with CF, the sensitivity of the ELISA was 98.1% and the specificity was 90%. The two samples negative by CF and positive by indirect ELISA were, however, also positive for antibody to serotype 5 capsule by ELISA. Immunization of normal pigs with whole cells or purified hemolysin boosted titers 4- to 128-fold within 4 weeks. Immunoblotting demonstrated that the affinity-purified immunoglobulin G to hemolysin used for capture in the assay recognized only a 110K protein of A. pleuropneumoniae serotypes 1 to 7, although the reactivity was quantitatively variable between serotypes. Therefore, the indirect ELISA is capable of identifying animals infected with or exposed to most, if not all, serotypes of A. pleuropneumoniae. If an indirect ELISA titer of 1:320 or greater is considered positive, the assay can be a valuable diagnostic tool in both clinical and research laboratories. Images PMID:2380363

  13. Alpha-synuclein lipid-dependent membrane binding and translocation through the α-hemolysin channel.

    PubMed

    Gurnev, Philip A; Yap, Thai Leong; Pfefferkorn, Candace M; Rostovtseva, Tatiana K; Berezhkovskii, Alexander M; Lee, Jennifer C; Parsegian, V Adrian; Bezrukov, Sergey M

    2014-02-01

    Gauging the interactions of a natively unfolded Parkinson disease-related protein, alpha-synuclein (α-syn) with membranes and its pathways between and within cells is important for understanding its pathogenesis. Here, to address these questions, we use a robust β-barrel channel, α-hemolysin, reconstituted into planar lipid bilayers. Transient, ~95% blockage of the channel current by α-syn was observed when 1), α-syn was added from the membrane side where the shorter (stem) part of the channel is exposed; and 2), the applied potential was lower on the side of α-syn addition. While the on-rate of α-syn binding to the channel strongly increased with the applied field, the off-rate displayed a turnover behavior. Statistical analysis suggests that at voltages >50 mV, a significant fraction of the α-syn molecules bound to the channel undergoes subsequent translocation. The observed on-rate varied by >100 times depending on the bilayer lipid composition. Removal of the last 25 amino acids from the highly negatively charged C-terminal of α-syn resulted in a significant decrease in the binding rates. Taken together, these results demonstrate that β-barrel channels may serve as sensitive probes of α-syn interactions with membranes as well as model systems for studies of channel-assisted protein transport.

  14. Alpha-Synuclein Lipid-Dependent Membrane Binding and Translocation through the α-Hemolysin Channel

    PubMed Central

    Gurnev, Philip A.; Yap, Thai Leong; Pfefferkorn, Candace M.; Rostovtseva, Tatiana K.; Berezhkovskii, Alexander M.; Lee, Jennifer C.; Parsegian, V. Adrian; Bezrukov, Sergey M.

    2014-01-01

    Gauging the interactions of a natively unfolded Parkinson disease-related protein, alpha-synuclein (α-syn) with membranes and its pathways between and within cells is important for understanding its pathogenesis. Here, to address these questions, we use a robust β-barrel channel, α-hemolysin, reconstituted into planar lipid bilayers. Transient, ∼95% blockage of the channel current by α-syn was observed when 1), α-syn was added from the membrane side where the shorter (stem) part of the channel is exposed; and 2), the applied potential was lower on the side of α-syn addition. While the on-rate of α-syn binding to the channel strongly increased with the applied field, the off-rate displayed a turnover behavior. Statistical analysis suggests that at voltages >50 mV, a significant fraction of the α-syn molecules bound to the channel undergoes subsequent translocation. The observed on-rate varied by >100 times depending on the bilayer lipid composition. Removal of the last 25 amino acids from the highly negatively charged C-terminal of α-syn resulted in a significant decrease in the binding rates. Taken together, these results demonstrate that β-barrel channels may serve as sensitive probes of α-syn interactions with membranes as well as model systems for studies of channel-assisted protein transport. PMID:24507596

  15. Fluctuating bottleneck model studies on kinetics of DNA escape from α-hemolysin nanopores.

    PubMed

    Bian, Yukun; Wang, Zilin; Chen, Anpu; Zhao, Nanrong

    2015-11-14

    We have proposed a fluctuation bottleneck (FB) model to investigate the non-exponential kinetics of DNA escape from nanometer-scale pores. The basic idea is that the escape rate is proportional to the fluctuating cross-sectional area of DNA escape channel, the radius r of which undergoes a subdiffusion dynamics subjected to fractional Gaussian noise with power-law memory kernel. Such a FB model facilitates us to obtain the analytical result of the averaged survival probability as a function of time, which can be directly compared to experimental results. Particularly, we have applied our theory to address the escape kinetics of DNA through α-hemolysin nanopores. We find that our theoretical framework can reproduce the experimental results very well in the whole time range with quite reasonable estimation for the intrinsic parameters of the kinetics processes. We believe that FB model has caught some key features regarding the long time kinetics of DNA escape through a nanopore and it might provide a sound starting point to study much wider problems involving anomalous dynamics in confined fluctuating channels. PMID:26567685

  16. Auto-Assembling Detoxified Staphylococcus aureus Alpha-Hemolysin Mimicking the Wild-Type Cytolytic Toxin.

    PubMed

    Fiaschi, Luigi; Di Palo, Benedetta; Scarselli, Maria; Pozzi, Clarissa; Tomaszewski, Kelly; Galletti, Bruno; Nardi-Dei, Vincenzo; Arcidiacono, Letizia; Mishra, Ravi P N; Mori, Elena; Pallaoro, Michele; Falugi, Fabiana; Torre, Antonina; Fontana, Maria Rita; Soriani, Marco; Bubeck Wardenburg, Juliane; Grandi, Guido; Rappuoli, Rino; Ferlenghi, Ilaria; Bagnoli, Fabio

    2016-06-01

    Staphylococcus aureus alpha-hemolysin (Hla) assembles into heptameric pores on the host cell membrane, causing lysis, apoptosis, and junction disruption. Herein, we present the design of a newly engineered S. aureus alpha-toxin, HlaPSGS, which lacks the predicted membrane-spanning stem domain. This protein is able to form heptamers in aqueous solution in the absence of lipophilic substrata, and its structure, obtained by transmission electron microscopy and single-particle reconstruction analysis, resembles the cap of the wild-type cytolytic Hla pore. HlaPSGS was found to be impaired in binding to host cells and to its receptor ADAM10 and to lack hemolytic and cytotoxic activity. Immunological studies using human sera as well as sera from mice convalescent from S. aureus infection suggested that the heptameric conformation of HlaPSGS mimics epitopes exposed by the cytolytic Hla pore during infection. Finally, immunization with this newly engineered Hla generated high protective immunity against staphylococcal infection in mice. Overall, this study provides unprecedented data on the natural immune response against Hla and suggests that the heptameric HlaPSGS is a highly valuable vaccine candidate against S. aureus.

  17. In vivo quantification of the secretion rates of the hemolysin A Type I secretion system.

    PubMed

    Lenders, Michael H H; Beer, Tobias; Smits, Sander H J; Schmitt, Lutz

    2016-01-01

    Type 1 secretion systems (T1SS) of Gram-negative bacteria secrete a broad range of substrates into the extracellular space. Common to all substrates is a C-terminal secretion sequence and nonapeptide repeats in the C-terminal part that bind Ca(2+) in the extracellular space, to trigger protein folding. Like all T1SS, the hemolysin A (HlyA) T1SS of Escherichia coli consists of an ABC transporter, a membrane fusion protein and an outer membrane protein allowing the one step translocation of the substrate across both membranes. Here, we analyzed the secretion rate of the HlyA T1SS. Our results demonstrate that the rate is independent of substrate-size and operates at a speed of approximately 16 amino acids per transporter per second. We also demonstrate that the rate is independent of the extracellular Ca(2+) concentration raising the question of the driving force of substrate secretion by T1SS in general. PMID:27616645

  18. Bacterial Delivery of Staphylococcus aureus α-Hemolysin Causes Regression and Necrosis in Murine Tumors

    PubMed Central

    St. Jean, Adam T; Swofford, Charles A; Panteli, Jan T; Brentzel, Zachary J; Forbes, Neil S

    2014-01-01

    Bacterial therapies, designed to manufacture therapeutic proteins directly within tumors, could eliminate cancers that are resistant to other therapies. To be effective, a payload protein must be secreted, diffuse through tissue, and efficiently kill cancer cells. To date, these properties have not been shown for a single protein. The gene for Staphylococcus aureus α-hemolysin (SAH), a pore-forming protein, was cloned into Escherichia coli. These bacteria were injected into tumor-bearing mice and volume was measured over time. The location of SAH relative to necrosis and bacterial colonies was determined by immunohistochemistry. In culture, SAH was released and killed 93% of cancer cells in 24 hours. Injection of SAH-producing bacteria reduced viable tissue to 9% of the original tumor volume. By inducing cell death, SAH moved the boundary of necrosis toward the tumor edge. SAH diffused 6.8 ± 0.3 µm into tissue, which increased the volume of affected tissue from 48.6 to 3,120 µm3. A mathematical model of molecular transport predicted that SAH efficacy is primarily dependent on colony size and the rate of protein production. As a payload protein, SAH will enable effective bacterial therapy because of its ability to diffuse in tissue, kill cells, and expand tumor necrosis. PMID:24590046

  19. Auto-Assembling Detoxified Staphylococcus aureus Alpha-Hemolysin Mimicking the Wild-Type Cytolytic Toxin

    PubMed Central

    Fiaschi, Luigi; Di Palo, Benedetta; Scarselli, Maria; Pozzi, Clarissa; Tomaszewski, Kelly; Galletti, Bruno; Nardi-Dei, Vincenzo; Arcidiacono, Letizia; Mishra, Ravi P. N.; Mori, Elena; Pallaoro, Michele; Falugi, Fabiana; Torre, Antonina; Fontana, Maria Rita; Soriani, Marco; Bubeck Wardenburg, Juliane; Grandi, Guido; Rappuoli, Rino

    2016-01-01

    Staphylococcus aureus alpha-hemolysin (Hla) assembles into heptameric pores on the host cell membrane, causing lysis, apoptosis, and junction disruption. Herein, we present the design of a newly engineered S. aureus alpha-toxin, HlaPSGS, which lacks the predicted membrane-spanning stem domain. This protein is able to form heptamers in aqueous solution in the absence of lipophilic substrata, and its structure, obtained by transmission electron microscopy and single-particle reconstruction analysis, resembles the cap of the wild-type cytolytic Hla pore. HlaPSGS was found to be impaired in binding to host cells and to its receptor ADAM10 and to lack hemolytic and cytotoxic activity. Immunological studies using human sera as well as sera from mice convalescent from S. aureus infection suggested that the heptameric conformation of HlaPSGS mimics epitopes exposed by the cytolytic Hla pore during infection. Finally, immunization with this newly engineered Hla generated high protective immunity against staphylococcal infection in mice. Overall, this study provides unprecedented data on the natural immune response against Hla and suggests that the heptameric HlaPSGS is a highly valuable vaccine candidate against S. aureus. PMID:27030589

  20. Hybrid pore formation by directed insertion of alpha hemolysin into solid-state nanopores

    PubMed Central

    Hall, Adam R.; Scott, Andrew; Rotem, Dvir; Mehta, Kunal K.; Bayley, Hagan; Dekker, Cees

    2011-01-01

    Nanopores hold great potential for genomic screening and sequencing technologies. Thus far, most studies have concentrated on the Staphylococcus aureus pore-forming protein alpha hemolysin (αHL)1 and artificial pores in solid-state (SS) membranes2. While biological pores offer an atomically precise structure3 and genetic engineering potential4, SS-pores offer durability, size and shape control5 and integratability. Each system, however, also has significant limitations: αHL is difficult to integrate because it relies on delicate lipid bilayers for mechanical support, and the fabrication of SS-pores at precise dimensions remains challenging. Here we show that these limitations may be overcome by inserting a single αHL pore into a SS-nanopore. A double-stranded DNA attached to a protein pore is threaded into a SS-nanopore by electrophoretic translocation. Protein insertion is observed in 30-40% of our attempts and translocation of single-stranded DNA demonstrates that the hybrid nanopore remains functional. The resulting hybrid structure offers a platform to create wafer-scale device arrays for genomic analysis including sequencing6. PMID:21113160

  1. Increased sensitivity to staphylococcal beta hemolysins of erythrocytes from chickens during aflatoxicosis.

    PubMed

    Doerr, J A; Huff, W E; Hamilton, P B

    1987-12-01

    The size of the zones of beta-hemolysis surrounding staphylococcal colonies on blood agar was found to be related to the level of dietary aflatoxin consumed by the chickens donating the blood. Zone sizes on blood from chickens fed the highest level of aflatoxin (10 micrograms/g of diet) were about six-fold larger than those on blood from control birds. The percentage of staphylococcal isolates displaying beta-hemolysis was increased from about 15% in normal blood to about 90% in blood from chickens fed aflatoxin (10 micrograms/g) whereas the time required for detection of beta-hemolysis was decreased by about one-half. The hemolytic activity of purified staphylococcal beta-hemolysin against suspensions of washed erythrocytes increased as the level of aflatoxin consumed by the donor chickens increased. These data imply a new mechanism for enhanced susceptibility of animals to infectious agents during mycotoxicoses whereby the animal is made more sensitive to the virulence factors of pathogens.

  2. Phospholipase C (heat-labile hemolysin) of Pseudomonas aeruginosa: purification and preliminary characterization.

    PubMed Central

    Berka, R M; Vasil, M L

    1982-01-01

    Phospholipase C (heat-labile hemolysin) was purified from Pseudomonas aeruginosa culture supernatants to near homogeneity by ammonium sulfate precipitation followed by a novel application of DEAE-Sephacel chromatography. Enzymatic activity remained associated with DEAE-Sephacel even in the presence of 1 M NaCl, but was eluted with a linear gradient of 0 to 5% tetradecyltrimethylammonium bromide. Elution from DEAE-Sephacel was also obtained with 2% lysophosphatidylcholine, and to a lesser extent with 2% phosphorylcholine, but not at all with choline. The enzyme was highly active toward phospholipids possessing substituted ammonium groups (e.g., phosphatidycholine, lysophosphatidylcholine, and sphingomyelin); however, it had little if any activity toward phospholipids lacking substituted ammonium groups (e.g., phosphatidylethanolamine, phosphatidylserine, and phosphaditylglycerol). Collectively, these data suggest that phospholipase C from P. aeruginosa exhibits high affinity for substituted ammonium groups, but requires an additional hydrophobic moiety for optimum binding. The specific activity of the purified enzyme preparation increased 1,900-fold compared with that of culture supernatants. The molecular weight of the phospholipase C was estimated to be 78,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Sephacryl S-200 column chromatography and was 76,000 by high-performance size exclusion chromatography. The isoelectric point was 5.5. Amino acid analysis showed that phospholipase C was rich in glycine, serine, threonine, aspartyl, glutamyl, and aromatic amino acids, but was cystine free. Images PMID:6811552

  3. Fluctuating bottleneck model studies on kinetics of DNA escape from α-hemolysin nanopores

    NASA Astrophysics Data System (ADS)

    Bian, Yukun; Wang, Zilin; Chen, Anpu; Zhao, Nanrong

    2015-11-01

    We have proposed a fluctuation bottleneck (FB) model to investigate the non-exponential kinetics of DNA escape from nanometer-scale pores. The basic idea is that the escape rate is proportional to the fluctuating cross-sectional area of DNA escape channel, the radius r of which undergoes a subdiffusion dynamics subjected to fractional Gaussian noise with power-law memory kernel. Such a FB model facilitates us to obtain the analytical result of the averaged survival probability as a function of time, which can be directly compared to experimental results. Particularly, we have applied our theory to address the escape kinetics of DNA through α-hemolysin nanopores. We find that our theoretical framework can reproduce the experimental results very well in the whole time range with quite reasonable estimation for the intrinsic parameters of the kinetics processes. We believe that FB model has caught some key features regarding the long time kinetics of DNA escape through a nanopore and it might provide a sound starting point to study much wider problems involving anomalous dynamics in confined fluctuating channels.

  4. Driven diffusion against electrostatic or effective energy barrier across α-hemolysin

    SciTech Connect

    Ansalone, Patrizio; Chinappi, Mauro; Rondoni, Lamberto; Cecconi, Fabio

    2015-10-21

    We analyze the translocation of a charged particle across an α-Hemolysin (αHL) pore in the framework of a driven diffusion over an extended energy barrier generated by the electrical charges of the αHL. A one-dimensional electrostatic potential is extracted from the full 3D solution of the Poisson’s equation. We characterize the particle transport under the action of a constant forcing by studying the statistics of the translocation time. We derive an analytical expression of translocation time average that compares well with the results from Brownian dynamic simulations of driven particles over the electrostatic potential. Moreover, we show that the translocation time distributions can be perfectly described by a simple theory which replaces the true barrier by an equivalent structureless square barrier. Remarkably, our approach maintains its accuracy also for low-applied voltage regimes where the usual inverse-Gaussian approximation fails. Finally, we discuss how the comparison between the simulated time distributions and their theoretical prediction results to be greatly simplified when using the notion of the empirical Laplace transform technique.

  5. Lectin, hemolysin and protease inhibitors in seed fractions with ovicidal activity against Haemonchus contortus.

    PubMed

    Salles, Hévila Oliveira; Braga, Ana Carolina Linhares; Nascimento, Maria Thayana dos Santos Canuto do; Sousa, Ana Márjory Paiva; Lima, Adriano Rodrigues; Vieira, Luiz da Silva; Cavalcante, Antônio Cézar Rocha; Egito, Antonio Silvio do; Andrade, Lúcia Betânia da Silva

    2014-01-01

    Bioactive molecules of plant species are promising alternatives for the chemical control of gastrointestinal nematodes in ruminants. Extracts of native and exotic seed species from Brazil's semi-arid region were tested in vitro in an egg hatch assay and the bioactivity of their proteins was investigated. Each seed species was subjected to three extractions with three types of solvents. All the seeds showed ovicidal activity, which varied according to the solvents. Higher ovicidal activity was found in the molecule fractions of low molecular weight (<12 kDa) for Albizia lebbeck, Ipomoea asarifolia, Jatropha curcas, Libidibia ferrea, Moringa oleifera and Ricinus communis (P<0.05, Bonferroni test). The two fractions of Crotalaria spectabilis showed the same ovicidal activity (P>0.05, Bonferroni test). Hemagglutinating activity was detected in the fractions of C. spectabilis and M. oleifera fractions, hemolysin activity in the A. lebbeck and M. oleifera fractions, serine protease inhibitory activity in the A. lebbeck, I. asarifolia, J. curcas, M. oleifera and R. communis fractions, cysteine protease inhibitor activity in the M. oleifera fraction, and no protein activity in the L. ferrea fraction. The results of this work reveal new plant species with a potential for use in controlling nematode parasites in goats, thus opening a new field of research involving plant protein molecules with ovicidal properties. PMID:25054490

  6. Electroosmosis through α-Hemolysin That Depends on Alkali Cation Type.

    PubMed

    Piguet, Fabien; Discala, Francoise; Breton, Marie-France; Pelta, Juan; Bacri, Laurent; Oukhaled, Abdelghani

    2014-12-18

    We demonstrate experimentally the existence of an electroosmotic flow (EOF) through the wild-type nanopore of α-hemolysin in a large range of applied voltages and salt concentrations for two different salts, LiCl and KCl. EOF controls the entry frequency and residence time of small neutral molecules (β-cyclodextrins, βCD) in the nanopore. The strength of EOF depends on the applied voltage, on the salt concentration, and, interestingly, on the nature of the cations in solution. In particular, EOF is stronger in the presence of LiCl than KCl. We interpret our results with a simple theoretical model that takes into account the pore selectivity and the solvation of ions. A stronger EOF in the presence of LiCl is found to originate essentially in a stronger anionic selectivity of the pore. Our work provides a new and easy way to control EOF in protein nanopores, without resorting to chemical modifications of the pore. PMID:26273988

  7. Potent leukocidal action of Escherichia coli hemolysin mediated by permeabilization of target cell membranes

    PubMed Central

    1989-01-01

    The contribution of Escherichia coli hemolysin (ECH) to bacterial virulence has been considered mainly in context with its hemolytic properties. We here report that this prevalent bacterial cytolysin is the most potent leukocidin known to date. Very low concentrations (approximately 1 ng/ml) of ECH evoke membrane permeability defects in PMN (2-10 x 10(6) cells/ml) leading to an efflux of cellular ATP and influx of propidium iodide. The attacked cells do not appear to repair the membrane lesions. Human serum albumin, high density and low density lipoprotein, and IgG together protect erythrocytes and platelets against attack by even high doses (5-25 micrograms/ml) of ECH. In contrast, PMN are still permeabilized by ECH at low doses (50-250 ng/ml) in the presence of these plasma inactivators. Thus, PMN become preferred targets for attack by ECH in human blood and protein-rich body fluids. Kinetic studies demonstrate that membrane permeabilization is a rapid process, ATP-release commencing within seconds after application of toxin to leukocytes. It is estimated that membrane permeabilization ensues upon binding of approximately 300 molecules ECH/PMN. This process is paralleled by granule exocytosis, and by loss of phagocytic killing capacity of the cells. The recognition that ECH directly counteracts a major immune defence mechanism of the human organism through its attack on granulocytes under physiological conditions sheds new light on its possible role and potential importance as a virulence factor of E. coli. PMID:2538544

  8. In vivo quantification of the secretion rates of the hemolysin A Type I secretion system

    PubMed Central

    Lenders, Michael H. H.; Beer, Tobias; Smits, Sander H. J.; Schmitt, Lutz

    2016-01-01

    Type 1 secretion systems (T1SS) of Gram-negative bacteria secrete a broad range of substrates into the extracellular space. Common to all substrates is a C-terminal secretion sequence and nonapeptide repeats in the C-terminal part that bind Ca2+ in the extracellular space, to trigger protein folding. Like all T1SS, the hemolysin A (HlyA) T1SS of Escherichia coli consists of an ABC transporter, a membrane fusion protein and an outer membrane protein allowing the one step translocation of the substrate across both membranes. Here, we analyzed the secretion rate of the HlyA T1SS. Our results demonstrate that the rate is independent of substrate-size and operates at a speed of approximately 16 amino acids per transporter per second. We also demonstrate that the rate is independent of the extracellular Ca2+ concentration raising the question of the driving force of substrate secretion by T1SS in general. PMID:27616645

  9. Identification of the Serratia marcescens hemolysin determinant by cloning into Escherichia coli

    SciTech Connect

    Braun, V.; Neuss, B.; Ruan, Y.; Schiebel, E.; Schoeffler, H.; Jander, G.

    1987-05-01

    A cosmid bank of Serratia marcescens was established from which DNA fragments were cloned into the plasmid pBR322, which conferred the chromosomally encoded hemolytic activity to Escherichia coli K-12. By transposon mutagenesis with Tn1000 and Tn5 IS50/sub L/::phoA (TnphoA), the coding region was assigned to a DNA fragment, designated hyl, comprising approximately 7 kilobases. Two proteins with molecular weights of 61,000 (61K protein) and 160,000 (160K protein) were expressed by the pBR322 derivatives and by a plasmid which contained the hly genes under the control of a phage T7 promoter and the T7 RNA polymerase. When strongly overexpressed the 160K protein was released by E. coli cells into the extracellular medium concomitant with hemolytic activity. The genes encoding the 61K and the 160K proteins were transcribed in the same direction. Mutants expressing a 160K protein truncated at the carboxyl-terminal end were partially hemolytic. Hemolysis was progressively inhibited by saccharides with increasing molecular weights from maltotriose (M/sub r/ 504) to maltoheptaose (M/sub r/ 1152) and as totally abolished by dextran 4 (M/sub r/ 4000). This result and the observed influx of (/sup 14/C)sucrose into erythrocytes in the presence of hemolytic E. coli transformants under osmotically protective conditions suggest the formation of defined transmembrane channels by the hemolysin.

  10. Iron Regulates Expression of Bacillus cereus Hemolysin II via Global Regulator Fur

    PubMed Central

    Shadrin, Andrey; Rodikova, Ekaterina A.; Andreeva-Kovalevskaya, Zhanna I.; Protsenko, Alexey S.; Mayorov, Sergey G.; Galaktionova, Darya Yu; Magelky, Erica

    2012-01-01

    The capacity of pathogens to respond to environmental signals, such as iron concentration, is key to bacterial survival and establishment of a successful infection. Bacillus cereus is a widely distributed bacterium with distinct pathogenic properties. Hemolysin II (HlyII) is one of its pore-forming cytotoxins and has been shown to be involved in bacterial pathogenicity in a number of cell and animal models. Unlike many other B. cereus pathogenicity factors, HlyII is not regulated by pleiotropic transcriptional regulator PlcR but is controlled by its own regulator, HlyIIR. Using a combination of in vivo and in vitro techniques, we show that hlyII expression is also negatively regulated by iron by the global regulator Fur via direct interaction with the hlyII promoter. DNase I footprinting and in vitro transcription experiments indicate that Fur prevents RNA polymerase binding to the hlyII promoter. HlyII expression profiles demonstrate that both HlyIIR and Fur regulate HlyII expression in a concerted fashion, with the effect of Fur being maximal in the early stages of bacterial growth. In sum, these results show that Fur serves as a transcriptional repressor for hlyII expression. PMID:22522892

  11. Python erythrocytes are resistant to α-hemolysin from Escherichia coli.

    PubMed

    Larsen, Casper K; Skals, Marianne; Wang, Tobias; Cheema, Muhammad U; Leipziger, Jens; Praetorius, Helle A

    2011-12-01

    α-Hemolysin (HlyA) from Escherichia coli lyses mammalian erythrocytes by creating nonselective cation pores in the membrane. Pore insertion triggers ATP release and subsequent P2X receptor and pannexin channel activation. Blockage of either P2X receptors or pannexin channels reduces HlyA-induced hemolysis. We found that erythrocytes from Python regius and Python molurus are remarkably resistant to HlyA-induced hemolysis compared to human and Trachemys scripta erythrocytes. HlyA concentrations that induced maximal hemolysis of human erythrocytes did not affect python erythrocytes, but increasing the HlyA concentration 40-fold did induce hemolysis. Python erythrocytes were more resistant to osmotic stress than human erythrocytes, but osmotic stress tolerance per se did not confer HlyA resistance. Erythrocytes from T. scripta, which showed higher osmotic resistance than python erythrocytes, were as susceptible to HlyA as human erythrocytes. Therefore, we tested whether python erythrocytes lack the purinergic signalling known to amplify HlyA-induced hemolysis in human erythrocytes. P. regius erythrocytes increased intracellular Ca²⁺ concentration and reduced cell volume when exposed to 3 mM ATP, indicating the presence of a P2X₇-like receptor. In addition, scavenging extracellular ATP or blocking P2 receptors or pannexin channels reduced the HlyA-induced hemolysis. We tested whether the low HlyA sensitivity resulted from low affinity of HlyA to the python erythrocyte membrane. We found comparable incorporation of HlyA into human and python erythrocyte membranes. Taken together, the remarkable HlyA resistance of python erythrocytes was not explained by increased osmotic resistance, lack of purinergic hemolysis amplification, or differences in HlyA affinity.

  12. Acylation of Escherichia coli Hemolysin: A Unique Protein Lipidation Mechanism Underlying Toxin Function

    PubMed Central

    Stanley, Peter; Koronakis, Vassilis; Hughes, Colin

    1998-01-01

    The pore-forming hemolysin (HlyA) of Escherichia coli represents a unique class of bacterial toxins that require a posttranslational modification for activity. The inactive protoxin pro-HlyA is activated intracellularly by amide linkage of fatty acids to two internal lysine residues 126 amino acids apart, directed by the cosynthesized HlyC protein with acyl carrier protein as the fatty acid donor. This action distinguishes HlyC from all bacterial acyltransferases such as the lipid A, lux-specific, and nodulation acyltransferases, and from eukaryotic transferases such as N-myristoyl transferases, prenyltransferases, and thioester palmitoyltransferases. Most lipids directly attached to proteins may be classed as N-terminal amide-linked and internal ester-linked acyl groups and C-terminal ether-linked isoprenoid groups. The acylation of HlyA and related toxins does not equate to these but does appear related to a small number of eukaryotic proteins that include inflammatory cytokines and mitogenic and cholinergic receptors. While the location and structure of lipid moieties on proteins vary, there are common effects on membrane affinity and/or protein-protein interactions. Despite being acylated at two residues, HlyA does not possess a “double-anchor” motif and does not have an electrostatic switch, although its dependence on calcium binding for activity suggests that the calcium-myristoyl switch may have relevance. The acyl chains on HlyA may provide anchorage points onto the surface of the host cell lipid bilayer. These could then enhance protein-protein interactions either between HlyA and components of a host signal transduction pathway to influence cytokine production or between HlyA monomers to bring about oligomerization during pore formation. PMID:9618444

  13. Host Response Signature to Staphylococcus aureus Alpha-Hemolysin Implicates Pulmonary Th17 Response

    PubMed Central

    Zhou, Tong; Moreno-Vinasco, Liliana; Hollett, Brian; Garcia, Joe G. N.

    2012-01-01

    Staphylococcus aureus pneumonia causes significant morbidity and mortality. Alpha-hemolysin (Hla), a pore-forming cytotoxin of S. aureus, has been identified through animal models of pneumonia as a critical virulence factor that induces lung injury. In spite of considerable molecular knowledge of how this cytotoxin injures the host, the precise host response to Hla in the context of infection remains poorly understood. We employed whole-genome expression profiling of infected lungs to define the host response to wild-type S. aureus compared with the response to an Hla-deficient isogenic mutant in experimental pneumonia. These data provide a complete expression profile at 4 and at 24 h postinfection, revealing a unique response to the toxin-expressing strain. Gene ontogeny analysis revealed significant differences in the extracellular matrix and cardiomyopathy pathways, both of which govern cellular interactions in the tissue microenvironment. Evaluation of individual transcript responses to Hla-secreting staphylococci was notable for upregulation of host cytokine and chemokine genes, including the p19 subunit of interleukin-23. Consistent with this observation, the cellular immune response to infection was characterized by a prominent Th17 response to the wild-type pathogen. These findings define specific host mRNA responses to Hla-producing S. aureus, coupling the pulmonary Th17 response to the secretion of this cytotoxin. Expression profiling to define the host response to a single virulence factor proved to be a valuable tool in identifying pathways for further investigation in S. aureus pneumonia. This approach may be broadly applicable to the study of bacterial toxins, defining host pathways that can be targeted to mitigate toxin-induced disease. PMID:22733574

  14. Secretion and activation of the Serratia marcescens hemolysin by structurally defined ShlB mutants.

    PubMed

    Pramanik, Avijit; Könninger, Ulrich; Selvam, Arun; Braun, Volkmar

    2014-05-01

    The ShlA hemolysin of Serratia marcescens is secreted across the outer membrane by the ShlB protein; ShlB belongs to the two-partner secretion system (type Vb), a subfamily of the Omp85 outer membrane protein assembly and secretion superfamily. During secretion, ShlA is converted from an inactive non-hemolytic form into an active hemolytic form. The structure of ShlB is predicted to consist of the N-terminal α-helix H1, followed by the two polypeptide-transport-associated domains POTRA P1 and P2, and the β-barrel of 16 β-strands. H1 is inserted into the pore of the β-barrel in the outer membrane; P1 and P2 are located in the periplasm. To obtain insights into the secretion and activation of ShlA by ShlB, we isolated ShlB mutants impaired in secretion and/or activation. The triple H1 P1 P2 mutant did not secrete ShlA. The P1 and P2 deletion derivatives secreted reduced amounts of ShlA, of which P1 showed some hemolysis, whereas P2 was inactive. Deletion of loop 6 (L6), which is conserved among exporters of the Omp85 family, compromised activation but retained low secretion. Secretion-negative mutants generated by random mutagenesis were located in loop 6. The inactive secreted ShlA derivatives were complemented in vitro to active ShlA by an N-terminal ShlA fragment (ShlA242) secreted by ShlB. Deletion of H1 did not impair secretion of hemolytic ShlA. The study defines domains of ShlB which are important for ShlA secretion and activation.

  15. Voltage-controlled insertion of single α-hemolysin and Mycobacterium smegmatis nanopores into lipid bilayer membranes

    NASA Astrophysics Data System (ADS)

    Renner, Stephan; Bessonov, Andrey; Simmel, Friedrich C.

    2011-02-01

    One of the prerequisites for single molecule nanopore translocation experiments is the availability of a single nanopore embedded into a lipid bilayer membrane. Using two alternative experimental setups, microdroplets, and a classical planar lipid bilayer setup, we here show that at near-neutral pH and high salt concentration the incorporation rates of the pore-forming proteins α-hemolysin and porin A from Mycobacterium smegmatis are exponentially enhanced at elevated voltages. This fact can be utilized to establish an experimental procedure by which a voltage-controlled insertion of single pores into lipid membranes can be achieved.

  16. Five birds, one stone: neutralization of α-hemolysin and 4 bi-component leukocidins of Staphylococcus aureus with a single human monoclonal antibody.

    PubMed

    Rouha, Harald; Badarau, Adriana; Visram, Zehra C; Battles, Michael B; Prinz, Bianka; Magyarics, Zoltán; Nagy, Gábor; Mirkina, Irina; Stulik, Lukas; Zerbs, Manuel; Jägerhofer, Michaela; Maierhofer, Barbara; Teubenbacher, Astrid; Dolezilkova, Ivana; Gross, Karin; Banerjee, Srijib; Zauner, Gerhild; Malafa, Stefan; Zmajkovic, Jakub; Maier, Sabine; Mabry, Robert; Krauland, Eric; Wittrup, K Dane; Gerngross, Tillman U; Nagy, Eszter

    2015-01-01

    Staphylococcus aureus is a major human pathogen associated with high mortality. The emergence of antibiotic resistance and the inability of antibiotics to counteract bacterial cytotoxins involved in the pathogenesis of S. aureus call for novel therapeutic approaches, such as passive immunization with monoclonal antibodies (mAbs). The complexity of staphylococcal pathogenesis and past failures with single mAb products represent considerable barriers for antibody-based therapeutics. Over the past few years, efforts have focused on neutralizing α-hemolysin. Recent findings suggest that the concerted actions of several cytotoxins, including the bi-component leukocidins play important roles in staphylococcal pathogenesis. Therefore, we aimed to isolate mAbs that bind to multiple cytolysins by employing high diversity human IgG1 libraries presented on the surface of yeast cells. Here we describe cross-reactive antibodies with picomolar affinity for α-hemolysin and 4 different bi-component leukocidins that share only ∼26% overall amino acid sequence identity. The molecular basis of cross-reactivity is the recognition of a conformational epitope shared by α-hemolysin and F-components of gamma-hemolysin (HlgAB and HlgCB), LukED and LukSF (Panton-Valentine Leukocidin). The amino acids predicted to form the epitope are conserved and known to be important for cytotoxic activity. We found that a single cross-reactive antibody prevented lysis of human phagocytes, epithelial and red blood cells induced by α-hemolysin and leukocidins in vitro, and therefore had superior effectiveness compared to α-hemolysin specific antibodies to protect from the combined cytolytic effect of secreted S. aureus toxins. Such mAb afforded high levels of protection in murine models of pneumonia and sepsis.

  17. Five birds, one stone: Neutralization of α-hemolysin and 4 bi-component leukocidins of Staphylococcus aureus with a single human monoclonal antibody

    PubMed Central

    Rouha, Harald; Badarau, Adriana; Visram, Zehra C; Battles, Michael B; Prinz, Bianka; Magyarics, Zoltán; Nagy, Gábor; Mirkina, Irina; Stulik, Lukas; Zerbs, Manuel; Jägerhofer, Michaela; Maierhofer, Barbara; Teubenbacher, Astrid; Dolezilkova, Ivana; Gross, Karin; Banerjee, Srijib; Zauner, Gerhild; Malafa, Stefan; Zmajkovic, Jakub; Maier, Sabine; Mabry, Robert; Krauland, Eric; Wittrup, K Dane; Gerngross, Tillman U; Nagy, Eszter

    2015-01-01

    Staphylococcus aureus is a major human pathogen associated with high mortality. The emergence of antibiotic resistance and the inability of antibiotics to counteract bacterial cytotoxins involved in the pathogenesis of S. aureus call for novel therapeutic approaches, such as passive immunization with monoclonal antibodies (mAbs). The complexity of staphylococcal pathogenesis and past failures with single mAb products represent considerable barriers for antibody-based therapeutics. Over the past few years, efforts have focused on neutralizing α-hemolysin. Recent findings suggest that the concerted actions of several cytotoxins, including the bi-component leukocidins play important roles in staphylococcal pathogenesis. Therefore, we aimed to isolate mAbs that bind to multiple cytolysins by employing high diversity human IgG1 libraries presented on the surface of yeast cells. Here we describe cross-reactive antibodies with picomolar affinity for α-hemolysin and 4 different bi-component leukocidins that share only ∼26% overall amino acid sequence identity. The molecular basis of cross-reactivity is the recognition of a conformational epitope shared by α-hemolysin and F-components of gamma-hemolysin (HlgAB and HlgCB), LukED and LukSF (Panton-Valentine Leukocidin). The amino acids predicted to form the epitope are conserved and known to be important for cytotoxic activity. We found that a single cross-reactive antibody prevented lysis of human phagocytes, epithelial and red blood cells induced by α-hemolysin and leukocidins in vitro, and therefore had superior effectiveness compared to α-hemolysin specific antibodies to protect from the combined cytolytic effect of secreted S. aureus toxins. Such mAb afforded high levels of protection in murine models of pneumonia and sepsis. PMID:25523282

  18. Five birds, one stone: neutralization of α-hemolysin and 4 bi-component leukocidins of Staphylococcus aureus with a single human monoclonal antibody.

    PubMed

    Rouha, Harald; Badarau, Adriana; Visram, Zehra C; Battles, Michael B; Prinz, Bianka; Magyarics, Zoltán; Nagy, Gábor; Mirkina, Irina; Stulik, Lukas; Zerbs, Manuel; Jägerhofer, Michaela; Maierhofer, Barbara; Teubenbacher, Astrid; Dolezilkova, Ivana; Gross, Karin; Banerjee, Srijib; Zauner, Gerhild; Malafa, Stefan; Zmajkovic, Jakub; Maier, Sabine; Mabry, Robert; Krauland, Eric; Wittrup, K Dane; Gerngross, Tillman U; Nagy, Eszter

    2015-01-01

    Staphylococcus aureus is a major human pathogen associated with high mortality. The emergence of antibiotic resistance and the inability of antibiotics to counteract bacterial cytotoxins involved in the pathogenesis of S. aureus call for novel therapeutic approaches, such as passive immunization with monoclonal antibodies (mAbs). The complexity of staphylococcal pathogenesis and past failures with single mAb products represent considerable barriers for antibody-based therapeutics. Over the past few years, efforts have focused on neutralizing α-hemolysin. Recent findings suggest that the concerted actions of several cytotoxins, including the bi-component leukocidins play important roles in staphylococcal pathogenesis. Therefore, we aimed to isolate mAbs that bind to multiple cytolysins by employing high diversity human IgG1 libraries presented on the surface of yeast cells. Here we describe cross-reactive antibodies with picomolar affinity for α-hemolysin and 4 different bi-component leukocidins that share only ∼26% overall amino acid sequence identity. The molecular basis of cross-reactivity is the recognition of a conformational epitope shared by α-hemolysin and F-components of gamma-hemolysin (HlgAB and HlgCB), LukED and LukSF (Panton-Valentine Leukocidin). The amino acids predicted to form the epitope are conserved and known to be important for cytotoxic activity. We found that a single cross-reactive antibody prevented lysis of human phagocytes, epithelial and red blood cells induced by α-hemolysin and leukocidins in vitro, and therefore had superior effectiveness compared to α-hemolysin specific antibodies to protect from the combined cytolytic effect of secreted S. aureus toxins. Such mAb afforded high levels of protection in murine models of pneumonia and sepsis. PMID:25523282

  19. The Staphylococcal Toxins γ-Hemolysin AB and CB Differentially Target Phagocytes by Employing Specific Chemokine Receptors

    PubMed Central

    Spaan, András N.; Vrieling, Manouk; Wallet, Pierre; Badiou, Cédric; Reyes-Robles, Tamara; Ohneck, Elizabeth A.; Benito, Yvonne; de Haas, Carla J.C.; Day, Christopher J.; Jennings, Michael P.; Lina, Gérard; Vandenesch, François; van Kessel, Kok P.M.; Torres, Victor J.; van Strijp, Jos A.G.; Henry, Thomas

    2014-01-01

    Evasion of the host phagocyte response by Staphylococcus aureus is crucial to successful infection with the pathogen. γ-Hemolysin AB and CB (HlgAB, HlgCB) are bicomponent pore-forming toxins present in almost all human S. aureus isolates. Cellular tropism and contribution of the toxins to S. aureus pathophysiology are poorly understood. Here, we identify the chemokine receptors CXCR1, CXCR2 and CCR2 as targets for HlgAB, and the complement receptors C5aR and C5L2 as targets for HlgCB. The receptor expression patterns allow the toxins to efficiently and differentially target phagocytic cells. Murine neutrophils are resistant to HlgAB and HlgCB. CCR2 is the sole murine receptor orthologue compatible with γ-Hemolysin. In a murine peritonitis model, HlgAB contributes to S. aureus bacteremia in a CCR2-dependent manner. HlgAB-mediated targeting of CCR2+ cells highlights the involvement of inflammatory macrophages during S. aureus infection. Functional quantification identifies HlgAB and HlgCB as major secreted staphylococcal leukocidins. PMID:25384670

  20. Hemolysin coregulated protein 1 as a molecular gluing unit for the assembly of nanoparticle hybrid structures.

    PubMed

    Pham, Tuan Anh; Schreiber, Andreas; Sturm Née Rosseeva, Elena V; Schiller, Stefan; Cölfen, Helmut

    2016-01-01

    Hybrid nanoparticle (NP) structures containing organic building units such as polymers, peptides, DNA and proteins have great potential in biosensor and electronic applications. The nearly free modification of the polymer chain, the variation of the protein and DNA sequence and the implementation of functional moieties provide a great platform to create inorganic structures of different morphology, resulting in different optical and magnetic properties. Nevertheless, the design and modification of a protein structure with functional groups or sequences for the assembly of biohybrid materials is not trivial. This is mainly due to the sensitivity of its secondary, tertiary and quaternary structure to the changes in the interaction (e.g., hydrophobic, hydrophilic, electrostatic, chemical groups) between the protein subunits and the inorganic material. Here, we use hemolysin coregulated protein 1 (Hcp1) from Pseudomonas aeruginosa as a building and gluing unit for the formation of biohybrid structures by implementing cysteine anchoring points at defined positions on the protein rim (Hcp1_cys3). We successfully apply the Hcp1_cys3 gluing unit for the assembly of often linear, hybrid structures of plasmonic gold (Au NP), magnetite (Fe3O4 NP), and cobalt ferrite nanoparticles (CoFe2O4 NP). Furthermore, the assembly of Au NPs into linear structures using Hcp1_cys3 is investigated by UV-vis spectroscopy, TEM and cryo-TEM. One key parameter for the formation of Au NP assembly is the specific ionic strength in the mixture. The resulting network-like structure of Au NPs is characterized by Raman spectroscopy, showing surface-enhanced Raman scattering (SERS) by a factor of 8·10(4) and a stable secondary structure of the Hcp1_cys3 unit. In order to prove the catalytic performance of the gold hybrid structures, they are used as a catalyst in the reduction reaction of 4-nitrophenol showing similar catalytic activity as the pure Au NPs. To further extend the functionality of the

  1. Hemolysin of enterohemorrhagic Escherichia coli: structure, transport, biological activity and putative role in virulence.

    PubMed

    Bielaszewska, Martina; Aldick, Thomas; Bauwens, Andreas; Karch, Helge

    2014-07-01

    Enterohemorrhagic Escherichia coli (EHEC) cause diarrhea, bloody diarrhea and hemolytic-uremic syndrome (HUS), a thrombotic microangiopathy affecting the renal glomeruli, the intestine, and the brain. The pathogenesis of EHEC-mediated diseases is incompletely understood. In addition to Shiga toxins, the major virulence factors of EHEC, the contribution of EHEC hemolysin (EHEC-Hly), also designated EHEC toxin (Ehx), which is a member of the RTX (repeats-in-toxin) family, is increasingly recognized. The toxin and its activation and secretion machinery are encoded by the EHEC-hlyCABD operon, in which EHEC-hlyA is the structural gene for EHEC-Hly and the EHEC-hlyC product mediates post-translational activation of EHEC-Hly; the EHEC-hlyB- and EHEC-hlyD-encoded proteins form, together with genetically unlinked TolC, the type I secretion system that transports EHEC-Hly out of the bacterial cell. EHEC-Hly exists in two biologically active forms: as a free EHEC-Hly, and an EHEC-Hly associated with outer membrane vesicles (OMVs) that are released by EHEC during growth. The OMV-associated form results from a rapid binding of free EHEC-Hly to OMVs upon its extracellular secretion. The OMV association stabilizes EHEC-Hly and thus substantially prolongs its hemolytic activity compared to the free toxin. The two EHEC-Hly forms differ by their mechanism of toxicity toward human intestinal epithelial and microvascular endothelial cells, which are the major targets during EHEC infection. The free EHEC-Hly lyses human microvascular endothelial cells, presumably by pore formation in the cell membrane. In contrast, the OMV-associated EHEC-Hly does not lyse any of these cell types, but after its cellular internalization via OMVs it targets mitochondria and triggers caspase-9-mediated apoptosis. The proinflammatory potential of EHEC-Hly, in particular its ability to elicit secretion of interleukin-1β from human monocytes/macrophages, might be an additional mechanism of its putative

  2. Hemolysin coregulated protein 1 as a molecular gluing unit for the assembly of nanoparticle hybrid structures

    PubMed Central

    Pham, Tuan Anh; Schreiber, Andreas; Sturm (née Rosseeva), Elena V

    2016-01-01

    Summary Hybrid nanoparticle (NP) structures containing organic building units such as polymers, peptides, DNA and proteins have great potential in biosensor and electronic applications. The nearly free modification of the polymer chain, the variation of the protein and DNA sequence and the implementation of functional moieties provide a great platform to create inorganic structures of different morphology, resulting in different optical and magnetic properties. Nevertheless, the design and modification of a protein structure with functional groups or sequences for the assembly of biohybrid materials is not trivial. This is mainly due to the sensitivity of its secondary, tertiary and quaternary structure to the changes in the interaction (e.g., hydrophobic, hydrophilic, electrostatic, chemical groups) between the protein subunits and the inorganic material. Here, we use hemolysin coregulated protein 1 (Hcp1) from Pseudomonas aeruginosa as a building and gluing unit for the formation of biohybrid structures by implementing cysteine anchoring points at defined positions on the protein rim (Hcp1_cys3). We successfully apply the Hcp1_cys3 gluing unit for the assembly of often linear, hybrid structures of plasmonic gold (Au NP), magnetite (Fe3O4 NP), and cobalt ferrite nanoparticles (CoFe2O4 NP). Furthermore, the assembly of Au NPs into linear structures using Hcp1_cys3 is investigated by UV–vis spectroscopy, TEM and cryo-TEM. One key parameter for the formation of Au NP assembly is the specific ionic strength in the mixture. The resulting network-like structure of Au NPs is characterized by Raman spectroscopy, showing surface-enhanced Raman scattering (SERS) by a factor of 8·104 and a stable secondary structure of the Hcp1_cys3 unit. In order to prove the catalytic performance of the gold hybrid structures, they are used as a catalyst in the reduction reaction of 4-nitrophenol showing similar catalytic activity as the pure Au NPs. To further extend the functionality

  3. Characterization of Plp, a phosphatidylcholine-specific phospholipase and hemolysin of Vibrio anguillarum

    PubMed Central

    2013-01-01

    Background Vibrio anguillarum is the causative agent of vibriosis in fish. Several extracellular proteins secreted by V. anguillarum have been shown to contribute to virulence. While two hemolysin gene clusters, vah1-plp and rtxACHBDE, have been previously identified and described, the activities of the protein encoded by the plp gene were not known. Here we describe the biochemical activities of the plp-encoded protein and its role in pathogenesis. Results The plp gene, one of the components in vah1 cluster, encodes a 416-amino-acid protein (Plp), which has homology to lipolytic enzymes containing the catalytic site amino acid signature SGNH. Hemolytic activity of the plp mutant increased 2-3-fold on sheep blood agar indicating that plp represses vah1; however, hemolytic activity of the plp mutant decreased by 2-3-fold on fish blood agar suggesting that Plp has different effects against erythrocytes from different species. His6-tagged recombinant Plp protein (rPlp) was over-expressed in E. coli. Purified and re-folded active rPlp exhibited phospholipase A2 activity against phosphatidylcholine and no activity against phosphatidylserine, phosphatidylethanolamine, or sphingomyelin. Characterization of rPlp revealed broad optimal activities at pH 5–9 and at temperatures of 30-64°C. Divalent cations and metal chelators did not affect activity of rPlp. We also demonstrated that Plp was secreted using thin layer chromatography and immunoblot analysis. Additionally, rPlp had strong hemolytic activity towards rainbow trout erythrocytes, but not to sheep erythrocytes suggesting that rPlp is optimized for lysis of phosphatidylcholine-rich fish erythrocytes. Further, only the loss of the plp gene had a significant effect on hemolytic activity of culture supernatant on fish erythrocytes, while the loss of rtxA and/or vah1 had little effect. However, V. anguillarum strains with mutations in plp or in plp and vah1 exhibited no significant reduction in virulence compared to

  4. Evaluation of an immunochromatographic assay for direct identification of thermostable direct hemolysin-producing Vibrio parahaemolyticus colonies on selective agar plates.

    PubMed

    Kawatsu, Kentaro; Sakata, Junko; Yonekita, Taro; Kumeda, Yuko

    2015-12-01

    We evaluated the utility of an immunochromatographic assay (NH IC TDH) in identifying thermostable direct hemolysin (TDH)-producing Vibrio parahaemolyticus colonies on selective agar plates. The sensitivity of the NH IC TDH assay was 100% (189 samples) and its specificity was 100% (41 samples) compared with the presence of tdh.

  5. A Single Residue Change in Vibrio harveyi Hemolysin Results in the Loss of Phospholipase and Hemolytic Activities and Pathogenicity for Turbot (Scophthalmus maximus)▿

    PubMed Central

    Sun, Boguang; Zhang, Xiao-Hua; Tang, Xuexi; Wang, Shushan; Zhong, Yingbin; Chen, Jixiang; Austin, Brian

    2007-01-01

    Vibrio harveyi hemolysin, an important virulence determinant in fish pathogenesis, was further characterized, and the enzyme was identified as a phospholipase B by gas chromatography. Site-directed mutagenesis revealed that a specific residue, Ser153, was critical for its enzymatic activity and for its virulence in fish. PMID:17220231

  6. Urea facilitates the translocation of single-stranded DNA and RNA through the alpha-hemolysin nanopore.

    PubMed

    Japrung, Deanpen; Henricus, Marsiyana; Li, Qiuhong; Maglia, Giovanni; Bayley, Hagan

    2010-05-19

    The staphylococcal alpha-hemolysin (alphaHL) protein nanopore is under investigation as a fast, cheap detector for nucleic acid analysis and sequencing. Although discrimination of all four bases of DNA by the alphaHL pore has been demonstrated, analysis of single-stranded DNAs and RNAs containing secondary structure mediated by basepairing is prevented because these nucleic acids cannot be translocated through the pore. Here, we show that a structured 95-nucleotide single-stranded DNA and its RNA equivalent are translocated through the alphaHL pore in the presence of 4 M urea, a concentration that denatures the secondary structure of the polynucleotides. The alphaHL pore is functional even in 7 M urea, and therefore it is easily stable enough for analyses of challenging DNA and RNA species. PMID:20441749

  7. Staphylococcus aureus Hemolysins, bi-component Leukocidins, and Cytolytic Peptides: A Redundant Arsenal of Membrane-Damaging Virulence Factors?

    PubMed Central

    Vandenesch, François; Lina, G.; Henry, Thomas

    2012-01-01

    One key aspect of the virulence of Staphylococcus aureus lies in its ability to target the host cell membrane with a large number of membrane-damaging toxins and peptides. In this review, we describe the hemolysins, the bi-component leukocidins (which include the Panton Valentine leukocidin, LukAB/GH, and LukED), and the cytolytic peptides (phenol soluble modulins). While at first glance, all of these factors might appear redundant, it is now clear that some of these factors play specific roles in certain S. aureus life stages and diseases or target specific cell types or species. In this review, we present an update of the literature on toxin receptors and their cell type and species specificities. Furthermore, we review epidemiological studies and animal models illustrating the role of these membrane-damaging factors in various diseases. Finally, we emphasize the interplay of these factors with the host immune system and highlight all their non-lytic functions. PMID:22919604

  8. Detection of benzo[a]pyrene-guanine adducts in single-stranded DNA using the α-hemolysin nanopore.

    PubMed

    Perera, Rukshan T; Fleming, Aaron M; Johnson, Robert P; Burrows, Cynthia J; White, Henry S

    2015-02-20

    The carcinogenic precursor benzo[a]pyrene (BP), a polycyclic aromatic hydrocarbon, is released into the environment through the incomplete combustion of hydrocarbons. Metabolism of BP in the human body yields a potent alkylating agent (benzo[a]pyrene diol epoxide, BPDE) that reacts with guanine (G) in DNA to form an adduct implicated in cancer initiation. We report that the α-hemolysin (αHL) nanopore platform can be used to detect a BPDE adduct to G in synthetic oligodeoxynucleotides. Translocation of a 41-mer poly-2'-deoxycytidine strand with a centrally located BPDE adduct to G through αHL in 1 M KCl produces a unique multi-level current signature allowing the adduct to be detected. This readily distinguishable current modulation was observed when the BPDE-adducted DNA strand translocated from either the 5' or 3' directions. This study suggests that BPDE adducts and other large aromatic biomarkers can be detected with αHL, presenting opportunities for the monitoring, quantification, and sequencing of mutagenic compounds from cellular DNA samples.

  9. Detection of benzo[a]pyrene-guanine adducts in single-stranded DNA using the α-hemolysin nanopore

    NASA Astrophysics Data System (ADS)

    Perera, Rukshan T.; Fleming, Aaron M.; Johnson, Robert P.; Burrows, Cynthia J.; White, Henry S.

    2015-02-01

    The carcinogenic precursor benzo[a]pyrene (BP), a polycyclic aromatic hydrocarbon, is released into the environment through the incomplete combustion of hydrocarbons. Metabolism of BP in the human body yields a potent alkylating agent (benzo[a]pyrene diol epoxide, BPDE) that reacts with guanine (G) in DNA to form an adduct implicated in cancer initiation. We report that the α-hemolysin (αHL) nanopore platform can be used to detect a BPDE adduct to G in synthetic oligodeoxynucleotides. Translocation of a 41-mer poly-2‧-deoxycytidine strand with a centrally located BPDE adduct to G through αHL in 1 M KCl produces a unique multi-level current signature allowing the adduct to be detected. This readily distinguishable current modulation was observed when the BPDE-adducted DNA strand translocated from either the 5‧ or 3‧ directions. This study suggests that BPDE adducts and other large aromatic biomarkers can be detected with αHL, presenting opportunities for the monitoring, quantification, and sequencing of mutagenic compounds from cellular DNA samples.

  10. Curcumin protects mice from Staphylococcus aureus pneumonia by interfering with the self-assembly process of α-hemolysin

    PubMed Central

    Wang, Jianfeng; Zhou, Xuan; Li, Wenhua; Deng, Xuming; Deng, Yanhong; Niu, Xiaodi

    2016-01-01

    α-hemolysin (Hla) is a self-assembling extracellular protein secreted as a soluble monomer by most Staphylococcus aureus strains and is an essential virulence factor for the pathogenesis of various S. aureus infections. Here, we show that curcumin (CUR), a natural compound with weak anti-S. aureus activity, can inhibit the hemolysis induced by Hla. Molecular dynamics simulations, free energy calculations, and mutagenesis assays were further employed for the Hla-CUR complex to determine the mechanism of such inhibition. The analysis of this combined approach indicated that the direct binding CUR to Hla blocks the conformational transition of Hla from the monomer to the oligomer, leading to an inhibition of Hla hemolytic activity. We also found that the addition of CUR significantly attenuated Hla-mediated injury of human alveolar cell (A549) co-cultured with S. aureus. The in vivo data further demonstrated that treatment with CUR protects mice from pneumonia caused by S. aureus, including methicillin-resistant strains (MRSA). These findings suggest that CUR inhibits the pore-forming activity of Hla through a novel mechanism, which would pave the way for the development of new and more effective antibacterial agents to combat S. aureus pneumonia. PMID:27345357

  11. Genome-wide CRISPR screen reveals novel host factors required for Staphylococcus aureus α-hemolysin-mediated toxicity

    PubMed Central

    Virreira Winter, Sebastian; Zychlinsky, Arturo; Bardoel, Bart W.

    2016-01-01

    Staphylococcus aureus causes a wide variety of infections and antibiotic resistant strains are a major problem in hospitals. One of the best studied virulence factors of S. aureus is the pore-forming toxin alpha hemolysin (αHL) whose mechanism of action is incompletely understood. We performed a genome-wide loss-of-function screen using CRISPR/Cas9 technology to identify host targets required for αHL susceptibility in human myeloid cells. We found gRNAs for ten genes enriched after intoxication with αHL and focused on the top five hits. Besides a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), the host receptor for αHL, we identified three proteins, Sys1 golgi trafficking protein (SYS1), ADP-ribosylation factor 1 (ARFRP1), and tetraspanin-14 (TSPAN14) which regulate the presentation of ADAM10 on the plasma membrane post-translationally. Interestingly, we also showed that cells lacking sphingomyelin synthase 1 (SGMS1) resist αHL intoxication, but have only a slightly reduced ADAM10 surface expression. SGMS1 regulates lipid raft formation, suggesting that αHL requires these membrane microdomains for attachment and cytotoxicity. PMID:27066838

  12. Phloretin derived from apple can reduce alpha-hemolysin expression in methicillin-resistant Staphylococcus aureus USA300.

    PubMed

    Zhou, Xuan; Liu, Shui; Li, Wenhua; Zhang, Bing; Liu, Bowen; Liu, Yan; Deng, Xuming; Peng, Liping

    2015-08-01

    Methicillin-resistant Staphylococcus aureus (MRSA) has become increasingly important because it is the most common cause of hospital-acquired infections, which have become globally epidemic. Our study specifically focused on the MRSA strain USA300, which was shown in 2014 to be responsible for the most current pandemic of highly virulent MRSA in the United States. We aimed to evaluate the in vitro effect of phloretin on USA300. Susceptibility testing, western blotting assays, hemolysis assays and real-time RT-PCR were employed to examine the in vitro effects of phloretin on alpha-hemolysin (Hla) production when the bacterium was co-cultured with phloretin. The protective effect of phloretin against the USA300-mediated injury of human alveolar epithelial cells (A549) was tested using the live/dead analysis and cytotoxicity assays. We showed that sub-inhibitory concentrations of phloretin have no effect on bacterial viability; however, they can markedly inhibit the production of Hla in culture supernatants and the transcriptional levels of hla (the gene encoding Hla) and agrA (the accessory gene regulator). Phloretin, at a final concentration of 16 µg/ml, could protect A549 cells from injury caused by USA300 in the co-culture system. Our study suggests that phloretin might have a potential application in the development of treatment for MRSA infections. PMID:26026280

  13. Existence of a new protein component with the same function as the LukF component of leukocidin or gamma-hemolysin and its gene in Staphylococcus aureus P83.

    PubMed

    Choorit, W; Kaneko, J; Muramoto, K; Kamio, Y

    1995-01-01

    Staphylococcal toxins, leukocidin and gamma-hemolysin, consist of two protein components, i.e. LukF and LukS for leukocodin and H gamma I and H gamma II for gamma-hemolysin. From a culture fluid of Staphylococcus aureus strain P83, a new protein component of leukocidin or gamma-hemolysin which was designated as LukM was isolated. This component showed the same biological activity as that of LukF component for leukocidin or H gamma I component of gamma-hemolysin in combination with LukS or H gamma II. However, the LukM component cross-reacted with the anti-LukS antibodies but not with the anti-LukF antibodies. On the basis of chemical analysis of the LukM component and the cloning and nucleotide sequencing of the lukM gene of S. aureus P83, we have demonstrated that LukM is an entirely new protein component of leukocidin or gamma-hemolysin. The deduced amino acid sequence of LukM from the lukM gene showed 66.7% and 67% identity to that of LukS and H gamma II, respectively. However, the amino acid sequence of LukM and LukF showed only 29% homology.

  14. Enterohemorrhagic Escherichia coli Hemolysin Employs Outer Membrane Vesicles to Target Mitochondria and Cause Endothelial and Epithelial Apoptosis

    PubMed Central

    Kunsmann, Lisa; Greune, Lilo; Bauwens, Andreas; Zhang, Wenlan; Kuczius, Thorsten; Kim, Kwang Sik; Mellmann, Alexander; Schmidt, M. Alexander; Karch, Helge

    2013-01-01

    Enterohemorrhagic Escherichia coli (EHEC) strains cause diarrhea and hemolytic uremic syndrome resulting from toxin-mediated microvascular endothelial injury. EHEC hemolysin (EHEC-Hly), a member of the RTX (repeats-in-toxin) family, is an EHEC virulence factor of increasingly recognized importance. The toxin exists as free EHEC-Hly and as EHEC-Hly associated with outer membrane vesicles (OMVs) released by EHEC during growth. Whereas the free toxin is lytic towards human endothelium, the biological effects of the OMV-associated EHEC-Hly on microvascular endothelial and intestinal epithelial cells, which are the major targets during EHEC infection, are unknown. Using microscopic, biochemical, flow cytometry and functional analyses of human brain microvascular endothelial cells (HBMEC) and Caco-2 cells we demonstrate that OMV-associated EHEC-Hly does not lyse the target cells but triggers their apoptosis. The OMV-associated toxin is internalized by HBMEC and Caco-2 cells via dynamin-dependent endocytosis of OMVs and trafficked with OMVs into endo-lysosomal compartments. Upon endosome acidification and subsequent pH drop, EHEC-Hly is separated from OMVs, escapes from the lysosomes, most probably via its pore-forming activity, and targets mitochondria. This results in decrease of the mitochondrial transmembrane potential and translocation of cytochrome c to the cytosol, indicating EHEC-Hly-mediated permeabilization of the mitochondrial membranes. Subsequent activation of caspase-9 and caspase-3 leads to apoptotic cell death as evidenced by DNA fragmentation and chromatin condensation in the intoxicated cells. The ability of OMV-associated EHEC-Hly to trigger the mitochondrial apoptotic pathway in human microvascular endothelial and intestinal epithelial cells indicates a novel mechanism of EHEC-Hly involvement in the pathogenesis of EHEC diseases. The OMV-mediated intracellular delivery represents a newly recognized mechanism for a bacterial toxin to enter host cells in

  15. Single pyrimidine discrimination during voltage-driven translocation of osmylated oligodeoxynucleotides via the α-hemolysin nanopore.

    PubMed

    Ding, Yun; Kanavarioti, Anastassia

    2016-01-01

    The influence of an electric field on an isolated channel or nanopore separating two compartments filled with electrolytes produces a constant ion flux through the pore. Nucleic acids added to one compartment traverse the pore, and modulate the current in a sequence-dependent manner. While translocation is faster than detection, the α-hemolysin nanopore (α-HL) successfully senses base modifications in ssDNA immobilized within the pore. With the assistance of a processing enzyme to slow down translocation, nanopore-based DNA sequencing is now a commercially available platform. However, accurate base calling is challenging because α-HL senses a sequence, and not a single nucleotide. Osmylated DNA was recently proposed as a surrogate for nanopore-based sequencing. Osmylation is the addition of osmium tetroxide 2,2'-bipyridine (OsBp) to the C5-C6 pyrimidine double bond. The process is simple, selective for deoxythymidine (dT) over deoxycytidine (dC), unreactive towards the purines, practically 100% effective, and strikingly independent of length, sequence, and composition. Translocation of an oligodeoxynucleotide (oligo) dA10XdA9 via α-HL is relatively slow, and exhibits distinct duration as well as distinct residual current when X = dA, dT(OsBp), or dC(OsBp). The data indicate that the α-HL constriction zone/β-barrel interacts strongly with both OsBp and the base. A 23 nucleotide long oligo with four dT(OsBp) traverses 18-times slower, and the same oligo with nine (dT+dC)(OsBp) moieties traverses 84-times slower compared to dA20, suggesting an average rate of 40 or 180 μs/base, respectively. These translocation speeds are well above detection limits, may be further optimized, and clear the way for nanopore-based sequencing using osmylated DNA. PMID:26925357

  16. Brief heat treatment causes a structural change and enhances cytotoxicity of the Escherichia coli α-hemolysin.

    PubMed

    Aulik, Nicole A; Atapattu, Dhammika N; Czuprynski, Charles J; McCaslin, Darrel R

    2013-02-01

    α-Hemolysin (HLY) is an important virulence factor for uropathogenic Escherichia coli. HLY is a member of the RTX family of exotoxins secreted by a number of Gram-negative bacteria. Recently, it was reported that a related RTX toxin, the Mannheimia haemolytica leukotoxin, exhibits increased cytotoxicity following brief heat treatment. In this article, we show that brief heat treatment (1 min at 100°C) increases cytotoxicity of HLY for human bladder cells, kidney epithelial cells (A498) and neutrophils. Heat treatment also increased hemolysis of human red blood cells (RBCs). Furthermore, heat treatment of previously inactived HLY restored its cytotoxicity. Heat-activated and native HLY both required glycophorin A to lyse RBCs. Native and heat-activated HLY appeared to bind equally well to the surface of A498 cells; although, Western blot analyses demonstrated binding to different proteins on the surface. Confocal microscopy revealed that heat-activated HLY bound more extensively to internal structures of permeabilized A498 cells than did native HLY. Several lines of spectroscopic evidence demonstrate irreversible changes in the structure of heat activated compared to native HLY. We show changes in secondary structure, increased exposure of tryptophan residues to the aqueous environment, an increase in molecular dimension and an increase in hydrophobic surface area. These properties are among the most common characteristics described for the molten globule state, first identified as an intermediate in protein folding. We hypothesize that brief heat treatment of HLY causes a conformational change leading to significant differences in protein-protein interactions that result in increased cytotoxicity for target cells. PMID:22994841

  17. Slow translocation of polynucleotides and their discrimination by α-hemolysin inside a single track-etched nanopore designed by atomic layer deposition.

    PubMed

    Cabello-Aguilar, Simon; Balme, Sébastien; Chaaya, Adib Abou; Bechelany, Mikhael; Balanzat, Emmanuel; Janot, Jean-Marc; Pochat-Bohatier, Celine; Miele, Philippe; Dejardin, Philippe

    2013-10-21

    We report the formation of a hybrid biological/artificial nanopore by the direct insertion of non-modified α-hemolysin at the entrance of a high aspect ratio (length/diameter) biomimetic nanopore. In this robust hybrid system, the protein exhibits the same polynucleotide discrimination properties as in the biological membrane and the polynucleotide dwell time is strongly increased. This nanopore is very promising for DNA sequencing applications where the high DNA translocation velocity and the fragility of the support are the main bottlenecks.

  18. Detection of Hemolysin Variants of Shiga Toxin-Producing Escherichia coli by PCR and Culture on VancomycinCefixime-Cefsulodin Blood Agar

    PubMed Central

    Lehmacher, Anselm; Meier, Heidi; Aleksic, Stojanka; Bockemühl, Jochen

    1998-01-01

    The presence of a hemolysin-encoding gene, elyA or hlyA, from Shiga toxin-producing Escherichia coli (STEC) was detected by PCR in each of 95 strains tested. PCR products of elyA from human STEC isolates of serovars frequently detected in Germany, such as O157:H−, O103:H2, O103:H−, O26:H11, and O26:H−, showed nucleotide sequences identical to previously reported ones for O157:H7 and O111:H− strains. Compared to them, four elyA amplicons derived from human isolates of rare STEC serovars showed identity of about 98% but lacked an AluI restriction site. However, the nucleotide sequence of an amplicon derived from a porcine O138:K81:H− STEC strain was identical to the corresponding region of hlyA, encoding alpha-hemolysin, from E. coli. This hlyA amplicon showed 68% identity with the nucleotide sequence of the corresponding elyA fragment. It differed from the elyA PCR product in restriction fragments generated by AluI, EcoRI, and MluI. Of the 95 representative STEC strains, 88 produced hemolysin on blood agar supplemented with vancomycin (30 mg/liter), cefixime (20 μg/liter), and cefsulodin (3 mg/liter) (BVCC). The lowest added numbers of two to six STEC CFU per g of stool or per ml of raw milk were detectable on BVCC plates after seeding of the preenrichment broth, modified tryptic soy broth (mTSB) supplemented with novobiocin (10 mg/liter), with 16 STEC strains. These strains represented the seven prevailing serovars diagnosed from German patients. However, with ground-beef samples, PCR was essential to identify the lowest added numbers of two to six STEC CFU among colonies of hemolyzing Enterobacteriaceae, such as Serratia spp. and alpha-hemolysin-producing E. coli. We conclude that preenrichment of stool and food samples in mTSB for 6 h followed by overnight culturing on BVCC is a simple method for the isolation and presumptive identification of STEC. PMID:9647814

  19. Slow translocation of polynucleotides and their discrimination by α-hemolysin inside a single track-etched nanopore designed by atomic layer deposition

    NASA Astrophysics Data System (ADS)

    Cabello-Aguilar, Simon; Balme, Sébastien; Chaaya, Adib Abou; Bechelany, Mikhael; Balanzat, Emmanuel; Janot, Jean-Marc; Pochat-Bohatier, Celine; Miele, Philippe; Dejardin, Philippe

    2013-09-01

    We report the formation of a hybrid biological/artificial nanopore by the direct insertion of non-modified α-hemolysin at the entrance of a high aspect ratio (length/diameter) biomimetic nanopore. In this robust hybrid system, the protein exhibits the same polynucleotide discrimination properties as in the biological membrane and the polynucleotide dwell time is strongly increased. This nanopore is very promising for DNA sequencing applications where the high DNA translocation velocity and the fragility of the support are the main bottlenecks.We report the formation of a hybrid biological/artificial nanopore by the direct insertion of non-modified α-hemolysin at the entrance of a high aspect ratio (length/diameter) biomimetic nanopore. In this robust hybrid system, the protein exhibits the same polynucleotide discrimination properties as in the biological membrane and the polynucleotide dwell time is strongly increased. This nanopore is very promising for DNA sequencing applications where the high DNA translocation velocity and the fragility of the support are the main bottlenecks. Electronic supplementary information (ESI) available: Materials, nanopore fabrication and characterization. See DOI: 10.1039/c3nr03683a

  20. Crystal structure of the octameric pore of staphylococcal γ-hemolysin reveals the β-barrel pore formation mechanism by two components

    PubMed Central

    Yamashita, Keitaro; Kawai, Yuka; Tanaka, Yoshikazu; Hirano, Nagisa; Kaneko, Jun; Tomita, Noriko; Ohta, Makoto; Kamio, Yoshiyuki; Yao, Min; Tanaka, Isao

    2011-01-01

    Staphylococcal γ-hemolysin is a bicomponent pore-forming toxin composed of LukF and Hlg2. These proteins are expressed as water-soluble monomers and then assemble into the oligomeric pore form on the target cell. Here, we report the crystal structure of the octameric pore form of γ-hemolysin at 2.5 Å resolution, which is the first high-resolution structure of a β-barrel transmembrane protein composed of two proteins reported to date. The octameric assembly consists of four molecules of LukF and Hlg2 located alternately in a circular pattern, which explains the biochemical data accumulated over the past two decades. The structure, in combination with the monomeric forms, demonstrates the elaborate molecular machinery involved in pore formation by two different molecules, in which interprotomer electrostatic interactions using loops connecting β2 and β3 (loop A: Asp43-Lys48 of LukF and Lys37-Lys43 of Hlg2) play pivotal roles as the structural determinants for assembly through unwinding of the N-terminal β-strands (amino-latch) of the adjacent protomer, releasing the transmembrane stem domain folded into a β-sheet in the monomer (prestem), and interaction with the adjacent protomer. PMID:21969538

  1. Restriction fragment length polymorphism and multiple copies of DNA sequences homologous with probes for P-fimbriae and hemolysin genes among uropathogenic Escherichia coli.

    PubMed

    Hull, S I; Bieler, S; Hull, R A

    1988-03-01

    Hemolysin and P-fimbriae are two virulence traits frequently found together in uropathogenic Escherichia coli. Previous studies have discovered evidence both for linkage between the genes for these traits and for their duplication in the chromosomes of a limited number of strains. To test whether these observations are characteristic of uropathogenic Escherichia coli, the method of DNA hybridization to DNA restriction fragments separated by electrophoresis and transferred to nylon was used to determine copy number of genes for P-fimbriae (pap) among 51 E. coli strains isolated from symptomatic urinary tract infections. Twenty percent of the strains had more than one copy of pap homologous sequences. Fifteen strains, each representing a unique clone, were examined for the presence of sequences homologous with cloned hemolysin genes (hly). Samples of DNA from 14 of the 15 strains hybridized with hly probes. In eight strains the number of copies of pap equalled the number of copies of hly, including one strain with two apparent copies of each. Five strains appeared to have one more copy of pap than of hly, and one strain had an extra copy of hly.

  2. The UPEC pore-forming toxin α-hemolysin triggers proteolysis of host proteins to disrupt cell adhesion, inflammatory, and survival pathways.

    PubMed

    Dhakal, Bijaya K; Mulvey, Matthew A

    2012-01-19

    Uropathogenic Escherichia coli (UPEC), which are the leading cause of both acute and chronic urinary tract infections, often secrete a labile pore-forming toxin known as α-hemolysin (HlyA). We show that stable insertion of HlyA into epithelial cell and macrophage membranes triggers degradation of the cytoskeletal scaffolding protein paxillin and other host regulatory proteins, as well as components of the proinflammatory NFκB signaling cascade. Proteolysis of these factors requires host serine proteases, and paxillin degradation specifically involves the serine protease mesotrypsin. The induced activation of mesotrypsin by HlyA is preceded by redistribution of mesotrypsin precursors from the cytosol into foci along microtubules and within nuclei. HlyA intoxication also stimulated caspase activation, which occurred independently of effects on host serine proteases. HlyA-induced proteolysis of host proteins likely allows UPEC to not only modulate epithelial cell functions, but also disable macrophages and suppress inflammatory responses.

  3. A critical role for hemolysin in Vibrio fluvialis-induced IL-1β secretion mediated by the NLRP3 inflammasome in macrophages

    PubMed Central

    Song, Liqiong; Huang, Yuanming; Zhao, Meng; Wang, Zhihao; Wang, Shujing; Sun, Hui; Kan, Biao; Meng, Guangxun; Liang, Weili; Ren, Zhihong

    2015-01-01

    Vibrio fluvialis causes human diarrhea, but the pathogenesis is not well-studied. We hypothesized that V. fluvialis-secreted hemolysin (VFH) may induce IL-1β secretion through the activation of the NLRP3 inflammasome and contribute to the pathogenicity of V. fluvialis. To examine this possibility, we constructed VFH mutant and complement strains and demonstrated that V. fluvialis-induced IL-1β production and cytotoxicity in human monocytic THP-1 cells and mouse macrophages is attributed to VFH. To evaluate the role of VFH in vivo, we infected adult C57BL/6 mice intraperitoneally and suckling C57/B6 mice orally with various strains. The mice treated with 108 CFU wild-type V. fluvialis or cell-free supernatant containing VFH induced significantly higher IL-1β production in peritoneal lavage fluid or in colon compared with those infected with the mutant strain, while no effect on TNF and IL-6 production was observed at day 5 or 24 h post-infection. VFH contributed to pathological changes and IL-1β release independent of colonization of V. fluvialis in the colon. VFH has no effect on the synthesis of pro-IL-1β, but rather it triggers the processing of pro-IL-1β into IL-1β. Furthermore, using deficient mouse strains, we verified that V. fluvialis-induced IL-1β is mediated through activation of Caspase-1 and the NLRP3 inflammasome ex vivo. Confocal microscopy suggests that VFH contributes to cathepsin B release. Furthermore, V. fluvialis-induced IL-1β secretion requires potassium (K+) efflux and reactive oxygen species production. Our results provide new evidence for the role of VFH in the activation of the NLRP3 inflammasome and pathogenesis in response to V. fluvialis infection. Summary Sentence: Vibrio fluvialis-secreted hemolysin induces IL-1β secretion through the activation of the NLRP3 inflammasome and contributes to the pathogenicity of V. fluvialis. PMID:26052324

  4. A critical role for hemolysin in Vibrio fluvialis-induced IL-1β secretion mediated by the NLRP3 inflammasome in macrophages.

    PubMed

    Song, Liqiong; Huang, Yuanming; Zhao, Meng; Wang, Zhihao; Wang, Shujing; Sun, Hui; Kan, Biao; Meng, Guangxun; Liang, Weili; Ren, Zhihong

    2015-01-01

    Vibrio fluvialis causes human diarrhea, but the pathogenesis is not well-studied. We hypothesized that V. fluvialis-secreted hemolysin (VFH) may induce IL-1β secretion through the activation of the NLRP3 inflammasome and contribute to the pathogenicity of V. fluvialis. To examine this possibility, we constructed VFH mutant and complement strains and demonstrated that V. fluvialis-induced IL-1β production and cytotoxicity in human monocytic THP-1 cells and mouse macrophages is attributed to VFH. To evaluate the role of VFH in vivo, we infected adult C57BL/6 mice intraperitoneally and suckling C57/B6 mice orally with various strains. The mice treated with 10(8) CFU wild-type V. fluvialis or cell-free supernatant containing VFH induced significantly higher IL-1β production in peritoneal lavage fluid or in colon compared with those infected with the mutant strain, while no effect on TNF and IL-6 production was observed at day 5 or 24 h post-infection. VFH contributed to pathological changes and IL-1β release independent of colonization of V. fluvialis in the colon. VFH has no effect on the synthesis of pro-IL-1β, but rather it triggers the processing of pro-IL-1β into IL-1β. Furthermore, using deficient mouse strains, we verified that V. fluvialis-induced IL-1β is mediated through activation of Caspase-1 and the NLRP3 inflammasome ex vivo. Confocal microscopy suggests that VFH contributes to cathepsin B release. Furthermore, V. fluvialis-induced IL-1β secretion requires potassium (K(+)) efflux and reactive oxygen species production. Our results provide new evidence for the role of VFH in the activation of the NLRP3 inflammasome and pathogenesis in response to V. fluvialis infection. Summary Sentence: Vibrio fluvialis-secreted hemolysin induces IL-1β secretion through the activation of the NLRP3 inflammasome and contributes to the pathogenicity of V. fluvialis.

  5. Delivery of Large Heterologous Polypeptides across the Cytoplasmic Membrane of Antigen-Presenting Cells by the Bordetella RTX Hemolysin Moiety Lacking the Adenylyl Cyclase Domain

    PubMed Central

    Holubova, Jana; Jelinek, Jiri; Tomala, Jakub; Masin, Jiri; Kosova, Martina; Stanek, Ondrej; Bumba, Ladislav; Michalek, Jaroslav; Kovar, Marek; Sebo, Peter

    2012-01-01

    The Bordetella adenylate cyclase toxin-hemolysin (CyaA; also called ACT or AC-Hly) targets CD11b-expressing phagocytes and translocates into their cytosol an adenylyl cyclase (AC) that hijacks cellular signaling by conversion of ATP to cyclic AMP (cAMP). Intriguingly, insertion of large passenger peptides removes the enzymatic activity but not the cell-invasive capacity of the AC domain. This has repeatedly been exploited for delivery of heterologous antigens into the cytosolic pathway of CD11b-expressing dendritic cells by CyaA/AC− toxoids, thus enabling their processing and presentation on major histocompatibility complex (MHC) class I molecules to cytotoxic CD8+ T lymphocytes (CTLs). We produced a set of toxoids with overlapping deletions within the first 371 residues of CyaA and showed that the structure of the AC enzyme does not contain any sequences indispensable for its translocation across target cell membrane. Moreover, replacement of the AC domain (residues 1 to 371) with heterologous polypeptides of 40, 146, or 203 residues yielded CyaAΔAC constructs that delivered passenger CTL epitopes into antigen-presenting cells (APCs) and induced strong antigen-specific CD8+ CTL responses in vivo in mice and ex vivo in human peripheral blood mononuclear cell cultures. This shows that the RTX (repeats in toxin) hemolysin moiety, consisting of residues 374 to 1706 of CyaA, harbors all structural information involved in translocation of the N-terminal AC domain across target cell membranes. These results decipher the extraordinary capacity of the AC domain of CyaA to transport large heterologous cargo polypeptides into the cytosol of CD11b+ target cells and pave the way for the construction of CyaAΔAC-based polyvalent immunotherapeutic T cell vaccines. PMID:22215742

  6. Substitution of lysine for arginine in the N-terminal 217th amino acid residue of the H gamma II of Staphylococcal gamma-hemolysin lowers the activity of the toxin.

    PubMed

    Sudo, K; Choorit, W; Asami, I; Kaneko, J; Muramoto, K; Kamio, Y

    1995-09-01

    The staphylococcal toxin gamma-hemolysin consists of two protein components, LukF and H gamma II. Staphylococcus aureus P83 was found to have five components, LukF, LukF-PV, LukM, LukS, and H gamma II for leukocidin or gamma-hemolysin. H gamma II of S. aureus P83 was demonstrated to be a naturally-occurring analogous molecule of H gamma II [H gamma II(P83)], in which the 217th arginine residue was replaced by lysine. The H gamma II(P83) showed about 50% of the hemolytic activity of normal H gamma II in the presence of LukF.

  7. Substrate-triggered recruitment of the TolC channel-tunnel during type I export of hemolysin by Escherichia coli.

    PubMed

    Balakrishnan, L; Hughes, C; Koronakis, V

    2001-10-26

    A defining event in type I export of hemolysin by Escherichia coli is the substrate-triggered recruitment of the TolC channel-tunnel by an inner membrane complex. This complex comprises a traffic ATPase (HlyB) and the 478 residue adaptor protein (HlyD), which contacts TolC during recruitment. HlyD has a large periplasmic domain (amino acid residues 81-478) linked by a single transmembrane helix to a small N-terminal cytosolic domain (1-59). Export was disabled by deletion of the ca 60 amino acid residue cytosolic domain of HlyD, even though the truncated HlyD (HlyDDelta45) was, like the wild-type, able to trimerise in the cytosolic membrane, and interact with the traffic ATPase. The mutant HlyB/HlyDDelta45 inner membrane complex engaged the hemolysin substrate, but this substrate-engaged complex failed to trigger recruitment of TolC. Further analyses showed that HlyDDelta45 was specifically unable to bind the substrate. The result suggests that substrate engagement by the traffic ATPase alone is insufficient to trigger TolC recruitment, and that substrate binding to the HlyD cytosolic domain is essential. Analysis of three further N-terminal deletion variants, HlyDDelta26, HlyDDelta26-45 and HlyDDelta34-38, indicated that an extreme N-terminal amphipathic helix and a cytosolic cluster of charged residues are central to the cytosolic domain function. The cytosolic amphipathic helix was not essential for substrate engagement or TolC recruitment, but export was impaired without it. In contrast, when the charged amino acid residues were deleted, the substrate was still engaged by HlyD but engagement was unproductive, i.e. TolC recruitment was not triggered. Our results are compatible with the HlyD cytosolic domain mediating transduction of the substrate binding signal directly, presumably to the HlyD periplasmic domain, to trigger recruitment of TolC and assemble the type I export complex.

  8. Comparative Prevalence of Immune Evasion Complex Genes Associated with β-Hemolysin Converting Bacteriophages in MRSA ST5 Isolates from Swine, Swine Facilities, Humans with Swine Contact, and Humans with No Swine Contact.

    PubMed

    Hau, Samantha J; Sun, Jisun; Davies, Peter R; Frana, Timothy S; Nicholson, Tracy L

    2015-01-01

    Livestock associated methicillin-resistant Staphylococcus aureus (LA-MRSA) draws concern from the public health community because in some countries these organisms may represent the largest reservoir of MRSA outside hospital settings. Recent studies indicate LA-MRSA strains from swine are more genetically diverse than the first reported sequence type ST398. In the US, a diverse population of LA-MRSA is found including organisms of the ST398, ST9, and ST5 lineages. Occurrence of ST5 MRSA in swine is of particular concern since ST5 is among the most prevalent lineages causing clinical infections in humans. The prominence of ST5 in clinical disease is believed to result from acquisition of bacteriophages containing virulence or host-adapted genes including the immune-evasion cluster (IEC) genes carried by β-hemolysin converting bacteriophages, whose absence in LA-MRSA ST398 is thought to contribute to reduced rates of human infection and transmission associated with this lineage. The goal of this study was to investigate the prevalence of IEC genes associated with β-hemolysin converting bacteriophages in MRSA ST5 isolates obtained from agricultural sources, including swine, swine facilities, and humans with short- or long-term swine exposure. To gain a broader perspective, the prevalence of these genes in LA-MRSA ST5 strains was compared to the prevalence in clinical MRSA ST5 strains from humans with no known exposure to swine. IEC genes were not present in any of the tested MRSA ST5 strains from agricultural sources and the β-hemolysin gene was intact in these strains, indicating the bacteriophage's absence. In contrast, the prevalence of the β-hemolysin converting bacteriophage in MRSA ST5 strains from humans with no exposure to swine was 90.4%. The absence of β-hemolysin converting bacteriophage in LA-MRSA ST5 isolates is consistent with previous reports evaluating ST398 strains and provides genetic evidence indicating LA-MRSA ST5 isolates may harbor a reduced

  9. Comparative Prevalence of Immune Evasion Complex Genes Associated with β-Hemolysin Converting Bacteriophages in MRSA ST5 Isolates from Swine, Swine Facilities, Humans with Swine Contact, and Humans with No Swine Contact

    PubMed Central

    Hau, Samantha J.; Sun, Jisun; Davies, Peter R.; Frana, Timothy S.; Nicholson, Tracy L.

    2015-01-01

    Livestock associated methicillin-resistant Staphylococcus aureus (LA-MRSA) draws concern from the public health community because in some countries these organisms may represent the largest reservoir of MRSA outside hospital settings. Recent studies indicate LA-MRSA strains from swine are more genetically diverse than the first reported sequence type ST398. In the US, a diverse population of LA-MRSA is found including organisms of the ST398, ST9, and ST5 lineages. Occurrence of ST5 MRSA in swine is of particular concern since ST5 is among the most prevalent lineages causing clinical infections in humans. The prominence of ST5 in clinical disease is believed to result from acquisition of bacteriophages containing virulence or host-adapted genes including the immune-evasion cluster (IEC) genes carried by β-hemolysin converting bacteriophages, whose absence in LA-MRSA ST398 is thought to contribute to reduced rates of human infection and transmission associated with this lineage. The goal of this study was to investigate the prevalence of IEC genes associated with β-hemolysin converting bacteriophages in MRSA ST5 isolates obtained from agricultural sources, including swine, swine facilities, and humans with short- or long-term swine exposure. To gain a broader perspective, the prevalence of these genes in LA-MRSA ST5 strains was compared to the prevalence in clinical MRSA ST5 strains from humans with no known exposure to swine. IEC genes were not present in any of the tested MRSA ST5 strains from agricultural sources and the β-hemolysin gene was intact in these strains, indicating the bacteriophage’s absence. In contrast, the prevalence of the β-hemolysin converting bacteriophage in MRSA ST5 strains from humans with no exposure to swine was 90.4%. The absence of β-hemolysin converting bacteriophage in LA-MRSA ST5 isolates is consistent with previous reports evaluating ST398 strains and provides genetic evidence indicating LA-MRSA ST5 isolates may harbor a

  10. Serodiagnosis of Acute Typhoid Fever in Nigerian Pediatric Cases by Detection of Serum IgA and IgG Against Hemolysin E and Lipopolysaccharide.

    PubMed

    Davies, D Huw; Jain, Aarti; Nakajima, Rie; Liang, Li; Jasinskis, Algis; Supnet, Medalyn; Felgner, Philip L; Teng, Andy; Pablo, Jozelyn; Molina, Douglas M; Obaro, Stephen K

    2016-08-01

    Inexpensive, easy-to-use, and highly sensitive diagnostic tests are currently unavailable for typhoid fever. To identify candidate serodiagnostic markers, we have probed microarrays displaying the full Salmonella enterica serovar Typhi (S. Typhi) proteome of 4,352 different proteins + lipopolysaccharides (LPSs), with sera from Nigerian pediatric typhoid and other febrile cases, Nigerian healthy controls, and healthy U.S. adults. Nigerian antibody profiles were broad (∼500 seropositive antigens) and mainly low level, with a small number of stronger "hits," whereas the profile in U.S. adults was < 1/5 as broad, consistent with endemic exposure in Nigeria. Nigerian profiles were largely unaffected by clinical diagnosis, although the response against t1477 (hemolysin E) consistently emerged as stronger in typhoid cases. The response to LPS was also a strong discriminator of healthy controls and typhoid, although LPS did not discriminate between typhoid and nontyphoidal Salmonella (NTS) disease. As a first step toward the development of a point-of-care diagnostic, t1477 and LPS were evaluated on immunostrips. Both provided good discrimination between healthy controls and typhoid/NTS disease. Such a test could provide a useful screen for salmonellosis (typhoid and NTS disease) in suspected pediatric cases that present with undefined febrile disease. PMID:27215295

  11. Stochastic Assembly of Two-Component Staphylococcal γ-Hemolysin into Heteroheptameric Transmembrane Pores with Alternate Subunit Arrangements in Ratios of 3:4 and 4:3

    PubMed Central

    Sugawara-Tomita, Noriko; Tomita, Toshio; Kamio, Yoshiyuki

    2002-01-01

    Self-assembling, pore-forming toxins from Staphylococcus aureus are illustrative molecules for the study of the assembly and membrane insertion of oligomeric transmembrane proteins. On the basis of previous studies, we have shown that the two-component γ-hemolysin assembles from LukF (or Hlg1, 34 kDa) and Hlg2 (32 kDa) to form ring-shaped transmembrane pores of ca. 200 kDa. Here we show that LukF and Hlg2 assemble in a stochastic manner to form alternate complexes with subunit stoichiometries of 3:4 and 4:3. High-resolution electron microscopic images of negatively stained pore complexes clearly revealed a heptameric structure. When adjacent monomers in the pore complexes were randomly cross-linked by using glutaraldehyde, LukF-LukF, LukF-Hlg2, and Hlg2-Hlg2 dimers were detected in an approximate ratio of 1:12:1, suggesting that LukF and Hlg2 were alternately arranged in the pore complex in molar ratios of 3:4 and 4:3. The alternate arrangements of LukF and Hlg2 in molar ratios of 3:4 and 4:3 were also visualized under electron microscope with the pore complexes consisting of glutathione S-transferase fusion protein of LukF or Hlg2 and wild-type protein of Hlg2 or LukF, respectively. PMID:12169599

  12. Prevalence of pandemic thermostable direct hemolysin-producing Vibrio parahaemolyticus O3:K6 in seafood and the coastal environment in Japan.

    PubMed

    Hara-Kudo, Yukiko; Sugiyama, Kanji; Nishibuchi, Mitsuaki; Chowdhury, Ashrafuzzaman; Yatsuyanagi, Jun; Ohtomo, Yoshimitsu; Saito, Akinobu; Nagano, Hidetoshi; Nishina, Tokuhiro; Nakagawa, Hiroshi; Konuma, Hirotaka; Miyahara, Michiko; Kumagai, Susumu

    2003-07-01

    Although thermostable direct hemolysin (TDH)-producing Vibrio parahaemolyticus has caused many infections in Asian countries, the United States, and other countries, it has been difficult to detect the same pathogen in seafoods and other environmental samples. In this study, we detected and enumerated tdh gene-positive V. parahaemolyticus in Japanese seafoods with a tdh-specific PCR method, a chromogenic agar medium, and a most-probable-number method. The tdh gene was detected in 33 of 329 seafood samples (10.0%). The number of tdh-positive V. parahaemolyticus ranged from <3 to 93/10 g. The incidence of tdh-positive V. parahaemolyticus tended to be high in samples contaminated with relatively high levels of total V. parahaemolyticus. TDH-producing strains of V. parahaemolyticus were isolated from 11 of 33 tdh-positive samples (short-necked clam, hen clam, and rock oyster). TDH-producing strains of V. parahaemolyticus were also isolated from the sediments of rivers near the coast in Japan. Representative strains of the seafood and sediment isolates were examined for the O:K serovar and by the PCR method specific to the pandemic clone and arbitrarily primed PCR and pulsed-field gel electrophoresis techniques. The results indicated that most O3:K6 tdh-positive strains belonged to the pandemic O3:K6 clone and suggested that serovariation took place in the Japanese environment.

  13. Ion Selectivity of α-Hemolysin with β-Cyclodextrin Adapter: I. Single Ion Potential of Mean Force and Diffusion Coefficient

    PubMed Central

    Luo, Yun; Egwolf, Bernhard; Walters, D. Eric; Roux, Benoît

    2010-01-01

    The α-hemolysin (αHL) is a self-assembling exotoxin that binds to the membrane of a susceptible host cell and causes its death. Experimental studies show that electrically neutral β-cyclodextrin (βCD) can insert into the αHL channel and significantly increase its anion selectivity. To understand how βCD can affect ion selectivity, molecular dynamics (MD) simulations potential of mean force (PMF) calculations are carried out for different αHL channels with and without βCD adapter. A multiscale approach based on the Generalized Solvent Boundary Potential (GSBP) is used to reduce the size of the simulated system. The PMF profiles reveal that βCD has no anion selectivity by itself, but can increase the Cl− selectivity of the αHL channel when lodged into the pore lumen. Analysis shows that βCD causes a partial desolvation of ions and affects the orientation of nearby charged residues. The ion selectivity appears to result from increased electrostatic interaction between the ion and the channel due to a reduction in dielectric shielding by the solvent. These observations suggest a reasonable explanation of the ion selectivity and provide important information for further ion channel modification. PMID:20041673

  14. Group B Streptococcus β-hemolysin/Cytolysin Breaches Maternal-Fetal Barriers to Cause Preterm Birth and Intrauterine Fetal Demise in Vivo

    PubMed Central

    Randis, Tara M.; Gelber, Shari E.; Hooven, Thomas A.; Abellar, Rosanna G.; Akabas, Leor H.; Lewis, Emma L.; Walker, Lindsay B.; Byland, Leah M.; Nizet, Victor; Ratner, Adam J.

    2014-01-01

    Background. Maternal vaginal colonization with Streptococcus agalactiae (Group B Streptococcus [GBS]) is a precursor to chorioamnionitis, fetal infection, and neonatal sepsis, but the understanding of specific factors in the pathogenesis of ascending infection remains limited. Methods. We used a new murine model to evaluate the contribution of the pore-forming GBS β-hemolysin/cytolysin (βH/C) to vaginal colonization, ascension, and fetal infection. Results. Competition assays demonstrated a marked advantage to βH/C-expressing GBS during colonization. Intrauterine fetal demise and/or preterm birth were observed in 54% of pregnant mice colonized with wild-type (WT) GBS and 0% of those colonized with the toxin-deficient cylE knockout strain, despite efficient colonization and ascension by both strains. Robust placental inflammation, disruption of maternal-fetal barriers, and fetal infection were more frequent in animals colonized with WT bacteria. Histopathologic examination revealed bacterial tropism for fetal lung and liver. Conclusions. Preterm birth and fetal demise are likely the direct result of toxin-induced damage and inflammation rather than differences in efficiency of ascension into the upper genital tract. These data demonstrate a distinct contribution of βH/C to GBS chorioamnionitis and subsequent fetal infection in vivo and showcase a model for this most proximal step in GBS pathogenesis. PMID:24474814

  15. RIP-V improves murine survival in a sepsis model by down-regulating RNAIII expression and α-hemolysin release of methicillin-resistant Staphylococcus aureus.

    PubMed

    Ma, Bo; Zhou, Ying; Li, Mingkai; Yu, Qian; Xue, Xiaoyan; Li, Zhi; Da, Fei; Hou, Zheng; Luo, Xiaoxing

    2015-02-01

    Staphylococcus aureus is associated with serious invasive infections and high mortality rates due to a large number of toxins released. The persistent increasing resistance of S. aureus has driven the need for new anti-infection agents and innovative therapeutic strategies. RNAIII-inhibiting peptide (RIP) has been reported to reduce bacterial virulence by interfering with S. aureus quorum sensing system. The present study aimed to investigate whether two new RIP derivatives (RIP-V and RIP-L) could improve the survival rate of mice in a MRSA sepsis model. We found that neither anti-bacterial nor cell toxicity were displayed by all RIPs in vitro. In vivo protective effects were observed using a MRSA-induced mice sepsis model. Among RIPs, RIP-V exhibited the strongest protection function on mice survival and inhibition of pathological damages. Our studies firstly verified that RIPs could inhibited the RNAIII expression of S. aurues isolated from liver tissue of BALB/c mice. Moreover, RIP-V exhibited the strongest inhibitory effect on RNAIII and can decrease markedly the secretion of o-hemolysin in liver. These findings indicate that RIP-V might be considered as a potential and specific drug candidate for treating S. aureus infections, especially for MRSA.

  16. Sequencing of leucocidin R from Staphylococcus aureus P83 suggests that staphylococcal leucocidins and gamma-hemolysin are members of a single, two-component family of toxins.

    PubMed

    Supersac, G; Prevost, G; Piemont, Y

    1993-02-01

    A 2,813-bp HincII-ClaI DNA fragment encodes the two S and F components (LukS-R and LukF-R) of leucocidin R (Luk-R) which are secreted by Staphylococcus aureus P83. The two genes (lukS-R and lukF-R) belong to a single operon. Two peptidic sequences were deduced: LukS-R is a 35,721-Da polypeptide of 315 amino acids, including a signal sequence of 29 residues, and LukF-R is a 36,838-Da polypeptide of 325 amino acids, including a signal sequence of 25 residues. LukS-R and LukF-R were expressed in Escherichia coli and purified from the periplasmic space. Luk-R exerts biological activities on polymorphonuclear cells and on erythrocytes from various animals. Comparison of the amino acid sequence of LukF-R with that of the B component of gamma-hemolysin (HlgB), those of the F and S components of another recently sequenced staphylococcal leucocidin, and those of a few peptides of the F component from Panton-Valentine leucocidin suggests that all four toxins belong to a single, two-component family of toxins.

  17. Pathogenesis of Streptococcus urinary tract infection depends on bacterial strain and β-hemolysin/cytolysin that mediates cytotoxicity, cytokine synthesis, inflammation and virulence

    PubMed Central

    Leclercq, Sophie Y.; Sullivan, Matthew J.; Ipe, Deepak S.; Smith, Joshua P.; Cripps, Allan W.; Ulett, Glen C.

    2016-01-01

    Streptococcus agalactiae can cause urinary tract infection (UTI) including cystitis and asymptomatic bacteriuria (ABU). The early host-pathogen interactions that occur during S. agalactiae UTI and subsequent mechanisms of disease pathogenesis are poorly defined. Here, we define the early interactions between human bladder urothelial cells, monocyte-derived macrophages, and mouse bladder using uropathogenic S. agalactiae (UPSA) 807 and ABU-causing S. agalactiae (ABSA) 834 strains. UPSA 807 adhered, invaded and killed bladder urothelial cells more efficiently compared to ABSA 834 via mechanisms including low-level caspase-3 activation, and cytolysis, according to lactate dehydrogenase release measures and cell viability. Severe UPSA 807-induced cytotoxicity was mediated entirely by the bacterial β-hemolysin/cytolysin (β-H/C) because an β-H/C-deficient UPSA 807 isogenic mutant, UPSA 807ΔcylE, was not cytotoxic in vitro; the mutant was also significantly attenuated for colonization in the bladder in vivo. Analysis of infection-induced cytokines, including IL-8, IL-1β, IL-6 and TNF-α in vitro and in vivo revealed that cytokine and chemokine responses were dependent on expression of β-H/C that also elicited severe bladder neutrophilia. Thus, virulence of UPSA 807 encompasses adhesion to, invasion of and killing of bladder cells, pro-inflammatory cytokine/chemokine responses that elicit neutrophil infiltration, and β-H/C-mediated subversion of innate immune-mediated bacterial clearance from the bladder. PMID:27383371

  18. cAMP and EPAC Are Key Players in the Regulation of the Signal Transduction Pathway Involved in the α-Hemolysin Autophagic Response

    PubMed Central

    Mestre, María Belén; Colombo, María Isabel

    2012-01-01

    Staphylococcus aureus is a microorganism that causes serious diseases in the human being. This microorganism is able to escape the phagolysosomal pathway, increasing intracellular bacterial survival and killing the eukaryotic host cell to spread the infection. One of the key features of S. aureus infection is the production of a series of virulence factors, including secreted enzymes and toxins. We have shown that the pore-forming toxin α-hemolysin (Hla) is the S. aureus–secreted factor responsible for the activation of the autophagic pathway and that this response occurs through a PI3K/Beclin1-independent form. In the present report we demonstrate that cAMP has a key role in the regulation of this autophagic response. Our results indicate that cAMP is able to inhibit the autophagy induced by Hla and that PKA, the classical cAMP effector, does not participate in this regulation. We present evidence that EPAC and Rap2b, through calpain activation, are the proteins involved in the regulation of Hla-induced autophagy. Similar results were obtained in cells infected with different S. aureus strains. Interestingly, in this report we show, for the first time to our knowledge, that both EPAC and Rap2b are recruited to the S. aureus–containing phagosome. We believe that our findings have important implications in understanding innate immune processes involved in intracellular pathogen invasion of the host cell. PMID:22654658

  19. Gene detection and toxin production evaluation of hemolysin BL of Bacillus cereus isolated from milk and dairy products marketed in Brazil.

    PubMed

    Reis, Andre L S; Montanhini, Maike T M; Bittencourt, Juliana V M; Destro, Maria T; Bersot, Luciano S

    2013-12-01

    Bacillus cereusis an ubiquitous, spore-forming bacteria that can survive pasteurization and the majority of the heating processes used in the dairy industry. Besides, it is a pathogen responsible for different types of food poisoning. One type of foodborne disease caused by B.cereusis the diarrheal syndrome, which is caused by the ingestion of vegetative cells producing toxins in the small intestine. One virulence factor for the diarrheal syndrome is the toxin hemolysin BL (HBL), a three-component protein formed by the L1, L2 and B components. In order to evaluate the presence of diarrheal strains isolated from milk and dairy products, 63 B. cereus isolates were obtained from 260 samples of UHT milk, pasteurized milk and powdered milk, sold in commercial establishments and from different brands. The isolates were subjected to the Polymerase Chain Reaction (PCR) for the detection of the encoding genes for the L1, L2 and B components and the toxin production capacity were evaluated with an immunoassay. A total of 23 [36.5%] isolates were identified carrying simultaneously the three tested genes, from which, 20 [86.9%] showed toxigenic capacity. 26 [41.3%] isolates did not carry any of genes tested and the other 14 [22.2%] were positive for one or two of them. The results showed a high toxigenic capacity among the B. cereus isolates able to produce the HBL, indicating a potential risk for consumers.

  20. Molecular characterization of enterohemorrhagic Escherichia coli hemolysin gene (EHEC-hlyA)-harboring isolates from cattle reveals a diverse origin and hybrid diarrheagenic strains.

    PubMed

    Askari Badouei, Mahdi; Morabito, Stefano; Najafifar, Arash; Mazandarani, Emad

    2016-04-01

    In the present study we investigated the occurrence of Escherichia coli strains harboring the gene encoding enterohemorrhagic E. coli hemolysin (EHEC-HlyA) in cattle and the association of this gene with various diarrheagenic E. coli (DEC) pathotypes. First, the bovine E. coli isolates were screened for EHEC-hlyA gene by PCR, and then they were characterized for the phylogenetic groups and the presence of the major virulence genes of different DEC pathotypes. In total, 25 virulence gene profiles were observed in 54 EHEC-hlyA+ isolates that reflect a considerable heterogeneity. The EHEC-hlyA+ strains were mostly associated with EHEC (72%), while only 7.4% were enteropathogenic E. coli (EPEC). We also showed the presence of estA gene of enterotoxigenic E. coli (ETEC) in 6 isolates (11.1%). Interestingly, two of the estA+ strains showed hybrid pathotypes with one carrying eae/estA (EPEC/ETEC), and the other one stx2/astA/estA (EHEC/ETEC). None of the isolates were related to enteroaggregative E. coli (EAggEC), enteroinvasive E. coli (EIEC), and necrotoxigenic E. coli (NTEC). The EHEC-plasmid encoded genes occurred in seven different combinations with EHEC-hlyA/saa/subA/espP being the most prevalent (46.3%). All stx-/eae+ strains carried O island 57 (OI-57) molecular marker(s) that may indicate these to be the progenitors of EHEC or strains losing stx. The most prevalent phylogroup was B1 (61.1%), but the most heterogeneous strains including the hybrid strains belonged to A phylogroup. Overall, our results indicate that cattle EHEC-hlyA encoding E. coli isolates consist of diverse diarrheagenic strains with the possible existence of hybrid pathotypes. Future studies are required to clarify the evolutionary aspects and clinical significance of these strains in humans and domestic animals.

  1. Structural Characterization of Humanized Nanobodies with Neutralizing Activity against the Bordetella pertussis CyaA-Hemolysin: Implications for a Potential Epitope of Toxin-Protective Antigen

    PubMed Central

    Malik, Aijaz Ahmad; Imtong, Chompounoot; Sookrung, Nitat; Katzenmeier, Gerd; Chaicumpa, Wanpen; Angsuthanasombat, Chanan

    2016-01-01

    Previously, the 126-kDa CyaA-hemolysin (CyaA-Hly) fragment cloned from Bordetella pertussis—the causative agent of whooping cough—and functionally expressed in Escherichia coli was revealed as a key determinant for CyaA-mediated hemolysis against target erythrocytes. Here, phagemid-transfected E. coli clones producing nanobodies capable of binding to CyaA-Hly were selected from a humanized-camel VH/VHH phage-display library. Subsequently verified for binding activities by indirect ELISA and Western blotting, four CyaA-Hly-specific nanobodies were obtained and designated according to the presence/absence of VHH-hallmark amino acids as VHH2, VH5, VH18 and VHH37. In vitro neutralization assay revealed that all four ~17-kDa His-tagged VH/VHH nanobodies, in particular VHH37, which were over-expressed as inclusions and successfully unfolded-refolded, were able to effectively inhibit CyaA-Hly-mediated hemolysis. Phage-mimotope searching revealed that only peptides with sequence homologous to Linker 1 connecting Blocks I and II within the CyaA-RTX subdomain were able to bind to these four CyaA-Hly-specific nanobodies. Structural analysis of VHH37 via homology modeling and intermolecular docking confirmed that this humanized nanobody directly interacts with CyaA-RTX/Linker 1 through multiple hydrogen and ionic bonds. Altogether, our present data demonstrate that CyaA-RTX/Linker 1 could serve as a potential epitope of CyaA-protective antigen that may be useful for development of peptide-based pertussis vaccines. Additionally, such toxin-specific nanobodies have a potential for test-driven development of a ready-to-use therapeutic in passive immunization for mitigation of disease severity. PMID:27043627

  2. Structural Characterization of Humanized Nanobodies with Neutralizing Activity against the Bordetella pertussis CyaA-Hemolysin: Implications for a Potential Epitope of Toxin-Protective Antigen.

    PubMed

    Malik, Aijaz Ahmad; Imtong, Chompounoot; Sookrung, Nitat; Katzenmeier, Gerd; Chaicumpa, Wanpen; Angsuthanasombat, Chanan

    2016-04-01

    Previously, the 126-kDa CyaA-hemolysin (CyaA-Hly) fragment cloned from Bordetella pertussis--the causative agent of whooping cough--and functionally expressed in Escherichia coli was revealed as a key determinant for CyaA-mediated hemolysis against target erythrocytes. Here, phagemid-transfected E. coli clones producing nanobodies capable of binding to CyaA-Hly were selected from a humanized-camel VH/VHH phage-display library. Subsequently verified for binding activities by indirect ELISA and Western blotting, four CyaA-Hly-specific nanobodies were obtained and designated according to the presence/absence of VHH-hallmark amino acids as VHH2, VH5, VH18 and VHH37. In vitro neutralization assay revealed that all four ~17-kDa His-tagged VH/VHH nanobodies, in particular VHH37, which were over-expressed as inclusions and successfully unfolded-refolded, were able to effectively inhibit CyaA-Hly-mediated hemolysis. Phage-mimotope searching revealed that only peptides with sequence homologous to Linker 1 connecting Blocks I and II within the CyaA-RTX subdomain were able to bind to these four CyaA-Hly-specific nanobodies. Structural analysis of VHH37 via homology modeling and intermolecular docking confirmed that this humanized nanobody directly interacts with CyaA-RTX/Linker 1 through multiple hydrogen and ionic bonds. Altogether, our present data demonstrate that CyaA-RTX/Linker 1 could serve as a potential epitope of CyaA-protective antigen that may be useful for development of peptide-based pertussis vaccines. Additionally, such toxin-specific nanobodies have a potential for test-driven development of a ready-to-use therapeutic in passive immunization for mitigation of disease severity.

  3. The β-prism lectin domain of Vibrio cholerae hemolysin promotes self-assembly of the β-pore-forming toxin by a carbohydrate-independent mechanism.

    PubMed

    Ganguly, Sreerupa; Mukherjee, Amarshi; Mazumdar, Budhaditya; Ghosh, Amar N; Banerjee, Kalyan K

    2014-02-14

    Vibrio cholerae cytolysin/hemolysin (VCC) is an amphipathic 65-kDa β-pore-forming toxin with a C-terminal β-prism lectin domain. Because deletion or point mutation of the lectin domain seriously compromises hemolytic activity, it is thought that carbohydrate-dependent interactions play a critical role in membrane targeting of VCC. To delineate the contributions of the cytolysin and lectin domains in pore formation, we used wild-type VCC, 50-kDa VCC (VCC(50)) without the lectin domain, and mutant VCC(D617A) with no carbohydrate-binding activity. VCC and its two variants with no carbohydrate-binding activity moved to the erythrocyte stroma with apparent association constants on the order of 10(7) M(-1). However, loss of the lectin domain severely reduced the efficiency of self-association of the VCC monomer with the β-barrel heptamer in the synthetic lipid bilayer from ∼83 to 27%. Notably, inactivation of the carbohydrate-binding activity by the D617A mutation marginally reduced oligomerization to ∼77%. Oligomerization of VCC(50) was temperature-insensitive; by contrast, VCC self-assembly increased with increasing temperature, suggesting that the process is driven by entropy and opposed by enthalpy. Asialofetuin, the β1-galactosyl-terminated glycoprotein inhibitor of VCC-induced hemolysis, promoted oligomerization of 65-kDa VCC to a species that resembled the membrane-inserted heptamer in stoichiometry and morphology but had reduced global amphipathicity. In conclusion, we propose (i) that the β-prism lectin domain facilitated toxin assembly by producing entropy during relocation in the heptamer and (ii) that glycoconjugates inhibited VCC by promoting its assembly to a water-soluble, less amphipathic oligomer variant with reduced ability to penetrate the bilayer.

  4. Structural Characterization of Humanized Nanobodies with Neutralizing Activity against the Bordetella pertussis CyaA-Hemolysin: Implications for a Potential Epitope of Toxin-Protective Antigen.

    PubMed

    Malik, Aijaz Ahmad; Imtong, Chompounoot; Sookrung, Nitat; Katzenmeier, Gerd; Chaicumpa, Wanpen; Angsuthanasombat, Chanan

    2016-04-01

    Previously, the 126-kDa CyaA-hemolysin (CyaA-Hly) fragment cloned from Bordetella pertussis--the causative agent of whooping cough--and functionally expressed in Escherichia coli was revealed as a key determinant for CyaA-mediated hemolysis against target erythrocytes. Here, phagemid-transfected E. coli clones producing nanobodies capable of binding to CyaA-Hly were selected from a humanized-camel VH/VHH phage-display library. Subsequently verified for binding activities by indirect ELISA and Western blotting, four CyaA-Hly-specific nanobodies were obtained and designated according to the presence/absence of VHH-hallmark amino acids as VHH2, VH5, VH18 and VHH37. In vitro neutralization assay revealed that all four ~17-kDa His-tagged VH/VHH nanobodies, in particular VHH37, which were over-expressed as inclusions and successfully unfolded-refolded, were able to effectively inhibit CyaA-Hly-mediated hemolysis. Phage-mimotope searching revealed that only peptides with sequence homologous to Linker 1 connecting Blocks I and II within the CyaA-RTX subdomain were able to bind to these four CyaA-Hly-specific nanobodies. Structural analysis of VHH37 via homology modeling and intermolecular docking confirmed that this humanized nanobody directly interacts with CyaA-RTX/Linker 1 through multiple hydrogen and ionic bonds. Altogether, our present data demonstrate that CyaA-RTX/Linker 1 could serve as a potential epitope of CyaA-protective antigen that may be useful for development of peptide-based pertussis vaccines. Additionally, such toxin-specific nanobodies have a potential for test-driven development of a ready-to-use therapeutic in passive immunization for mitigation of disease severity. PMID:27043627

  5. Improved Protection in a Rabbit Model of Community-Associated Methicillin-Resistant Staphylococcus aureus Necrotizing Pneumonia upon Neutralization of Leukocidins in Addition to Alpha-Hemolysin.

    PubMed

    Diep, Binh An; Le, Vien T M; Visram, Zehra C; Rouha, Harald; Stulik, Lukas; Dip, Etyene Castro; Nagy, Gábor; Nagy, Eszter

    2016-10-01

    Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), especially the USA300 pulsotype, is a frequent cause of skin and soft tissue infections and severe pneumonia. Despite appropriate antibiotic treatment, complications are common and pneumonia is associated with high mortality. S. aureus strains express multiple cytotoxins, including alpha-hemolysin (Hla) and up to five bicomponent leukocidins that specifically target phagocytic cells for lysis. CA-MRSA USA300 strains carry the genes for all six cytotoxins. Species specificity of the leukocidins greatly contributes to the ambiguity regarding their role in S. aureus pathogenesis. We performed a comparative analysis of the leukocidin susceptibility of human, rabbit, and mouse polymorphonuclear leukocytes (PMNs) to assess the translational value of mouse and rabbit S. aureus models. We found that mouse PMNs were largely resistant to LukSF-PV, HlgAB, and HlgCB and susceptible only to LukED, whereas rabbit and human PMNs were highly sensitive to all these cytotoxins. In the rabbit pneumonia model with a USA300 CA-MRSA strain, passive immunization with a previously identified human monoclonal antibody (MAb), Hla-F#5, which cross-neutralizes Hla, LukSF-PV, HlgAB, HlgCB, and LukED, provided full protection, whereas an Hla-specific MAb was only partially protective. In the mouse USA300 CA-MRSA pneumonia model, both types of antibodies demonstrated full protection, suggesting that Hla, but not leukocidin(s), is the principal virulence determinant in mice. As the rabbit recapitulates the high susceptibility to leukocidins characteristic of humans, this species represents a valuable model for assessing novel, cytotoxin-targeting anti-S. aureus therapeutic approaches.

  6. [Ca2+]i Oscillations and IL-6 Release Induced by α-Hemolysin from Escherichia coli Require P2 Receptor Activation in Renal Epithelia.

    PubMed

    Christensen, Mette G; Fagerberg, Steen K; de Bruijn, Pauline I; Bjaelde, Randi G; Jakobsen, Helle; Leipziger, Jens; Skals, Marianne; Praetorius, Helle A

    2015-06-01

    Urinary tract infections are commonly caused by α-hemolysin (HlyA)-producing Escherichia coli. In erythrocytes, the cytotoxic effect of HlyA is strongly amplified by P2X receptors, which are activated by extracellular ATP released from the cytosol of the erythrocytes. In renal epithelia, HlyA causes reversible [Ca(2+)]i oscillations, which trigger interleukin-6 (IL-6) and IL-8 release. We speculate that this effect is caused by HlyA-induced ATP release from the epithelial cells and successive P2 receptor activation. Here, we demonstrate that HlyA-induced [Ca(2+)]i oscillations in renal epithelia were completely prevented by scavenging extracellular ATP. In accordance, HlyA was unable to inflict any [Ca(2+)]i oscillations in 132-1N1 cells, which lack P2R completely. After transfecting these cells with the hP2Y2 receptor, HlyA readily triggered [Ca(2+)]i oscillations, which were abolished by P2 receptor antagonists. Moreover, HlyA-induced [Ca(2+)]i oscillations were markedly reduced in medullary thick ascending limbs isolated from P2Y2 receptor-deficient mice compared with wild type. Interestingly, the following HlyA-induced IL-6 release was absent in P2Y2 receptor-deficient mice. This suggests that HlyA induces ATP release from renal epithelia, which via P2Y2 receptors is the main mediator of HlyA-induced [Ca(2+)]i oscillations and IL-6 release. This supports the notion that ATP signaling occurs early during bacterial infection and is a key player in the further inflammatory response.

  7. Characterization of the dapA-nlpB genetic locus involved in regulation of swarming motility, cell envelope architecture, hemolysin production, and cell attachment ability in Serratia marcescens.

    PubMed

    Soo, Po-Chi; Wei, Jun-Rong; Horng, Yu-Tze; Hsieh, Shang-Chen; Ho, Shen-Wu; Lai, Hsin-Chih

    2005-09-01

    Swarming migration of Serratia marcescens requires both flagellar motility and cellular differentiation and is a population-density-dependent behavior. While the flhDC and quorum-sensing systems have been characterized as important factors regulating S. marcescens swarming, the underlying molecular mechanisms are currently far from being understood. Serratia swarming is thermoregulated and is characterized by continuous surface migration on rich swarming agar surfaces at 30 degrees C but not at 37 degrees C. To further elucidate the mechanisms, identification of specific and conserved regulators that govern the initiation of swarming is essential. We performed transposon mutagenesis to screen for S. marcescens strain CH-1 mutants that swarmed at 37 degrees C. Analysis of a "precocious-swarming" mutant revealed that the defect in a conserved dapA(Sm)-nlpB(Sm) genetic locus which is closely related to the synthesis of bacterial cell wall peptidoglycan is responsible for the aberrant swarming phenotype. Further complementation and gene knockout studies showed that nlpB(Sm), which encodes a membrane lipoprotein, NlpB(Sm), but not dapA(Sm), is specifically involved in swarming regulation. On the other hand, dapA(Sm) but not nlpB(Sm) is responsible for the determination of cell envelope architecture, regulation of hemolysin production, and cellular attachment capability. While the nlpB(Sm) mutant showed similar cytotoxicity to its parent strain, the dapA(Sm) mutant significantly increased in cytotoxicity. We present evidence that DapA(Sm) is involved in the determination of cell-envelope-associated phenotypes and that NlpB(Sm) is involved in the regulation of swarming motility.

  8. The deletion of several amino acid stretches of Escherichia coli alpha-hemolysin (HlyA) suggests that the channel-forming domain contains beta-strands.

    PubMed

    Benz, Roland; Maier, Elke; Bauer, Susanne; Ludwig, Albrecht

    2014-01-01

    Escherichia coli α-hemolysin (HlyA) is a pore-forming protein of 110 kDa belonging to the family of RTX toxins. A hydrophobic region between the amino acid residues 238 and 410 in the N-terminal half of HlyA has previously been suggested to form hydrophobic and/or amphipathic α-helices and has been shown to be important for hemolytic activity and pore formation in biological and artificial membranes. The structure of the HlyA transmembrane channel is, however, largely unknown. For further investigation of the channel structure, we deleted in HlyA different stretches of amino acids that could form amphipathic β-strands according to secondary structure predictions (residues 71-110, 158-167, 180-203, and 264-286). These deletions resulted in HlyA mutants with strongly reduced hemolytic activity. Lipid bilayer measurements demonstrated that HlyAΔ71-110 and HlyAΔ264-286 formed channels with much smaller single-channel conductance than wildtype HlyA, whereas their channel-forming activity was virtually as high as that of the wildtype toxin. HlyAΔ158-167 and HlyAΔ180-203 were unable to form defined channels in lipid bilayers. Calculations based on the single-channel data indicated that the channels generated by HlyAΔ71-110 and HlyAΔ264-286 had a smaller size (diameter about 1.4 to 1.8 nm) than wildtype HlyA channels (diameter about 2.0 to 2.6 nm), suggesting that in these mutants part of the channel-forming domain was removed. Osmotic protection experiments with erythrocytes confirmed that HlyA, HlyAΔ71-110, and HlyAΔ264-286 form defined transmembrane pores and suggested channel diameters that largely agreed with those estimated from the single-channel data. Taken together, these results suggest that the channel-forming domain of HlyA might contain β-strands, possibly in addition to α-helical structures. PMID:25463653

  9. The deletion of several amino acid stretches of Escherichia coli alpha-hemolysin (HlyA) suggests that the channel-forming domain contains beta-strands.

    PubMed

    Benz, Roland; Maier, Elke; Bauer, Susanne; Ludwig, Albrecht

    2014-01-01

    Escherichia coli α-hemolysin (HlyA) is a pore-forming protein of 110 kDa belonging to the family of RTX toxins. A hydrophobic region between the amino acid residues 238 and 410 in the N-terminal half of HlyA has previously been suggested to form hydrophobic and/or amphipathic α-helices and has been shown to be important for hemolytic activity and pore formation in biological and artificial membranes. The structure of the HlyA transmembrane channel is, however, largely unknown. For further investigation of the channel structure, we deleted in HlyA different stretches of amino acids that could form amphipathic β-strands according to secondary structure predictions (residues 71-110, 158-167, 180-203, and 264-286). These deletions resulted in HlyA mutants with strongly reduced hemolytic activity. Lipid bilayer measurements demonstrated that HlyAΔ71-110 and HlyAΔ264-286 formed channels with much smaller single-channel conductance than wildtype HlyA, whereas their channel-forming activity was virtually as high as that of the wildtype toxin. HlyAΔ158-167 and HlyAΔ180-203 were unable to form defined channels in lipid bilayers. Calculations based on the single-channel data indicated that the channels generated by HlyAΔ71-110 and HlyAΔ264-286 had a smaller size (diameter about 1.4 to 1.8 nm) than wildtype HlyA channels (diameter about 2.0 to 2.6 nm), suggesting that in these mutants part of the channel-forming domain was removed. Osmotic protection experiments with erythrocytes confirmed that HlyA, HlyAΔ71-110, and HlyAΔ264-286 form defined transmembrane pores and suggested channel diameters that largely agreed with those estimated from the single-channel data. Taken together, these results suggest that the channel-forming domain of HlyA might contain β-strands, possibly in addition to α-helical structures.

  10. What controls open-pore and residual currents in the first sensing zone of alpha-hemolysin nanopore? Combined experimental and theoretical study

    NASA Astrophysics Data System (ADS)

    de Biase, Pablo M.; Ervin, Eric N.; Pal, Prithwish; Samoylova, Olga; Markosyan, Suren; Keehan, Michael G.; Barrall, Geoffrey A.; Noskov, Sergei Yu.

    2016-06-01

    The electrophoretic transport of single-stranded DNA through biological nanopores such as alpha-hemolysin (αHL) is a promising and cost-effective technology with the potential to revolutionize genomics. The rational design of pores with the controlled polymer translocation rates and high contrast between different nucleotides could improve significantly nanopore sequencing applications. Here, we apply a combination of theoretical and experimental methods in an attempt to elucidate several selective modifications in the pore which were proposed to be central for the effective discrimination between purines and pyrimidines. Our nanopore test set includes the wild type αHL and six mutants (E111N/M113X/K147N) in which the cross-section and chemical functionality of the first constriction zone of the pore are modified. Electrophysiological recordings were combined with all-atom Molecular Dynamics simulations (MD) and a recently developed Brownian Dynamics (BROMOC) protocol to investigate residual ion currents and pore-DNA interactions for two homo-polymers e.g. poly(dA)40 or poly(dC)40 blocking the pore. The calculated residual currents and contrast in the poly(dA)40/poly(dC)40 blocked pore are in qualitative agreement with the experimental recordings. We showed that a simple structural metric allows rationalization of key elements in the emergent contrast between purines and pyrimidines in the modified αHL mutants. The shape of the pore and its capacity for hydrogen bonding to a translocated polynucleotide are two essential parameters for contrast optimization. To further probe the impact of these two factors in the ssDNA sensing, we eliminated the effect of the primary constriction using serine substitutions (i.e. E111S/M113S/T145S/K147S) and increased the hydrophobic volume of the central residue in the secondary constriction (L135I). This pore modification sharply increased the contrast between Adenine (A) and Cytosine (C).The electrophoretic transport of single

  11. What controls open-pore and residual currents in the first sensing zone of alpha-hemolysin nanopore? Combined experimental and theoretical study

    NASA Astrophysics Data System (ADS)

    de Biase, Pablo M.; Ervin, Eric N.; Pal, Prithwish; Samoylova, Olga; Markosyan, Suren; Keehan, Michael G.; Barrall, Geoffrey A.; Noskov, Sergei Yu.

    2016-06-01

    The electrophoretic transport of single-stranded DNA through biological nanopores such as alpha-hemolysin (αHL) is a promising and cost-effective technology with the potential to revolutionize genomics. The rational design of pores with the controlled polymer translocation rates and high contrast between different nucleotides could improve significantly nanopore sequencing applications. Here, we apply a combination of theoretical and experimental methods in an attempt to elucidate several selective modifications in the pore which were proposed to be central for the effective discrimination between purines and pyrimidines. Our nanopore test set includes the wild type αHL and six mutants (E111N/M113X/K147N) in which the cross-section and chemical functionality of the first constriction zone of the pore are modified. Electrophysiological recordings were combined with all-atom Molecular Dynamics simulations (MD) and a recently developed Brownian Dynamics (BROMOC) protocol to investigate residual ion currents and pore-DNA interactions for two homo-polymers e.g. poly(dA)40 or poly(dC)40 blocking the pore. The calculated residual currents and contrast in the poly(dA)40/poly(dC)40 blocked pore are in qualitative agreement with the experimental recordings. We showed that a simple structural metric allows rationalization of key elements in the emergent contrast between purines and pyrimidines in the modified αHL mutants. The shape of the pore and its capacity for hydrogen bonding to a translocated polynucleotide are two essential parameters for contrast optimization. To further probe the impact of these two factors in the ssDNA sensing, we eliminated the effect of the primary constriction using serine substitutions (i.e. E111S/M113S/T145S/K147S) and increased the hydrophobic volume of the central residue in the secondary constriction (L135I). This pore modification sharply increased the contrast between Adenine (A) and Cytosine (C).The electrophoretic transport of single

  12. Human-to-Bovine Jump of Staphylococcus aureus CC8 Is Associated with the Loss of a β-Hemolysin Converting Prophage and the Acquisition of a New Staphylococcal Cassette Chromosome

    PubMed Central

    Resch, Grégory; François, Patrice; Morisset, Delphine; Stojanov, Milos; Bonetti, Eve J.; Schrenzel, Jacques; Sakwinska, Olga; Moreillon, Philippe

    2013-01-01

    Staphylococcus aureus can colonize and infect both humans and animals, but isolates from both hosts tend to belong to different lineages. Our recent finding of bovine-adapted S. aureus showing close genetic relationship to the human S. aureus clonal complex 8 (CC8) allowed us to examine the genetic basis of host adaptation in this particular CC. Using total chromosome microarrays, we compared the genetic makeup of 14 CC8 isolates obtained from cows suffering subclinical mastitis, with nine CC8 isolates from colonized or infected human patients, and nine S. aureus isolates belonging to typical bovine CCs. CC8 isolates were found to segregate in a unique group, different from the typical bovine CCs. Within this CC8 group, human and bovine isolates further segregated into three subgroups, among which two contained a mix of human and bovine isolates, and one contained only bovine isolates. This distribution into specific clusters and subclusters reflected major differences in the S. aureus content of mobile genetic elements (MGEs). Indeed, while the mixed human-bovine clusters carried commonly human-associated β-hemolysin converting prophages, the bovine-only isolates were devoid of such prophages but harbored an additional new non-mec staphylococcal cassette chromosome (SCC) unique to bovine CC8 isolates. This composite cassette carried a gene coding for a new LPXTG-surface protein sharing homologies with a protein found in the environmental bacterium Geobacillus thermoglucosidans. Thus, in contrast to human CC8 isolates, the bovine-only CC8 group was associated with the combined loss of β-hemolysin converting prophages and gain of a new SCC probably acquired in the animal environment. Remaining questions are whether the new LPXTG-protein plays a role in bovine colonization or infection, and whether the new SCC could further acquire antibiotic-resistance genes and carry them back to human. PMID:23505465

  13. Human-to-bovine jump of Staphylococcus aureus CC8 is associated with the loss of a β-hemolysin converting prophage and the acquisition of a new staphylococcal cassette chromosome.

    PubMed

    Resch, Grégory; François, Patrice; Morisset, Delphine; Stojanov, Milos; Bonetti, Eve J; Schrenzel, Jacques; Sakwinska, Olga; Moreillon, Philippe

    2013-01-01

    Staphylococcus aureus can colonize and infect both humans and animals, but isolates from both hosts tend to belong to different lineages. Our recent finding of bovine-adapted S. aureus showing close genetic relationship to the human S. aureus clonal complex 8 (CC8) allowed us to examine the genetic basis of host adaptation in this particular CC. Using total chromosome microarrays, we compared the genetic makeup of 14 CC8 isolates obtained from cows suffering subclinical mastitis, with nine CC8 isolates from colonized or infected human patients, and nine S. aureus isolates belonging to typical bovine CCs. CC8 isolates were found to segregate in a unique group, different from the typical bovine CCs. Within this CC8 group, human and bovine isolates further segregated into three subgroups, among which two contained a mix of human and bovine isolates, and one contained only bovine isolates. This distribution into specific clusters and subclusters reflected major differences in the S. aureus content of mobile genetic elements (MGEs). Indeed, while the mixed human-bovine clusters carried commonly human-associated β-hemolysin converting prophages, the bovine-only isolates were devoid of such prophages but harbored an additional new non-mec staphylococcal cassette chromosome (SCC) unique to bovine CC8 isolates. This composite cassette carried a gene coding for a new LPXTG-surface protein sharing homologies with a protein found in the environmental bacterium Geobacillus thermoglucosidans. Thus, in contrast to human CC8 isolates, the bovine-only CC8 group was associated with the combined loss of β-hemolysin converting prophages and gain of a new SCC probably acquired in the animal environment. Remaining questions are whether the new LPXTG-protein plays a role in bovine colonization or infection, and whether the new SCC could further acquire antibiotic-resistance genes and carry them back to human. PMID:23505465

  14. Unfolding Kinetics of the Human Telomere i-Motif Under a 10 pN Force Imposed by the α-Hemolysin Nanopore Identify Transient Folded-State Lifetimes at Physiological pH.

    PubMed

    Ding, Yun; Fleming, Aaron M; He, Lidong; Burrows, Cynthia J

    2015-07-22

    Cytosine (C)-rich DNA can adopt i-motif folds under acidic conditions, with the human telomere i-motif providing a well-studied example. The dimensions of this i-motif are appropriate for capture in the nanocavity of the α-hemolysin (α-HL) protein pore under an electrophoretic force. Interrogation of the current vs time (i-t) traces when the i-motif interacts with α-HL identified characteristic signals that were pH dependent. These features were evaluated from pH 5.0 to 7.2, a region surrounding the transition pH of the i-motif (6.1). When the i-motif without polynucleotide tails was studied at pH 5.0, the folded structure entered the nanocavity of α-HL from either the top or bottom face to yield characteristic current patterns. Addition of a 5' 25-mer poly-2'-deoxyadensosine tail allowed capture of the i-motif from the unfolded terminus, and this was used to analyze the pH dependency of unfolding. At pH values below the transition point, only folded strands were observed, and when the pH was increased above the transition pH, the number of folded events decreased, while the unfolded events increased. At pH 6.8 and 7.2 4% and 2% of the strands were still folded, respectively. The lifetimes for the folded states at pH 6.8 and 7.2 were 21 and 9 ms, respectively, at 160 mV electrophoretic force. These lifetimes are sufficiently long to affect enzymes operating on DNA. Furthermore, these transient lifetimes are readily obtained using the α-HL nanopore, a feature that is not easily achievable by other methods.

  15. Hemolysin EthA in Edwardsiella tarda is essential for fish invasion in vivo and in vitro and regulated by two-component system EsrA-EsrB and nucleoid protein HhaEt.

    PubMed

    Wang, Xin; Wang, Qiyao; Xiao, Jingfan; Liu, Qin; Wu, Haizhen; Zhang, Yuanxing

    2010-12-01

    Edwardsiella tarda is a Gram-negative pathogen for hemorrhagic septicemia in fish. Recently, two-component system (TCS) EsrA-EsrB in E. tarda has been found to play key roles in regulating type III secretion system (TTSS) and type VI secretion system (T6SS). In this study, a markedly attenuated ΔesrB mutant was investigated to exhibit enhanced cell-invasion capability, as well as the increased cytotoxicity of its extracellular products (ECPs). Compared with the parental strain, the ΔesrB mutant unexpectedly displayed the significantly increased hemolytic activity, and the restoration of hemolysin production was observed in the complemented strain esrB(+). A hemolysis-associated 147 kDa protein, EthA, was found to be up-regulated in the ECPs of ΔesrB. The deletion of ethA gene in E. tarda wild type and ΔesrB strains drastically decreased their capacities in internalization of epithelial papilloma of carp (EPC) cells. These results indicated that the increased production of EthA was responsible for the enhanced cell-invasion related capabilities in ΔesrB. Furthermore, the expression of EthA in ΔesrB exhibited a temperature-induced manner, and a nucleoid protein Hha(Et) was identified to mediate ethA expression by directly binding to its promoter. These results demonstrated that the virulence determinant EthA was fully required for invasion abilities of E. tarda and was subjected to the control of a complicated and precisely regulated network primed for its invasion, colonization and infection process in fish. PMID:20832475

  16. Genetic conservation of hlyA determinants and serological conservation of HlyA: basis for developing a broadly cross-reactive subunit Escherichia coli alpha-hemolysin vaccine.

    PubMed

    O'Hanley, P; Marcus, R; Baek, K H; Denich, K; Ji, G E

    1993-03-01

    The HlyA determinant among Escherichia coli isolates from patients with symptomatic urinary tract infection was compared in this report with a prototype HlyA encoded by pSF4000 by DNA-DNA hybridization tests with 20-base synthetic oligonucleotides and monoclonal antibody binding and neutralization assays. Hybridization results demonstrated that 349 (98%) of 357 definitive reactions among 54 hemolytic strains shared homology with seven DNA probes spanning many HlyA regions corresponding to residues (R) 41 to 47, 55 to 61, 248 to 254, 306 to 312, 336 to 343, 402 to 408, and 929 to 935. Genetic divergence was identified by lack of hybridization signals among 17 to 76% of the hemolytic strains within the distal portion of a predicted hydrophobic region corresponding to R491 to 319 and within a predicted hydrophilic region corresponding to R491 to 497 and R532 to 538. Serological studies demonstrated that 26 (81%) culture supernatants of 32 hemolytic strains were bound by all 12 monoclonal anti-HlyA antibodies. Among five of six remaining strains, the culture supernatants were bound by 3 to 11 monoclonal antibody preparations. There was only one hemolytic culture supernatant that failed to be bound by any monoclonal antibody, although the strain hybridized with nine hemolysin DNA probes. In addition, hemolytic activity of all 24 different culture supernatants tested was reduced by at least twofold by one monoclonal antibody specific for R2-161. These data extend and support previous views that the HlyA determinant is conserved among E. coli strains and suggest that a broadly cross-reactive HlyA subunit vaccine can be developed.

  17. POSSIBLE ROLES OF FUNGAL HEMOLYSINS IN SICK BUILDING SYNDROME

    EPA Science Inventory

    The World Health Organization (WHO) definition of SBS includes such symptoms in the occupants as headache, distraction, dizziness, fatigue, watery eyes, runny or blocked or bleeding nose, dry or sore throat and skin irritation. The WHO has set a criterion for a healthy building ...

  18. DNA translocation through α-hemolysin nanopores with potential application to macromolecular data storage

    NASA Astrophysics Data System (ADS)

    Khulbe, Pramod K.; Mansuripur, Masud; Gruener, Raphael

    2005-05-01

    Digital information can be encoded in the building-block sequence of macromolecules, such as RNA and single-stranded DNA. Methods of "writing" and "reading" macromolecular strands are currently available, but they are slow and expensive. In an ideal molecular data storage system, routine operations such as write, read, erase, store, and transfer must be done reliably and at high speed within an integrated chip. As a first step toward demonstrating the feasibility of this concept, we report preliminary results of DNA readout experiments conducted in miniaturized chambers that are scalable to even smaller dimensions. We show that translocation of a single-stranded DNA molecule (consisting of 50 adenosine bases followed by 100 cytosine bases) through an ion channel yields a characteristic signal that is attributable to the two-segment structure of the molecule. We also examine the dependence of the translocation rate and speed on the adjustable parameters of the experiment.

  19. All-Atom Molecular Dynamics Simulation of Protein Translocation through an α-Hemolysin Nanopore.

    PubMed

    Di Marino, Daniele; Bonome, Emma Letizia; Tramontano, Anna; Chinappi, Mauro

    2015-08-01

    Nanopore sensing is attracting the attention of a large and varied scientific community. One of the main issues in nanopore sensing is how to associate the measured current signals to specific features of the molecule under investigation. This is particularly relevant when the translocating molecule is a protein and the pore is sufficiently narrow to necessarily involve unfolding of the translocating protein. Recent experimental results characterized the cotranslocational unfolding of Thioredoxin (Trx) passing through an α-hemolisin pore, providing evidence for the existence of a multistep process. In this study we report the results of all-atom molecular dynamics simulations of the same system. Our data indicate that Trx translocation involves two main barriers. The first one is an unfolding barrier associated with a translocation intermediate where the N-terminal region of Trx is stuck at the pore entrance in a conformation that strongly resembles the native one. After the abrupt unfolding of the N-terminal region, the Trx enters the α-hemolisin vestibule. During this stage, the constriction is occupied not only by the translocating residue but also by a hairpin-like structure forming a tangle in the constriction. The second barrier is associated with the disentangling of this region.

  20. Genomic Analysis of Immune Response against Vibrio cholerae Hemolysin in Caenorhabditis elegans

    PubMed Central

    Sahu, Surasri N.; Bozdag, Serdar; Lee, Jeong H.; LeClerc, Joseph E.; Cinar, Hediye Nese

    2012-01-01

    Vibrio cholerae cytolysin (VCC) is among the accessory V. cholerae virulence factors that may contribute to disease pathogenesis in humans. VCC, encoded by hlyA gene, belongs to the most common class of bacterial toxins, known as pore-forming toxins (PFTs). V. cholerae infects and kills Caenorhabditis elegans via cholerae toxin independent manner. VCC is required for the lethality, growth retardation and intestinal cell vacuolation during the infection. However, little is known about the host gene expression responses against VCC. To address this question we performed a microarray study in C. elegans exposed to V. cholerae strains with intact and deleted hlyA genes. Many of the VCC regulated genes identified, including C-type lectins, Prion-like (glutamine [Q]/asparagine [N]-rich)-domain containing genes, genes regulated by insulin/IGF-1-mediated signaling (IIS) pathway, were previously reported as mediators of innate immune response against other bacteria in C. elegans. Protective function of the subset of the genes up-regulated by VCC was confirmed using RNAi. By means of a machine learning algorithm called FastMEDUSA, we identified several putative VCC induced immune regulatory transcriptional factors and transcription factor binding motifs. Our results suggest that VCC is a major virulence factor, which induces a wide variety of immune response- related genes during V. cholerae infection in C. elegans. PMID:22675448

  1. Complete Genome Sequence of Hemolysin-Containing Carnobacterium sp. Strain CP1 Isolated from the Antarctic

    PubMed Central

    Zhu, Sidong; Wang, Xing; Zhang, Di; Jing, Xiaohuan; Zhang, Ning

    2016-01-01

    Carnobacterium sp. strain CP1 was isolated from Antarctic sandy soil and predicted to be a novel species belonging to the genus Carnobacterium. Herein, we report the complete genome sequence, which consists of a circular 2,605,518-bp chromosome and an 8,883-bp plasmid with G+C contents of 38.13% and 31.63%, respectively. PMID:27445381

  2. PolyA Single Strand DNA Translocation Through an Alpha-Hemolysin Pore Stem

    NASA Technical Reports Server (NTRS)

    OKeeffe, James; Cozmuta, Ioana; Stolc, Viktor

    2003-01-01

    A new model for the polymer-pore interaction energy is introduced, based on an atomic-scale description of coulombic polymer-pore interaction. The enhanced drift velocity, experimentally observed for short polymers, is successfully accounted for, using this interaction energy model. For R/R(sub 0)>4 (R(sub 0)=7 angstroms) the translocation velocity approaches the free space drift velocity v(sub 0). This motivates the need to appropriately derivatize artificial nanopores, where R>R(sub 0).

  3. Shiga Toxin, Cytolethal Distending Toxin, and Hemolysin Repertoires in Clinical Escherichia coli O91 Isolates▿

    PubMed Central

    Bielaszewska, Martina; Stoewe, Franziska; Fruth, Angelika; Zhang, Wenlan; Prager, Rita; Brockmeyer, Jens; Mellmann, Alexander; Karch, Helge; Friedrich, Alexander W.

    2009-01-01

    Shiga toxin (Stx)-producing Escherichia coli (STEC) strains of serogroup O91 are the most common human pathogenic eae-negative STEC strains. To facilitate diagnosis and subtyping of these pathogens, we genotypically and phenotypically characterized 100 clinical STEC O91 isolates. Motile strains expressed flagellar antigens H8 (1 strain), H10 (2 strains), H14 (52 strains), and H21 (20 strains) or were H nontypeable (Hnt) (10 strains); 15 strains were nonmotile. All nonmotile and Hnt strains possessed the fliC gene encoding the flagellin subunit of the H14 antigen (fliCH14). Most STEC O91 strains possessed enterohemorrhagic E. coli hlyA and expressed an enterohemolytic phenotype. Among seven stx alleles identified, stx2dact, encoding mucus- and elastase-activatable Stx2d, was present solely in STEC O91:H21, whereas most strains of the other serotypes possessed stx1. Moreover, only STEC O91:H21 possessed the cdt-V cluster, encoding cytolethal distending toxin V; the toxin was regularly expressed and was lethal to human microvascular endothelial cells. Infection with STEC O91:H21 was associated with hemolytic-uremic syndrome (P = 0.0015), whereas strains of the other serotypes originated mostly in patients with nonbloody diarrhea. We conclude that STEC O91 clinical isolates belong to at least four lineages that differ by H antigens/fliC types, stx genotypes, and non-stx putative virulence factors, with accumulation of virulence determinants in the O91:H21 lineage. Isolation of STEC O91 from patients' stools on enterohemolysin agar and the rapid initial subtyping of these isolates using fliC genotyping facilitate the identification of these emerging pathogens in clinical and epidemiological studies and enable prediction of the risk of a severe clinical outcome. PMID:19403777

  4. Immunization with recombinant aerolysin and hemolysin protected channel catfish against virulent Aeromonas hydrophila

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aeromonas hydrophila is emerging as one of the major concerns in catfish aquaculture in the Southeastern United States due to recent outbreaks of motile aeromonad septicemia (MAS) caused by virulent clonal isolates. There is no effective vaccine currently available for the prevention of MAS. In this...

  5. Real-time PCR detection of the thermostable direct hemolysin and thermolabile hemolysin genes in a Vibrio parahaemolyticus cultured from mussels and mussel homogenate associated with a foodborne outbreak.

    PubMed

    Davis, Carisa R; Heller, Loree C; Peak, K Kealy; Wingfield, David L; Goldstein-Hart, Cynthia L; Bodager, Dean W; Cannons, Andrew C; Amuso, Philip T; Cattanii, Jacqueline

    2004-05-01

    Molecular methods have become vital epidemiological tools in the detection and characterization of bacteria associated with a foodborne outbreak. We used both culture and real-time PCR to detect a Vibrio parahaemolyticus isolate associated with a foodborne outbreak. The outbreak occurred in July 2002 in Polk County, Florida, and originated at a Chinese buffet, with one person being hospitalized. The hospital isolated V. parahaemolyticus from the patient but destroyed the sample after diagnosis. From an onsite visit of the restaurant, food samples that possibly contributed to the outbreak were collected and sent to the Florida Department of Health, Tampa Branch Laboratory. Crab legs, crabsticks, and mussel samples were homogenized and incubated according to the Food and Drug Administration Bacteriological Analytical Manual culture protocol. Three sets of primers and a TaqMan probe were designed to target the tdh, trh, and tlh genes and used for real-time PCR. This study was successful in isolating V. parahaemolyticus from a mussel sample and detecting two of its genes (tdh and tlh) in food and pure culture by real-time PCR. PMID:15151240

  6. The Serratia marcescens hemolysin is secreted but not activated by stable protoplast-type L-forms of Proteus mirabilis.

    PubMed

    Sieben, S; Hertle, R; Gumpert, J; Braun, V

    1998-10-01

    The outer-membrane protein ShlB of Serratia marcescens activates and secretes hemolytic ShlA into the culture medium. Without ShlB, inactive ShlA (termed ShlA*) remains in the periplasm. Since Proteus mirabilis L-form cells lack an outer membrane and a periplasm, it was of interest to determine in which compartment recombinant ShlA* and ShlB are localized and whether ShlB activates ShlA*. The cloned shlB and shlA genes were transcribed in P. mirabilis stable L-form cells by the temperature-inducible phage T7 RNA polymerase. Radiolabeling, Western blotting, and complementation with C-terminally truncated ShlA (ShlA255) identified inactive ShlA* in the culture supernatant. ShlB remained cell-bound and did not activate ShlA without integration in an outer membrane. Although hemolytic ShlA added to L-form cells had access to the cytoplasmic membrane, it did not affect L-form cells. Synthesis of the large ShlA protein (165 kDa) in P. mirabilis L-form cells under phage T7 promoter control demonstrates that L-form cells are suitable for the synthesis and secretion of large recombinant proteins. This property and the easy isolation of released proteins make L-form cells suitable for the biotechnological production of proteins.

  7. Incomplete activation of Escherichia coli hemolysin (HlyA) due to mutations in the 3' region of hlyC.

    PubMed Central

    Guzmán-Verri, C; García, F; Arvidson, S

    1997-01-01

    Mutational analysis of the carboxy-terminal region of Escherichia coli HlyC was performed by site-directed mutagenesis. Replacement of residue Val-127 or Lys-129 reduced the activity of HlyC to about 30 or 60%, respectively, of that of the wild type, while replacement of Gly-128 reduced the activity to less than 1% of the wild-type level. Complete inactivation of HlyC was caused by a double mutation, replacement of Gly-128 with valine and of Lys-129 with isoleucine. Analysis of culture supernatants from mutants with reduced hemolytic activity by two-dimensional gel electrophoresis revealed the production and simultaneous secretion of nonacylated, monoacylated, and fully acylated HlyA forms, demonstrating impairment of the acylation reaction, possibly due to a decreased affinity of HlyC for the individual HlyA acylation sites. PMID:9294460

  8. Comparative prevalence of immune evasion complex genes associated with beta-hemolysin converting bacteriophages in MRSA ST5 isolates from swine, swine facilities, humans with swine contact, and humans with no swine contact

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Livestock associated methicillin-resistant Staphylococcus aureus (LA-MRSA) draws concern from the public health community because in some countries these organisms may represent the largest reservoir of MRSA outside hospital settings. Recent studies indicate LA-MRSA strains from swine are more genet...

  9. Effect of sodium chloride and citric acid on growth and toxin production by A. caviae and A. sobria at moderate and low temperatures.

    PubMed

    Abu-Ghazaleh, B M

    2000-10-01

    The effect of sodium chloride and citric acid on hemolysin and caseinase production by Aeromonas caviae and Aeromonas sobria at 32 degrees C and 5 degrees C was investigated. At 32 degrees C, although both strains were tolerant to 3% NaCl in TSB, the production of caseinase was decreased in the presence of 1-3% NaCl, and the production of hemolysin was abolished by 2-3% NaCl. Citric acid (0.03%) was less effective than NaCl in reducing hemolysin and caseinase production by both strains at 32 degrees C. A combination of low temperature (5 degrees C) and citric acid treatment reduced hemolysin and caseinase production by both strains. A combination of low temperature (5 degrees C) and NaCl (3%) treatment was the most effective procedure in reducing growth and hemolysin and caseinase production by the tested strains.

  10. Thiol-independent activity of a cholesterol-binding enterohemolysin produced by enteropathogenic Escherichia coli.

    PubMed

    Figueirêdo, P M S; Catani, C F; Yano, T

    2003-11-01

    Enterohemolysin produced by Escherichia coli associated with infant diarrhea showed characteristics similar to those of thiol-activated hemolysins produced by Gram-positive bacteria, including inactivation by cholesterol, lytic activity towards eukaryotic cells and thermoinstability. However, enterohemolysin activity was not inactivated by oxidation or by SH group-blocking agents (1 mM HgCl2, 1 mM iodoacetic acid) and the hemolysin (100 microg/ml) was not lethal to mice, in contrast to the lethality of the thiol-activated hemolysin family to animals. Earlier reports showed that intravenous injection of partially purified streptolysin O preparations (0.2 microg) was rapidly lethal to mice. These results suggest that E. coli enterohemolysin is not a thiol-activated hemolysin, despite its ability to bind cholesterol, probably due to the absence of free thiol-group(s) that characterize the active form of the thiol-activated hemolysin molecule.

  11. FACTORS INFLUENCING IN VITRO KILLING OF BACTERIA BY HEMOCYTES OF THE EASTERN OYSTER (CRASSOSTREA VIRGINICA)

    EPA Science Inventory

    Vibrio parahaemolyticus strains altered in motility or colonial morphology (opaque versus translucent), Listeria monocytogenes mutants lacking catalase, superoxide dismutase, hemolysin, or phospholipase activities, and Vibrio vulnificus strains, possessing and lacking capsules we...

  12. Aeromonas Caviae Strain Induces Th1 Cytokine Response in Mouse Intestinal Tract

    EPA Science Inventory

    Aeromonas caviae has been associated with human gastrointestinal disease. Strains of this species typically lack virulence factors (VFs) such as enterotoxins and hemolysins that are produced by other human pathogens of the Aeromonas genus. Microarray profiling of murine small i...

  13. 78 FR 33692 - Implementation of the Understandings Reached at the 2012 Australia Group (AG) Plenary Meeting and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-05

    ... that the controls on ``Staphylococcus aureus toxins'' apply to ``Staphylococcus aureus enterotoxins, hemolysin alpha toxin, and toxic shock syndrome toxin (formerly known as Staphylococcus enterotoxin F... Biological Agents for Export Control,'' as follows, and ``subunits'' thereof: * * * * * d.14....

  14. Ham test

    MedlinePlus

    Acid hemolysin test; Paroxysmal nocturnal hemoglobinuria - Ham test; PNH - Ham test ... BJ. In: Chernecky CC, Berger BJ, eds. Laboratory Tests and Diagnostic Procedures . 6th ed. Philadelphia, PA: Elsevier ...

  15. Quorum sensing inhibitors of Staphylococcus aureus from Italian medicinal plants.

    PubMed

    Quave, Cassandra L; Plano, Lisa R W; Bennett, Bradley C

    2011-01-01

    Morbidity and mortality estimates due to methicillin-resistant Staphylococcus aureus (MRSA) infections continue to rise. Therapeutic options are limited by antibiotic resistance. Anti-pathogenic compounds, which inhibit quorum sensing (QS) pathways, may be a useful alternative to antibiotics. Staphylococcal QS is encoded by the AGR locus and is responsible for the production of δ-hemolysin. Quantification of δ-hemolysin found in culture supernatants permits the analysis of AGR activity at the translational rather than transcriptional level. We employed reversed phase high performance chromatographic (RP-HPLC) techniques to investigate the anti-QS activity of 168 extracts from 104 Italian plants through quantification of δ-hemolysin. Extracts from three medicinal plants (Ballota nigra, Castanea sativa, and Sambucus ebulus) exhibited a dose-dependent response in the production of δ-hemolysin, indicating anti-QS activity in a pathogenic MRSA isolate.

  16. Quorum Sensing Inhibitors for Staphylococcus aureus from Italian Medicinal Plants

    PubMed Central

    Quave, Cassandra L.; Plano, Lisa R.W.; Bennett, Bradley C.

    2010-01-01

    Morbidity and mortality estimates due to methicillin-resistant Staphylococcus aureus (MRSA) infections continue to rise. Therapeutic options are limited by antibiotic resistance. Anti-pathogenic compounds, which inhibit quorum sensing (QS) pathways, may be a useful alternative to antibiotics. Staphylococcal QS is encoded by the agr locus and is responsible for the production of δ-hemolysin. Quantification of δ-hemolysin found in culture supernatants permits the analysis of agr activity at the translational, rather than transcriptional, level. We employed RP-HPLC techniques to investigate the anti-QS activity of 168 extracts from 104 Italian plants through quantification of δ-hemolysin. Extracts from three medicinal plants (Ballota nigra, Castanea sativa, and Sambucus ebulus) exhibited a dose-dependent response in the production of δ-hemolysin, indicating strong anti-QS activity in a pathogenic MRSA isolate. PMID:20645243

  17. VIRULENCE CHARACTERISTICS OF HETEROTROPHIC BACTERIA COMMONLY ISOLATED FROM POTABLE WATER

    EPA Science Inventory

    Heterotrophic bacteria isolated from drinking water distribution systems were examined to determine if they possessed putative virulence factors such as hemolysins, proteases, or cytotoxins. Representative samples of colonies from several different distribution systems indicated ...

  18. CHARACTERIZATION OF STAPHYLOCOCCI ISOLATED FROM RAW MILK.

    PubMed

    ZEMELMAN, R; LONGERI, L

    1965-03-01

    To evaluate the pathogenicity of staphylococci from bovine raw milk, the general characteristics of 775 strains isolated from 798 samples of milk were studied. The coagulase test was performed by use of rabbit plasma. Chromogenesis, mannitol fermentation, and gelatin liquefaction were investigated on Chapman's Medium 110, after 48 hr of incubation. Production of beta-hemolysin, which has been considered indicative of pathogenic staphylococci of animal origin, was determined by streaking different strains on sheep blood-agar plates in the presence of a strain of Lancefield group B streptococci. Plates were incubated at 37 C for 24 hr, and strong hemolysis was produced in the zone of interaction of beta-hemolysin and some substance liberated by streptococcus (CAMP test). Of 404 strains found to be coagulase-positive, 95.8% exhibited a deep-orange pigment, 76.5% produced beta-hemolysin, 91.8% fermented mannitol, and 75% liquefield gelatin. Of 371 strains which gave a negative coagulase test, about 16% fermented mannitol and liquefied gelatin; none of these strains produced beta-hemolysin. When results are grouped according to pigmentation and coagulase production, beta-hemolysin seems to be developed by pathogenic strains of Staphylococcus aureus only. If suitability of these tests for investigation of pathogenicity is compared, production of beta-hemolysin appears to be the most useful one, since no "false positive" results were found. The use of the CAMP test as a simple and rapid technique to determine production of beta-hemolysin by pathogenic strains of animal staphylococci during routine bacteriological work is suggested.

  19. Formation of individual protein channels in lipid bilayers suspended in nanopores.

    PubMed

    Studer, André; Han, Xiaojun; Winkler, Fritz K; Tiefenauer, Louis X

    2009-10-15

    Free-standing lipid bilayers are formed in regularly arranged nanopores of 200, 400 and 800 nm in a 300 nm thin hydrophobic silicon nitride membrane separating two fluid compartments. The extraordinary stability of the lipid bilayers allows us to monitor channel formation of the model peptide melittin and alpha-hemolysin from Staphylococcus aureus using electrochemical impedance spectroscopy and chronoamperometry. We observed that melittin channel formation is voltage-dependent and transient, whereas transmembrane heptameric alpha-hemolysin channels in nano-BLMs persist for hours. The onset of alpha-hemolysin-mediated conduction depends on the applied protein concentration and strongly on the diameter of the nanopores. Heptameric channel formation from adsorbed alpha-hemolysin monomers needs more time in bilayers suspended in 200 nm pores compared to bilayers in pores of 400 and 800 nm diameters. Diffusion of sodium ions across alpha-hemolysin channels present in a sufficiently high number in the bilayers was quantitatively and specifically determined using ion selective electrodes. The results demonstrate that relatively small variations of nano-dimensions have a tremendous effect on observable dynamic biomolecular processes. Such nanopore chips are potentially useful as supports for stable lipid bilayers to establish functional assays of membrane proteins needed in basic research and drug discovery.

  20. Biological and Immunological Properties of Encapsulated Strains of Staphylococcus aureus from Human Sources

    PubMed Central

    Yoshida, Kosaku; Smith, Melvin R.; Naito, Yoshiko

    1970-01-01

    Of 875 strains of Staphylococcus aureus isolated from human source clinical specimens, 37 (4.2%) were encapsulated strains. These were all negative for clumping factor and could not be typed with bacteriophages or by serology. Twenty-one of these did not produce any hemolysins, 15 produced alpha hemolysin, 1 produced beta hemolysin, and 1 produced both beta and delta hemolysins. After one or two subcultures, 27 of the encapsulated strains converted to the compact variant form, all became positive for clumping factor, 12 became phage-typable, and 24 became sero-typable. In addition, 7 strains converted from negative to alpha hemolysin production. Comparison of phage- and sero-types did not reveal any relationships. Immunologically, mice challenged with heat-killed encapsulated strains were protected against a challenge infection with the Smith diffuse strain. Protective antibodies in rabbit anti-Smith diffuse strain antisera were removed by adsorption using the encapsulated organisms isolated in this study. The adsorbed sera no longer protected against challenge infection in mice with the Smith diffuse strain. From these results, it appears that the encapsulated strains isolated were immunologically and biologically similar to the classical Smith diffuse strain. PMID:16557873

  1. Variation in hemolytic activity of Brachyspira hyodysenteriae strains from pigs.

    PubMed

    Mahu, Maxime; De Pauw, Nele; Vande Maele, Lien; Verlinden, Marc; Boyen, Filip; Ducatelle, Richard; Haesebrouck, Freddy; Martel, An; Pasmans, Frank

    2016-01-01

    Brachyspira hyodysenteriae is the primary cause of swine dysentery, which is responsible for major economic losses to the pig industry worldwide. The hemolytic activity of 10 B. hyodysenteriae strains isolated from stools of pigs with mild to mucohemorrhagic diarrhea was compared and seven hemolysis associated genes were sequenced. Hemolysis induced by these strains varied from strong to near absent. One weakly hemolytic B. hyodysenteriae strain showed sequence changes in five hemolysis associated genes (tlyA, tlyB, hemolysin III, hemolysin activation protein and hemolysin III channel protein) resulting in amino acid substitutions. The occurrence of weakly hemolytic strains identifiable as B. hyodysenteriae should be taken into account in swine dysentery diagnostics. The presence of these strains may affect herd dysentery status, with great impact on a farms trading opportunities. PMID:27338265

  2. Distribution of virulence genes in clinical and environmental isolates of Aeromonas spp.

    PubMed

    Chacón, M R; Figueras, M J; Castro-Escarpulli, G; Soler, L; Guarro, J

    2003-01-01

    The distribution and phenotypic activity of the genes encoding for serine protease, glycerophospholipid-cholesterol acyltransferase, lipases, aerolysin/hemolysin and DNases were investigated in 234 isolates identified by 16S rDNA-RFLP representing all the species of Aeromonas. The former three genes were found to be highly conserved among the genus. Aerolysin/hemolysin and DNase genes and beta-hemolytic activity were significantly more frequent in clinical than in environmental isolates. Aerolysin/hemolysin and serine protease genes were present in all beta-hemolytic strains supporting serine protease as possibly important for the activation of the former gene. The high prevalence of virulence factors in clinical isolates indicates that they may play a role in the mechanisms of pathogenesis of these microorganisms.

  3. Vibrio parahaemolyticus: a review on the pathogenesis, prevalence, and advance molecular identification techniques

    PubMed Central

    Letchumanan, Vengadesh; Chan, Kok-Gan; Lee, Learn-Han

    2014-01-01

    Vibrio parahaemolyticus is a Gram-negative halophilic bacterium that is found in estuarine, marine and coastal environments. V. parahaemolyticus is the leading causal agent of human acute gastroenteritis following the consumption of raw, undercooked, or mishandled marine products. In rare cases, V. parahaemolyticus causes wound infection, ear infection or septicaemia in individuals with pre-existing medical conditions. V. parahaemolyticus has two hemolysins virulence factors that are thermostable direct hemolysin (tdh)-a pore-forming protein that contributes to the invasiveness of the bacterium in humans, and TDH-related hemolysin (trh), which plays a similar role as tdh in the disease pathogenesis. In addition, the bacterium is also encodes for adhesions and type III secretion systems (T3SS1 and T3SS2) to ensure its survival in the environment. This review aims at discussing the V. parahaemolyticus growth and characteristics, pathogenesis, prevalence and advances in molecular identification techniques. PMID:25566219

  4. Probing Peptide and Protein Insertion in a Biomimetic S-Layer Supported Lipid Membrane Platform

    PubMed Central

    Damiati, Samar; Schrems, Angelika; Sinner, Eva-Kathrin; Sleytr, Uwe B.; Schuster, Bernhard

    2015-01-01

    The most important aspect of synthetic lipid membrane architectures is their ability to study functional membrane-active peptides and membrane proteins in an environment close to nature. Here, we report on the generation and performance of a biomimetic platform, the S-layer supported lipid membrane (SsLM), to investigate the structural and electrical characteristics of the membrane-active peptide gramicidin and the transmembrane protein α-hemolysin in real-time using a quartz crystal microbalance with dissipation monitoring in combination with electrochemical impedance spectroscopy. A shift in membrane resistance is caused by the interaction of α-hemolysin and gramicidin with SsLMs, even if only an attachment onto, or functional channels through the lipid membrane, respectively, are formed. Moreover, the obtained results did not indicate the formation of functional α-hemolysin pores, but evidence for functional incorporation of gramicidin into this biomimetic architecture is provided. PMID:25633104

  5. Urovirulence determinants in Escherichia coli isolates causing first-episode and recurrent cystitis in women.

    PubMed

    Stapleton, A; Moseley, S; Stamm, W E

    1991-04-01

    To assess the prevalence of urovirulence determinants among Escherichia coli isolates from women with acute uncomplicated cystitis, 121 isolates from 87 women with first-episode or recurrent cystitis and 156 fecal isolates from 52 women without recent urinary tract infection were tested using DNA probes for P fimbriae, hemolysin, aerobactin, and diffuse adhesin and for expression of hemolysin and P and F adhesins. P fimbrial genotype (P = .002), hemolysin phenotype (P = .007), and the diffuse adhesin determinant (P = .03), but not aerobactin, were found more frequently in E. coli from women with acute cystitis, and expression of the F adhesin (41%) was more common than the P adhesin (24%; P = .001). E. coli isolates that caused cystitis in women using diaphragms had fewer virulence determinants than those from nonusers (P = .04), suggesting that diaphragm use may allow infection with less virulent E. coli.

  6. Isolation of extraintestinal pathogenic Escherichia coli from diarrheic dogs and their antimicrobial resistance profile

    PubMed Central

    de Cleber Jacob Silva Paula; Marin, José Moacir

    2008-01-01

    From January to December 2006, 92 Escherichia coli isolates from 25 diarrheic dogs were analyzed by screening for the presence of adhesin-encoding genes (pap, sfa, afa), hemolysin and aerobactin genes. Virulence gene frequencies detected in those isolates were: 12% pap, 1% sfa, 10% hemolysin and 6.5% aerobactin. Ten isolates were characterized as extraintestinal pathogenic E. coli (ExPEC) strains; all showed a multidrug resistance phenotype that may represent a reason for concern due the risk of dissemination of antimicrobial resistant genes to the microbiota of human beings. PMID:24031253

  7. Aeromonas caviae strain induces Th1 cytokine response in mouse intestinal tract

    EPA Science Inventory

    Aeromonas caviae has been associated with human gastrointestinal disease. Strains of this species typically lack virulence factors (VFs) such as enterotoxins and hemolysins that are produced by other human pathogens of the Aeromonas genus,. Microarray profiling of...

  8. Ecology of Vibrio parahaemolyticus and Vibrio vulnificus in the Coastal and Estuarine Waters of Louisiana, Maryland, Mississippi, and Washington (United States)

    PubMed Central

    Bowers, John C.; Griffitt, Kimberly J.; Molina, Vanessa; Clostio, Rachel W.; Pei, Shaofeng; Laws, Edward; Paranjpye, Rohinee N.; Strom, Mark S.; Chen, Arlene; Hasan, Nur A.; Huq, Anwar; Noriea, Nicholas F.; Grimes, D. Jay; Colwell, Rita R.

    2012-01-01

    Vibrio parahaemolyticus and Vibrio vulnificus, which are native to estuaries globally, are agents of seafood-borne or wound infections, both potentially fatal. Like all vibrios autochthonous to coastal regions, their abundance varies with changes in environmental parameters. Sea surface temperature (SST), sea surface height (SSH), and chlorophyll have been shown to be predictors of zooplankton and thus factors linked to vibrio populations. The contribution of salinity, conductivity, turbidity, and dissolved organic carbon to the incidence and distribution of Vibrio spp. has also been reported. Here, a multicoastal, 21-month study was conducted to determine relationships between environmental parameters and V. parahaemolyticus and V. vulnificus populations in water, oysters, and sediment in three coastal areas of the United States. Because ecologically unique sites were included in the study, it was possible to analyze individual parameters over wide ranges. Molecular methods were used to detect genes for thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and tdh-related hemolysin (trh) as indicators of V. parahaemolyticus and the hemolysin gene vvhA for V. vulnificus. SST and suspended particulate matter were found to be strong predictors of total and potentially pathogenic V. parahaemolyticus and V. vulnificus. Other predictors included chlorophyll a, salinity, and dissolved organic carbon. For the ecologically unique sites included in the study, SST was confirmed as an effective predictor of annual variation in vibrio abundance, with other parameters explaining a portion of the variation not attributable to SST. PMID:22865080

  9. Cloning, expressing, and hemolysis of tdh, trh and tlh genes of Vibrio parahaemolyticus

    NASA Astrophysics Data System (ADS)

    Zhao, Yonggang; Tang, Xiaoqian; Zhan, Wenbin

    2011-09-01

    Vibrio parahaemolyticus (VP) is one of the pathogenic vibrios endangering net-cage cultured Pseudosciaena crocea, Fennerpenaeus chinensis, and shellfish in coastal areas of China. Several types of hemolysins produced by Vp have been characterized as major virulence factors. They are thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH) and thermolabile hemolysin (TLH). In this study, we cloned tdh, trh, and tlh genes from the genome DNA of VP by polymerase chain reaction (PCR). We ligated the three genes into prokaryotic expression vector pET-28a (+), and transformed the recombinant plasmids into Escherichia coli BL21 (DE3). The expression of recombinant proteins was induced by isopropyl-β-D-thiogalacto-pyranoside (IPTG). The recombinant proteins were expressed in a form of inclusion bodies and thus purified with Ni-NTA affinity chromatography. Western blotting results showed that recombinant proteins, TDH, TRH and TLH, could be recognized by rabbit anti-VP serum. The three purified proteins were renatured by gradient dialysis. The renatured proteins exhibited hemolytic activity except for TLH in the presence of phosphatidylcholine. These results not only are helpful for better understanding these genes' functions under a single factor level, but also provide evidence for VP vaccine engineering.

  10. Comparison of the hemolytic activity between C. albicans and non-albicans Candida species.

    PubMed

    Rossoni, Rodnei Dennis; Barbosa, Júnia Oliveira; Vilela, Simone Furgeri Godinho; Jorge, Antonio Olavo Cardoso; Junqueira, Juliana Campos

    2013-01-01

    The ability to produce enzymes, such as hemolysins, is an important virulence factor for the genus Candida.The objective of this study was to compare the hemolytic activity between C. albicansand non-albicans Candida species. Fifty strains of Candida species, isolated from the oral cavity of patients infected with HIV were studied. The isolates included the following species: C. albicans, C. dubliniensis, C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, C. dubliniensis, C. norvegensis, C. lusitaniae, and C. guilliermondii. Hemolysin production was evaluated on Sabouraud dextrose agar containing chloramphenicol, blood, and glucose. A loop-full of pure Candidaculture was spot-inoculated onto plates and incubated at 37 ºC for 24 h in a 5% CO2 atmosphere. Hemolytic activity was defined as the formation of a translucent halo around the colonies. All C. albicansstrains that were studied produced hemolysins. Among the non-albicans Candidaspecies, 86% exhibited hemolytic activity. Only C. guilliermondiiand some C. parapsilosis isolates were negative for this enzyme. In conclusion, most non-albicans Candidaspecies had a similar ability to produce hemolysins when compared to C. albicans.

  11. RssAB-FlhDC-ShlBA as a major pathogenesis pathway in Serratia marcescens.

    PubMed

    Lin, Chuan-Sheng; Horng, Jim-Tong; Yang, Chun-Hung; Tsai, Yu-Huan; Su, Lin-Hui; Wei, Chia-Fong; Chen, Chang-Chieh; Hsieh, Shang-Chen; Lu, Chia-Chen; Lai, Hsin-Chih

    2010-11-01

    Serratia marcescens has long been recognized as an important opportunistic pathogen, but the underlying pathogenesis mechanism is not completely clear. Here, we report a key pathogenesis pathway in S. marcescens comprising the RssAB two-component system and its downstream elements, FlhDC and the dominant virulence factor hemolysin ShlBA. Expression of shlBA is under the positive control of FlhDC, which is repressed by RssAB signaling. At 37°C, functional RssAB inhibits swarming, represses hemolysin production, and promotes S. marcescens biofilm formation. In comparison, when rssBA is deleted, S. marcescens displays aberrant multicellularity favoring motile swarming with unbridled hemolysin production. Cellular and animal infection models further demonstrate that loss of rssBA transforms this opportunistic pathogen into hypervirulent phenotypes, leading to extensive inflammatory responses coupled with destructive and systemic infection. Hemolysin production is essential in this context. Collectively, a major virulence regulatory pathway is identified in S. marcescens.

  12. Aerobactin and other virulence factor genes among strains of Escherichia coli causing urosepsis: association with patient characteristics.

    PubMed

    Johnson, J R; Moseley, S L; Roberts, P L; Stamm, W E

    1988-02-01

    To assess the role of aerobactin as a virulence factor among uropathogenic Escherichia coli, we determined the prevalence, location, and phenotypic expression of aerobactin determinants among 58 E. coli strains causing bacteremic urinary tract infections. We correlated the presence of the aerobactin system with antimicrobial-agent resistance, the presence and phenotypic expression of other uropathogenic virulence factor determinants (P fimbriae, hemolysin, and type 1 fimbriae), and characteristics of patients. Colony and Southern hybridization of total and plasmid DNA with DNA probes for each virulence factor showed that aerobactin determinants were present in 78% of the strains and were plasmid associated in 21%, whereas P fimbria, hemolysin, and type 1 fimbria determinants were present in 74, 43, and 98% of the strains, respectively, and were always chromosomal. Chromosomal aerobactin, P fimbria, and hemolysin determinants occurred together on the chromosome more often in strains from patients without predisposing urological or medical conditions (P = 0.04). Strains with plasmid-encoded aerobactin lacked determinants for P fimbriae (P = 0.004) and hemolysin (P = 0.0004), were resistant to multiple antimicrobial agents (P = 0.0001), and were found only in compromised patients. Mating experiments demonstrated that some aerobactin plasmids also encoded antimicrobial-agent resistance. These findings suggest that the determinants for aerobactin, P fimbriae, and hemolysin are conserved on the chromosome of the antimicrobial-agent-susceptible uropathogenic strains of E. coli which invade noncompromised patients. In contrast, these chromosomal virulence factors are often absent from E. coli strains causing urosepsis in compromised hosts; these strains may acquire plasmid aerobactin in conjunction with antimicrobial-agent resistance genes.

  13. Influence of growth temperature and lipopolysaccharide on hemolytic activity of Serratia marcescens.

    PubMed

    Poole, K; Braun, V

    1988-11-01

    Log-phase cells of Serratia marcescens cultured at 30 degrees C were approximately 10-fold more hemolytic than those grown at 37 degrees C. By using a cloned gene fusion of the promoter-proximal part of the hemolysin gene (shlA) to the Escherichia coli alkaline phosphatase gene (phoA), hemolysin gene expression as a function of alkaline phosphatase activity was measured at 30 and 37 degrees C. No difference in alkaline phosphatase activity was observed as a function of growth temperature, although more hemolysin was detectable immunologically in whole-cell extracts of cells grown at 30 degrees C. The influence of temperature was, however, growth phase dependent, because the hemolytic activities of cells cultured to early log phase at 30 and 37 degrees C were comparable. Given the outer membrane location of the hemolysin, lipopolysaccharide (LPS) was examined as a candidate for mediating the temperature effect on hemolytic activity. Silver staining of LPS in polyacrylamide gels revealed a shift towards shorter O-antigen molecules at 37 degrees C relative to 30 degrees C. Moreover, there was less binding of O-antigen-specific bacteriophage to S. marcescens with increasing growth temperature, a finding consistent with temperature-mediated changes in LPS structure. Smooth strains of S. marcescens were 20- to 30-fold more hemolytic than rough derivatives, a result confirming that changes in LPS structure can influence hemolytic activity. The alkaline phosphatase activity of rough strains harboring the shlA-phoA fusion was threefold lower than that of smooth strains harboring the fusion plasmids, a result consistent with a decrease in hemolysin gene expression in rough strains. The absence of a similar effect of temperature on gene expression may be related to less-marked changes in LPS structure as a function of temperature compared with a smooth-to-rough mutational change.

  14. Virulence markers in Shiga toxin-producing Escherichia coli isolated from cattle.

    PubMed Central

    Sandhu, K S; Clarke, R C; Gyles, C L

    1999-01-01

    This study identified potential virulence markers in 93 eae-positive and 179 eae-negative Shiga toxin-producing Escherichia coli (STEC), isolated from a random sampling of healthy cattle in southwestern Ontario. PCR amplification was used to identify genes for enterohemorrhagic E. coli (EHEC)-hemolysin, the EAF plasmid, and bundle-forming pili (Bfp); adherence to HEp-2 cells and to bovine colonocytes, and the fluorescent actin staining (FAS) test were used to characterize interaction of the bacteria with epithelial cells. The EHEC-hemolysin sequences were detected in 98% of eae-positive isolates compared with 34% of eae-negative isolates. All isolates were negative for EAF and bfp sequences. There was 100% correlation between localized adherence (LA) to HEp-2 cells and the FAS test. Forty-eight (52%) of the eae-positive isolates were LA/FAS-positive, whereas none of the 179 eae-negative isolates was positive in either test. Among the eae-negative isolates, 20 (11%) showed diffuse adherence and 5 (2.8%) showed enteroaggregative adherence to HEp-2 cells. Seventy-three percent of the eae-positive isolates adhered to bovine colonocytes, whereas only 26% of 120 eae-negative isolates that were tested adhered. All 13 O157:H7 isolates were positive for eae and EHEC-hemolysin gene sequences, LA/FAS, and adherence to bovine colonocytes. It is concluded that possession of genes for eae and EHEC hemolysin is correlated with the serotype of STEC, that production of EHEC hemolysin was highly correlated with serotypes implicated in human disease, and that none of the potential markers that were examined can be used to predict the potential virulence of an isolate. Images Figure 1. Figure 2. Figure 3. PMID:10480459

  15. Structure based virtual screening of novel inhibitors against multidrug resistant superbugs.

    PubMed

    Skariyachan, Sinosh; Mahajanakatti, Arpitha Badarinath; Sharma, Narasimha; Karanth, Shraddha; Rao, Shruthi; Rajeswari, Narayanappa

    2012-01-01

    Pathogenic microorganisms are persistently expressing resistance towards present generation antibiotics and are on the verge of joining the superbug family. Recent studies revealed that, notorious pathogens such as Salmonella typhi, Shigella dysenteriae and Vibrio cholerae have acquired multiple drug resistance and the treatment became a serious concern. This necessitates an alternative therapeutic solution. Present study investigates the utility of computer aided method to study the mechanism of receptor-ligand interactions and thereby inhibition of virulence factors (shiga toxin of Shigella dysenteriae, cholera toxin of Vibrio cholerae and hemolysin-E of Salmonella typhi) by novel phytoligands. The rational designs of improved therapeutics require the crystal structure for the drug targets. The structures of the virulent toxins were identified as probable drug targets. However, out of the three virulent factors, the structure for hemolysin-E is not yet available in its native form. Thus, we tried to model the structure by homology modeling using Modeller 9v9. After extensive literature survey, we selected 50 phytoligands based on their medicinal significance and drug likenesses. The receptor-ligands interactions between selected leads and toxins were studied by molecular docking using Auto Dock 4.0. We have identified two novel sesquiterpenes, Cadinane [(1S, 4S, 4aS, 6S, 8aS)- 4- Isopropyl- 1, 6- dimethyldecahydronaphthalene] and Cedrol [(8α)-Cedran-8-ol] against Shiga (binding energy -5.56 kcal/mol) and cholera toxins (binding energy -5.33 kcal/mol) respectively which have good inhibitory properties. Similarly, a natural Xanthophyll, Violaxanthin [3S, 3'S, 5R, 5'R, 6S, 6'S)-5, 5', 6, 6'-Tetrahydro-5, 6:5', 6'-diepoxy-β, β-carotene-3, 3'-diol] was identified as novel therapeutic lead for hemolysin-E (binding energy of -5.99 kcal/mol). This data provide an insight for populating the pool of novel inhibitors against various drug targets of superbugs when all

  16. The function of PlcR in Bacillus anthracis vaccine strain A16R.

    PubMed

    Xiaolin, Jia; Dongshu, Wang; Zhiqi, Gao; Erling, Feng; Jiping, Zheng; Hengliang, Wang; Guiying, Guo; Xiankai, Liu

    2015-05-01

    Bacillus anthracis, B. thuringiensis and B. cereus are members of the B. cereus group. They share high genetic similarity. Whereas plcR (Phospholipase C regulator) usually encodes a functional pleiotropic activator protein in B. cereus and B. thuringiensis isolates, a characteristic nonsense mutation is found in all B. anthracis strains investigated, making the gene dysfunctional. To study the function of PlcR in B. anthracis, we used the B. cereus CMCC63301 genome as a template and constructed a recombinant expression plasmid pBE2A-plcR, and introduced it into the B. anthracis vaccine strain A16R, and then analyzed the activity of the hemolysin and sphingomyelinase. The results showed that transformation of B. anthracis with plasmid pBE2A-plcR carrying the native B. cereus plcR gene active the expression of sphingomyelinase gene, but did not activate expression of hemolysin genes of B. anthracis A16R.

  17. Long-term human serum antibody responses after immunization with whole-cell pertussis vaccine in France.

    PubMed Central

    Grimprel, E; Bégué, P; Anjak, I; Njamkepo, E; François, P; Guiso, N

    1996-01-01

    Three hundred sixty children were tested for pertussis serology 0.5 to 1.58 months after complete whole-cell pertussis vaccination. An immunoblot assay was used to detect serum antibodies to pertussis toxin, filamentous hemagglutinin, adenylate cyclase-hemolysin, and pertactin, and agglutination was used for detection of anti-agglutinogen antibodies. Antibodies against pertussis toxin, pertactin, and agglutinogens decreased rapidly after vaccination but increased secondarily, suggesting exposure to infected persons. In contrast, anti-filamentous hemagglutinin antibodies persisted and anti-adenylate cyclase-hemolysin antibodies increased continuously, suggesting either cross-reaction with non-Bordetella antigens or exposure to Bordetella isolates expressing these two antigens, including Bordetella pertussis. These data suggest that unrecognized pertussis is common in France despite massive and sustained immunization in infants and that vaccinated children become susceptible to infection more than 6 years after their last vaccination. PMID:8770511

  18. In situ and in vitro impacts of the Deepwater Horizon oil spill on Vibrio parahaemolyticus.

    PubMed

    Stephens, Erica L; Molina, Vanessa; Cole, Krystal M; Laws, Edward; Johnson, Crystal N

    2013-10-15

    Most established virulence genes in Vibrio parahaemolyticus (Vp), e.g., thermostable direct hemolysin (tdh), tdh-related hemolysin (trh), and type three secretion system 2 (TTSS2), are on the chromosome 2 pathogenicity island, which also possesses numerous uncharacterized genes. We hypothesized the 2010 Deepwater Horizon (DH) oil spill would cause an increase in populations of Vibrio parahaemolyticus carrying environmental adaptation genes. Vp isolated pre- and post-spill were analyzed for TTSS2 genes, and impacts of DH oil on Vp were examined in vitro. There was no change in TTSS2 in situ, but tdh and V. vulnificus levels were higher post-spill. In vitro exposure of water samples to DH oil produced no changes in Vp densities. Two years post-spill, total Vp remained low; tdh and trh increased. These results indicate the effects of the DH oil spill on potentially pathogenic Vp subpopulations were complex and difficult to discern from other concurrent anthropogenic and natural events.

  19. Escherichia vulneris: an unusual cause of complicated diarrhoea and sepsis in an infant. A case report and review of literature.

    PubMed

    Jain, S; Nagarjuna, D; Gaind, R; Chopra, S; Debata, P K; Dawar, R; Sardana, R; Yadav, M

    2016-09-01

    Escherichia vulneris is an opportunistic human pathogen. It has been primarily reported in adult patients and invasive infections have been observed in immune-suppressed individuals. This is the first report of E. vulneris causing complicated diarrhoea and sepsis in an infant. Two month old sick infant, born full-term, was admitted to the paediatrics department with loose motions and refusal to feed for four days. E. vulneris was isolated from blood in pure culture. The isolate was characterized for diarrhoeal virulence markers: heat labile and heat stable toxins (LT, ST) and hemolysin (hlyA) by PCR. The presence of LT enterotoxin and hemolysin provides strong evidence of the diarrhoeagenic potential of E. vulneris, further leading to the invasive infection triggering sepsis. As E. vulneris can lead to serious complications, an attempt should be made in clinical laboratories to identify and further characterize this new Escherichia species. PMID:27536376

  20. Perivascular macrophages mediate neutrophil recruitment during bacterial skin infection

    PubMed Central

    Abtin, Arby; Jain, Rohit; Mitchell, Andrew J.; Roediger, Ben; Brzoska, Anthony J.; Tikoo, Shweta; Cheng, Qiang; Ng, Lai Guan; Cavanagh, Lois L.; von Andrian, Ulrich H.; Hickey, Michael J.; Firth, Neville; Weninger, Wolfgang

    2014-01-01

    Transendothelial migration of neutrophils in post-capillary venules is a key event in the inflammatory response against pathogens and tissue damage. The precise regulation of this process is incompletely understood. We report that perivascular macrophages are critical for neutrophil migration into skin infected with the pathogen Staphylococcus aureus. Using multiphoton intravital microscopy we show that neutrophils extravasate from inflamed dermal venules in close proximity to perivascular macrophages, which are a major source of neutrophil chemoattractants. The virulence factor alpha-hemolysin lyses perivascular macrophages leading to decreased neutrophil transmigration. Our data illustrate a previously unrecognized role for perivascular macrophages in neutrophil recruitment to inflamed skin, and indicate that Staphylococcus aureus uses hemolysin-dependent killing of these cells as an immune evasion strategy. PMID:24270515

  1. A subset of two adherence systems, acute pro-inflammatory pap genes and invasion coding dra, fim, or sfa, increases the risk of Escherichia coli translocation to the bloodstream.

    PubMed

    Szemiako, K; Krawczyk, B; Samet, A; Śledzińska, A; Nowicki, B; Nowicki, S; Kur, J

    2013-12-01

    An analysis of the phylogenetic distribution and virulence genes of Escherichia coli isolates which predispose this bacteria to translocate from the urinary tract to the bloodstream is presented. One-dimensional analysis indicated that the occurrence of P fimbriae and α-hemolysin coding genes is more frequent among the E. coli which cause bacteremia. However, a two-dimensional analysis revealed that a combination of genes coding two adherence factors, namely, P + Dr, P + S, S + Dr, S + fim, and hemolysin + one adherence factor, were associated with bacteremia and, therefore, with the risk of translocation to the vascular system. The frequent and previously unrecognized co-existence of pro-inflammatory P fimbriae with the invasion promoting Dr adhesin in the same E. coli isolate may represent high-risk and potentially lethal pathogens.

  2. Escherichia vulneris: an unusual cause of complicated diarrhoea and sepsis in an infant. A case report and review of literature.

    PubMed

    Jain, S; Nagarjuna, D; Gaind, R; Chopra, S; Debata, P K; Dawar, R; Sardana, R; Yadav, M

    2016-09-01

    Escherichia vulneris is an opportunistic human pathogen. It has been primarily reported in adult patients and invasive infections have been observed in immune-suppressed individuals. This is the first report of E. vulneris causing complicated diarrhoea and sepsis in an infant. Two month old sick infant, born full-term, was admitted to the paediatrics department with loose motions and refusal to feed for four days. E. vulneris was isolated from blood in pure culture. The isolate was characterized for diarrhoeal virulence markers: heat labile and heat stable toxins (LT, ST) and hemolysin (hlyA) by PCR. The presence of LT enterotoxin and hemolysin provides strong evidence of the diarrhoeagenic potential of E. vulneris, further leading to the invasive infection triggering sepsis. As E. vulneris can lead to serious complications, an attempt should be made in clinical laboratories to identify and further characterize this new Escherichia species.

  3. Penicillinase Studies on L-Phase Variants, G-Phase Variants, and Reverted Strains of Staphylococcus aureus.

    PubMed

    Simon, H J; Yin, E J

    1970-11-01

    L-phase variants and small colony (G-phase) variants derived from penicillinase-producing Staphylococcus aureus strains were tested for penicillinase (beta lactamase) production. A refined variation of the modified Gots test for penicillinase was used to demonstrate penicillinase synthesis. Penicillinase synthesis was reduced in L-phase variants and G-phase variants when compared to parental strains. After reversion of variants to vegetative stages had been induced, revertants were tested for production of penicillinase, coagulase, and alpha hemolysin, mannitol fermentation, and pigment production, and comparisons were made between parent and reverted vegetative forms. All revertants of G-phase variants retained penicillinase activity. Most revertants of L-phase variants showed reduction or loss of penicillinase activity. Retention of coagulase activity, alpha hemolysin production, mannitol fermentation, pigmentation, and phage type varied among revertants.

  4. Perivascular macrophages mediate neutrophil recruitment during bacterial skin infection.

    PubMed

    Abtin, Arby; Jain, Rohit; Mitchell, Andrew J; Roediger, Ben; Brzoska, Anthony J; Tikoo, Shweta; Cheng, Qiang; Ng, Lai Guan; Cavanagh, Lois L; von Andrian, Ulrich H; Hickey, Michael J; Firth, Neville; Weninger, Wolfgang

    2014-01-01

    Transendothelial migration of neutrophils in postcapillary venules is a key event in the inflammatory response against pathogens and tissue damage. The precise regulation of this process is incompletely understood. We report that perivascular macrophages are critical for neutrophil migration into skin infected with the pathogen Staphylococcus aureus. Using multiphoton intravital microscopy we showed that neutrophils extravasate from inflamed dermal venules in close proximity to perivascular macrophages, which are a major source of neutrophil chemoattractants. The virulence factor α-hemolysin produced by S. aureus lyses perivascular macrophages, which leads to decreased neutrophil transmigration. Our data illustrate a previously unrecognized role for perivascular macrophages in neutrophil recruitment to inflamed skin and indicate that S. aureus uses hemolysin-dependent killing of these cells as an immune evasion strategy. PMID:24270515

  5. Comparison of the pathogenic potentials of environmental and clinical vibrio parahaemolyticus strains indicates a role for temperature regulation in virulence.

    PubMed

    Mahoney, Jennifer C; Gerding, Matthew J; Jones, Stephen H; Whistler, Cheryl A

    2010-11-01

    Although the presence of pathogenic Vibrio spp. in estuarine environments of northern New England has been known for some time (C. H. Bartley and L. W. Slanetz, Appl. Microbiol. 21: 965-966, 1971, and K. R. O'Neil, S. H. Jones, and D. J. Grimes, FEMS Microbiol. Lett. 60:163-167, 1990), their virulence and the relative threat they may pose to human health has yet to be evaluated. In this study, the virulence potential of 33 Vibrio parahaemolyticus isolates collected from the Great Bay Estuary of New Hampshire was assessed in comparison to that of clinical strains. The environmental isolates lack thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH), which are encoded by tdh and trh, respectively. Though not hemolytic, they do possess putative virulence factors, such type III secretion system 1, and are highly cytotoxic to human gastrointestinal cells. The expression of known and putative virulence-associated traits, including hemolysin, protease, motility, biofilm formation, and cytotoxicity, by clinical reference isolates correlated with increased temperature from 28°C to 37°C. In contrast, the environmental isolates did not induce their putative virulence-associated traits in response to a temperature of 37°C. We further identified a significant correlation between hemolytic activity and growth phase among clinical strains, whereby hemolysin production decreases with increasing cell density. The introduction of a tdh::gfp promoter fusion into the environmental strains revealed that they regulate this virulence-associated gene appropriately in response to temperature, indicating that their existing regulatory mechanisms are primed to manage newly acquired virulence genes.

  6. Differences in biofilm formation and virulence factors between clinical and fecal enterococcal isolates of human and animal origin.

    PubMed

    Tsikrikonis, Giorgos; Maniatis, Antonios N; Labrou, Maria; Ntokou, Eleni; Michail, Giorgos; Daponte, Alexandros; Stathopoulos, Constantinos; Tsakris, Athanassios; Pournaras, Spyros

    2012-06-01

    The present study investigated the possible correlation between carriage of the virulence genes esp and fsrb, production of hemolysin and gelatinase and biofilm formation in human vs. animal enterococcal isolates. A collection of 219 enterococcal isolates recovered from clinical and fecal surveillance samples of hospitalized patients and 132 isolates from animal feces were studied. Isolates were tested for hemolysin and gelatinase phenotypically and for quantitative biofilm production by a microtitre method. Genes esp and fsrb were detected by PCR. Human Enterococcus faecium and Enterococcus faecalis isolates from both surveillance and clinical samples produced biofilm significantly more often than animal isolates (P < 0.0001 for both species). The quantity of biofilm did not differ significantly between human and animal isolates, while was significantly higher in esp-positive compared with esp-negative human E. faecium isolates (P < 0.0001). The frequency of esp gene carriage was significantly higher in human compared with animal E. faecium and E. faecalis isolates (P < 0.0001). The gene fsrb was detected significantly more often in animal than human E. faecium isolates (P 0.004). Hemolysin production was significantly more common in human clinical compared with animal E. faecalis isolates (P < 0.0001). Similar proportions of animal and human E. faecalis produced gelatinase, which was significantly correlated with the presence of fsrb gene (P < 0.0001) in both human clinical and animal E. faecalis isolates. The hemolysin trait did not exhibit any correlation with the presence of esp and fsrb genes, but appeared to be linked with enhanced quantity of biofilm production in both human clinical and animal E. faecalis isolates. Production of gelatinase was associated with the proportion and the degree of biofilm production mainly in animal E. faecalis isolates.

  7. Detection of hemolytic Listeria monocytogenes by using DNA colony hybridization

    SciTech Connect

    Datta, A.R.; Wentz, B.A.; Hill, W.E.

    1987-09-01

    A fragment of about 500 base pairs of the beta-hemolysin gene from Listeria monocytogenes was used to screen different bacterial strains by DNA colony hybridization. The cells in the colonies were lysed by microwaves in the presence of sodium hydroxide. Of 52 different strains of Listeria species screened, only the DNA from beta-hemolytic (CAMP-positive) strains of L. monocytogenes hybridized with this probe.

  8. Single-Molecule Study of Proteins by Biological Nanopore Sensors

    PubMed Central

    Wu, Dongmei; Bi, Sheng; Zhang, Liyu; Yang, Jun

    2014-01-01

    Nanopore technology has been developed for detecting properties of proteins through monitoring of ionic current modulations as protein passes via a nanosize pore. As a real-time, sensitive, selective and stable technology, biological nanopores are of widespread concern. Here, we introduce the background of nanopore researches in the area of α-hemolysin (α-HL) nanopores in protein conformation detections and protein–ligand interactions. Moreover, several original biological nanopores are also introduced with various features and functions. PMID:25268917

  9. The Abi-domain Protein Abx1 Interacts with the CovS Histidine Kinase to Control Virulence Gene Expression in Group B Streptococcus

    PubMed Central

    Firon, Arnaud; Tazi, Asmaa; Da Cunha, Violette; Brinster, Sophie; Sauvage, Elisabeth; Dramsi, Shaynoor; Golenbock, Douglas T.; Glaser, Philippe; Poyart, Claire; Trieu-Cuot, Patrick

    2013-01-01

    Group B Streptococcus (GBS), a common commensal of the female genital tract, is the leading cause of invasive infections in neonates. Expression of major GBS virulence factors, such as the hemolysin operon cyl, is regulated directly at the transcriptional level by the CovSR two-component system. Using a random genetic approach, we identified a multi-spanning transmembrane protein, Abx1, essential for the production of the GBS hemolysin. Despite its similarity to eukaryotic CaaX proteases, the Abx1 function is not involved in a post-translational modification of the GBS hemolysin. Instead, we demonstrate that Abx1 regulates transcription of several virulence genes, including those comprising the hemolysin operon, by a CovSR-dependent mechanism. By combining genetic analyses, transcriptome profiling, and site-directed mutagenesis, we showed that Abx1 is a regulator of the histidine kinase CovS. Overexpression of Abx1 is sufficient to activate virulence gene expression through CovS, overcoming the need for an additional signal. Conversely, the absence of Abx1 has the opposite effect on virulence gene expression consistent with CovS locked in a kinase-competent state. Using a bacterial two-hybrid system, direct interaction between Abx1 and CovS was mapped specifically to CovS domains involved in signal processing. We demonstrate that the CovSR two-component system is the core of a signaling pathway integrating the regulation of CovS by Abx1 in addition to the regulation of CovR by the serine/threonine kinase Stk1. In conclusion, our study reports a regulatory function for Abx1, a member of a large protein family with a characteristic Abi-domain, which forms a signaling complex with the histidine kinase CovS in GBS. PMID:23436996

  10. Photobacterium damselae subsp. damselae Major Virulence Factors Dly, Plasmid-Encoded HlyA, and Chromosome-Encoded HlyA Are Secreted via the Type II Secretion System

    PubMed Central

    Rivas, Amable J.; Vences, Ana; Husmann, Matthias; Lemos, Manuel L.

    2015-01-01

    Photobacterium damselae subsp. damselae is a marine bacterium that causes septicemia in marine animals and in humans. Previously, we had determined a major role of pPHDD1 plasmid-encoded Dly (damselysin) and HlyA (HlyApl) and the chromosome-encoded HlyA (HlyAch) hemolysins in virulence. However, the mechanisms by which these toxins are secreted remain unknown. In this study, we found that a mini-Tn10 transposon mutant in a plasmidless strain showing an impaired hemolytic phenotype contained an insertion in epsL, a component of a type II secretion system (T2SS). Reconstruction of the mutant by allelic exchange confirmed the specific involvement of epsL in HlyAch secretion. In addition, mutation of epsL in a pPHDD1-harboring strain caused an almost complete abolition of hemolytic activity against sheep erythrocytes, indicating that epsL plays a major role in secretion of the plasmid-encoded HlyApl and Dly. This was further demonstrated by analysis of different combinations of hemolysin gene mutants and by strain-strain complementation assays. We also found that mutation of the putative prepilin peptidase gene pilD severely affected hemolysis, which dropped at levels inferior to those of epsL mutants. Promoter expression analyses suggested that impairment of hemolysin secretion in epsL and pilD mutants might constitute a signal that affects hemolysin and T2SS gene expression at the transcriptional level. In addition, single epsL and pilD mutations caused a drastic decrease in virulence for mice, demonstrating a major role of T2SS and pilD in P. damselae subsp. damselae virulence. PMID:25583529

  11. Electrically sensing protease activity with nanopores

    NASA Astrophysics Data System (ADS)

    Kukwikila, Mikiembo; Howorka, Stefan

    2010-11-01

    The enzymatic activity of a protease was electrically detected using nanopore recordings. A peptide substrate was tethered to microscale beads, and cleavage by the enzyme trypsin released a soluble fragment that was electrophoretically driven through the α-hemolysin protein pore, leading to detectable blockades in the ionic current. Owing to its simplicity, this approach to sense enzymatic activity may be applied to other proteases.

  12. Complete genome sequence of Vibrio parahaemolyticus FORC_023 isolated from raw fish storage water.

    PubMed

    Chung, Han Young; Na, Eun Jung; Lee, Kyu-Ho; Ryu, Sangryeol; Yoon, Hyunjin; Lee, Ju-Hoon; Kim, Hyeun Bum; Kim, Heebal; Choi, Sang Ho; Kim, Bong-Soo

    2016-06-01

    Vibrio parahaemolyticusis a Gram-negative halophilic bacterium that causes food-borne gastroenteritis in humans who consumeV. parahaemolyticus-contaminated seafood.The FORC_023 strain was isolated from raw fish storage water, containing live fish at a sashimi restaurant. Here, we aimed to sequence and characterize the genome of the FORC_023 strain. The genome of the FORC_023 strain showed two circular chromosomes, which contained 4227 open reading frames (ORFs), 131 tRNA genes and 37 rRNA genes. Although the genome of FORC_023 did not include major virulence genes, such as genes encoding thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH), it contained genes encoding other hemolysins, secretion systems, iron uptake-related proteins and severalV. parahaemolyticusislands. The highest average nucleotide identity value was obtained between the FORC_023 strain and UCM-V493 (CP007004-6). Comparative genomic analysis of FORC_023 with UCM-V493 revealed that FORC_023 carried an additional genomic region encoding virulence factors, such as repeats-in-toxin and type II secretion factors. Furthermore,in vitrocytotoxicity testing showed that FORC_023 exhibited a high level of cytotoxicity toward INT-407 human epithelial cells. These results suggested that the FORC_023 strain may be a food-borne pathogen. PMID:27073252

  13. Pathogenicity of Vibrio parahaemolyticus in Different Food Matrices.

    PubMed

    Wang, Rundong; Sun, Lijun; Wang, Yaling; Deng, Yijia; Liu, Ying; Xu, Defeng; Liu, Huanming; Ye, Riying; Gooneratne, Ravi

    2016-02-01

    The pathogenicity and virulence factors of Vibrio parahaemolyticus in four food matrices--shrimp, freshwater fish, pork, and egg-fried rice--were compared by measuring the thermostable direct hemolysin activity and total hemolytic titer. Significantly high thermostable direct hemolysin and also hemolytic titers (P < 0.05) were produced by V. parahaemolyticus in egg-fried rice > shrimp > freshwater fish > pork. Filtrates of V. parahaemolyticus in shrimp given intraperitoneally induced marked liver and kidney damage and were highly lethal to adult mice compared with filtrates of V. parahaemolyticus in freshwater fish > egg-fried rice > pork. From in vitro and in vivo pathogenicity tests, it seems the type of food matrix has a significant impact on the virulence of V. parahaemolyticus. These results suggest that hemolysin may not necessarily be the only virulence factor for pathogenicity of V. parahaemolyticus. This is the first report that shows that virulence factors produced by V. parahaemolyticus in seafood such as shrimp are more toxic in vivo than in nonseafood.

  14. Coral-Associated Bacteria as a Promising Antibiofilm Agent against Methicillin-Resistant and -Susceptible Staphylococcus aureus Biofilms

    PubMed Central

    Gowrishankar, Shanmugaraj; Duncun Mosioma, Nyagwencha; Karutha Pandian, Shunmugiah

    2012-01-01

    The current study deals with the evaluation of two coral-associated bacterial (CAB) extracts to inhibit the biofilm synthesis in vitro as well as the virulence production like hemolysin and exopolysaccharide (EPS), and also to assess their ability to modify the adhesion properties, that is cell surface hydrophobicity (CSH) of methicillin-resistant (MRSA) and -susceptible Staphylococcus aureus (MSSA). Out of nine CAB screened, the ethyl acetate extract of CAB-E2 (Bacillus firmus) and CAB-E4 (Vibrio parahemolyticus) have shown excellent antibiofilm activity against S. aureus. CAB-E2 reduced the production of EPS (57–79%) and hemolysin (43–70%), which ultimately resulted in the significant inhibition of biofilms (80–87%) formed by both MRSA and MSSA. Similarly, CAB-E4 was also found to decrease the production of EPS (43–57%), hemolysin (43–57%) and biofilms (80–85%) of test pathogens. CLSM analysis also proved the antibiofilm efficacy of CAB extracts. Furthermore, the CAB extracts strongly decreased the CSH of S. aureus. Additionally, FT-IR analysis of S. aureus treated with CAB extracts evidenced the reduction in cellular components compared to their respective controls. Thus, the present study reports for the first time, B. firmus—a coral-associated bacterium, as a promising source of antibiofilm agent against the recalcitrant biofilms formed by multidrug resistant S. aureus. PMID:22988476

  15. Interactions of Neuropathogenic Escherichia coli K1 (RS218) and Its Derivatives Lacking Genomic Islands with Phagocytic Acanthamoeba castellanii and Nonphagocytic Brain Endothelial Cells

    PubMed Central

    Yousuf, Farzana Abubakar; Yousuf, Zuhair; Iqbal, Junaid; Siddiqui, Ruqaiyyah; Khan, Hafsa; Khan, Naveed Ahmed

    2014-01-01

    Here we determined the role of various genomic islands in E. coli K1 interactions with phagocytic A. castellanii and nonphagocytic brain microvascular endothelial cells. The findings revealed that the genomic islands deletion mutants of RS218 related to toxins (peptide toxin, α-hemolysin), adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin), protein secretion system (T1SS for hemolysin), invasins (IbeA, CNF1), metabolism (D-serine catabolism, dihydroxyacetone, glycerol, and glyoxylate metabolism) showed reduced interactions with both A. castellanii and brain microvascular endothelial cells. Interestingly, the deletion of RS218-derived genomic island 21 containing adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin), protein secretion system (T1SS for hemolysin), invasins (CNF1), metabolism (D-serine catabolism) abolished E. coli K1-mediated HBMEC cytotoxicity in a CNF1-independent manner. Therefore, the characterization of these genomic islands should reveal mechanisms of evolutionary gain for E. coli K1 pathogenicity. PMID:24818136

  16. Phobalysin, a Small β-Pore-Forming Toxin of Photobacterium damselae subsp. damselae

    PubMed Central

    Rivas, Amable J.; von Hoven, Gisela; Neukirch, Claudia; Meyenburg, Martina; Qin, Qianqian; Füser, Sabine; Boller, Klaus; Lemos, Manuel L.; Osorio, Carlos R.

    2015-01-01

    Photobacterium damselae subsp. damselae, an important pathogen of marine animals, may also cause septicemia or hyperaggressive necrotizing fasciitis in humans. We previously showed that hemolysin genes are critical for virulence of this organism in mice and fish. In the present study, we characterized the hlyA gene product, a putative small β-pore-forming toxin, and termed it phobalysin P (PhlyP), for “photobacterial lysin encoded on a plasmid.” PhlyP formed stable oligomers and small membrane pores, causing efflux of K+, with no significant leakage of lactate dehydrogenase but entry of vital dyes. The latter feature distinguished PhlyP from the related Vibrio cholerae cytolysin. Attack by PhlyP provoked a loss of cellular ATP, attenuated translation, and caused profound morphological changes in epithelial cells. In coculture experiments with epithelial cells, Photobacterium damselae subsp. damselae led to rapid hemolysin-dependent membrane permeabilization. Unexpectedly, hemolysins also promoted the association of P. damselae subsp. damselae with epithelial cells. The collective observations of this study suggest that membrane-damaging toxins commonly enhance bacterial adherence. PMID:26303391

  17. Detection, Identification, and Prevalence of Pathogenic Vibrio parahaemolyticus in Fish and Coastal Environment in Jordan.

    PubMed

    Alaboudi, Akram R; Ababneh, Mustafa; Osaili, Tareq M; Al Shloul, Khalaf

    2016-01-01

    Vibrio parahaemolyticus is widely distributed in the marine environments and considered the leading cause of human gastroenteritis in Asian countries. A total of 150 marketed fish and 50 water and sediment samples from the Gulf of Aqaba were examined for the prevalence of pathogenic strains of V. parahaemolyticus. A total of 132 typical isolates obtained from the primary selective medium (thiosulfate-citrate bile salt sucrose agar) and showed positive biochemical properties were subjected to confirmation by polymerase chain reaction targeting the gyrB and toxR genes. These genes were confirmed at rates of 82% (108 isolates) and 72% (95 isolates), respectively. The toxR positive isolates were tested for the presence of thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and tdh-related hemolysin (trh) virulence genes. Accordingly, the prevalence rates of pathogenic V. parahaemolyticus were 4%, 8%, and 12% in sediment, water, and fish samples, respectively. The 16S rRNA amplification and sequences were conducted for confirmation of the isolates and showing the relatedness among these isolates. The results showed that both 16S rRNA and toxR assays had same sensitivity and tested isolates had high nucleotide similarity irrespective of their sources.

  18. Genetic characterization of clinical and environmental Vibrio parahaemolyticus from the Northeast USA reveals emerging resident and non-indigenous pathogen lineages.

    PubMed

    Xu, Feng; Ilyas, Saba; Hall, Jeffrey A; Jones, Stephen H; Cooper, Vaughn S; Whistler, Cheryl A

    2015-01-01

    Gastric infections caused by the environmentally transmitted pathogen, Vibrio parahaemolyticus, have increased over the last two decades, including in many parts of the United States (US). However, until recently, infections linked to shellfish from the cool northeastern US waters were rare. Cases have risen in the Northeast, consistent with changes in local V. parahaemolyticus populations toward greater abundance or a shift in constituent pathogens. We examined 94 clinical isolates from a period of increasing disease in the region and compared them to 200 environmental counterparts to identify resident and non-indigenous lineages and to gain insight into the emergence of pathogenic types. Genotyping and multi-locus sequence analysis (MLSA) of clinical isolates collected from 2010 to 2013 in Massachusetts, New Hampshire, and Maine revealed their polyphyletic nature. Although 80% of the clinical isolates harbored the trh hemolysin either alone or with tdh, and were urease positive, 14% harbored neither hemolysin exposing a limitation for these traits in pathogen detection. Resident sequence type (ST) 631 strains caused seven infections, and show a relatively recent history of recombination with other clinical and environmental lineages present in the region. ST34 and ST674 strains were each linked to a single infection and these strain types were also identified from the environment as isolates harboring hemolysin genes. Forty-two ST36 isolates were identified from the clinical collection, consistent with reports that this strain type caused a rise in regional infections starting in 2012. Whole-genome phylogenies that included three ST36 outbreak isolates traced to at least two local sources demonstrated that the US Atlantic coastal population of this strain type was indeed derived from the Pacific population. This study lays the foundation for understanding dynamics within natural populations associated with emergence and invasion of pathogenic strain types in the

  19. Genetic characterization of clinical and environmental Vibrio parahaemolyticus from the Northeast USA reveals emerging resident and non-indigenous pathogen lineages

    PubMed Central

    Xu, Feng; Ilyas, Saba; Hall, Jeffrey A.; Jones, Stephen H.; Cooper, Vaughn S.; Whistler, Cheryl A.

    2015-01-01

    Gastric infections caused by the environmentally transmitted pathogen, Vibrio parahaemolyticus, have increased over the last two decades, including in many parts of the United States (US). However, until recently, infections linked to shellfish from the cool northeastern US waters were rare. Cases have risen in the Northeast, consistent with changes in local V. parahaemolyticus populations toward greater abundance or a shift in constituent pathogens. We examined 94 clinical isolates from a period of increasing disease in the region and compared them to 200 environmental counterparts to identify resident and non-indigenous lineages and to gain insight into the emergence of pathogenic types. Genotyping and multi-locus sequence analysis (MLSA) of clinical isolates collected from 2010 to 2013 in Massachusetts, New Hampshire, and Maine revealed their polyphyletic nature. Although 80% of the clinical isolates harbored the trh hemolysin either alone or with tdh, and were urease positive, 14% harbored neither hemolysin exposing a limitation for these traits in pathogen detection. Resident sequence type (ST) 631 strains caused seven infections, and show a relatively recent history of recombination with other clinical and environmental lineages present in the region. ST34 and ST674 strains were each linked to a single infection and these strain types were also identified from the environment as isolates harboring hemolysin genes. Forty-two ST36 isolates were identified from the clinical collection, consistent with reports that this strain type caused a rise in regional infections starting in 2012. Whole-genome phylogenies that included three ST36 outbreak isolates traced to at least two local sources demonstrated that the US Atlantic coastal population of this strain type was indeed derived from the Pacific population. This study lays the foundation for understanding dynamics within natural populations associated with emergence and invasion of pathogenic strain types in the

  20. Investigation of whether the acute hemolysis associated with Rho(D) immune globulin intravenous (human) administration for treatment of immune thrombocytopenic purpura is consistent with the acute hemolytic transfusion reaction model

    PubMed Central

    Gaines, Ann Reed; Lee-Stroka, Hallie; Byrne, Karen; Scott, Dorothy E.; Uhl, Lynne; Lazarus, Ellen; Stroncek, David F.

    2012-01-01

    BACKGROUND Immune thrombocytopenic purpura and secondary thrombocytopenia patients treated with Rho(D) immune globulin intravenous (human; anti-D IGIV) have experienced acute hemolysis, which is inconsistent with the typical presentation of extravascular hemolysis—the presumed mechanism of action of anti-D IGIV. Although the mechanism of anti-D-IGIV–associated acute hemolysis has not been established, the onset, signs/symptoms, and complications appear consistent with the intravascular hemolysis of acute hemolytic transfusion reactions (AHTRs). In transfusion medicine, the red blood cell (RBC) antigen-antibody incompatibility(-ies) that precipitate AHTRs can be detected in vitro with compatibility testing. Under the premise that anti-D-IGIV–associated acute hemolysis results from RBC antigen-antibody–mediated complement activation, this study evaluated whether the incompatibility(-ies) could be detected in vitro with a hemolysin assay, which would support the AHTR model as the hemolytic mechanism. STUDY DESIGN AND METHODS Seven anti-D IGIV lots were tested to determine the RBC antibody identities in those lots, including four lots that had been implicated in acute hemolytic episodes. Hemolysin assays were performed that tested each of 73 RBC specimens against each lot, including the RBCs of one patient who had experienced acute hemolysis after anti-D IGIV administration. RESULTS Only two anti-D IGIV lots contained RBC antibodies beyond those expected. No hemolysis endpoint was observed in any of the hemolysin assays. CONCLUSION Although the findings did not support the AHTR model, the results are reported to contribute knowledge about the mechanism of anti-D-IGIV–associated acute hemolysis and to prompt continued investigation into cause(s), prediction, and prevention of this potentially serious adverse event. PMID:19220820

  1. Glycerol Monolaurate Inhibits the Effects of Gram Positive Select Agents on Eukaryotic Cells†

    PubMed Central

    Peterson, Marnie L.; Schlievert, Patrick M.

    2008-01-01

    Many exotoxins of gram positive bacteria, such as superantigens (staphylococcal enterotoxins, toxic shock syndrome toxin-1 [TSST-1], and streptococcal pyrogenic exotoxins) and anthrax toxin are bioterrorism agents that cause diseases by immunostimulation or cytotoxicity. Glycerol monolaurate (GML), a fatty acid monoester found naturally in humans, has been reported to prevent synthesis of gram positive bacterial exotoxins. This study explored the ability of GML to inhibit the effects of exotoxins on mammalian cells and prevent rabbit lethality from TSS. GML (≥10 ug/ml) inhibited superantigen (5 ug/ml) immunoproliferation, as determined by inhibition of 3H-thymidine incorporation into DNA of human peripheral blood mononuclear cells (1 × 106 cells/ml) as well as phospholipase Cγ1, suggesting inhibition of signal transduction. The compound (20 ug/ml) prevented superantigen (100 ug/ml) induced cytokine secretion by human vaginal epithelial cells (HVECs) as measured by ELISA. GML (250 ug) inhibited rabbit lethality due to TSST-1 administered vaginally. GML (10 ug/ml) inhibited HVEC and macrophage cytotoxicity by anthrax toxin, prevented erythrocyte lysis by purified hemolysins (staphylococcal α and β) and culture fluids containing streptococcal and Bacillus anthracis hemolysins, and was non-toxic to mammalian cells (up to 100 ug/ml) and rabbits (250 ug). GML stabilized mammalian cell membranes, as erythrocyte lysis was reduced in the presence of hypotonic aqueous solutions (0 to 0.05 M saline) or staphylococcal α and β-hemolysins when erythrocytes were pretreated with GML. GML may be useful in management of gram positive exotoxin illnesses; its action appears to be membrane stabilization with inhibition of signal transduction. PMID:16475828

  2. Identification of the Staphylococcus aureus vfrAB Operon, a Novel Virulence Factor Regulatory Locus

    PubMed Central

    Daly, Seth M.; Hall, Pamela R.; Bayles, Kenneth W.

    2014-01-01

    During a screen of the Nebraska Transposon Mutant Library, we identified 71 mutations in the Staphylococcus aureus genome that altered hemolysis on blood agar medium. Although many of these mutations disrupted genes known to affect the production of alpha-hemolysin, two of them were associated with an apparent operon, designated vfrAB, that had not been characterized previously. Interestingly, a ΔvfrB mutant exhibited only minor effects on the transcription of the hla gene, encoding alpha-hemolysin, when grown in broth, as well as on RNAIII, a posttranscriptional regulatory RNA important for alpha-hemolysin translation, suggesting that VfrB may function at the posttranscriptional level. Indeed, a ΔvfrB mutant had increased aur and sspAB protease expression under these conditions. However, disruption of the known secreted proteases in the ΔvfrB mutant did not restore hemolytic activity in the ΔvfrB mutant on blood agar. Further analysis revealed that, in contrast to the minor effects of VfrB on hla transcription when strains were cultured in liquid media, the level of hla transcription was decreased 50-fold in the absence of VfrB on solid media. These results demonstrate that while VfrB represses protease expression when strains are grown in broth, hla regulation is highly responsive to factors associated with growth on solid media. Intriguingly, the ΔvfrB mutant displayed increased pathogenesis in a model of S. aureus dermonecrosis, further highlighting the complexity of VfrB-dependent virulence regulation. The results of this study describe a phenotype associated with a class of highly conserved yet uncharacterized proteins found in Gram-positive bacteria, and they shed new light on the regulation of virulence factors necessary for S. aureus pathogenesis. PMID:24549328

  3. Identification of the Staphylococcus aureus vfrAB operon, a novel virulence factor regulatory locus.

    PubMed

    Bose, Jeffrey L; Daly, Seth M; Hall, Pamela R; Bayles, Kenneth W

    2014-05-01

    During a screen of the Nebraska Transposon Mutant Library, we identified 71 mutations in the Staphylococcus aureus genome that altered hemolysis on blood agar medium. Although many of these mutations disrupted genes known to affect the production of alpha-hemolysin, two of them were associated with an apparent operon, designated vfrAB, that had not been characterized previously. Interestingly, a ΔvfrB mutant exhibited only minor effects on the transcription of the hla gene, encoding alpha-hemolysin, when grown in broth, as well as on RNAIII, a posttranscriptional regulatory RNA important for alpha-hemolysin translation, suggesting that VfrB may function at the posttranscriptional level. Indeed, a ΔvfrB mutant had increased aur and sspAB protease expression under these conditions. However, disruption of the known secreted proteases in the ΔvfrB mutant did not restore hemolytic activity in the ΔvfrB mutant on blood agar. Further analysis revealed that, in contrast to the minor effects of VfrB on hla transcription when strains were cultured in liquid media, the level of hla transcription was decreased 50-fold in the absence of VfrB on solid media. These results demonstrate that while VfrB represses protease expression when strains are grown in broth, hla regulation is highly responsive to factors associated with growth on solid media. Intriguingly, the ΔvfrB mutant displayed increased pathogenesis in a model of S. aureus dermonecrosis, further highlighting the complexity of VfrB-dependent virulence regulation. The results of this study describe a phenotype associated with a class of highly conserved yet uncharacterized proteins found in Gram-positive bacteria, and they shed new light on the regulation of virulence factors necessary for S. aureus pathogenesis. PMID:24549328

  4. Virulence Markers of Vancomycin Resistant Enterococci Isolated from Infected and Colonized Patients

    PubMed Central

    Biswas, Priyanka Paul; Dey, Sangeeta; Adhikari, Luna; Sen, Aninda

    2014-01-01

    Background: The aim of study was to find out the potential pathogenic role of virulence factors elaborated by strains of vancomycin resistant enterococci (VRE) isolated from clinical samples and VRE colonizing the gastrointestinal tract of hospitalized patients. Materials and Methods: Enterococci were isolated from various clinical samples and also from fecal specimens of colonized patients at the time of admission, after 48 h and after 5 days of admission. Various virulence determinants were detected by phenotypic tests. Vancomycin susceptibility in enterococci was detected by disc diffusion and agar screen method. Minimum inhibitory concentration was determined by agar dilution method. Results: Out of all the clinical and fecal samples processed, 12.0% isolates were either vancomycin resistant or vancomycin intermediate. Hemagglutinating activity against rabbit red blood cells was seen with 27.8% and 25.0% of clinical and fecal strains, respectively. Slime layer formation was seen with fecal VRE strains (37.5%) when compared to clinical VRE (27.8%). Among the clinical VRE strains the most prolific biofilm producers were Enterococcus. fecalis (92.9%) when compared to Enterococcus. faecium (52.9%). Biofilm formation/(presence of adhesions) was also seen in (29.2%) of the fecal VREs. In wound infection production of gelatinase, deoxyribonuclease (DNase), and caseinase (70.0% each) were the major virulence factors. The predominant virulence factors seen in the blood stream infection were adhesin, and hemolysin (44.4% each) and in catheter induced infection were DNase and adhesins (75.0% each). Adhesin (29.2%), slime layer (37.6%), DNAse (33.3%), gelatinase (25.0%), lipase (20.8%) and caseinase (16.6%) and hemolysin (8.3%) were produced the fecal isolates. Conclusion: An association between adhesin (as detected by biofilm formation) and urinary tract infection, adhesion and hemolysin with BSI, as also between DNase gelatinase & caseinase with wound infection was noted

  5. The sae locus of Staphylococcus aureus controls exoprotein synthesis at the transcriptional level.

    PubMed

    Giraudo, A T; Cheung, A L; Nagel, R

    1997-07-01

    Agr and sar are known regulatory loci of Staphylococcus aureus that control the production of several extracellular and cell-wall-associated proteins. A pleiotropic insertional mutation in S. aureus, designated sae, that leads to the production of drastically diminished levels of alpha- and beta-hemolysins and coagulase and slightly reduced levels of protein A has been described. The study of the expression of the genes coding for these exoproteins in the sae::Tn551 mutant (carried out in this work by Northern blot analyses) revealed that the genes for alpha- and beta-hemolysins (hla and hlb) and coagulase (coa) are not transcribed and that the gene for protein A (spa) is transcribed at a somewhat reduced level. These results indicate that the sae locus regulates these exoprotein genes at the transcriptional level. Northern blot analyses also show that the sae mutation does not affect the expression of agr or sar regulatory loci. An sae::Tn551 agr::tetM double mutant has been phenotypically characterized as producing reduced or null levels of alpha-, beta-, and delta-hemolysins, coagulase, and high levels of protein A. Northern blot analyses carried out in this work with the double mutant revealed that hla, hlb, hld, and coa genes are not transcribed, while spa is transcribed at high levels. The fact that coa is not expressed in the sae agr mutant, as in the sae parental strain, while spa is expressed at the high levels characteristic of the agr parental strain, suggests that sae and agr interact in a complex way in the control of the expression of the genes of several exoproteins.

  6. Structurally designed attenuated subunit vaccines for S. aureus LukS-PV and LukF-PV confer protection in a mouse bacteremia model.

    PubMed

    Karauzum, Hatice; Adhikari, Rajan P; Sarwar, Jawad; Devi, V Sathya; Abaandou, Laura; Haudenschild, Christian; Mahmoudieh, Mahta; Boroun, Atefeh R; Vu, Hong; Nguyen, Tam; Warfield, Kelly L; Shulenin, Sergey; Aman, M Javad

    2013-01-01

    Previous efforts towards S. aureus vaccine development have largely focused on cell surface antigens to induce opsonophagocytic killing aimed at providing sterile immunity, a concept successfully applied to other Gram-positive pathogens such as Streptococcus pneumoniae. However, these approaches have largely failed, possibly in part due to the remarkable diversity of the staphylococcal virulence factors such as secreted immunosuppressive and tissue destructive toxins. S. aureus produces several pore-forming toxins including the single subunit alpha hemolysin as well as bicomponent leukotoxins such as Panton-Valentine leukocidin (PVL), gamma hemolysins (Hlg), and LukED. Here we report the generation of highly attenuated mutants of PVL subunits LukS-PV and LukF-PV that were rationally designed, based on an octameric structural model of the toxin, to be deficient in oligomerization. The attenuated subunit vaccines were highly immunogenic and showed significant protection in a mouse model of S. aureus USA300 sepsis. Protection against sepsis was also demonstrated by passive transfer of rabbit immunoglobulin raised against LukS-PV. Antibodies to LukS-PV inhibited the homologous oligomerization of LukS-PV with LukF-PV as well heterologous oligomerization with HlgB. Importantly, immune sera from mice vaccinated with the LukS mutant not only inhibited the PMN lytic activity produced by the PVL-positive USA300 but also blocked PMN lysis induced by supernatants of PVL-negative strains suggesting a broad protective activity towards other bicomponent toxins. These findings strongly support the novel concept of an anti-virulence, toxin-based vaccine intended for prevention of clinical S. aureus invasive disease, rather than achieving sterile immunity. Such a multivalent vaccine may include attenuated leukotoxins, alpha hemolysin, and superantigens. PMID:23762356

  7. Fungal proteinaceous compounds with multiple biological activities.

    PubMed

    Ng, Tzi Bun; Cheung, Randy Chi Fai; Wong, Jack Ho; Chan, Yau Sang; Dan, Xiuli; Pan, Wenliang; Wang, Hexiang; Guan, Suzhen; Chan, Ki; Ye, Xiuyun; Liu, Fang; Xia, Lixin; Chan, Wai Yee

    2016-08-01

    Fungi comprise organisms like molds, yeasts and mushrooms. They have been used as food or medicine for a long time. A large number of fungal proteins or peptides with diverse biological activities are considered as antibacterial, antifungal, antiviral and anticancer agents. They encompass proteases, ribosome inactivating proteins, defensins, hemolysins, lectins, laccases, ribonucleases, immunomodulatory proteins, and polysaccharopeptides. The target of the present review is to update the status of the various bioactivities of these fungal proteins and peptides and discuss their therapeutic potential. PMID:27338574

  8. [Evaluation of drug sensitivity and biochemical properties of coagulase-negative S. aureus strains isolated from clinical specimens].

    PubMed

    Szymanowska, A; Sawicka-Grzelak, A; Młynarczyk, A; Młynarczyk, G

    1993-01-01

    20-25% of strains isolated in our hospital in 1991 from clinical specimens and identified as S. aureus were coagulase-negative. These strains were characterized in respect of biochemical properties and resistance to antibacterial drugs. It was shown that the investigated group of strains displayed high drug resistance and particularly high percent of strains were resistant to methicillin (60%). 100% strains were resistant to penicillin and tetracyclines and most of them were resistant to aminocyclitol antibiotics. Coagulase-negative strains, in comparison with coagulase-positive, less frequently produced hemolysins and more frequently staphylokinase.

  9. Effect of solar irradiation on extracellular enzymes of Aeromonas proteolytica

    NASA Technical Reports Server (NTRS)

    Foster, B. G.

    1973-01-01

    The bacterium Aeromonas proteolytica was selected for studying the effects of solar irradiation on extracellular enzymes because it produces an endopeptidase that is capable of degrading proteins and a hemolysin that is active in lysing human erythrocytes. Possible alterations in the rate of enzyme production in response to the test conditions are currently underway and are not available for this preliminary report. Completed viability studies are indicative that little difference exists among the survival curves derived for cells exposed to various components of ultraviolet irradiation in space.

  10. The genome of Brucella melitensis.

    PubMed

    DelVecchio, Vito G; Kapatral, Vinayak; Elzer, Philip; Patra, Guy; Mujer, Cesar V

    2002-12-20

    The genome of Brucella melitensis strain 16M was sequenced and contained 3,294,931 bp distributed over two circular chromosomes. Chromosome I was composed of 2,117,144 bp and chromosome II has 1,177,787 bp. A total of 3,198 ORFs were predicted. The origins of replication of the chromosomes are similar to each other and to those of other alpha-proteobacteria. Housekeeping genes such as those that encode for DNA replication, protein synthesis, core metabolism, and cell-wall biosynthesis were found on both chromosomes. Genes encoding adhesins, invasins, and hemolysins were also identified.

  11. Uropathogenic Escherichia coli-Associated Exotoxins.

    PubMed

    Welch, Rodney A

    2016-06-01

    Escherichia coli are a common cause of infectious disease outside of the gastrointestinal tract. Several independently evolved E. coli clades are common causes of urinary tract and bloodstream infections. There is ample epidemiological and in vitro evidence that several different protein toxins common to many, but not all, of these strains are likely to aid the colonization and immune-evasion ability of these bacteria. This review discusses our current knowledge and areas of ignorance concerning the contribution of the hemolysin; cytotoxic-necrotizing factor-1; and the autotransporters, Sat, Pic, and Vat, to extraintestinal human disease. PMID:27337488

  12. Uropathogenic Escherichia coli-associated exotoxins

    PubMed Central

    Welch, Rodney A.

    2015-01-01

    Escherichia coli are a common cause of infectious disease outside of the gastrointestinal tract. Several independently evolved E. coli clades are common causes of urinary tract and blood stream infections. There is ample epidemiological and in vitro evidence that several different protein toxins common to many but not all of these strains are likely to aid the colonization and immune evasion ability of these bacteria. This review discusses our current knowledge and areas of ignorance concerning the contribution of the hemolysin, cytotoxic necrotizing factor-1 and the autotransporters, Sat, Pic and Vat to extraintestinal human disease. PMID:27337488

  13. Individual RNA Base Recognition in Immobilized Oligonucleotides using a Protein Nanopore

    PubMed Central

    Ayub, Mariam; Bayley, Hagan

    2012-01-01

    Protein nanopores are under investigation as key components of rapid, low-cost platforms to sequence DNA molecules. Previously, it has been shown that the α-hemolysin (αHL) nanopore contains three recognition sites, capable of discriminating between individual DNA bases when oligonucleotides are immobilized within the nanopore. However, the direct sequencing of RNA is also of critical importance. Here, we achieve sharply defined current distributions that enable clear discrimination of the four nucleobases, guanine, cytosine, adenine and uracil, in RNA. Further, the modified bases, inosine, N6-methyladenosine and N5-methylcytosine, can be distinguished. PMID:23043363

  14. Presence of pathogenic Vibrio parahaemolyticus in waters and seafood from the Tunisian Sea.

    PubMed

    Khouadja, Sadok; Suffredini, Elisabetta; Spagnoletti, Matteo; Croci, Luciana; Colombo, Mauro M; Amina, Bakhrouf

    2013-08-01

    The occurrence of the hemolysin genes, tdh and trh, in Vibrio parahaemolyticus strains isolated from environmental samples collected from various exported seafood products comprising of fishes and shellfish (Mytilus edulis and Crassostrea gigas) or seawater, was studied. Eight strains were confirmed as V. parahaemolyticus by toxR -based polymerase chain reaction and only one strain out of these 8 strains was positive for tdh and trh genes. Toxigenic V. parahaemolyticus isolates are present in Tunisian coastal areas and they may also be present in Tunisian exported seafood products. PMID:23430717

  15. [Avian Escherichia coli virulence factors associated with coli septicemia in broiler chickens].

    PubMed

    Ramirez Santoyo, R M; Moreno Sala, A; Almanza Marquez, Y

    2001-01-01

    In order to detect phenotypic characteristics associated with pathogenicity, 25 strains of Escherichia coli, isolated from clinical cases of colisepticemia in broiler chickens, were examined to determine the following properties: colicinogenicity, colicin V production, type 1 fimbriae, hemolysin expression and motility. Colicinogenicity occurred in 72% of the strains, 56% of all strains produced colicin V, 84% were positive for type 1 fimbriae and 80% were positive for motility. None of the strains had hemolytic activity; however, all of them, expressed at least one of the other characteristics studied. These results suggest that the diversity of phenotypes detected partially explain the multifactorial nature of avian colisepticemia.

  16. Bacillus cereus and related species.

    PubMed

    Drobniewski, F A

    1993-10-01

    Bacillus cereus is a gram-positive aerobic or facultatively anaerobic spore-forming rod. It is a cause of food poisoning, which is frequently associated with the consumption of rice-based dishes. The organism produces an emetic or diarrheal syndrome induced by an emetic toxin and enterotoxin, respectively. Other toxins are produced during growth, including phospholipases, proteases, and hemolysins, one of which, cereolysin, is a thiol-activated hemolysin. These toxins may contribute to the pathogenicity of B. cereus in nongastrointestinal disease. B. cereus isolated from clinical material other than feces or vomitus was commonly dismissed as a contaminant, but increasingly it is being recognized as a species with pathogenic potential. It is now recognized as an infrequent cause of serious nongastrointestinal infection, particularly in drug addicts, the immunosuppressed, neonates, and postsurgical patients, especially when prosthetic implants such as ventricular shunts are inserted. Ocular infections are the commonest types of severe infection, including endophthalmitis, panophthalmitis, and keratitis, usually with the characteristic formation of corneal ring abscesses. Even with prompt surgical and antimicrobial agent treatment, enucleation of the eye and blindness are common sequelae. Septicemia, meningitis, endocarditis, osteomyelitis, and surgical and traumatic wound infections are other manifestations of severe disease. B. cereus produces beta-lactamases, unlike Bacillus anthracis, and so is resistant to beta-lactam antibiotics; it is usually susceptible to treatment with clindamycin, vancomycin, gentamicin, chloramphenicol, and erythromycin. Simultaneous therapy via multiple routes may be required.

  17. Negatively charged residues of the segment linking the enzyme and cytolysin moieties restrict the membrane-permeabilizing capacity of adenylate cyclase toxin

    PubMed Central

    Masin, Jiri; Osickova, Adriana; Sukova, Anna; Fiser, Radovan; Halada, Petr; Bumba, Ladislav; Linhartova, Irena; Osicka, Radim; Sebo, Peter

    2016-01-01

    The whooping cough agent, Bordetella pertussis, secretes an adenylate cyclase toxin-hemolysin (CyaA) that plays a crucial role in host respiratory tract colonization. CyaA targets CR3-expressing cells and disrupts their bactericidal functions by delivering into their cytosol an adenylate cyclase enzyme that converts intracellular ATP to cAMP. In parallel, the hydrophobic domain of CyaA forms cation-selective pores that permeabilize cell membrane. The invasive AC and pore-forming domains of CyaA are linked by a segment that is unique in the RTX cytolysin family. We used mass spectrometry and circular dichroism to show that the linker segment forms α-helical structures that penetrate into lipid bilayer. Replacement of the positively charged arginine residues, proposed to be involved in target membrane destabilization by the linker segment, reduced the capacity of the toxin to translocate the AC domain across cell membrane. Substitutions of negatively charged residues then revealed that two clusters of negative charges within the linker segment control the size and the propensity of CyaA pore formation, thereby restricting the cell-permeabilizing capacity of CyaA. The ‘AC to Hly-linking segment’ thus appears to account for the smaller size and modest cell-permeabilizing capacity of CyaA pores, as compared to typical RTX hemolysins. PMID:27581058

  18. Bordetella pertussis and Bordetella parapertussis: two immunologically distinct species.

    PubMed Central

    Khelef, N; Danve, B; Quentin-Millet, M J; Guiso, N

    1993-01-01

    Bordetella pertussis and Bordetella parapertussis are closely related species. Both are responsible for outbreaks of whooping cough in humans and produce similar virulence factors, with the exception of pertussis toxin, specific to B. pertussis. Current pertussis whole-cell vaccine will soon be replaced by acellular vaccines containing major adhesins (filamentous hemagglutinin and pertactin) and major toxin (pertussis toxin). All of these factors are antigens that stimulate a protective immune response in the murine respiratory model and in clinical assays. In the present study, we examined the protective efficacies of these factors, and that of adenylate cyclase-hemolysin, another B. pertussis toxin, against B. parapertussis infection in a murine respiratory model. As expected, pertussis toxin did not protect against B. parapertussis infection, since this bacterium did not express this protein, but the surprising result was that none of the other factors were protective against B. parapertussis infection. Furthermore, B. parapertussis adenylate cyclase-hemolysin, although it protected against B. parapertussis infection, did not protect against B. pertussis infection. Despite a high degree of homology between both B. pertussis and B. parapertussis species, no cross-protection was observed. Our results outline the fact that, as in other gram-negative bacteria, Bordetella surface proteins vary immunologically. Images PMID:8423077

  19. Interaction of Heparins and Dextran Sulfates with a Mesoscopic Protein Nanopore

    PubMed Central

    Teixeira, Luciana R.; Merzlyak, Petr G.; Valeva, Angela; Krasilnikov, Oleg V.

    2009-01-01

    Abstract A mechanism of how polyanions influence the channel formed by Staphylococcus aureus α-hemolysin is described. We demonstrate that the probability of several types of polyanions to block the ion channel depends on the presence of divalent cations and the polyanion molecular weight and concentration. For heparins, a 10-fold increase in molecular weight decreases the half-maximal inhibitory concentration, IC50, nearly 104-fold. Dextran sulfates were less effective at blocking the channel. The polyanions are significantly more effective at reducing the conductance when added to the trans side of this channel. Lastly, the effectiveness of heparins on the channel conductance correlated with their influence on the ζ-potential of liposomes. A model that includes the binding of polyanions to the channel-membrane complex via Ca2+-bridges and the asymmetry of the channel structure describes the data adequately. Analysis of the single channel current noise of wild-type and site-directed mutant versions of α-hemolysin channels suggests that a single polyanion enters the pore due to electrostatic forces and physically blocks the ion conduction path. The results might be of interest for pharmacology, biomedicine, and research aiming to design mesoscopic pore blockers. PMID:19948118

  20. Effect of detergents on streptolysin S precursor.

    PubMed

    Calandra, G B

    1980-08-01

    Group A streptococci which produce streptolysin S contain a cellular precursor to streptolysin S in the membranes and cytoplasm which is activatable by blending in a Vortex mixer with glass beads and ribonucleic acid (RNA)-core (RNA preparation from yeast). Although no activation of precursor occurred when it was mixed with detergents, it was activated when blended with glass beads and detergents such as Tergitol NP-40 and Brij 35. Maximum activation of precursor was achieved in 1 to 2% detergent, in pH 6.5 buffer, and after 8 min of blending. Detergents Tween 20, 40, 60, and 80, Brij 56, and Lubrol WX also activated precursor, but, of all the hemolysin preparations, those with Tween 40 or 60 or Lubrol WX were the most stable. The addition of RNA-core during or after blending of precursor with detergents enhanced the titer and stability of the hemolysin. This was due in part to the association of the hemolytic moiety with RNA-core. Activation of precursor in the membrane was better with a detergent, whereas that in the cytoplasm was better with RNA-core. Therefore, precursor from two different cellular locations can be differentiated by the effects of RNA-core and detergents on precursor titer.

  1. Diminished virulence of a sar-/agr- mutant of Staphylococcus aureus in the rabbit model of endocarditis.

    PubMed Central

    Cheung, A L; Eberhardt, K J; Chung, E; Yeaman, M R; Sullam, P M; Ramos, M; Bayer, A S

    1994-01-01

    Microbial pathogenicity in Staphylococcus aureus is a complex process involving a number of virulence genes that are regulated by global regulatory systems including sar and agr. To evaluate the roles of these two loci in virulence, we constructed sar-/agr- mutants of strains RN6390 and RN450 and compared their phenotypic profiles to the corresponding single sar- and agr- mutants and parents. The secretion of all hemolysins was absent in the sar-/agr- mutants while residual beta-hemolysin activity remained in single agr- mutants. The fibronectin binding capacity was significantly diminished in both single sar- mutants and double mutants when compared with parents while the reduction in fibrinogen binding capacity in the double mutants was modest. In the rabbit endocarditis model, there was a significant decrease in both infectivity rates and intravegetation bacterial densities with the double mutant as compared to the parent (RN6390) at 10(3)-10(6) CFU inocula despite comparable levels of early bacteremia among various challenge groups. Notably, fewer bacteria in the double mutant group adhered to valvular vegetations at 30 min after challenge (10(6) CFU) than the parent group. These studies suggest that both the sar and agr loci are involved in initial valvular adherence, intravegetation persistence and multiplication of S. aureus in endocarditis. Images PMID:7962526

  2. Genetic diversity of Vibrio parahaemolyticus strains isolated from farmed Pacific white shrimp and ambient pond water affected by acute hepatopancreatic necrosis disease outbreak in Thailand.

    PubMed

    Chonsin, Kaknokrat; Matsuda, Shigeaki; Theethakaew, Chonchanok; Kodama, Toshio; Junjhon, Jiraphan; Suzuki, Yasuhiko; Suthienkul, Orasa; Iida, Tetsuya

    2016-01-01

    Acute hepatopancreatic necrosis disease (AHPND) is an emerging shrimp disease that causes massive die-offs in farmed shrimps. Recent outbreaks of AHPND in Asia have been causing great losses for shrimp culture and have become a serious socioeconomic problem. The causative agent of AHPND is Vibrio parahaemolyticus, which is typically known to cause food-borne gastroenteritis in humans. However, there have been few reports of the epidemiology of V. parahaemolyticus AHPND strains, and the genetic relationship among AHPND strains is unclear. Here, we report the genetic characterization of V. parahaemolyticus strains isolated from AHPND outbreaks in Thailand. We found eight isolates from AHPND-suspected shrimps and pond water that were positive for AHPND markers AP1 and AP2. PCR analysis confirmed that none of these eight AP-positive AHPND strains possesses the genes for the conventional virulence factors affecting to humans, such as thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH) and type III secretion system 2. Phylogenetic analysis by multilocus sequence typing showed that the AHPND strains are genetically diverse, suggesting that AHPND strains were not derived from a single genetic lineage. Our study represents the first report of molecular epidemiology of AHPND-causing V. parahaemolyticus strains using multilocus sequence typing, and provides an insight into their evolutionary mechanisms.

  3. Cellular and humoral antibody responses of normal pastel and sapphire mink to goat erythrocytes.

    PubMed

    Lodmell, D L; Bergman, R K; Hadlow, W J; Munoz, J J

    1971-02-01

    This study was undertaken to determine whether normal sapphire and royal pastel mink differ immunologically at the cellular and humoral levels. Two days after primary intraperitoneal (ip) inoculation of goat erythrocytes (GE), essentially no 19 or 7S plaque-forming cells (PFC) per 10(6) cells were detected in spleen or in abdominal and peripheral lymph nodes of either color phase. On the 4th day, more 19S PFC were detected in pastel than in sapphire tissues; pastel tissues also contained 7S PFC, whereas essentially none was present in sapphires until the 6th day. After an ip booster inoculation, the number of PFC was markedly different between the two color phases. These differences were most apparent in spleen and peripheral lymph nodes. In parallel with differences observed in PFC responses between the color phases, total hemolysin and 2-mercaptoethanol-resistant hemolysin titers of pastels exceeded those of sapphires in all but one interval after the primary, and at every interval after the booster, inoculation. These data indicate that sapphire mink are not immunological cripples, nor are they immunologically hyperactive, but that differences do exist between sapphire and royal pastel mink, especially in the response to booster injections of GE. PMID:16557957

  4. Bacillus thuringiensis membrane-damaging toxins acting on mammalian cells.

    PubMed

    Celandroni, Francesco; Salvetti, Sara; Senesi, Sonia; Ghelardi, Emilia

    2014-12-01

    Bacillus thuringiensis is widely used as a biopesticide in forestry and agriculture, being able to produce potent species-specific insecticidal toxins and considered nonpathogenic to other animals. More recently, however, repeated observations are documenting the association of this microorganism with various infectious diseases in humans, such as food-poisoning-associated diarrheas, periodontitis, bacteremia, as well as ocular, burn, and wound infections. Similar to B. cereus, B. thuringiensis produces an array of virulence factors acting against mammalian cells, such as phosphatidylcholine- and phosphatidylinositol-specific phospholipase C (PC-PLC and PI-PLC), hemolysins, in particular hemolysin BL (HBL), and various enterotoxins. The contribution of some of these toxins to B. thuringiensis pathogenicity has been studied in animal models of infection, following intravitreous, intranasal, or intratracheal inoculation. These studies lead to the speculation that the activities of PC-PLC, PI-PLC, and HBL are responsible for most of the pathogenic properties of B. thuringiensis in nongastrointestinal infections in mammals. This review summarizes data regarding the biological activity, the genetic basis, and the structural features of these membrane-damaging toxins.

  5. Quantitative Analysis of the Nanopore Translocation Dynamics of Simple Structured Polynucleotides

    PubMed Central

    Schink, Severin; Renner, Stephan; Alim, Karen; Arnaut, Vera; Simmel, Friedrich C.; Gerland, Ulrich

    2012-01-01

    Nanopore translocation experiments are increasingly applied to probe the secondary structures of RNA and DNA molecules. Here, we report two vital steps toward establishing nanopore translocation as a tool for the systematic and quantitative analysis of polynucleotide folding: 1), Using α-hemolysin pores and a diverse set of different DNA hairpins, we demonstrate that backward nanopore force spectroscopy is particularly well suited for quantitative analysis. In contrast to forward translocation from the vestibule side of the pore, backward translocation times do not appear to be significantly affected by pore-DNA interactions. 2), We develop and verify experimentally a versatile mesoscopic theoretical framework for the quantitative analysis of translocation experiments with structured polynucleotides. The underlying model is based on sequence-dependent free energy landscapes constructed using the known thermodynamic parameters for polynucleotide basepairing. This approach limits the adjustable parameters to a small set of sequence-independent parameters. After parameter calibration, the theoretical model predicts the translocation dynamics of new sequences. These predictions can be leveraged to generate a baseline expectation even for more complicated structures where the assumptions underlying the one-dimensional free energy landscape may no longer be satisfied. Taken together, backward translocation through α-hemolysin pores combined with mesoscopic theoretical modeling is a promising approach for label-free single-molecule analysis of DNA and RNA folding. PMID:22225801

  6. Occurrence of Vibrio spp. in fish and shellfish collected from the Swiss market.

    PubMed

    Schärer, K; Savioz, S; Cernela, N; Saegesser, G; Stephan, R

    2011-08-01

    The genus Vibrio includes gram-negative bacteria that inhabit estuarine ecosystems. V. cholerae, V. parahaemolyticus, and V. vulnificus pose a considerable public health threat as agents of sporadic and epidemic foodborne infections associated with the consumption of raw or undercooked contaminated fish or shellfish. In this study, we analyzed 138 fish and shellfish samples collected from the Swiss market (fish fillets [n = 102], bivalves [n = 34], and squid [n = 2]). Microbiological analysis was done according to International Organization for Standardization method 21872-1/21872-2:2007, using thiosulfate citrate bile sucrose agar and chromID Vibrio agar as selective agar. Presumptive-positive colonies on thiosulfate citrate bile sucrose agar or chromID Vibrio agar were picked and were identified by the API 20E and species-specific PCR systems. V. cholerae isolates were tested further by PCR for the presence of the cholera toxin A subunit gene (ctxA). V. parahaemolyticus isolates were tested by PCR for genes encoding for thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh). V. cholerae was isolated from three samples and V. parahaemolyticus from eight samples. None of these strains harbored species-specific virulence factors. Further, V. alginolyticus was isolated from 40 samples, and V. fluvialis was isolated from 1 sample. Our study provides, for the first time, data for the assessment of exposure to Vibrio spp. in raw fish and bivalves consumed in Switzerland.

  7. A hemolytic pigment of Group B Streptococcus allows bacterial penetration of human placenta.

    PubMed

    Whidbey, Christopher; Harrell, Maria Isabel; Burnside, Kellie; Ngo, Lisa; Becraft, Alexis K; Iyer, Lakshminarayan M; Aravind, L; Hitti, Jane; Waldorf, Kristina M Adams; Rajagopal, Lakshmi

    2013-06-01

    Microbial infection of the amniotic fluid is a significant cause of fetal injury, preterm birth, and newborn infections. Group B Streptococcus (GBS) is an important human bacterial pathogen associated with preterm birth, fetal injury, and neonatal mortality. Although GBS has been isolated from amniotic fluid of women in preterm labor, mechanisms of in utero infection remain unknown. Previous studies indicated that GBS are unable to invade human amniotic epithelial cells (hAECs), which represent the last barrier to the amniotic cavity and fetus. We show that GBS invades hAECs and strains lacking the hemolysin repressor CovR/S accelerate amniotic barrier failure and penetrate chorioamniotic membranes in a hemolysin-dependent manner. Clinical GBS isolates obtained from women in preterm labor are hyperhemolytic and some are associated with covR/S mutations. We demonstrate for the first time that hemolytic and cytolytic activity of GBS is due to the ornithine rhamnolipid pigment and not due to a pore-forming protein toxin. Our studies emphasize the importance of the hemolytic GBS pigment in ascending infection and fetal injury.

  8. Development of a novel multiplex electrochemiluminescent-based immunoassay for quantification of human serum IgG against 10 Staphylococcus aureus toxins.

    PubMed

    Adhikari, Rajan P; Haudenschild, Christian; Sterba, Patricia M; Sahandi, Sara; Enterlein, Sven; Holtsberg, Frederick W; Aman, M Javad

    2016-03-01

    An electrochemiluminescent (ECL)-based multiplex immunoassay using Meso-Scale Discovery (MSD) technology was developed for detecting antibody response toward 10 Staphylococcus aureus (S. aureus) exotoxins. These 10 antigens included three different groups of toxins: 1) single component pore-forming toxins such as alpha- and delta-hemolysins, 2) the bicomponent pore-forming toxin Panton-Valentine leukocidin (PVL), comprised of LukS-PV and LukF-PV subunits, and 3) enterotoxin/superantigens - Staphylococcal enterotoxins A (SEA), B (SEB), C1 (SEC1), D (SED), K (SEK) and Toxic shock syndrome toxin-1 (TSST-1). Assay development included optimization steps with a conventional SEB ELISA-based serological assay and then optimized parameters were transferred and re-optimized in a singleplex ECL format. Finally, two pentaplex solid-phase ECL formats were developed. As proof of concept, one set of pentaplex ECL data was compared with conventional ELISA results. During the assay development controls were screened and developed for both the singleplex and multiplex assays. ECL-based multiplex assays were more sensitive with a wide dynamic range and proved more time-efficient than conventional ELISAs. Using the newly developed ECL method we showed, for the first time, that delta-hemolysin toxin can induce an immune response as antibody titers could be detected.

  9. Bacillus cereus and related species.

    PubMed Central

    Drobniewski, F A

    1993-01-01

    Bacillus cereus is a gram-positive aerobic or facultatively anaerobic spore-forming rod. It is a cause of food poisoning, which is frequently associated with the consumption of rice-based dishes. The organism produces an emetic or diarrheal syndrome induced by an emetic toxin and enterotoxin, respectively. Other toxins are produced during growth, including phospholipases, proteases, and hemolysins, one of which, cereolysin, is a thiol-activated hemolysin. These toxins may contribute to the pathogenicity of B. cereus in nongastrointestinal disease. B. cereus isolated from clinical material other than feces or vomitus was commonly dismissed as a contaminant, but increasingly it is being recognized as a species with pathogenic potential. It is now recognized as an infrequent cause of serious nongastrointestinal infection, particularly in drug addicts, the immunosuppressed, neonates, and postsurgical patients, especially when prosthetic implants such as ventricular shunts are inserted. Ocular infections are the commonest types of severe infection, including endophthalmitis, panophthalmitis, and keratitis, usually with the characteristic formation of corneal ring abscesses. Even with prompt surgical and antimicrobial agent treatment, enucleation of the eye and blindness are common sequelae. Septicemia, meningitis, endocarditis, osteomyelitis, and surgical and traumatic wound infections are other manifestations of severe disease. B. cereus produces beta-lactamases, unlike Bacillus anthracis, and so is resistant to beta-lactam antibiotics; it is usually susceptible to treatment with clindamycin, vancomycin, gentamicin, chloramphenicol, and erythromycin. Simultaneous therapy via multiple routes may be required. PMID:8269390

  10. Effect of Tyrosol and Farnesol on Virulence and Antibiotic Resistance of Clinical Isolates of Pseudomonas aeruginosa

    PubMed Central

    Hassan Abdel-Rhman, Shaymaa; Mostafa El-Mahdy, Areej; El-Mowafy, Mohammed

    2015-01-01

    Mixed-species biofilms could create a protected environment that allows for survival to external antimicrobials and allows different bacterial-fungal interactions. Pseudomonas aeruginosa-Candida albicans coexistence is an example for such mixed-species community. Numerous reports demonstrated how P. aeruginosa or its metabolites could influence the growth, morphogenesis, and virulence of C. albicans. In this study, we investigated how the C. albicans quorum sensing compounds, tyrosol and farnesol, might affect Egyptian clinical isolates of P. aeruginosa regarding growth, antibiotic sensitivity, and virulence. We could demonstrate that tyrosol possesses an antibacterial activity against P. aeruginosa (10 µM inhibited more than 50% of growth after 16 h cultivation). Moreover, we could show for the first time that tyrosol strongly inhibits the production of the virulence factors hemolysin and protease in P. aeruginosa, whereas farnesol inhibits, to lower extent, hemolysin production in this bacterial pathogen. Cumulatively, tyrosol is expected to strongly affect P. aeruginosa in mixed microbial biofilm. PMID:26844228

  11. Allicin from garlic inhibits the biofilm formation and urease activity of Proteus mirabilis in vitro.

    PubMed

    Ranjbar-Omid, Mahsa; Arzanlou, Mohsen; Amani, Mojtaba; Shokri Al-Hashem, Seyyedeh Khadijeh; Amir Mozafari, Nour; Peeri Doghaheh, Hadi

    2015-05-01

    Several virulence factors contribute to the pathogenesis of Proteus mirabilis. This study determined the inhibitory effects of allicin on urease, hemolysin and biofilm of P. mirabilis ATCC 12453 and its antimicrobial activity against 20 clinical isolates of P. mirabilis. Allicin did not inhibit hemolysin, whereas it did inhibit relative urease activity in both pre-lysed (half-maximum inhibitory concentration, IC50 = 4.15 μg) and intact cells (IC50 = 21 μg) in a concentration-dependent manner. Allicin at sub-minimum inhibitory concentrations (2-32 μg mL(-1)) showed no significant effects on the growth of the bacteria (P > 0.05), but it reduced biofilm development in a concentration-dependent manner (P < 0.001). A higher concentration of allicin was needed to inhibit the established biofilms. Using the microdilution technique, the MIC90 and MBC90 values of allicin against P. mirabilis isolates were determined to be 128 and 512 μg mL(-1), respectively. The results suggest that allicin could have clinical applications in controlling P. mirabilis infections. PMID:25837813

  12. Cellular and humoral antibody responses of normal pastel and sapphire mink to goat erythrocytes.

    PubMed

    Lodmell, D L; Bergman, R K; Hadlow, W J; Munoz, J J

    1971-02-01

    This study was undertaken to determine whether normal sapphire and royal pastel mink differ immunologically at the cellular and humoral levels. Two days after primary intraperitoneal (ip) inoculation of goat erythrocytes (GE), essentially no 19 or 7S plaque-forming cells (PFC) per 10(6) cells were detected in spleen or in abdominal and peripheral lymph nodes of either color phase. On the 4th day, more 19S PFC were detected in pastel than in sapphire tissues; pastel tissues also contained 7S PFC, whereas essentially none was present in sapphires until the 6th day. After an ip booster inoculation, the number of PFC was markedly different between the two color phases. These differences were most apparent in spleen and peripheral lymph nodes. In parallel with differences observed in PFC responses between the color phases, total hemolysin and 2-mercaptoethanol-resistant hemolysin titers of pastels exceeded those of sapphires in all but one interval after the primary, and at every interval after the booster, inoculation. These data indicate that sapphire mink are not immunological cripples, nor are they immunologically hyperactive, but that differences do exist between sapphire and royal pastel mink, especially in the response to booster injections of GE.

  13. Bacterial nanofluidic structures for medicine and engineering.

    PubMed

    Hesse, William R; Freedman, Kevin J; Yi, Dong Kee; Ahn, Chi Won; Kim, Minjun

    2010-04-23

    Bacteria are microscopic, single-celled organisms that utilize a variety of nanofluidic structures. One of the best known and widely used nanofluidic structures that are derived from bacteria is the alpha-hemolysin pore. This pore, which self-assembles in lipid bilayers, has been used for a wide variety of sensing applications, most notably, DNA sensing. Synthetic pores drilled in a wide variety of materials, such as silicon nitride and polymers have been developed that use inspiration from the alpha-hemolysin pore. Higher-aspect-ratio nanofluidic structures, akin to nanotubes, are also synthesized by bacteria. Examples of such structures include those that are associated with bacterial transport apparatus, such as pili, and are used by bacteria to facilitate the transfer of genetic material from one bacterium to another. Flagella, and its associated structures, such as the rod and hook, are also tubular nanostructures, through which the protein, flagellin, travels to assemble the flagellum. Genetic engineering allows for the creation of modified bacterial nanopores and nanotubes that can be used for a variety of medical and engineering purposes.

  14. Cellular and Humoral Antibody Responses of Normal Pastel and Sapphire Mink to Goat Erythrocytes

    PubMed Central

    Lodmell, D. L.; Bergman, R. K.; Hadlow, W. J.; Munoz, J. J.

    1971-01-01

    This study was undertaken to determine whether normal sapphire and royal pastel mink differ immunologically at the cellular and humoral levels. Two days after primary intraperitoneal (ip) inoculation of goat erythrocytes (GE), essentially no 19 or 7S plaque-forming cells (PFC) per 106 cells were detected in spleen or in abdominal and peripheral lymph nodes of either color phase. On the 4th day, more 19S PFC were detected in pastel than in sapphire tissues; pastel tissues also contained 7S PFC, whereas essentially none was present in sapphires until the 6th day. After an ip booster inoculation, the number of PFC was markedly different between the two color phases. These differences were most apparent in spleen and peripheral lymph nodes. In parallel with differences observed in PFC responses between the color phases, total hemolysin and 2-mercaptoethanol-resistant hemolysin titers of pastels exceeded those of sapphires in all but one interval after the primary, and at every interval after the booster, inoculation. These data indicate that sapphire mink are not immunological cripples, nor are they immunologically hyperactive, but that differences do exist between sapphire and royal pastel mink, especially in the response to booster injections of GE. PMID:16557957

  15. Variable expressions of Staphylococcus aureus bicomponent leucotoxins semiquantified by competitive reverse transcription-PCR.

    PubMed

    Bronner, S; Stoessel, P; Gravet, A; Monteil, H; Prévost, G

    2000-09-01

    A competitive reverse transcription-PCR method was developed for the semiquantitation of the expression of genes encoding bicomponent leucotoxins of Staphylococcus aureus, e.g., Panton-Valentine leucocidin (lukPV), gamma-hemolysin (hlgA and hlgCB), and LukE-LukD (lukED). The optimization procedure included RNA preparation; reverse transcription; the use of various amounts of enzymes, antisense primer, and RNA; and the final amplification chain reaction. Reproducible results were obtained, with sensitivity for detection of cDNA within the range of 1 mRNA/10(4) CFU to 10(2) mRNA/CFU, depending on the gene. Both specific mRNAs were more significantly expressed at the late-exponential phase of growth. Expression was about 100-fold higher in yeast extract-Casamino Acids-pyruvate medium than in heart infusion medium. Expression of the widely distributed gamma-hemolysin locus in the NTCC 8178 strain was around 10-fold diminished compared with that in the ATCC 49775 strain. Because of the lower level of hlgA expression, the corresponding protein, which is generally not abundant in culture supernatant, should be investigated for its contribution to the leucotoxin-associated virulence. The agr, sar, and agr sar mutant strains revealed a great dependence with regard to leucotoxin expression on the global regulatory system in S. aureus, except that expression of hlgA was not affected in the agr mutant.

  16. Stilbenes reduce Staphylococcus aureus hemolysis, biofilm formation, and virulence.

    PubMed

    Lee, Kayeon; Lee, Jin-Hyung; Ryu, Shi Yong; Cho, Moo Hwan; Lee, Jintae

    2014-09-01

    Stilbenoids have a broad range of beneficial health effects. On the other hand, the emergence of antibiotic-resistant Staphylococcus aureus presents a worldwide problem that requires new antibiotics or nonantibiotic strategies. S. aureus produces α-hemolysin (a pore-forming cytotoxin) that has been implicated in the pathogenesis of sepsis and pneumonia. Furthermore, the biofilms formed by S. aureus constitute a mechanism of antimicrobial resistance. In this study, we investigated the hemolytic and antibiofilm activities of 10 stilbene-related compounds against S. aureus. trans-Stilbene and resveratrol at 10 μg/mL were found to markedly inhibit human blood hemolysis by S. aureus, and trans-stilbene also inhibited S. aureus biofilm formation without affecting its bacterial growth. Furthermore, trans-stilbene and resveratrol attenuated S. aureus virulence in vivo in the nematode Caenorhabditis elegans, which is normally killed by S. aureus. Transcriptional analysis showed that trans-stilbene repressed the α-hemolysin hla gene and the intercellular adhesion locus (icaA and icaD) in S. aureus, and this finding was in line with observed reductions in virulence and biofilm formation. In addition, vitisin B, a stilbenoid tetramer, at 1 μg/mL was observed to significantly inhibit human blood hemolysis by S. aureus.

  17. A vesicle bioreactor as a step toward an artificial cell assembly

    NASA Astrophysics Data System (ADS)

    Noireaux, Vincent; Libchaber, Albert

    2004-12-01

    An Escherichia coli cell-free expression system is encapsulated in a phospholipid vesicle to build a cell-like bioreactor. Large unilamellar vesicles containing extracts are produced in an oil-extract emulsion. To form a bilayer the vesicles are transferred into a feeding solution that contains ribonucleotides and amino acids. Transcription-translation of plasmid genes is isolated in the vesicles. Whereas in bulk solution expression of enhanced GFP stops after 2 h, inside the vesicle permeability of the membrane to the feeding solution prolongs the expression for up to 5 h. To solve the energy and material limitations and increase the capacity of the reactor, the -hemolysin pore protein from Staphylococcus aureus is expressed inside the vesicle to create a selective permeability for nutrients. The reactor can then sustain expression for up to 4 days with a protein production of 30 µM after 4 days. Oxygen diffusion and osmotic pressure are critical parameters to maintain expression and avoid vesicle burst. -hemolysin | cell-free protein expression | membrane-anchoring polypeptide

  18. Massively Parallel, Molecular Analysis Platform Developed Using a CMOS Integrated Circuit With Biological Nanopores

    PubMed Central

    Roever, Stefan

    2012-01-01

    A massively parallel, low cost molecular analysis platform will dramatically change the nature of protein, molecular and genomics research, DNA sequencing, and ultimately, molecular diagnostics. An integrated circuit (IC) with 264 sensors was fabricated using standard CMOS semiconductor processing technology. Each of these sensors is individually controlled with precision analog circuitry and is capable of single molecule measurements. Under electronic and software control, the IC was used to demonstrate the feasibility of creating and detecting lipid bilayers and biological nanopores using wild type α-hemolysin. The ability to dynamically create bilayers over each of the sensors will greatly accelerate pore development and pore mutation analysis. In addition, the noise performance of the IC was measured to be 30fA(rms). With this noise performance, single base detection of DNA was demonstrated using α-hemolysin. The data shows that a single molecule, electrical detection platform using biological nanopores can be operationalized and can ultimately scale to millions of sensors. Such a massively parallel platform will revolutionize molecular analysis and will completely change the field of molecular diagnostics in the future.

  19. Characterization of staphylococci isolated from mastitic cows in Spain.

    PubMed

    Garcia, M L; Moreno, B; Bergdoll, M S

    1980-03-01

    A total of 57 gram-positive, catalase-positive cocci, considered etiological agents of clinical and subclinical bovine mastitis, were tested for glucose and mannitol fermentation, coagulase and thermonuclease production, sensitivity to lysostaphin, gelatin hydrolysis, lysozyme, phosphatase and egg yolk factor production, hemolytic properties, antibiotic sensitivity, susceptibility to human and bovine phages, and enterotoxin production. All 57 strains were identified as staphylococci. A good correlation was found between 3+ and 4+ coagulase reactions, thermonuclease production, and high sensitivity to lysostaphin. Neither mannitol fermentation nor production of other enzymes appeared to be a specific property of bovine Staphylococcus aureus strains. beta- and delta-hemolysins were more frequently found than alpha-hemolysin. Nearly 40% of the strains were penicillin resistant. Strains were lysed by phage 42E from the human phage set more frequently than by phage 42D, whereas with the bovine set, strains were more sensitive to specific bovine phages. Three strains produced enterotoxin C, and one strain produced enterotoxin D.

  20. Evolution in Fast Forward: a Potential Role for Mutators in Accelerating Staphylococcus aureus Pathoadaptation

    PubMed Central

    Canfield, Gregory S.; Schwingel, Johanna M.; Foley, Matthew H.; Vore, Kelly L.; Boonanantanasarn, Kanitsak; Gill, Ann L.; Sutton, Mark D.

    2013-01-01

    Pathogen evolution and subsequent phenotypic heterogeneity during chronic infection are proposed to enhance Staphylococcus aureus survival during human infection. We tested this theory by genetically and phenotypically characterizing strains with mutations constructed in the mismatch repair (MMR) and oxidized guanine (GO) system, termed mutators, which exhibit increased spontaneous-mutation frequencies. Analysis of these mutators revealed not only strain-dependent increases in the spontaneous-mutation frequency but also shifts in mutational type and hot spots consistent with loss of GO or MMR functions. Although the GO and MMR systems are relied upon in some bacterial species to prevent reactive oxygen species-induced DNA damage, no deficit in hydrogen peroxide sensitivity was found when either of these DNA repair pathways was lost in S. aureus. To gain insight into the contribution of increased mutation supply to S. aureus pathoadaptation, we measured the rate of α-hemolysin and staphyloxanthin inactivation during serial passage. Detection of increased rates of α-hemolysin and staphyloxanthin inactivation in GO and MMR mutants suggests that these strains are capable of modifying virulence phenotypes implicated in mediating infection. Accelerated derivation of altered virulence phenotypes, combined with the absence of increased ROS sensitivity, highlights the potential of mutators to drive pathoadaptation in the host and serve as catalysts for persistent infections. PMID:23204459

  1. Nanopore Detection of 8-Oxo-7,8-dihydro-2′-deoxyguanosine in Immobilized Single-stranded DNA via Adduct Formation to the DNA Damage Site

    PubMed Central

    Schibel, Anna E. P.; An, Na; Jin, Qian; Fleming, Aaron M.; Burrows, Cynthia J.; White, Henry S.

    2010-01-01

    The ability to detect DNA damage within the context of the surrounding sequence is an important goal in medical diagnosis and therapies, but there are no satisfactory methods available to detect a damaged base while providing sequence information. One of the most common base lesions is 8-oxo-7,8-dihydroguanine that occurs during oxidation of guanine. In the work presented here, we demonstrate the detection of a single oxidative damage site using ion channel nanopore methods employing α-hemolysin. Hydantoin lesions produced from further oxidation of 8-oxo-7,8-dihydroguanine, as well as spirocyclic adducts produced from covalently attaching a primary amine to the spiroiminodihydantoin lesion, were detected by tethering the damaged DNA to streptavidin via a biotin linkage, and capturing the DNA inside an α-hemolysin ion channel. Spirocyclic adducts, in both homo- and hetero-polymer background single-stranded DNA sequences, produced current blockage levels differing by almost 10% from those of native base current blockage levels. These preliminary studies show the applicability of ion channel recordings not only for DNA sequencing, which has recently received much attention, but also to detecting DNA damage, which will be an important component to any sequencing efforts. PMID:21138270

  2. Remodeling of the Streptococcus agalactiae Transcriptome in Response to Growth Temperature

    PubMed Central

    Mereghetti, Laurent; Sitkiewicz, Izabela; Green, Nicole M.; Musser, James M.

    2008-01-01

    Background To act as a commensal bacterium and a pathogen in humans and animals, Streptococcus agalactiae (group B streptococcus, GBS) must be able to monitor and adapt to different environmental conditions. Temperature variation is a one of the most commonly encountered variables. Methodology/Principal Findings To understand the extent to which GBS modify gene expression in response to temperatures encountered in the various hosts, we conducted a whole genome transcriptome analysis of organisms grown at 30°C and 40°C. We identified extensive transcriptome remodeling at various stages of growth, especially in the stationary phase (significant transcript changes occurred for 25% of the genes). A large proportion of genes involved in metabolism was up-regulated at 30°C in stationary phase. Conversely, genes up-regulated at 40°C relative to 30°C include those encoding virulence factors such as hemolysins and extracellular secreted proteins with LPXTG motifs. Over-expression of hemolysins was linked to larger zones of hemolysis and enhanced hemolytic activity at 40°C. A key theme identified by our study was that genes involved in purine metabolism and iron acquisition were significantly up-regulated at 40°C. Conclusion/Significance Growth of GBS in vitro at different temperatures resulted in extensive remodeling of the transcriptome, including genes encoding proven and putative virulence genes. The data provide extensive new leads for molecular pathogenesis research. PMID:18665215

  3. A hemolytic pigment of Group B Streptococcus allows bacterial penetration of human placenta

    PubMed Central

    Whidbey, Christopher; Harrell, Maria Isabel; Burnside, Kellie; Ngo, Lisa; Becraft, Alexis K.; Iyer, Lakshminarayan M.; Aravind, L.; Hitti, Jane

    2013-01-01

    Microbial infection of the amniotic fluid is a significant cause of fetal injury, preterm birth, and newborn infections. Group B Streptococcus (GBS) is an important human bacterial pathogen associated with preterm birth, fetal injury, and neonatal mortality. Although GBS has been isolated from amniotic fluid of women in preterm labor, mechanisms of in utero infection remain unknown. Previous studies indicated that GBS are unable to invade human amniotic epithelial cells (hAECs), which represent the last barrier to the amniotic cavity and fetus. We show that GBS invades hAECs and strains lacking the hemolysin repressor CovR/S accelerate amniotic barrier failure and penetrate chorioamniotic membranes in a hemolysin-dependent manner. Clinical GBS isolates obtained from women in preterm labor are hyperhemolytic and some are associated with covR/S mutations. We demonstrate for the first time that hemolytic and cytolytic activity of GBS is due to the ornithine rhamnolipid pigment and not due to a pore-forming protein toxin. Our studies emphasize the importance of the hemolytic GBS pigment in ascending infection and fetal injury. PMID:23712433

  4. Detection of toxigenic Bacillus cereus strains isolated from vegetables in Mexico City.

    PubMed

    Flores-Urbán, Karen A; Natividad-Bonifacio, Iván; Vázquez-Quiñones, Carlos R; Vázquez-Salinas, Carlos; Quiñones-Ramírez, Elsa Irma

    2014-12-01

    Bacillus cereus can cause diarrhea and emetic syndromes after ingestion of food contaminated with it. This ability is due to the production of enterotoxins by this microorganism, these being the hemolysin BL complex, which is involved in the diarrheal syndrome, and cereulide, which is responsible for the emetic syndrome. The detection of genes associated with the production of these toxins can predict the virulence of strains isolated from contaminated food. In this paper, we analyzed 100 samples of vegetables, 25 of each kind (broccoli, coriander, carrot, and lettuce) obtained from different markets in Mexico City and its metropolitan area. B. cereus was isolated in 32, 44, 84, and 68% of the samples of broccoli, carrot, lettuce, and coriander, respectively. The hblA gene (encoding one of the three subunits of hemolysin BL) was amplified in 100% of the B. cereus isolates, and the ces gene (encoding the cereulide) could not be amplified from any of them. This is the first report of B. cereus isolation from the vegetables analyzed in this work and, also, the first report in Mexico of the isolation from vegetables of strains with potential virulence. The results should serve as evidence of the potential risk of consuming these foods without proper treatment. PMID:25474064

  5. Comparative pathogenicity of Escherichia coli O157 and intimin-negative non-O157 Shiga toxin-producing E coli strains in neonatal pigs.

    PubMed

    Dean-Nystrom, Evelyn A; Melton-Celsa, Angela R; Pohlenz, Joachim F L; Moon, Harley W; O'Brien, Alison D

    2003-11-01

    We compared the pathogenicity of intimin-negative non-O157:H7 Shiga toxin (Stx)-producing Escherichia coli (STEC) O91:H21 and O104:H21 strains with the pathogenicity of intimin-positive O157:H7 and O157:H(-) strains in neonatal pigs. We also examined the role of Stx2d-activatable genes and the large hemolysin-encoding plasmid of O91:H21 strain B2F1 in the pathogenesis of STEC disease in pigs. We found that all E. coli strains that made wild-type levels of Stx caused systemic illness and histological lesions in the brain and intestinal crypts, whereas none of the control Stx-negative E. coli strains evoked comparable central nervous system signs or intestinal lesions. By contrast, the absence of intimin, hemolysin, or motility had little impact on the overall pathogenesis of systemic disease during STEC infection. The most striking differences between pigs inoculated with non-O157 STEC strains and pigs inoculated with O157 STEC strains were the absence of attaching and effacing intestinal lesions in pigs inoculated with non-O157:H7 strains and the apparent association between the level of Stx2d-activatable toxin produced by an STEC strain and the severity of lesions. PMID:14573674

  6. Negatively charged residues of the segment linking the enzyme and cytolysin moieties restrict the membrane-permeabilizing capacity of adenylate cyclase toxin.

    PubMed

    Masin, Jiri; Osickova, Adriana; Sukova, Anna; Fiser, Radovan; Halada, Petr; Bumba, Ladislav; Linhartova, Irena; Osicka, Radim; Sebo, Peter

    2016-01-01

    The whooping cough agent, Bordetella pertussis, secretes an adenylate cyclase toxin-hemolysin (CyaA) that plays a crucial role in host respiratory tract colonization. CyaA targets CR3-expressing cells and disrupts their bactericidal functions by delivering into their cytosol an adenylate cyclase enzyme that converts intracellular ATP to cAMP. In parallel, the hydrophobic domain of CyaA forms cation-selective pores that permeabilize cell membrane. The invasive AC and pore-forming domains of CyaA are linked by a segment that is unique in the RTX cytolysin family. We used mass spectrometry and circular dichroism to show that the linker segment forms α-helical structures that penetrate into lipid bilayer. Replacement of the positively charged arginine residues, proposed to be involved in target membrane destabilization by the linker segment, reduced the capacity of the toxin to translocate the AC domain across cell membrane. Substitutions of negatively charged residues then revealed that two clusters of negative charges within the linker segment control the size and the propensity of CyaA pore formation, thereby restricting the cell-permeabilizing capacity of CyaA. The 'AC to Hly-linking segment' thus appears to account for the smaller size and modest cell-permeabilizing capacity of CyaA pores, as compared to typical RTX hemolysins. PMID:27581058

  7. Genetic characterization of trh positive Vibrio spp. isolated from Norway

    PubMed Central

    Ellingsen, Anette B.; Olsen, Jaran S.; Granum, Per E.; Rørvik, Liv M.; González-Escalona, Narjol

    2013-01-01

    The thermostable direct hemolysin (TDH) and/or TDH-related hemolysin (TRH) genes are carried by most virulent Vibrio parahaemolyticus serovars. In Norway, trh+ V. parahaemolyticus constitute 4.4 and 4.5% of the total number of V. parahaemolyticus isolated from blue mussel (Mytilus edulis) and water, respectively. The trh gene is located in a region close to the gene cluster for urease production (ure). This region was characterized in V. parahaemolyticus strain TH3996 and it was found that a nickel transport operon (nik) was located between the first gene (ureR) and the rest of the ure cluster genes. The organization of the trh-ureR-nik-ure gene cluster in the Norwegian trh+ isolates was unknown. In this study, we explore the gene organization within the trh-ureR-nik-ure cluster for these isolates. PCR analyses revealed that the genes within the trh-ureR-nik-ure gene cluster of Norwegian trh+ isolates were organized in a similar fashion as reported previously for TH33996. Additionally, the phylogenetic relationship among these trh+ isolates was investigated using Multilocus Sequence Typing (MLST). Analysis by MLST or ureR-trh sequences generated two different phylogenetic trees for the same strains analyzed, suggesting that ureR-trh genes have been acquired at different times in Norwegian V. parahaemolyticus isolates. MLST results revealed that some pathogenic and non-pathogenic V. parahaemolyticus isolates in Norway appear to be highly genetically related. PMID:24400227

  8. Quinolones Induce Partial or Total Loss of Pathogenicity Islands in Uropathogenic Escherichia coli by SOS-Dependent or -Independent Pathways, Respectively

    PubMed Central

    Soto, S. M.; Jimenez de Anta, M. T.; Vila, J.

    2006-01-01

    Escherichia coli is the most common microorganism causing urinary tract infections. Quinolone-resistant E. coli strains have fewer virulence factors than quinolone-susceptible strains. Several urovirulence genes are located in pathogenicity islands (PAIs). We investigated the capacity of quinolones to induce loss of virulence factors such as hemolysin, cytotoxic necrotizing factor 1, P fimbriae, and autotransporter Sat included in PAIs in three uropathogenic E. coli strains. In a multistep selection, all strains lost hemolytic capacity at between 1 and 4 passages when they were incubated with subinhibitory concentrations of ciprofloxacin, showing a partial or total loss of the PAI containing the hly (hemolysin) and cnf-1 (cytotoxic necrotizing factor 1) genes. RecA− mutants were obtained from the two E. coli strains with partial or total loss of the PAI. The inactivation of the RecA protein affected only the partial loss of the PAI induced by quinolones. No spontaneous loss of PAIs was observed on incubation in the absence of quinolones in either the wild-type or mutant E. coli strains. Quinolones induce partial or total loss of PAIs in vitro in uropathogenic E. coli by SOS-dependent or -independent pathways, respectively. PMID:16436722

  9. Characteristics of Vibrio parahaemolyticus O3:K6 from Asia

    PubMed Central

    Wong, Hin-Chung; Liu, Shu-Hui; Wang, Tien-Kuei; Lee, Chih-Lung; Chiou, Chien-Shun; Liu, Ding-Ping; Nishibuchi, Mitsuaki; Lee, Bok-Kwon

    2000-01-01

    A variety of serovars of the food-borne pathogen Vibrio parahaemolyticus normally cause infection. Since 1996, the O3:K6 strains of this pathogen have caused pandemics in many Asian countries, including Taiwan. For a better understanding of these pandemic strains, the recently isolated clinical O3:K6 strains from India, Japan, Korea, and Taiwan were examined in terms of pulsed-field gel electrophoresis (PFGE) typing and other biological characteristics. After PFGE and cluster analysis, all the O3:K6 strains were grouped into two unrelated groups. The recently isolated O3:K6 strains were all in one group, consisting of eight closely related patterns, with I1(81%) and I5(13%) being the most frequent patterns. Pattern I1 was the major one for strains from Japan, Korea, and Taiwan. All recently isolated O3:K6 strains carried the thermostable direct hemolysin (tdh) gene. No significant difference was observed between recently isolated O3:K6 strains and either non-O3:K6 reference strains or old O3:K6 strains isolated before 1996 with respect to antibiotic susceptibility, the level of thermostable direct hemolysin, and the susceptibility to environmental stresses. Results in this study confirmed that the recently isolated O3:K6 strains of V. parahaemolyticus are genetically close to each other, while the other biological traits examined were usually strain dependent, and no unique trait was found in the recently isolated O3:K6 strains. PMID:10966418

  10. Accumulation of Pyrimidine Intermediate Orotate Decreases Virulence Factor Production in Pseudomonas aeruginosa.

    PubMed

    Niazy, Abdurahman; Hughes, Lee E

    2015-08-01

    The impact of orotate accumulation in the medically important bacterium Pseudomonas aeruginosa was studied by deleting pyrE, the gene encoding orotate phosphoribosyltransferase and responsible for converting orotate into orotate monophosphate within the de novo pyrimidine synthesis pathway. The pyrE mutant accumulated orotate and exhibited decreased production of hemolysin, casein protease, and elastase. Feeding orotate at a concentration of 51.25 μM to the wild type, PAO1, likewise decreased production of these factors except for hemolysin, which was not affected. A significant increase in the pigments pyocyanin and pyoverdin was also observed. Pyocyanin increase in the pyrE mutant was heightened when the mutant was supplemented with orotate. Although pyoverdin production in the wild-type PAO1 was unaffected by orotate supplementation, a decrease in the mutant's production was observed when supplemented with orotate. These results indicate a significant reduction in virulence factor production in the pyrE mutant and reduction in some virulence factors in the wild type when supplemented with orotate. PMID:25917504

  11. Single-molecule investigation of G-quadruplex folds of the human telomere sequence in a protein nanocavity

    PubMed Central

    An, Na; Fleming, Aaron M.; Middleton, Eric G.; Burrows, Cynthia J.

    2014-01-01

    Human telomeric DNA consists of tandem repeats of the sequence 5′-TTAGGG-3′ that can fold into various G-quadruplexes, including the hybrid, basket, and propeller folds. In this report, we demonstrate use of the α-hemolysin ion channel to analyze these subtle topological changes at a nanometer scale by providing structure-dependent electrical signatures through DNA–protein interactions. Whereas the dimensions of hybrid and basket folds allowed them to enter the protein vestibule, the propeller fold exceeds the size of the latch region, producing only brief collisions. After attaching a 25-mer poly-2′-deoxyadenosine extension to these structures, unraveling kinetics also were evaluated. Both the locations where the unfolding processes occur and the molecular shapes of the G-quadruplexes play important roles in determining their unfolding profiles. These results provide insights into the application of α-hemolysin as a molecular sieve to differentiate nanostructures as well as the potential technical hurdles DNA secondary structures may present to nanopore technology. PMID:25225404

  12. Genetic diversity of Vibrio parahaemolyticus strains isolated from farmed Pacific white shrimp and ambient pond water affected by acute hepatopancreatic necrosis disease outbreak in Thailand.

    PubMed

    Chonsin, Kaknokrat; Matsuda, Shigeaki; Theethakaew, Chonchanok; Kodama, Toshio; Junjhon, Jiraphan; Suzuki, Yasuhiko; Suthienkul, Orasa; Iida, Tetsuya

    2016-01-01

    Acute hepatopancreatic necrosis disease (AHPND) is an emerging shrimp disease that causes massive die-offs in farmed shrimps. Recent outbreaks of AHPND in Asia have been causing great losses for shrimp culture and have become a serious socioeconomic problem. The causative agent of AHPND is Vibrio parahaemolyticus, which is typically known to cause food-borne gastroenteritis in humans. However, there have been few reports of the epidemiology of V. parahaemolyticus AHPND strains, and the genetic relationship among AHPND strains is unclear. Here, we report the genetic characterization of V. parahaemolyticus strains isolated from AHPND outbreaks in Thailand. We found eight isolates from AHPND-suspected shrimps and pond water that were positive for AHPND markers AP1 and AP2. PCR analysis confirmed that none of these eight AP-positive AHPND strains possesses the genes for the conventional virulence factors affecting to humans, such as thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH) and type III secretion system 2. Phylogenetic analysis by multilocus sequence typing showed that the AHPND strains are genetically diverse, suggesting that AHPND strains were not derived from a single genetic lineage. Our study represents the first report of molecular epidemiology of AHPND-causing V. parahaemolyticus strains using multilocus sequence typing, and provides an insight into their evolutionary mechanisms. PMID:26590959

  13. Hemolytic E. coli Promotes Colonic Tumorigenesis in Females.

    PubMed

    Jin, Ye; Tang, Senwei; Li, Weilin; Ng, Siew Chien; Chan, Michael W Y; Sung, Joseph J Y; Yu, Jun

    2016-05-15

    Bacterial infection is linked to colorectal carcinogenesis, but the species that contribute to a protumorigenic ecology are ill-defined. Here we report evidence that α-hemolysin-positive (hly(+)) type I Escherichia coli (E. coli) drives adenomagenesis and colorectal cancer in human females but not males. We classified E. coli into four types using a novel typing method to monitor fimH mutation patterns of fecal isolates from adenoma patients (n= 59), colorectal cancer patients (n= 83), and healthy subjects (n= 85). hly(+) type I E. coli was found to be relatively more prevalent in stools from females with adenoma and colorectal cancer, correlating with poor survival in colorectal cancer patients. In mechanistic studies in female mice, we found that hly(+) type 1 E. coli activated expression of the glucose transporter GLUT1 and repressed expression of the tumor suppressor BIM. hly-encoded alpha hemolysin partially accounted for these effects by elevating the levels of HIF1α. Notably, colon tumorigenesis in mice could be promoted by feeding hly(+) type I E. coli to female but not male subjects. Collectively, our findings point to hemolytic type I E. coli as a candidate causative factor of colorectal cancer in human females, with additional potential as a biomarker of disease susceptibility. Cancer Res; 76(10); 2891-900. ©2016 AACR.

  14. Novel Nucleoside Diphosphatase Contributes to Staphylococcus aureus Virulence.

    PubMed

    Imae, Kenta; Saito, Yuki; Kizaki, Hayato; Ryuno, Hiroki; Mao, Han; Miyashita, Atsushi; Suzuki, Yutaka; Sekimizu, Kazuhisa; Kaito, Chikara

    2016-09-01

    We identified SA1684 as a Staphylococcus aureus virulence gene using a silkworm infection model. The SA1684 gene product carried the DUF402 domain, which is found in RNA-binding proteins, and had amino acid sequence similarity with a nucleoside diphosphatase, Streptomyces coelicolor SC4828 protein. The SA1684-deletion mutant exhibited drastically decreased virulence, in which the LD50 against silkworms was more than 10 times that of the parent strain. The SA1684-deletion mutant also exhibited decreased exotoxin production and colony-spreading ability. Purified SA1684 protein had Mn(2+)- or Co(2+)-dependent hydrolyzing activity against nucleoside diphosphates. Alanine substitutions of Tyr-88, Asp-106, and Asp-123/Glu-124, which are conserved between SA1684 and SC4828, diminished the nucleoside diphosphatase activity. Introduction of the wild-type SA1684 gene restored the hemolysin production of the SA1684-deletion mutant, whereas none of the alanine-substituted SA1684 mutant genes restored the hemolysin production. RNA sequence analysis revealed that SA1684 is required for the expression of the virulence regulatory genes agr, sarZ, and sarX, as well as metabolic genes involved in glycolysis and fermentation pathways. These findings suggest that the novel nucleoside diphosphatase SA1684 links metabolic pathways and virulence gene expression and plays an important role in S. aureus virulence. PMID:27422825

  15. Detection of toxigenic Bacillus cereus strains isolated from vegetables in Mexico City.

    PubMed

    Flores-Urbán, Karen A; Natividad-Bonifacio, Iván; Vázquez-Quiñones, Carlos R; Vázquez-Salinas, Carlos; Quiñones-Ramírez, Elsa Irma

    2014-12-01

    Bacillus cereus can cause diarrhea and emetic syndromes after ingestion of food contaminated with it. This ability is due to the production of enterotoxins by this microorganism, these being the hemolysin BL complex, which is involved in the diarrheal syndrome, and cereulide, which is responsible for the emetic syndrome. The detection of genes associated with the production of these toxins can predict the virulence of strains isolated from contaminated food. In this paper, we analyzed 100 samples of vegetables, 25 of each kind (broccoli, coriander, carrot, and lettuce) obtained from different markets in Mexico City and its metropolitan area. B. cereus was isolated in 32, 44, 84, and 68% of the samples of broccoli, carrot, lettuce, and coriander, respectively. The hblA gene (encoding one of the three subunits of hemolysin BL) was amplified in 100% of the B. cereus isolates, and the ces gene (encoding the cereulide) could not be amplified from any of them. This is the first report of B. cereus isolation from the vegetables analyzed in this work and, also, the first report in Mexico of the isolation from vegetables of strains with potential virulence. The results should serve as evidence of the potential risk of consuming these foods without proper treatment.

  16. Characterization of trh2 Harbouring Vibrio parahaemolyticus Strains Isolated in Germany

    PubMed Central

    Bechlars, Silke; Jäckel, Claudia; Diescher, Susanne; Wüstenhagen, Doreen A.; Kubick, Stefan; Dieckmann, Ralf; Strauch, Eckhard

    2015-01-01

    Background Vibrio parahaemolyticus is a recognized human enteropathogen. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) as well as the type III secretion system 2 (T3SS2) are considered as major virulence factors. As tdh positive strains are not detected in coastal waters of Germany, we focused on the characterization of trh positive strains, which were isolated from mussels, seawater and patients in Germany. Results Ten trh harbouring V. parahaemolyticus strains from Germany were compared to twenty-one trh positive strains from other countries. The complete trh sequences revealed clustering into three different types: trh1 and trh2 genes and a pseudogene Ψtrh. All German isolates possessed alleles of the trh2 gene. MLST analysis indicated a close relationship to Norwegian isolates suggesting that these strains belong to the autochthonous microflora of Northern Europe seawaters. Strains carrying the pseudogene Ψtrh were negative for T3SS2β effector vopC. Transcription of trh and vopC genes was analyzed under different growth conditions. Trh2 gene expression was not altered by bile while trh1 genes were inducible. VopC could be induced by urea in trh2 bearing strains. Most trh1 carrying strains were hemolytic against sheep erythrocytes while all trh2 positive strains did not show any hemolytic activity. TRH variants were synthesized in a prokaryotic cell-free system and their hemolytic activity was analyzed. TRH1 was active against sheep erythrocytes while TRH2 variants were not active at all. Conclusion Our study reveals a high diversity among trh positive V. parahaemolyticus strains. The function of TRH2 hemolysins and the role of the pseudogene Ψtrh as pathogenicity factors are questionable. To assess the pathogenic potential of V. parahaemolyticus strains a differentiation of trh variants and the detection of T3SS2β components like vopC would improve the V. parahaemolyticus diagnostics and could lead to a refinement of the risk

  17. Mutagenesis of Bordetella pertussis with transposon Tn5tac1: conditional expression of virulence-associated genes.

    PubMed Central

    Cookson, B T; Berg, D E; Goldman, W E

    1990-01-01

    The Tn5tac1 transposon contains a strong outward-facing promoter, Ptac, a lacI repressor gene, and a selectable Kanr gene. Transcription from Ptac is repressed by the lacI protein unless an inducer (isopropyl-beta-D-thiogalactopyranoside [IPTG]) is present. Thus, Tn5tac1 generates insertion mutations in Escherichia coli with conditional phenotypes because it is polar on distal gene expression when IPTG is absent and directs transcription of these genes when the inducer is present. To test the usefulness of Tn5tac1 in Bordetella pertussis, a nonenteric gram-negative bacterial pathogen, we chose the bifunctional adenylate cyclase-hemolysin determinant as an easily scored marker to monitor insertional mutagenesis. Tn5tac1 delivered to B. pertussis on conjugal suicide plasmids resulted in Kanr exconjugants at a frequency of 10(-3) per donor cell, and nonhemolytic (Hly-) mutants were found among the Kanr colonies at a frequency of about 1%. Of eight independent Kanr Hly- mutants, two were conditional and exhibited an Hly+ phenotype only in the presence of IPTG. Using a new quantitative assay for adenylate cyclase based on high-pressure liquid chromatography, we found that enzymatic activity in these two strains was specifically induced at least 500-fold in a dose-dependent fashion over the range of 0 to 125 microM IPTG. These data show that Ptac serves as a promoter, lacI is expressed and is functional, and IPTG can induce Ptac transcription in B. pertussis. Adenylate cyclase expression in whole cells, culture supernatants, and cell extracts from these strains depended upon IPTG, suggesting that the insertions do not merely alter secretion of adenylate cyclase-hemolysin. Other virulence determinants under control of the vir locus are expressed normally, implying that these Tn5tac1 insertions specifically regulate adenylate cyclase-hemolysin expression. We conclude that Tn5tac1 insertion mutations permit sensitive, exogenous control over the expression of genes of

  18. Antibodies to S. aureus LukS-PV Attenuated Subunit Vaccine Neutralize a Broad Spectrum of Canonical and Non-Canonical Bicomponent Leukotoxin Pairs

    PubMed Central

    Adhikari, Rajan P.; Kort, Thomas; Shulenin, Sergey; Kanipakala, Tulasikumari; Ganjbaksh, Nader; Roghmann, Mary-Claire; Holtsberg, Frederick W.; Aman, M. Javad

    2015-01-01

    S. aureus vaccine development has proven particularly difficult. The conventional approach to achieve sterile immunity through opsonophagocytic killing has been largely unsuccessful. S. aureus is highly toxigenic and a great body of evidence suggests that a successful future vaccine for this organism should target extracellular toxins which are responsible for host tissue destruction and immunosuppression. Major staphylococcal toxins are alpha toxin (a single subunit hemolysin) along with a group of bicomponent pore-forming toxins (BCPFT), namely Panton-Valentine leukocidin (PVL), gamma hemolysins (HlgCB and AB), LukAB and LukED. In our previous report, an attenuated mutant of LukS-PV (PVL- S subunit) named as “LukS-mut9” elicited high immunogenic response as well as provided a significant protection in a mouse sepsis model. Recent discovery of PVL receptors shows that mice lack receptors for this toxin, thus the reported protection of mice with the PVL vaccine may relate to cross protective responses against other homologous toxins. This manuscript addresses this issue by demonstrating that polyclonal antibody generated by LukS-mut9 can neutralize other canonical and non-canonical leukotoxin pairs. In this report, we also demonstrated that several potent toxins can be created by non-canonical pairing of subunits. Out of 5 pairs of canonical and 8 pairs of non-canonical toxins tested, anti-LukS-mut9 polyclonal antibodies neutralized all except for LukAB. We also studied the potential hemolytic activities of canonical and noncanonical pairs among biocomponent toxins and discovered that a novel non-canonical pair consisting of HlgA and LukD is a highly toxic combination. This pair can lyse RBC from different species including human blood far better than alpha hemolysin. Moreover, to follow-up our last report, we explored the correlation between the levels of pre-existing antibodies to new sets of leukotoxins subunits and clinical outcomes in adult patients with S

  19. 10′(Z),13′(E)-Heptadecadienylhydroquinone Inhibits Swarming and Virulence Factors and Increases Polymyxin B Susceptibility in Proteus mirabilis

    PubMed Central

    Wang, Won-Bo; Yuan, Yu-Han; Hsueh, Po-Ren; Liaw, Shwu-Jen

    2012-01-01

    In this study, we demonstrated that 10′(Z), 13′(E)-heptadecadienylhydroquinone (HQ17-2), isolated from the lacquer tree, could decrease swarming motility and hemolysin activity but increase polymyxin B (PB) susceptibilityof Proteus mirabilis which is intrinsically highly-resistant to PB. The increased PB susceptibility induced by HQ17-2 was also observed in clinical isolates and biofilm-grown cells. HQ17-2 could inhibit swarming in the wild-type and rppA mutant but not in the rcsB mutant, indicating that HQ17-2 inhibits swarming through the RcsB-dependent pathway, a two-component signaling pathway negatively regulating swarming and virulence factor expression. The inhibition of hemolysin activity by HQ17-2 is also mediated through the RcsB-dependent pathway, because HQ17-2 could not inhibit hemolysin activity in the rcsB mutant. Moreover, the finding that HQ17-2 inhibits the expression of flhDC gene in the wild-type and rcsB-complemented strain but not in the rcsB mutant supports the notion. By contrast, HQ17-2 could increase PB susceptibility in the wild-type and rcsB mutant but not in the rppA mutant, indicating that HQ17-2 increases PB susceptibility through the RppA-dependent pathway, a signaling pathway positively regulating PB resistance. In addition, HQ17-2 could inhibit the promoter activities of rppA and pmrI, a gene positively regulated by RppA and involved in PB resistance, in the wild-type but not in the rppA mutant. The inhibition of rppA and pmrI expression caused lipopolysaccharide purified from HQ17-2-treated cells to have higher affinity for PB. Altogether, this study uncovers new biological effects of HQ17-2 and provides evidence for the potential of HQ17-2 in clinical applications. PMID:23029100

  20. Semisynthetic Nanoreactor for Reversible Single-Molecule Covalent Chemistry.

    PubMed

    Lee, Joongoo; Boersma, Arnold J; Boudreau, Marc A; Cheley, Stephen; Daltrop, Oliver; Li, Jianwei; Tamagaki, Hiroko; Bayley, Hagan

    2016-09-27

    Protein engineering has been used to remodel pores for applications in biotechnology. For example, the heptameric α-hemolysin pore (αHL) has been engineered to form a nanoreactor to study covalent chemistry at the single-molecule level. Previous work has been confined largely to the chemistry of cysteine side chains or, in one instance, to an irreversible reaction of an unnatural amino acid side chain bearing a terminal alkyne. Here, we present four different αHL pores obtained by coupling either two or three fragments by native chemical ligation (NCL). The synthetic αHL monomers were folded and incorporated into heptameric pores. The functionality of the pores was validated by hemolysis assays and by single-channel current recording. By using NCL to introduce a ketone amino acid, the nanoreactor approach was extended to an investigation of reversible covalent chemistry on an unnatural side chain at the single-molecule level. PMID:27537396

  1. A minimal generic model of bacteria-induced intracellular Ca2+ oscillations in epithelial cells.

    PubMed

    Oxhamre, Camilla; Richter-Dahlfors, Agneta; Zhdanov, Vladimir P; Kasemo, Bengt

    2005-04-01

    The toxin alpha-hemolysin expressed by uropathogenic Escherichia coli bacteria was recently shown as the first pathophysiologically relevant protein to induce oscillations of the intracellular Ca(2+) concentration in target cells. Here, we propose a generic three-variable kinetic model describing the Ca(2+) oscillations induced in single rat renal epithelial cells by this toxin. Specifically, we take into account the interplay between 1), the cytosolic Ca(2+) concentration; 2), IP(3)-sensitive Ca(2+) channels located in the membrane separating the cytosol and endoplasmic reticulum; and 3), toxin-related activation of production of IP(3) by phospholipase C. With these ingredients, the predicted response of cells exposed to the toxin is in good agreement with the results of experiments.

  2. Semisynthetic Nanoreactor for Reversible Single-Molecule Covalent Chemistry

    PubMed Central

    2016-01-01

    Protein engineering has been used to remodel pores for applications in biotechnology. For example, the heptameric α-hemolysin pore (αHL) has been engineered to form a nanoreactor to study covalent chemistry at the single-molecule level. Previous work has been confined largely to the chemistry of cysteine side chains or, in one instance, to an irreversible reaction of an unnatural amino acid side chain bearing a terminal alkyne. Here, we present four different αHL pores obtained by coupling either two or three fragments by native chemical ligation (NCL). The synthetic αHL monomers were folded and incorporated into heptameric pores. The functionality of the pores was validated by hemolysis assays and by single-channel current recording. By using NCL to introduce a ketone amino acid, the nanoreactor approach was extended to an investigation of reversible covalent chemistry on an unnatural side chain at the single-molecule level. PMID:27537396

  3. Aeromonas proteolyrica bacteria in aerospace environments. [possible genetic alterations and effects on man

    NASA Technical Reports Server (NTRS)

    Foster, B. G.

    1974-01-01

    Preflight studies on Aeromonas proteolytica are reported to investigate the possibility of genetic alterations resulting in increased proteolysis in spacecraft environments. This organism may be present on human tissue and could pose medical problems if its endopeptidase and a hemolysin were to be produced in ususually high quantities or altered in such a way as to be more effective in their activities. Considered are: (1) Development of a nutrative holding medium for suspension of organisms; (2) the establishment of baseline information for the standardization of the assay for endopeptidase levels and hemolytic titers; (3) formulation of a method by which intracutaneous hemorrhage could be quantitated in guinea pig tissue; and (4) the responses of these organisms to parameters of spaceflight and experimentation.

  4. Identification of genetic bases of vibrio fluvialis species-specific biochemical pathways and potential virulence factors by comparative genomic analysis.

    PubMed

    Lu, Xin; Liang, Weili; Wang, Yunduan; Xu, Jialiang; Zhu, Jun; Kan, Biao

    2014-03-01

    Vibrio fluvialis is an important food-borne pathogen that causes diarrheal illness and sometimes extraintestinal infections in humans. In this study, we sequenced the genome of a clinical V. fluvialis strain and determined its phylogenetic relationships with other Vibrio species by comparative genomic analysis. We found that the closest relationship was between V. fluvialis and V. furnissii, followed by those with V. cholerae and V. mimicus. Moreover, based on genome comparisons and gene complementation experiments, we revealed genetic mechanisms of the biochemical tests that differentiate V. fluvialis from closely related species. Importantly, we identified a variety of genes encoding potential virulence factors, including multiple hemolysins, transcriptional regulators, and environmental survival and adaptation apparatuses, and the type VI secretion system, which is indicative of complex regulatory pathways modulating pathogenesis in this organism. The availability of V. fluvialis genome sequences may promote our understanding of pathogenic mechanisms for this emerging pathogen.

  5. Effect of confinement on DNA, solvent and counterion dynamics in a model biological nanopore

    NASA Astrophysics Data System (ADS)

    Markosyan, Suren; de Biase, Pablo M.; Czapla, Luke; Samoylova, Olga; Singh, Gurpreet; Cuervo, Javier; Tieleman, D. Peter; Noskov, Sergei Yu.

    2014-07-01

    The application of recent advances in nanopore technology to high-throughput DNA sequencing requires a more detailed understanding of solvent, ion and DNA interactions occurring within these pores. Here we present a combination of atomistic and coarse-grained modeling studies of the dynamics of short single-stranded DNA (ssDNA) homopolymers within the alpha-hemolysin pore, for the two single-stranded homopolymers poly(dA)40 and poly(dC)40. Analysis of atomistic simulations along with the per-residue decomposition of protein-DNA interactions in these simulations gives new insight into the very complex issues that have yet to be fully addressed with detailed MD simulations. We discuss a modification of the solvent properties and ion distribution around DNA within nanopore confinement and put it into the general framework of counterion condensation theory. There is a reasonable agreement in computed properties from our all-atom simulations and the resulting predictions from analytical theories with experimental data, and our equilibrium results here support the conclusions from our previous non-equilibrium Brownian dynamics studies with a recently developed BROMOC protocol that cations are the primary charge carriers through alpha-hemolysin nanopores under an applied voltage in the presence of ssDNA. Clustering analysis led to an identification of distinct conformational states of captured polymer and depth of the current blockade. Therefore, our data suggest that confined polymer may act as a flickering gate, thus contributing to excess noise phenomena. We also discuss the extent of water structuring due to nanopore confinement and the relationship between the conformational dynamics of a captured polymer and the distribution of blocked current.The application of recent advances in nanopore technology to high-throughput DNA sequencing requires a more detailed understanding of solvent, ion and DNA interactions occurring within these pores. Here we present a

  6. Elderly infection in the community due to ST5/SCCmecII methicillin-resistant Staphylococcus aureus (the New York/Japan clone) in Japan: Panton-Valentine leukocidin-negative necrotizing pneumonia.

    PubMed

    Khokhlova, Olga; Tomita, Yusuke; Hung, Wei-Chun; Takano, Tomomi; Iwao, Yasuhisa; Higuchi, Wataru; Nishiyama, Akihito; Reva, Ivan; Yamamoto, Tatsuo

    2015-06-01

    An 89-year-old man suffered from and died of necrotizing pneumonia with rapid progression and cavity formation due to methicillin-resistant Staphylococcus aureus (MRSA). He was at no risk for hospital-acquired MRSA infection. His MRSA exhibited genotype ST5/spa2(t002)/agr2/SCCmecII/coagulaseII and was negative for Panton-Valentine leukocidin, indicating the New York/Japan clone (the predominant epidemic hospital-acquired MRSA clone in Japan). However, this strain expressed the cytolytic peptide (phenol-soluble modulin or δ-hemolysin) genes at high level, similar to USA300 (the most common community-acquired MRSA in the United States), indicating a variant of the New York/Japan clone with an important feature of community-acquired MRSA.

  7. Serological response to the P fimbriae of uropathogenic Escherichia coli in pyelonephritis.

    PubMed Central

    de Ree, J M; van den Bosch, J F

    1987-01-01

    Uropathogenic Escherichia coli strains isolated from four patients with pyelonephritis were characterized by their O:K serotype, hemolysin production, mannose-resistant hemagglutination, and the serotype of the P fimbriae. These P fimbriae were serotyped with specific monoclonal antibodies. Serum samples from the patients were analyzed for the presence of specific antibodies to the P fimbriae. In all cases antifimbrial antibodies were found, strongly suggesting that these P fimbriae are expressed in vivo. However, the antibodies in the patient sera were not able to inhibit the mannose-resistant hemagglutination. This finding suggests that these antibodies react with the fimbrial components and not with the minor components which are responsible for adhesion. PMID:2887515

  8. Aeromonas caviae strain induces Th1 cytokine response in mouse intestinal tract

    SciTech Connect

    Hayes, S L; Lye, D J; McKinstry, Craig A.; Vesper, Sephen J.

    2010-01-01

    Aeromonas caviae has been associated with human gastrointestinal disease. Strains of this species typically lack virulence factors (VFs) such as enterotoxins and hemolysins that are produced by other human pathogens of the Aeromonas genus. Microarray profiling of murine small intestinal extracts, 24 hours after oral infection with an A. caviae strain, provides evidence of a Th1 type immune response. A large number of gamma-interferon (γ-IFN) induced genes are up-regulated as well as several tumor necrosis factor-alpha (TNF-α) transcripts. A. caviae has always been considered as opportunistic pathogen because it lacks obvious virulence factors. This current effort suggests that an A. caviae strain can colonize the murine intestinal tract and cause what has been described by others as a dysregulatory cytokine response. This response could explain why a number of diarrheal waterborne disease cases have been attributed to A. caviae even though it lacks obvious enteropathogenic properties.

  9. Aeromonas caviae strain induces Th1 cytokine response in mouse intestinal tract.

    PubMed

    Hayes, Samuel L; Lye, Dennis J; McKinstry, Craig A; Vesper, Stephen J

    2010-01-01

    Aeromonas caviae has been associated with human gastrointestinal disease. Strains of this species typically lack virulence factors (VFs) such as enterotoxins and hemolysins that are produced by other human pathogens of the Aeromonas genus. Microarray profiling of murine small intestinal extracts, 24 h after oral infection with an A. caviae strain, provides evidence of a Th1 type immune response. A large number of gamma-interferon (gamma-IFN) induced genes are up-regulated as well as several tumor necrosis factor-alpha (TNF-alpha) transcripts. Aeromonas caviae has always been considered an opportunistic pathogen because it lacks obvious virulence factors. This current effort suggests that an A. caviae strain can colonize the murine intestinal tract and cause what has been described by others as a dysregulatory cytokine response. This response could explain why a number of diarrheal waterborne disease cases have been attributed to A. caviae even though it lacks obvious enteropathogenic properties.

  10. Surface proteins of Bordetella pertussis: comparison of virulent and avirulent strains and effects of phenotypic modulation.

    PubMed Central

    Armstrong, S K; Parker, C D

    1986-01-01

    The surface proteins of several Bordetella strains and their modulated derivatives were examined by surface radioiodination, cell fractionation, and Western blotting. A surface protein with a high Mr, missing in a mutant lacking the filamentous hemagglutinin, was identified in virulent Bordetella pertussis and Bordetella parapertussis cells and was absent in avirulent B. pertussis strains. The electrophoretic profiles of lipopolysaccharide and the 40,000-Mr anion-selective porin were not determinants which correlated with phase variation or phenotypic modulation. At least three envelope proteins (91,000, 32,000, and 30,000 molecular weight) were found only in virulent B. pertussis strains and were absent or diminished in the avirulent phase and most phenotypically modulated strains. Two transposon-induced mutants unable to produce hemolysin, dermonecrotic toxin, pertussis toxin, and filamentous hemagglutinin also lacked these three envelope proteins, confirming that virulence-associated envelope proteins were genetically regulated with other virulence-associated traits. Images PMID:2876957

  11. Identification of a Hydrophobic Cleft in the LytTR Domain of AgrA as a Locus for Small Molecule Interactions that Inhibit DNA Binding

    PubMed Central

    Leonard, Paul G.; Bezar, Ian F.; Sidote, David J.; Stock, Ann M.

    2012-01-01

    The AgrA transcription factor regulates the quorum-sensing response in Staphylococcus aureus, controlling the production of hemolysins and other virulence factors. AgrA binds to DNA via its C-terminal LytTR domain, a domain not found in humans but common in many pathogenic bacteria, making it a potential target for antimicrobial development. We have determined the crystal structure of the apo AgrA LytTR domain and screened a library of 500 fragment compounds to find inhibitors of AgrA DNA-binding activity. Using NMR, the binding site for five compounds has been mapped to a common locus at the C-terminal end of the LytTR domain, a site known to be important for DNA-binding activity. Three of these compounds inhibit AgrA DNA binding. These results provide the first evidence that LytTR domains can be targeted by small organic compounds. PMID:23181972

  12. Proteopolymersomes: in vitro production of a membrane protein in polymersome membranes.

    PubMed

    Nallani, Madhavan; Andreasson-Ochsner, Mirjam; Tan, Cherng-Wen Darren; Sinner, Eva-Kathrin; Wisantoso, Yudi; Geifman-Shochat, Susana; Hunziker, Walter

    2011-12-01

    Polymersomes are stable self-assembled architectures which mimic cell membranes. For characterization, membrane proteins can be incorporated into such bio-mimetic membranes by reconstitution methods, leading to so-called proteopolymersomes. In this work, we demonstrate the direct incorporation of a membrane protein into polymersome membranes by a cell-free expression system. Firstly, we demonstrate pore formation in the preformed polymersome membrane using α-hemolysin. Secondly, we use claudin-2, a protein involved in cell-cell interactions, to demonstrate the in vitro expression of a membrane protein into these polymersomes. Surface plasmon resonance (Biacore) binding studies with the claudin-2 proteopolymersomes and claudin-2 specific antibodies are performed to show the presence of the in vitro expressed protein in polymersome membranes.

  13. Nanopore detection of copper ions using a polyhistidine probe.

    PubMed

    Wang, Guihua; Wang, Liang; Han, Yujing; Zhou, Shuo; Guan, Xiyun

    2014-03-15

    We report a stochastic nanopore sensing method for the detection of Cu(2+) ions. By employing a polyhistidine molecule as a chelating agent, and based on the different signatures of the events produced by the translocation of the chelating agent through an α-hemolysin pore in the absence and presence of target analytes, trace amounts of copper ions could be detected with a detection limit of 40 nM. Importantly, although Co(2+), Ni(2+), and Zn(2+) also interacts with the polyhistidine molecule, since the event residence times and/or blockage amplitudes for these metal chelates are significantly different from those of copper chelates, these metal ions do not interfere with Cu(2+) detection. This chelating reaction approach should find useful application in the development of nanopore sensors for other metal ions.

  14. Staphylococcus schleiferi subsp. coagulans subsp. nov., isolated from the external auditory meatus of dogs with external ear otitis.

    PubMed

    Igimi, S; Takahashi, E; Mitsuoka, T

    1990-10-01

    A new subspecies, Staphylococcus schleiferi subsp. coagulans, was isolated from the external auditory meatus of dogs suffering from external ear otitis and is described on the basis of studies of 21 strains. Phenotypic studies showed that these strains are more closely related to Staphylococcus intermedius than to other staphylococci, but DNA hybridization studies indicated that they are closely related to Staphylococcus schleiferi N850274T. On the basis of biochemical distinctiveness (positive test tube coagulase test and different carbohydrate reactions) and the etiological importance (frequent isolation from otitis specimens from dogs) of these strains, we propose to classify them as a subspecies of S. schleiferi. The strains of this new subspecies are coagulase tube test, beta-hemolysin, and heat-stable nuclease positive but clumping factor negative. A simple scheme for the differentiation of S. schleiferi subsp. coagulans from the other coagulase-positive staphylococci is presented. The type strain is GA211 (= JCM 7470).

  15. Characteristics of Staphylococcus intermedius isolates from diseased and healthy dogs.

    PubMed

    Sasaki, Asako; Shimizu, Akira; Kawano, Junichi; Wakita, Yoshihisa; Hayashi, Toshikatsu; Ootsuki, Shigenobu

    2005-01-01

    Staphylococcus intermedius isolates from diseased and healthy dogs were examined for production of extracellular enzymes and toxins, and phage patterns. There were no significant differences between the two groups of isolates in the production rates of DNase, protease, lipase, gelatinase, hyaluronidase, hemolysins, protein A, and TSST-1, or in phage patterns. But the production rate of enterotoxins in isolates from diseased dogs was significantly higher than that in isolates from healthy dogs. PFGE analysis was performed with isolates from different body sites in individual dogs. In 3 of 6 healthy dogs, identical PFGE patterns were seen in isolates from the nares, external auditory meatus or skin. The remaining 3 dogs yielded isolates of different patterns. In 4 of 6 diseased dogs, identical patterns were seen in isolates from lesions as well as from the other normal sites.

  16. Staphylococcus aureus toxins.

    PubMed

    Otto, Michael

    2014-02-01

    Staphylococcus aureus is a dangerous pathogen that causes a variety of severe diseases. The virulence of S. aureus is defined by a large repertoire of virulence factors, among which secreted toxins play a preeminent role. Many S. aureus toxins damage biological membranes, leading to cell death. In particular, S. aureus produces potent hemolysins and leukotoxins. Among the latter, some were recently identified to lyse neutrophils after ingestion, representing an especially powerful weapon against bacterial elimination by innate host defense. Furthermore, S. aureus secretes many factors that inhibit the complement cascade or prevent recognition by host defenses. Several further toxins add to this multi-faceted program of S. aureus to evade elimination in the host. This review will give an overview over S. aureus toxins focusing on recent advances in our understanding of how leukotoxins work in receptor-mediated or receptor-independent fashions.

  17. An immunostimulatory polysaccharide (SCP-IIa) from the fruit of Schisandra chinensis (Turcz.) Baill.

    PubMed

    Chen, Yong; Tang, Jinbao; Wang, Xiaoke; Sun, Fengxiang; Liang, Shujuan

    2012-04-01

    A water-soluble polysaccharide named SCP-IIa was isolated from the water extract of the fruit of Schisandra chinensis (Turcz.) Baill by means of ethanol precipitation, deproteination, anion-exchange and gel-permeation chromatography. The molecular weight of SCP-IIa was ascertained via HPLC, and immuno-modulating effect was evaluated using the immunosuppressed model induced by cyclophosphamide. SCP-IIa was a homogeneous form of polysaccharide, with an average molecular weight of approximately 7700 Da. The detected parameters showed that SCP-IIa increased the thymus and spleen indices, as well as the pinocytic activity of the peritoneal macrophages in immunosuppressed mice. The splenocyte proliferation assay showed that SCP-IIa, in combination with Con A or LPS, positively affected splenocyte proliferation. Moreover, the polysaccharide promoted hemolysin formation. The results suggested that SCP-IIa was involved in immunomodulatory effects leading to the exploration for SCP-IIa as a potential immunostimulant.

  18. Identification of tdh-positive Vibrio parahaemolyticus from an outbreak associated with raw oyster consumption in Spain.

    PubMed

    Lozano-León, Antonio; Torres, Julio; Osorio, Carlos R; Martínez-Urtaza, Jaime

    2003-09-26

    Between August and September 1999, a total of 64 cases of illness were identified in three episodes of acute gastroenteritis associated with the consumption of live oysters from a typical outdoor street market in Galicia (northwest Spain). Nine case patients were hospitalized and analysis of their stool samples revealed the presence of Vibrio parahaemolyticus. The strains isolated from two stool samples were studied for antibiotic susceptibility, biochemical characteristics and presence of virulence factors. Both isolates were Kanagawa phenomenon positive and produced thermostable direct hemolysin, which is related to pathogenicity in humans. These results show the presence of pathogenic V. parahaemolyticus in mollusks harvested in Europe and reveal the risk of illness associated with their consumption, suggesting the revision of V. parahaemolyticus risk assessment associated with consumption of raw live shellfish.

  19. Sizing the Bacillus anthracis PA63 Channel with Nonelectrolyte Poly(Ethylene Glycols)

    PubMed Central

    Nablo, Brian J.; Halverson, Kelly M.; Robertson, Joseph W. F.; Nguyen, Tam L.; Panchal, Rekha G.; Gussio, Rick; Bavari, Sina; Krasilnikov, Oleg V.; Kasianowicz, John J.

    2008-01-01

    Nonelectrolyte polymers of poly(ethylene glycol) (PEG) were used to estimate the diameter of the ion channel formed by the Bacillus anthracis protective antigen 63 (PA63). Based on the ability of different molecular weight PEGs to partition into the pore and reduce channel conductance, the pore appears to be narrower than the one formed by Staphylococcus aureus α-hemolysin. Numerical integration of the PEG sample mass spectra and the channel conductance data were used to refine the estimate of the pore's PEG molecular mass cutoff (∼1400 g/mol). The results suggest that the limiting diameter of the PA63 pore is <2 nm, which is consistent with an all-atom model of the PA63 channel and previous experiments using large ions. PMID:18645196

  20. An Autonomously Reciprocating Transmembrane Nanoactuator.

    PubMed

    Watson, Matthew A; Cockroft, Scott L

    2016-01-22

    Biological molecular machines operate far from equilibrium by coupling chemical potential to repeated cycles of dissipative nanomechanical motion. This principle has been exploited in supramolecular systems that exhibit true machine behavior in solution and on surfaces. However, designed membrane-spanning assemblies developed to date have been limited to simple switches or stochastic shuttles, and true machine behavior has remained elusive. Herein, we present a transmembrane nanoactuator that turns over chemical fuel to drive autonomous reciprocating (back-and-forth) nanomechanical motion. Ratcheted reciprocating motion of a DNA/PEG copolymer threaded through a single α-hemolysin pore was induced by a combination of DNA strand displacement processes and enzyme-catalyzed reactions. Ion-current recordings revealed saw-tooth patterns, indicating that the assemblies operated in autonomous, asymmetric cycles of conformational change at rates of up to one cycle per minute. PMID:26661295

  1. Placement of oppositely charged aminoacids at a polypeptide termini determines the voltage-controlled braking of polymer transport through nanometer-scale pores

    PubMed Central

    Asandei, Alina; Chinappi, Mauro; Lee, Jong-kook; Ho Seo, Chang; Mereuta, Loredana; Park, Yoonkyung; Luchian, Tudor

    2015-01-01

    Protein and solid-state nanometer-scale pores are being developed for the detection, analysis, and manipulation of single molecules. In the simplest embodiment, the entry of a molecule into a nanopore causes a reduction in the latter’s ionic conductance. The ionic current blockade depth and residence time have been shown to provide detailed information on the size, adsorbed charge, and other properties of molecules. Here we describe the use of the nanopore formed by Staphylococcus aureus α-hemolysin and polypeptides with oppositely charged segments at the N- and C-termini to increase both the polypeptide capture rate and mean residence time of them in the pore, regardless of the polarity of the applied electrostatic potential. The technique provides the means to improve the signal to noise of single molecule nanopore-based measurements. PMID:26029865

  2. Microfluidic trapping of giant unilamellar vesicles to study transport through a membrane pore

    PubMed Central

    Robinson, T.; Kuhn, P.; Eyer, K.; Dittrich, P. S.

    2013-01-01

    We present a microfluidic platform able to trap single GUVs in parallel. GUVs are used as model membranes across many fields of biophysics including lipid rafts, membrane fusion, and nanotubes. While their creation is relatively facile, handling and addressing single vesicles remains challenging. The PDMS microchip used herein contains 60 chambers, each with posts able to passively capture single GUVs without compromising their integrity. The design allows for circular valves to be lowered from the channel ceiling to isolate the vesicles from rest of the channel network. GUVs containing calcein were trapped and by rapidly opening the valves, the membrane pore protein α-hemolysin (αHL) was introduced to the membrane. Confocal microscopy revealed the kinetics of the small molecule efflux for different protein concentrations. This microfluidic approach greatly improves the number of experiments possible and can be applied to a wide range of biophysical applications. PMID:24404039

  3. Formation of droplet interface bilayers in a Teflon tube

    PubMed Central

    Walsh, Edmond; Feuerborn, Alexander; Cook, Peter R.

    2016-01-01

    Droplet-interface bilayers (DIBs) have applications in disciplines ranging from biology to computing. We present a method for forming them manually using a Teflon tube attached to a syringe pump; this method is simple enough it should be accessible to those without expertise in microfluidics. It exploits the properties of interfaces between three immiscible liquids, and uses fluid flow through the tube to pack together drops coated with lipid monolayers to create bilayers at points of contact. It is used to create functional nanopores in DIBs composed of phosphocholine using the protein α-hemolysin (αHL), to demonstrate osmotically-driven mass transfer of fluid across surfactant-based DIBs, and to create arrays of DIBs. The approach is scalable, and thousands of DIBs can be prepared using a robot in one hour; therefore, it is feasible to use it for high throughput applications. PMID:27681313

  4. [Effects of bee pollen on lipid peroxides and immune response in aging and malnourished mice].

    PubMed

    Qian, B; Zang, X; Liu, X

    1990-05-01

    The results showed that the level of hemolysin (HC), the numbers of plaque forming cells (PFC) and specific rosette forming cells (SRFC) in primary response to sheep red blood cells (SRBC) were markedly lowered and the lipoperoxide level in brain, liver and serum was increased in aging (over 18 months) and malnourished mice fed with ground corn in comparison to normal controls, while HC and the numbers of PFC and SRFC were significantly increased and the lipoperoxide level was markedly decreased after treatment with bee pollen 10 g/kg/d orally for 3 months and with 20% bee pollen-containing ground corn for 3 weeks respectively. The reduction of total protein and albumin contents of serum, DNA, RNA and protein contents of spleen and thymus in mice fed with ground corn can be prevented by adding 20% bee pollen in ground corn diet.

  5. Purification, characterization and immunostimulatory activity of polysaccharide from Cipangopaludina chinensis.

    PubMed

    Xiong, Qingping; Jiao, Yunpeng; Zhao, Xirong; Chen, Xiaoming; Zhang, Qianghua; Jiang, Changxing

    2013-10-15

    In this study, we investigated the purification, preliminary characterization and immunostimulatory activity in vivo of polysaccharide from Cipangopaludina chinensis (CCPS). Firstly, crude CCPS was prepared by hot water extraction. And the crude CCPS was sequentially purified by chromatography of DEAE-52 and Sephadex G-100, resulting in two purified fractions of CCPS-1 and CCPS-2. We found the two fractions were homogeneous heteropolysaccharides mainly composed of rhamnose and glucose with the average molecular weight of 226 and 235 kDa, respectively. CCPS-2 was quite different from CCPS-1. It had much higher content of uronic acid and sulfuric radical. For immunostimulatory activity in vivo, crude CCPS could significantly increase the thymus and spleen indices, enhance the macrophage function, and increase the level of serum hemolysin in cyclophosphamide-treated mice, suggesting CCPS had a potent immunostimulatory activity and could be explored as a potential natural immunomodulatory agent.

  6. Beta-hemolytic activity of Trichomonas vaginalis correlates with virulence.

    PubMed Central

    Krieger, J N; Poisson, M A; Rein, M F

    1983-01-01

    The reasons that some women develop symptomatic trichomonal vaginitis, whereas many other infected women remain asymptomatic, are unclear, but it has been suggested that Trichomonas vaginalis strains vary in their intrinsic virulence. We describe beta-hemolytic activity in T. vaginalis which correlates with virulence in patients as well as in an animal model and in tissue culture. Fresh T. vaginalis isolates from four women with severe, symptomatic trichomoniasis had high-level (86.3 +/- 6.6%) hemolytic activity, whereas isolates from three completely asymptomatic women had low-level (45.3 +/- 8.4%) hemolytic activity (P less than 0.001). Hemolytic activity also correlated with the production of subcutaneous abscesses in mice (r = 0.74) and with destruction of CHO cell monolayers (r = 0.94). All of the 20 clinical isolates of T. vaginalis tested possessed hemolytic activity. The beta-hemolysin may be a virulence factor for T. vaginalis. Images PMID:6604026

  7. Immunomodulatory and hypoallergenic properties of milk protein hydrolysates in ICR mice.

    PubMed

    Pan, D D; Wu, Z; Liu, J; Cao, X Y; Zeng, X Q

    2013-08-01

    Approximately 2.5% of young children are allergic to cow milk. In this study, milk protein hydrolysates made from full-cream milk via enzymatic hydrolysis played a positive role in regulating the immune system of ICR mice. Milk protein hydrolysates enhanced immunity in mice by stimulating host immunity, probably by increasing the weight of certain immune system organs, improving the level of hemolysin in serum, and enhancing the phagocytosis of macrophages. Milk protein hydrolysates have the capability to reduce type I hypersensitivity by decreasing IgE levels, IL-4 in serum, and the release of histamine and bicarbonate in peritoneal mast cells, as well as enhancing transforming growth factor-β levels in the serum of ovalbumin-sensitized mice.

  8. Discrimination of Single Base Pair Differences Among Individual DNA Molecules Using a Nanopore

    NASA Technical Reports Server (NTRS)

    Vercoutere, Wenonah; DeGuzman, Veronica

    2003-01-01

    The protein toxin alpha-hemolysin form nanometer scale channels across lipid membranes. Our lab uses a single channel in an artificial lipid bilayer in a patch clamp device to capture and examine individual DNA molecules. This nanopore detector used with a support vector machine (SVM) can analyze DNA hairpin molecules on the millisecond time scale. We distinguish duplex stem length, base pair mismatches, loop length, and single base pair differences. The residual current fluxes also reveal structural molecular dynamics elements. DNA end-fraying (terminal base pair dissociation) can be observed as near full blockades, or spikes, in current. This technique can be used to investigate other biological processes dependent on DNA end-fraying, such as the processing of HIV DNA by HIV integrase.

  9. Nanoscale Bio-engineering Solutions for Space Exploration: The Nanopore Sequencer

    NASA Technical Reports Server (NTRS)

    Stolc, Viktor; Cozmuta, Ioana

    2004-01-01

    Characterization of biological systems at the molecular level and extraction of essential information for nano-engineering design to guide the nano-fabrication of solid-state sensors and molecular identification devices is a computational challenge. The alpha hemolysin protein ion channel is used as a model system for structural analysis of nucleic acids like DNA. Applied voltage draws a DNA strand and surrounding ionic solution through the biological nanopore. The subunits in the DNA strand block ion flow by differing amounts. Atomistic scale simulations are employed using NASA supercomputers to study DNA translocation, with the aim to enhance single DNA subunit identification. Compared to protein channels, solid-state nanopores offer a better temporal control of the translocation of DNA and the possibility to easily tune its chemistry to increase the signal resolution. Potential applications for NASA missions, besides real-time genome sequencing include astronaut health, life detection and decoding of various genomes.

  10. Characterisation of a synergohymenotropic toxin produced by Staphylococcus intermedius.

    PubMed

    Prevost, G; Bouakham, T; Piemont, Y; Monteil, H

    1995-12-01

    Staphylococcal synergohymenotropic (SHT) toxins damage membranes of host defence cells and erythrocytes by the synergy of two secreted and non-associated proteins: class S and class F components. Whereas Panton-Valentine leucocidin (PVL), gamma-hemolysin and Luk-M from Staphylococcus aureus are members of this toxin family, a new bi-component toxin (LukS-I + LukF-I) from Staphylococcus intermedius, a pathogen for small animals, was characterised and sequenced. It is encoded as a luk-I operon by two cotranscribed genes, like PVL, LukS-I + LukF-I shares a strong leukotoxicity of various PMNs, but only slight haemolytic properties on rabbit erythrocytes. When intradermally injected into rabbit skin, a 100 ng dose caused acute inflammatory reaction leading to tissue necrosis. The new SHT seemed to be largely distributed among various Staphylococcus intermedius strains.

  11. A droplet microfluidic system for sequential generation of lipid bilayers and transmembrane electrical recordings.

    PubMed

    Czekalska, Magdalena A; Kaminski, Tomasz S; Jakiela, Slawomir; Tanuj Sapra, K; Bayley, Hagan; Garstecki, Piotr

    2015-01-21

    This paper demonstrates a microfluidic system that automates i) formation of a lipid bilayer at the interface between a pair of nanoliter-sized aqueous droplets in oil, ii) exchange of one droplet of the pair to form a new bilayer, and iii) current measurements on single proteins. A new microfluidic architecture is introduced - a set of traps designed to localize the droplets with respect to each other and with respect to the recording electrodes. The system allows for automated execution of experimental protocols by active control of the flow on chip with the use of simple external valves. Formation of stable artificial lipid bilayers, incorporation of α-hemolysin into the bilayers and electrical measurements of ionic transport through the protein pore are demonstrated. PMID:25412368

  12. Candida bracarensis: Evaluation of Virulence Factors and its Tolerance to Amphotericin B and Fluconazole.

    PubMed

    Moreira, André; Silva, Sónia; Botelho, Cláudia; Sampaio, Paula; Pais, Célia; Henriques, Mariana

    2015-12-01

    Candida bracarensis is an uncommon Candida species found during an epidemiological study of candidiasis performed in Braga, Portugal. Initially, it was identified as C. glabrata, but recently detailed analyses pointed out their differences. So, little information is still available about C. bracarensis virulence factors and antifungal susceptibilities. Therefore, the main goal of this work is to evaluate the ability of C. bracarensis to form biofilms, to produce hydrolytic enzymes (proteases, phospholipases and hemolysins), as well as its susceptibility to amphotericin B and fluconazole. It was shown, for the first time, that all C. bracarensis strains were able to form biofilms and display proteinase and hemolytic activities. Moreover, although planktonic cells presented antifungal susceptibility, amphotericin B and fluconazole were unable to inhibit biofilm formation and eradicate pre-formed biofilms. Due to the propensity of C. bracarensis to display antifungal resistance and virulence attributes, the control of these emerging pathogens is recommended. PMID:26179982

  13. A heating-superfusion platform technology for the investigation of protein function in single cells.

    PubMed

    Xu, Shijun; Ainla, Alar; Jardemark, Kent; Jesorka, Aldo; Jeffries, Gavin D M

    2015-01-01

    Here, we report on a novel approach for the study of single-cell intracellular enzyme activity at various temperatures, utilizing a localized laser heating probe in combination with a freely positionable microfluidic perfusion device. Through directed exposure of individual cells to the pore-forming agent α-hemolysin, we have controlled the membrane permeability, enabling targeted delivery of the substrate. Mildly permeabilized cells were exposed to fluorogenic substrates to monitor the activity of intracellular enzymes, while adjusting the local temperature surrounding the target cells, using an infrared laser heating system. We generated quantitative estimates for the intracellular alkaline phosphatase activity at five different temperatures in different cell lines, constructing temperature-response curves of enzymatic activity at the single-cell level. Enzymatic activity was determined rapidly after cell permeation, generating five-point temperature-response curves within just 200 s.

  14. Clonal distribution of Streptococcus suis isolated from diseased pigs in the central region of Chile

    PubMed Central

    Morales, Bárbara; Ruiz, Álvaro; Lacouture, Sonia; Gottschalk, Marcelo

    2015-01-01

    The characteristics of 29 Chilean field strains of Streptococcus suis recovered between 2007 and 2011 from pigs with clinical signs at different farms were studied. Serotyping with use of the coagglutination test revealed that all but 1 strain belonged to serotype 6; the remaining strain was serotype 22. All the serotype-6 strains were suilysin (hemolysin)-negative; in addition, they were found to be genotypically homogeneous by enterobacterial repetitive intergenic consensus sequence-based polymerase chain reaction (ERIC-PCR) and sensitive to ampicillin, ceftiofur, penicillin, and trimethoprim/sulfamethoxazole. The results indicate that, in contrast to what is generally observed in other countries, a single clone of S. suis was isolated from diseased pigs in the central region of Chile. PMID:26424917

  15. Production of virulence factors in Candida strains isolated from patients with denture stomatitis and control individuals.

    PubMed

    Pereira, Cristiane Aparecida; Domingues, Nádia; Araújo, Maria Izabel Daniel Santos Alves; Junqueira, Juliana Campos; Back-Brito, Graziella Nuernberg; Jorge, Antonio Olavo Cardoso

    2016-05-01

    The aim of this study was to evaluate the production of virulence factors in Candida isolates from the oral cavities of 50 patients with different degrees of denture stomatitis (DS, type I, II and III) and 50 individuals without signs of DS. We evaluated the enzymatic and hemolytic activities, the biofilm formation, and the cell surface hydrophobicity (CSH) in all isolates. Germ tube (GT) production was also evaluated in Candida albicans and Candida dubliniensis isolates. In C. albicans and C. dubliniensis the secretion of hemolysin and GT production was significantly different between isolates from patients with DS and individuals without DS. No significant difference was observed in the production of virulence factors by Candida glabrata isolates. Candida isolates expressed a wide range of virulence factors. However, in the majority of isolates from the type III lesions, the production of the virulence factors was higher than for the other groups.

  16. Candida bracarensis: Evaluation of Virulence Factors and its Tolerance to Amphotericin B and Fluconazole.

    PubMed

    Moreira, André; Silva, Sónia; Botelho, Cláudia; Sampaio, Paula; Pais, Célia; Henriques, Mariana

    2015-12-01

    Candida bracarensis is an uncommon Candida species found during an epidemiological study of candidiasis performed in Braga, Portugal. Initially, it was identified as C. glabrata, but recently detailed analyses pointed out their differences. So, little information is still available about C. bracarensis virulence factors and antifungal susceptibilities. Therefore, the main goal of this work is to evaluate the ability of C. bracarensis to form biofilms, to produce hydrolytic enzymes (proteases, phospholipases and hemolysins), as well as its susceptibility to amphotericin B and fluconazole. It was shown, for the first time, that all C. bracarensis strains were able to form biofilms and display proteinase and hemolytic activities. Moreover, although planktonic cells presented antifungal susceptibility, amphotericin B and fluconazole were unable to inhibit biofilm formation and eradicate pre-formed biofilms. Due to the propensity of C. bracarensis to display antifungal resistance and virulence attributes, the control of these emerging pathogens is recommended.

  17. Gene of LukF-PV-like component of Panton-Valentine leukocidin in Staphylococcus aureus P83 is linked with lukM.

    PubMed

    Kaneko, J; Muramoto, K; Kamio, Y

    1997-03-01

    Staphylococcus aureus P83 (ATCC 31890) produces five components, I to V for synergistic toxins, leukocidin and gamma-hemolysin [Sudo et al., Biosci. Biotech. Biochem., 56, 1786-1789 (1995)]. We report here the identification of component II, which is designated LukF-PV(P83) and its gene (lukF-PV(P83)). The lukF-PV(P83) gene was found to be one base downstream of the stop codon of the lukM gene. The deduced amino acid sequence of LukF-PV(P83) showed 78.4% identity with that of LukF-PV. The lukM and lukF-PV(P83) genes were encoded as one operon like that of Panton-Valentine leukocidin.

  18. Interaction of DNA and Proteins with Single Nanopores

    NASA Astrophysics Data System (ADS)

    Kasianowicz, J. J.

    2006-03-01

    The bacterial toxins Staphylococcus aureus alpha-hemolysin and Bacillus anthracis protective antigen kill cells in part by forming ion channels in target membranes. We are using electrophysiology, molecular biology/protein biochemistry and computer modeling to study how biopolymers (e.g., single-stranded DNA and proteins) bind to and transport through these nanometer-scale pores. The results provide insight into the mechanism by which these toxins work and are the basis for several potential nanobiotechnology applications including ultra-rapid DNA sequencing, the sensitive and selective detection of a wide range of analytes and high throughput screening of therapeutic agents against several anthrax toxins. In collaboration with V.M. Stanford, M. Misakian, B. Nablo, S.E. Henrickson, NIST, EEEL, Gaithersburg, MD; T. Nguyen, R. Gussio, NCI, Ft. Detrick, MD; and K.M. Halverson, S. Bavari, R.G. Panchal, USAMRIID, Ft. Detrick, MD.

  19. Formation of droplet interface bilayers in a Teflon tube

    NASA Astrophysics Data System (ADS)

    Walsh, Edmond; Feuerborn, Alexander; Cook, Peter R.

    2016-09-01

    Droplet-interface bilayers (DIBs) have applications in disciplines ranging from biology to computing. We present a method for forming them manually using a Teflon tube attached to a syringe pump; this method is simple enough it should be accessible to those without expertise in microfluidics. It exploits the properties of interfaces between three immiscible liquids, and uses fluid flow through the tube to pack together drops coated with lipid monolayers to create bilayers at points of contact. It is used to create functional nanopores in DIBs composed of phosphocholine using the protein α-hemolysin (αHL), to demonstrate osmotically-driven mass transfer of fluid across surfactant-based DIBs, and to create arrays of DIBs. The approach is scalable, and thousands of DIBs can be prepared using a robot in one hour; therefore, it is feasible to use it for high throughput applications.

  20. Release of outer membrane vesicles from Bordetella pertussis.

    PubMed

    Hozbor, D; Rodriguez, M E; Fernández, J; Lagares, A; Guiso, N; Yantorno, O

    1999-05-01

    The aim of the study reported here was to investigate the production of Bordetella pertussis outer membrane vesicles (OMVs). Numerous vesicles released from cells grown in Stainer-Scholte liquid medium were observed. The formation of similar vesicle-like structures could also be artificially induced by sonication of concentrated bacterial suspensions. Immunoblot analysis showed that OMVs contain adenylate cyclase-hemolysin (AC-Hly), among other polypeptides, as well as the lipopolysaccharide (LPS). Experiments carried out employing purified AC-Hly and OMVs isolated from B. pertussis AC-Hly- showed that AC-Hly is an integral component of the vesicles. OMVs reported here contain several protective immunogens and might be considered a possible basic material for the development of acellular pertussis vaccines.

  1. The SaeRS Two-Component System of Staphylococcus aureus

    PubMed Central

    Liu, Qian; Yeo, Won-Sik; Bae, Taeok

    2016-01-01

    In the Gram-positive pathogenic bacterium Staphylococcus aureus, the SaeRS two-component system (TCS) plays a major role in controlling the production of over 20 virulence factors including hemolysins, leukocidins, superantigens, surface proteins, and proteases. The SaeRS TCS is composed of the sensor histidine kinase SaeS, response regulator SaeR, and two auxiliary proteins SaeP and SaeQ. Since its discovery in 1994, the sae locus has been studied extensively, and its contributions to staphylococcal virulence and pathogenesis have been well documented and understood; however, the molecular mechanism by which the SaeRS TCS receives and processes cognate signals is not. In this article, therefore, we review the literature focusing on the signaling mechanism and its interaction with other global regulators. PMID:27706107

  2. Simple and Stable Lipid Bilayer Formation: A Droplets Contacting Method using Parylene Micro-pores for Multiple Ion Channel Recordings

    NASA Astrophysics Data System (ADS)

    Tsuji, Yutaro; Kawano, Ryuji; Osaki, Toshihisa; Sasaki, Hirotaka; Miki, Norihisa; Takeuchi, Shoji

    This paper describes multiple ion-channel recordings through membrane proteins reconstituted in bilayer lipid membranes (BLMs) array. The BLMs array can be prepared by “Droplets Contacting Method” which forms BLMs at the interface of two lipid monolayers. Since this method does not require skilled techniques, it is highly reproducible and can be applied to automated system. We used a double well chip (DWC) for the droplets contacting method. We attempted to confine the BLMs forming areas with parylene micro-pore (parylene double well chip, PDWC) to augment the mechanical stability of BLMs. Subsequently, we arrayed the PDWC with electrodes for multiple recordings of channel proteins. We successfully demonstrated 14 channels simultaneous ion channel recordings through α-hemolysin.

  3. Potential virulence factors of Proteus bacilli.

    PubMed Central

    Rózalski, A; Sidorczyk, Z; Kotełko, K

    1997-01-01

    The object of this review is the genus Proteus, which contains bacteria considered now to belong to the opportunistic pathogens. Widely distributed in nature (in soil, water, and sewage), Proteus species play a significant ecological role. When present in the niches of higher macroorganisms, these species are able to evoke pathological events in different regions of the human body. The invaders (Proteus mirabilis, P. vulgaris, and P. penneri) have numerous factors including fimbriae, flagella, outer membrane proteins, lipopolysaccharide, capsule antigen, urease, immunoglobulin A proteases, hemolysins, amino acid deaminases, and, finally, the most characteristic attribute of Proteus, swarming growth, enabling them to colonize and survive in higher organisms. All these features and factors are described and commented on in detail. The questions important for future investigation of these facultatively pathogenic microorganisms are also discussed. PMID:9106365

  4. Responses of fish chromatophore-based cytosensor to a broad range of biological agents.

    PubMed

    Dierksen, Karen P; Mojovic, Ljiljana; Caldwell, Bruce A; Preston, R Ryan; Upson, Rosalyn; Lawrence, Jeannine; McFadden, Philip N; Trempy, Janine E

    2004-01-01

    A cytosensor based on living chromatophores from Betta splendens Siamese fighting fish was used to test several classes of biologically active agents. Tested agents include neurotransmitters, adenyl cyclase activators, cytoskeleton effectors, cell membrane effectors and protein synthesis inhibitors. Characteristic cell responses were analyzed, and potential cytosensor applications were considered. Streptococcus pyogenes toxins streptolysin S and streptolysin O, Clostridium tetani tetanolysin, Staphylococcus aureus alpha-toxin and Vibrio parahemolyticus hemolysin, all bacterial toxins that act on cell membranes, elicited a strong response from chromatophores. A comparison of purified toxin to actual bacterial culture from Vibrio parahemolyticus demonstrated a nearly identical chromatophore cell response pattern. This suggests that the cytosensor response is reflective of bacterial toxin production. PMID:15478182

  5. An engineered dimeric protein pore that spans adjacent lipid bilayers

    PubMed Central

    Mantri, Shiksha; Sapra, K. Tanuj; Cheley, Stephen; Sharp, Thomas H.; Bayley, Hagan

    2013-01-01

    The bottom-up construction of artificial tissues is an underexplored area of synthetic biology. An important challenge is communication between constituent compartments of the engineered tissue and between the engineered tissue and additional compartments, including extracellular fluids, further engineered tissue and living cells. Here we present a dimeric transmembrane pore that can span two adjacent lipid bilayers and thereby allow aqueous compartments to communicate. Two heptameric staphylococcal α-hemolysin (αHL) pores were covalently linked in an aligned cap-to-cap orientation. The structure of the dimer, (α7)2, was confirmed by biochemical analysis, transmission electron microscopy (TEM) and single-channel electrical recording. We show that one of two β barrels of (α7)2 can insert into the lipid bilayer of a small unilamellar vesicle, while the other spans a planar lipid bilayer. (α7)2 pores spanning two bilayers were also observed by TEM. PMID:23591892

  6. Hemolytic activity of plasma and urine from rabbits experimentally infected with Legionella pneumophila.

    PubMed

    Baine, W B; Rasheed, J K; Maca, H W; Kaufmann, A F

    1979-01-01

    Rabbits were infected with Legionella pneumophila by intravenous administration of allantoic fluid from eggs infected with this organism. Heated plasma from animals with severe illness caused by L. pneumophila lysed erythrocytes from guinea pigs in a radial hemolysis assay. Plasma from control rabbits did not lyse guinea pig erythrocytes in parallel assays. Urine from two of the infected animals also showed hemolytic activity. Attempts to induce illness in rabbits by intranasal administration of L. pneumohpila were less successful. Allantoic fluid from embrynated hen eggs developed hemolytic activity when maintained eithr in vitro at room temperature or in eggs whose embryos were killed by refrigeration. Hemolytic activity in filtrates of allantoic fluid from eggs infected with L. pneumophila, as previously reported, may not be due to the presence of bacterial hemolysins in the fluid. PMID:399383

  7. Lifesaving liver transplantation for multi-organ failure caused by Bacillus cereus food poisoning.

    PubMed

    Tschiedel, Eva; Rath, Peter-Michael; Steinmann, Jörg; Becker, Heinz; Dietrich, Rudolf; Paul, Andreas; Felderhoff-Müser, Ursula; Dohna-Schwake, Christian

    2015-02-01

    Bacillus cereus is a spore-forming, gram-positive bacterium that causes food poisoning presenting with either emesis or diarrhea. Diarrhea is caused by proteinaceous enterotoxin complexes, mainly hemolysin BL, non-hemolytic enterotoxin (NHE), and cytotoxin K. In contrast, emesis is caused by the ingestion of the depsipeptide toxin cereulide, which is produced in B. cereus contaminated food, particularly in pasta or rice. In general, the illness is mild and self-limiting. However, due to cereulide intoxication, nine severe cases with rhabdomyolysis and/or liver failure, five of them lethal, are reported in literature. Here we report the first case of life-threatening liver failure and severe rhabdomyolysis in this context that could not be survived without emergency hepatectomy and consecutive liver transplantation.

  8. Development of Two Animal Models To Study the Function of Vibrio parahaemolyticus Type III Secretion Systems▿

    PubMed Central

    Piñeyro, Pablo; Zhou, Xiaohui; Orfe, Lisa H.; Friel, Patrick J.; Lahmers, Kevin; Call, Douglas R.

    2010-01-01

    Vibrio parahaemolyticus is an emerging food- and waterborne pathogen that encodes two type III secretion systems (T3SSs). Previous studies have linked type III secretion system 1 (T3SS1) to cytotoxicity and T3SS2 to intestinal fluid accumulation, but animal challenge models needed to study these phenomena are limited. In this study we evaluated the roles of the T3SSs during infection using two novel animal models: a model in which piglets were inoculated orogastrically and a model in which mice were inoculated in their lungs (intrapulmonarily). The bacterial strains employed in this study had equivalent growth rates and beta-hemolytic activity based on in vitro assays. Inoculation of 48-h-old conventional piglets with 1011 CFU of the wild-type strain (NY-4) or T3SS1 deletion mutant strains resulted in acute, self-limiting diarrhea, whereas inoculation with a T3SS2 deletion mutant strain failed to produce any clinical symptoms. Intrapulmonary inoculation of C57BL/6 mice with the wild-type strain and T3SS2 deletion mutant strains (5 × 105 CFU) induced mortality or a moribund state within 12 h (80 to 100% mortality), whereas inoculation with a T3SS1 deletion mutant or a T3SS1 T3SS2 double deletion mutant produced no mortality. Bacteria were recovered from multiple organs regardless of the strain used in the mouse model, indicating that the mice were capable of clearing the lung infection in the absence of a functional T3SS1. Because all strains had a similar beta-hemolysin phenotype, we surmise that thermostable direct hemolysin (TDH) plays a limited role in these models. The two models introduced herein produce robust results and provide a means to determine how different T3SS1 and T3SS2 effector proteins contribute to pathogenesis of V. parahaemolyticus infection. PMID:20823199

  9. TetR-type transcriptional regulator VtpR functions as a global regulator in Vibrio tubiashii.

    PubMed

    Hasegawa, Hiroaki; Häse, Claudia C

    2009-12-01

    Vibrio tubiashii, a causative agent of severe shellfish larval disease, produces multiple extracellular proteins, including a metalloprotease (VtpA), as potential virulence factors. We previously reported that VtpA is toxic for Pacific oyster (Crassostrea gigas) larvae. In this study, we show that extracellular protease production by V. tubiashii was much reduced by elevated salt concentrations, as well as by elevated temperatures. In addition, V. tubiashii produced dramatically less protease in minimal salts medium supplemented with glucose or sucrose as the sole carbon source than with succinate. We identified a protein that belongs to the TetR family of transcriptional regulators, VtpR, which showed high homology with V. cholerae HapR. We conclude that VtpR activates VtpA production based on the following: (i) a VtpR-deficient V. tubiashii mutant did not produce extracellular proteases, (ii) the mutant showed reduced expression of a vtpA-lacZ fusion, and (iii) VtpR activated vtpA-lacZ in a V. cholerae heterologous background. Moreover, we show that VtpR activated the expression of an additional metalloprotease gene (vtpB). The deduced VtpB sequence showed high homology with a metalloprotease, VhpA, from V. harveyi. Furthermore, the vtpR mutant strain produced reduced levels of extracellular hemolysin, which is attributed to the lower expression of the V. tubiashii hemolysin genes (vthAB). The VtpR-deficient mutant also had negative effects on bacterial motility and did not demonstrate toxicity to oyster larvae. Together, these findings establish that the V. tubiashii VtpR protein functions as a global regulator controlling an array of potential virulence factors.

  10. Phenotypic and Genotypic Characterization of Canadian Clinical Isolates of Vibrio parahaemolyticus Collected from 2000 to 2009

    PubMed Central

    Kearney, Ashley K.; Nadon, Celine A.; Peterson, Christy-Lynn; Tyler, Kevin; Bakouche, Laurene; Clark, Clifford G.; Hoang, Linda; Gilmour, Matthew W.; Farber, Jeffrey M.

    2014-01-01

    Vibrio parahaemolyticus is the leading bacterial cause of food-borne illness due to the consumption of contaminated seafood. The aim of the present study was to determine the population of its subtypes and establish a better understanding of the various types of V. parahaemolyticus strains that are causing human illness in Canada. The subtypes for 100 human clinical isolates of V. parahaemolyticus collected between 2000 and 2009 were determined by performing serotyping, ribotyping, pulsed-field gel electrophoresis, and multilocus sequence typing. Within this panel of strains, there was a high level of diversity (between 22 and 53 subtypes per method), but the presence of predominant clones with congruent subtypes between the various methods was also observed. For example, all 32 isolates belonging to sequence type 36 (ST36) were from serogroup O4, while 31 of them were ribotype EcoVib235-287, and 24 of the 32 were SfiI pulsed-field gel electrophoresis (PFGE) pattern VPSF1.0001. With regard to the presence of known virulence genes, 74 of the 100 isolates were PCR positive for the presence of the thermostable direct hemolysin (tdh); and 59 of these 74 strains also contained the second virulence marker, the tdh-related hemolysin (trh). The detection of trh was more predominant (81%) among the clinical isolates, and only four (4%) of the clinical isolates tested negative for the presence of both tdh and trh. This database, comprising 100 clinical isolates of V. parahaemolyticus strains from Canada, forms a baseline understanding of subtype diversity for future source attribution and other epidemiologic studies. PMID:24452166

  11. Baicalein Inhibits Staphylococcus aureus Biofilm Formation and the Quorum Sensing System In Vitro

    PubMed Central

    Wang, Ke; Hou, Changchun; Cai, Shuangqi; Huang, Yingying; Du, Zhongye; Huang, Hong; Kong, Jinliang; Chen, Yiqiang

    2016-01-01

    Biofilm formed by Staphylococcus aureus significantly enhances antibiotic resistance by inhibiting the penetration of antibiotics, resulting in an increasingly serious situation. This study aimed to assess whether baicalein can prevent Staphylococcus aureus biofilm formation and whether it may have synergistic bactericidal effects with antibiotics in vitro. To do this, we used a clinically isolated strain of Staphylococcus aureus 17546 (t037) for biofilm formation. Virulence factors were detected following treatment with baicalein, and the molecular mechanism of its antibiofilm activity was studied. Plate counting, crystal violet staining, and fluorescence microscopy revealed that 32 μg/mL and 64 μg/mL baicalein clearly inhibited 3- and 7-day biofilm formation in vitro. Moreover, colony forming unit count, confocal laser scanning microscopy, and scanning electron microscopy showed that vancomycin (VCM) and baicalein generally enhanced destruction of biofilms, while VCM alone did not. Western blotting and real-time quantitative polymerase chain reaction analyses (RTQ-PCR) confirmed that baicalein treatment reduced staphylococcal enterotoxin A (SEA) and α-hemolysin (hla) levels. Most strikingly, real-time qualitative polymerase chain reaction data demonstrated that 32 μg/mL and 64 μg/mL baicalein downregulated the quorum-sensing system regulators agrA, RNAIII, and sarA, and gene expression of ica, but 16 μg/mL baicalein had no effect. In summary, baicalein inhibited Staphylococcus aureus biofilm formation, destroyed biofilms, increased the permeability of vancomycin, reduced the production of staphylococcal enterotoxin A and α-hemolysin, and inhibited the quorum sensing system. These results support baicalein as a novel drug candidate and an effective treatment strategy for Staphylococcus aureus biofilm-associated infections. PMID:27128436

  12. Evaluation of Virulence Factors and Antibiotic Sensitivity Pattern of Escherichia Coli Isolated from Extraintestinal Infections

    PubMed Central

    Vaish, Ritu; Pradeep, MSS; Setty, CR

    2016-01-01

    Introduction  Identification of virulence determinants among the clinically isolated microorganisms assumes greater significance in the patient management perspective. Among the hospitalized patients, extremes of age groups (neonatal and geriatric age patients), patients who are debilitated due to other associated medical conditions, patients taking immunosuppressive therapy, and patients undergoing major surgeries are prone to infections with previously nonpathogenic or opportunistic pathogens. Screening of the pathogenic potential of such bacteria and identifying their virulence factors and antimicrobial susceptibility patterns could be instrumental in better patient care and management. Materials & methods  In this study, we evaluated the virulence determinants and antimicrobial susceptibility patterns of 100 clinical isolates of E. coli collected from extraintestinal infections and 50 control strains of E. coli. Hemolysin production, serum resistance, cell surface hydrophobicity, and gelatinase production were tested using standard laboratory procedures. Results  Results showed that E. colistrains have a variable pattern of virulence markers that included hemolysin production (9%), cell surface hydrophobicity (9%), serum resistance (93%), and gelatinase production (2%). Antimicrobial susceptibility testing revealed a higher rate of resistance against cephalothin (84%) and ampicillin (98%). Susceptibility to amikacin (80%) and co-trimoxazole (47%) was variable and none of the test strains revealed resistance to imipenem. The control strains in contrast exhibited fewer virulence factors and the least resistance to antibiotics. Conclusion  In conclusion, the study results revealed that E. coli isolated from extraintestinal infections had demonstrated greater virulence and higher resistance to antibiotics as compared to the E. coli strains isolated from healthy individuals. PMID:27330872

  13. Baicalein Inhibits Staphylococcus aureus Biofilm Formation and the Quorum Sensing System In Vitro.

    PubMed

    Chen, Yan; Liu, Tangjuan; Wang, Ke; Hou, Changchun; Cai, Shuangqi; Huang, Yingying; Du, Zhongye; Huang, Hong; Kong, Jinliang; Chen, Yiqiang

    2016-01-01

    Biofilm formed by Staphylococcus aureus significantly enhances antibiotic resistance by inhibiting the penetration of antibiotics, resulting in an increasingly serious situation. This study aimed to assess whether baicalein can prevent Staphylococcus aureus biofilm formation and whether it may have synergistic bactericidal effects with antibiotics in vitro. To do this, we used a clinically isolated strain of Staphylococcus aureus 17546 (t037) for biofilm formation. Virulence factors were detected following treatment with baicalein, and the molecular mechanism of its antibiofilm activity was studied. Plate counting, crystal violet staining, and fluorescence microscopy revealed that 32 μg/mL and 64 μg/mL baicalein clearly inhibited 3- and 7-day biofilm formation in vitro. Moreover, colony forming unit count, confocal laser scanning microscopy, and scanning electron microscopy showed that vancomycin (VCM) and baicalein generally enhanced destruction of biofilms, while VCM alone did not. Western blotting and real-time quantitative polymerase chain reaction analyses (RTQ-PCR) confirmed that baicalein treatment reduced staphylococcal enterotoxin A (SEA) and α-hemolysin (hla) levels. Most strikingly, real-time qualitative polymerase chain reaction data demonstrated that 32 μg/mL and 64 μg/mL baicalein downregulated the quorum-sensing system regulators agrA, RNAIII, and sarA, and gene expression of ica, but 16 μg/mL baicalein had no effect. In summary, baicalein inhibited Staphylococcus aureus biofilm formation, destroyed biofilms, increased the permeability of vancomycin, reduced the production of staphylococcal enterotoxin A and α-hemolysin, and inhibited the quorum sensing system. These results support baicalein as a novel drug candidate and an effective treatment strategy for Staphylococcus aureus biofilm-associated infections.

  14. Incidence of Aeromonas spp. infection in fish and chicken meat and its related public health hazards: A review

    PubMed Central

    Praveen, Praveen Kumar; Debnath, Chanchal; Shekhar, Shashank; Dalai, Nirupama; Ganguly, Subha

    2016-01-01

    Aeromonas is recognized to cause a variety of diseases in man. In humans, they are associated with intestinal and extra-intestinal infections. With the growing importance of Aeromonas as an emerging pathogen, it is important to combat this organism. It is indisputable that Aeromonas strains may produce many different putative virulence factors such as enterotoxins, hemolysins or cytotoxins, and antibiotic resistance against different antibiotics. The ability of these bacteria to grow competitively at 5°C may be indicative of their potential as a public health hazard. Comprehensive enteric disease surveillance strategies, prevention and education are essential for meeting the challenges in the years ahead. It is important for us to promote the value of enteric cultures when patients have a gastrointestinal illness or bloody diarrhea or when multiple cases of enteric disease occur after a common exposure. With the growing importance of Aeromonas as an emerging pathogen, it is important to combat this organism. It is indisputable that Aeromonas strains may produce many different putative virulence factors, such as enterotoxins, hemolysins or cytotoxins. It has been established that aerolysin is a virulence factor contributing to the pathogenesis of Aeromonas hydrophila infection. Fish and chicken play an important role in the transmission of this pathogen to humans. In the present study, the high prevalence of toxin-producing strains was found among the Aeromonas isolates. The ability of these bacteria to grow competitively at 5°C may be indicative of their potential as a public health hazard. The present review was constructed with a view to highlight the zoonotic importance of Aeromonas pathogen in fish and chicken meat. PMID:27051177

  15. Adaptive change inferred from genomic population analysis of the ST93 epidemic clone of community-associated methicillin-resistant Staphylococcus aureus.

    PubMed

    Stinear, Timothy P; Holt, Kathryn E; Chua, Kyra; Stepnell, Justin; Tuck, Kellie L; Coombs, Geoffrey; Harrison, Paul Francis; Seemann, Torsten; Howden, Benjamin P

    2014-02-01

    Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has emerged as a major public health problem around the world. In Australia, ST93-IV[2B] is the dominant CA-MRSA clone and displays significantly greater virulence than other S. aureus. Here, we have examined the evolution of ST93 via genomic analysis of 12 MSSA and 44 MRSA ST93 isolates, collected from around Australia over a 17-year period. Comparative analysis revealed a core genome of 2.6 Mb, sharing greater than 99.7% nucleotide identity. The accessory genome was 0.45 Mb and comprised additional mobile DNA elements, harboring resistance to erythromycin, trimethoprim, and tetracycline. Phylogenetic inference revealed a molecular clock and suggested that a single clone of methicillin susceptible, Panton-Valentine leukocidin (PVL) positive, ST93 S. aureus likely spread from North Western Australia in the early 1970s, acquiring methicillin resistance at least twice in the mid 1990s. We also explored associations between genotype and important MRSA phenotypes including oxacillin MIC and production of exotoxins (α-hemolysin [Hla], δ-hemolysin [Hld], PSMα3, and PVL). High-level expression of Hla is a signature feature of ST93 and reduced expression in eight isolates was readily explained by mutations in the agr locus. However, subtle but significant decreases in Hld were also noted over time that coincided with decreasing oxacillin resistance and were independent of agr mutations. The evolution of ST93 S. aureus is thus associated with a reduction in both exotoxin expression and oxacillin MIC, suggesting MRSA ST93 isolates are under pressure for adaptive change. PMID:24482534

  16. Adaptive Change Inferred from Genomic Population Analysis of the ST93 Epidemic Clone of Community-Associated Methicillin-Resistant Staphylococcus aureus

    PubMed Central

    Stinear, Timothy P.; Holt, Kathryn E.; Chua, Kyra; Stepnell, Justin; Tuck, Kellie L.; Coombs, Geoffrey; Harrison, Paul Francis; Seemann, Torsten; Howden, Benjamin P.

    2014-01-01

    Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has emerged as a major public health problem around the world. In Australia, ST93-IV[2B] is the dominant CA-MRSA clone and displays significantly greater virulence than other S. aureus. Here, we have examined the evolution of ST93 via genomic analysis of 12 MSSA and 44 MRSA ST93 isolates, collected from around Australia over a 17-year period. Comparative analysis revealed a core genome of 2.6 Mb, sharing greater than 99.7% nucleotide identity. The accessory genome was 0.45 Mb and comprised additional mobile DNA elements, harboring resistance to erythromycin, trimethoprim, and tetracycline. Phylogenetic inference revealed a molecular clock and suggested that a single clone of methicillin susceptible, Panton-Valentine leukocidin (PVL) positive, ST93 S. aureus likely spread from North Western Australia in the early 1970s, acquiring methicillin resistance at least twice in the mid 1990s. We also explored associations between genotype and important MRSA phenotypes including oxacillin MIC and production of exotoxins (α-hemolysin [Hla], δ-hemolysin [Hld], PSMα3, and PVL). High-level expression of Hla is a signature feature of ST93 and reduced expression in eight isolates was readily explained by mutations in the agr locus. However, subtle but significant decreases in Hld were also noted over time that coincided with decreasing oxacillin resistance and were independent of agr mutations. The evolution of ST93 S. aureus is thus associated with a reduction in both exotoxin expression and oxacillin MIC, suggesting MRSA ST93 isolates are under pressure for adaptive change. PMID:24482534

  17. Lethal infection by Bordetella pertussis mutants in the infant mouse model.

    PubMed Central

    Weiss, A A; Goodwin, M S

    1989-01-01

    Different aspects of lethal infection of infant mice with Bordetella pertussis were examined. Mutants deficient in vir-regulated genes were tested for the ability to cause a lethal infection in the infant mouse model. Adenylate cyclase toxin-hemolysin and pertussis toxin were required to cause a lethal infection at low doses. Mixed infection caused by challenging the mice with an equal number of pertussis toxin and adenylate cyclase toxin-hemolysin mutants at a dose at which neither alone was lethal was also unable to cause a lethal infection. Production of the filamentous hemagglutinin and the dermonecrotic toxin was not required to cause a lethal infection. Nine other mutants in vir-regulated genes whose phenotypes have yet to be determined were also tested. Only two of these mutants were impaired in the ability to cause a lethal infection. Expression of fimbriae does not appear to affect the dose required to cause a lethal infection; however, fimbrial expression was correlated with the later stages of a nonlethal, persistent infection. Growth of the bacteria in MgSO4, a condition which reversibly suppresses expression of the genes required for virulence, did not alter the ability of the bacteria to cause a lethal infection. Auxotrophic mutants deficient in leucine biosynthesis were as virulent as the parental strain; however, mutants deficient in methionine biosynthesis were less virulent. A B. parapertussis strain was much less effective in promoting a lethal infection than any of the wild-type B. pertussis strains examined. A persistent infection in the lungs was observed for weeks after challenge for mice given a sublethal dose of B. pertussis, and transmission from infected infants to the mother was never observed. PMID:2572561

  18. Prevalence and antimicrobial susceptibility of Vibrio parahaemolyticus isolated from retail shrimps in Malaysia

    PubMed Central

    Letchumanan, Vengadesh; Yin, Wai-Fong; Lee, Learn-Han; Chan, Kok-Gan

    2015-01-01

    Vibrio parahaemolyticus is a marine and estuarine bacterium that has been the leading cause of foodborne outbreaks which leads to a significant threat to human health worldwide. Consumption of seafood contaminated with V. parahaemolyticus causes acute gastroenteritis in individuals. The bacterium poses two main virulence factor including the thermostable direct hemolysin (tdh) which is a pore-forming protein that contributes to the invasiveness of the bacterium in humans and TDH-related hemolysin (trh), which plays a similar role as tdh in the disease pathogenesis. This study aimed to investigate the antimicrobial resistance V. parahaemolyticus strains in shrimps purchased from wetmarkets and supermarkets. The toxR-based PCR assay indicated that a total of 57.8% (185/320) isolates were positive for V. parahaemolyticus. Only 10% (19/185) toxR-positive isolate exhibit the trh gene and none of the isolates were tested positive for tdh. The MAR index was measured for 14 common antimicrobial agents. The results indicated 98% of the isolates were highly susceptible to imipenem, ampicillin sulbactam (96%), chloramphenicol (95%), trimethoprim-sulfamethoxazole (93%), gentamicin (85%), levofloxacin (83%), and tetracycline (82%). The chloramphenicol (catA2) and kanamycin (aphA-3) resistance genes were detected in the resistant V. parahaemolyticus isolates. Our results demonstrate that shrimps are contaminated with V. parahaemolyticus, some of which carry the trh-gene thus being potential to cause food borne illness. The occurrence of multidrug resistance strains in the environment could be an indication of excessive usage of antibiotics in agriculture and aquaculture fields. PMID:25688239

  19. Distribution and dynamics of epidemic and pandemic Vibrio parahaemolyticus virulence factors.

    PubMed

    Ceccarelli, Daniela; Hasan, Nur A; Huq, Anwar; Colwell, Rita R

    2013-01-01

    Vibrio parahaemolyticus, autochthonous to estuarine, marine, and coastal environments throughout the world, is the causative agent of food-borne gastroenteritis. More than 80 serotypes have been described worldwide, based on antigenic properties of the somatic (O) and capsular (K) antigens. Serovar O3:K6 emerged in India in 1996 and subsequently was isolated worldwide, leading to the conclusion that the first V. parahaemolyticus pandemic had taken place. Most strains of V. parahaemolyticus isolated from the environment or seafood, in contrast to clinical strains, do not produce a thermostable direct hemolysin (TDH) and/or a TDH-related hemolysin (TRH). Type 3 secretion systems (T3SSs), needle-like apparatuses able to deliver bacterial effectors into host cytoplasm, were identified as triggering cytotoxicity and enterotoxicity. Type 6 secretion systems (T6SS) predicted to be involved in intracellular trafficking and vesicular transport appear to play a role in V. parahaemolyticus virulence. Recent advances in V. parahaemolyticus genomics identified several pathogenicity islands (VpaIs) located on either chromosome in both epidemic and pandemic strains and comprising additional colonization factors, such as restriction-modification complexes, chemotaxis proteins, classical bacterial surface virulence factors, and putative colicins. Furthermore, studies indicate strains lacking toxins and genomic regions associated with pathogenicity may also be pathogenic, suggesting other important virulence factors remain to be identified. The unique repertoire of virulence factors identified to date, their occurrence and distribution in both epidemic and pandemic strains worldwide are described, with the aim of highlighting the complexity of V. parahaemolyticus pathogenicity as well as its dynamic genome.

  20. Structural Insights into Clostridium perfringens Delta Toxin Pore Formation

    PubMed Central

    Huyet, Jessica; Naylor, Claire E.; Savva, Christos G.; Gibert, Maryse; Popoff, Michel R.; Basak, Ajit K.

    2013-01-01

    Clostridium perfringens Delta toxin is one of the three hemolysin-like proteins produced by C. perfringens type C and possibly type B strains. One of the others, NetB, has been shown to be the major cause of Avian Nectrotic Enteritis, which following the reduction in use of antibiotics as growth promoters, has become an emerging disease of industrial poultry. Delta toxin itself is cytotoxic to the wide range of human and animal macrophages and platelets that present GM2 ganglioside on their membranes. It has sequence similarity with Staphylococcus aureus β-pore forming toxins and is expected to heptamerize and form pores in the lipid bilayer of host cell membranes. Nevertheless, its exact mode of action remains undetermined. Here we report the 2.4 Å crystal structure of monomeric Delta toxin. The superposition of this structure with the structure of the phospholipid-bound F component of S. aureus leucocidin (LukF) revealed that the glycerol molecules bound to Delta toxin and the phospholipids in LukF are accommodated in the same hydrophobic clefts, corresponding to where the toxin is expected to latch onto the membrane, though the binding sites show significant differences. From structure-based sequence alignment with the known structure of staphylococcal α-hemolysin, a model of the Delta toxin pore form has been built. Using electron microscopy, we have validated our model and characterized the Delta toxin pore on liposomes. These results highlight both similarities and differences in the mechanism of Delta toxin (and by extension NetB) cytotoxicity from that of the staphylococcal pore-forming toxins. PMID:23805259

  1. Clinical and Molecular Characteristics of Infections with CO2-Dependent Small-Colony Variants of Staphylococcus aureus▿

    PubMed Central

    Gómez-González, Carmen; Acosta, Joshi; Villa, Jennifer; Barrado, Laura; Sanz, Francisca; Orellana, M. Ángeles; Otero, Joaquín R.; Chaves, Fernando

    2010-01-01

    Most Staphylococcus aureus small-colony variants (SCVs) are auxotrophs for menadione, hemin, or thymidine but rarely for CO2. We conducted a prospective investigation of all clinical cases of CO2-dependent S. aureus during a 3-year period. We found 14 CO2-dependent isolates of S. aureus from 14 patients that fulfilled all requirements to be considered SCVs, 9 of which were methicillin resistant. The clinical presentations included four cases of catheter-related bacteremia, one complicated by endocarditis; two deep infections (mediastinitis and spondylodiscitis); four wound infections; two respiratory infections; and two cases of nasal colonization. Pulsed-field gel electrophoresis typing showed that the 14 isolates were distributed into 4 types corresponding to sequence types ST125-agr group II (agrII), ST30-agrIII, ST34-agrIII, and ST45-agrI. An array hybridization technique performed on the 14 CO2-dependent isolates and 20 S. aureus isolates with normal phenotype and representing the same sequence types showed that all possessed the enterotoxin gene cluster egc, as well as the genes for α-hemolysin and δ-hemolysin; biofilm genes icaA, icaC, and icaD; several microbial surface components recognizing adhesive matrix molecules (MSCRAMM) genes (clfA, clfB, ebh, eno, fib, ebpS, sdrC, and vw); and the isaB gene. Our study confirms the importance of CO2-dependent SCVs of S. aureus as significant pathogens. Clinical microbiologists should be aware of this kind of auxotrophy because recovery and identification are challenging and not routine. Further studies are necessary to determine the incidence of CO2 auxotrophs of S. aureus, the factors that select these strains in the host, and the genetic basis of this type of auxotrophy. PMID:20554819

  2. Antifungal Susceptibility in Serum and Virulence Determinants of Candida Bloodstream Isolates from Hong Kong

    PubMed Central

    Seneviratne, Chaminda J.; Rajan, Suhasini; Wong, Sarah S. W.; Tsang, Dominic N. C.; Lai, Christopher K. C.; Samaranayake, Lakshman P.; Jin, Lijian

    2016-01-01

    Candida bloodstream infections (CBI) are one of the most common nosocomial infections globally, and they account for a high mortality rate. The increasing global prevalence of drug-resistant Candida strains has also been posing a challenge to clinicians. In this study, we comprehensively evaluated the biofilm formation and production of hemolysin and proteinase of 63 CBI isolates derived from a hospital setting in Hong Kong as well as their antifungal susceptibility both in the presence and in the absence of human serum, using standard methodology. Candida albicans was the predominant species among the 63 CBI isolates collected, and non-albicans Candida species accounted for approximately one third of the isolates (36.5%). Of them, Candida tropicalis was the most common non-albicans Candida species. A high proportion (31.7%) of the CBI isolates (40% of C. albicans isolates, 10% of C. tropicalis isolates, 11% of C. parapsilosis isolates, and 100% of C. glabrata isolates) were found to be resistant to fluconazole. One of the isolates (C. tropicalis) was resistant to amphotericin B. A rising prevalence of drug-resistance CBI isolates in Hong Kong was observed with reference to a previous study. Notably, all non-albicans Candida species, showed increased hemolytic activity relative to C. albicans, whilst C. albicans, C. tropicalis, and C. parapsilosis exhibited proteinase activities. Majority of the isolates were capable of forming mature biofilms. Interestingly, the presence of serum distorted the yeast sensitivity to fluconazole, but not amphotericin B. Taken together, our findings demonstrate that CBI isolates of Candida have the potential to express to varying extent their virulence attributes (e.g., biofilm formation, hemolysin production, and proteinase activity) and these, together with perturbations in their antifungal sensitivity in the presence of serum, may contribute to treatment complication in candidemia. The effect of serum on antifungal activity

  3. Prevalence, Molecular Characterization, and Antibiotic Susceptibility of Vibrio parahaemolyticus from Ready-to-Eat Foods in China

    PubMed Central

    Xie, Tengfei; Xu, Xiaoke; Wu, Qingping; Zhang, Jumei; Cheng, Jianheng

    2016-01-01

    Vibrio parahaemolyticus is the leading cause of foodborne outbreaks, particularly outbreaks associated with consumption of fish and shellfish, and represents a major threat to human health worldwide. This bacterium harbors two main virulence factors: the thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH). Additionally, various serotypes have been identified. The extensive use of antibiotics is a contributing factor to the increasing incidence of antimicrobial-resistant V. parahaemolyticus. In the current study, we aimed to determine the incidence and features of V. parahaemolyticus in ready-to-eat (RTE) foods in China. We found 39 V. parahaemolyticus strains on Chinese RTE foods through investigation of 511 RTE foods samples from 24 cities in China. All isolates were analyzed for the presence of tdh and trh gene by PCR, serotyping was performed using multiplex PCR, antibiotic susceptibility analysis was carried out using the disk diffusion method, and molecular typing was performed using enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) typing and multilocus sequence typing (MLST). The results showed that none of the isolates were positive for tdh and trh. Most of the isolates (33.3%) were serotype O2. Antimicrobial susceptibility results indicated that most strains were resistant to streptomycin (89.7%), cefazolin (51.3%), and ampicillin (51.3%). The isolates were grouped into five clusters by ERIC-PCR and four clusters by MLST. We updated 10 novel loci and 33 sequence types (STs) in the MLST database. Thus, our findings demonstrated the presence of V. parahaemolyticus in Chinese RTE foods, provided insights into the dissemination of antibiotic-resistant strains, and improved our knowledge of methods of microbiological risk assessment in RTE foods. PMID:27148231

  4. Effects of Dry Storage and Resubmersion of Oysters on Total Vibrio vulnificus and Total and Pathogenic (tdh+/trh+) Vibrio parahaemolyticus Levels.

    PubMed

    Kinsey, Thomas P; Lydon, Keri A; Bowers, John C; Jones, Jessica L

    2015-08-01

    Vibrio vulnificus (Vv) and Vibrio parahaemolyticus (Vp) are the two leading causes of bacterial illnesses associated with raw shellfish consumption. Levels of these pathogens in oysters can increase during routine antifouling aquaculture practices involving dry storage in ambient air conditions. After storage, common practice is to resubmerge these stored oysters to reduce elevated Vv and Vp levels, but evidence proving the effectiveness of this practice is lacking. This study examined the changes in Vv and in total and pathogenic (thermostable direct hemolysin gene and the tdh-related hemolysin gene, tdh+ and trh+) Vp levels in oysters after 5 or 24 h of dry storage (28 to 32°C), followed by resubmersion (27 to 32°C) for 14 days. For each trial, replicate oyster samples were collected at initial harvest, after dry storage, after 7 days, and after 14 days of resubmersion. Oysters not subjected to dry storage were collected and analyzed to determine natural undisturbed vibrio levels (background control). Vibrio levels were measured using a most-probable-number enrichment followed by real-time PCR. After storage, vibrio levels (excluding tdh+ and trh+ Vp during 5-h storage) increased significantly (P < 0.001) from initial levels. After 7 days of resubmersion, Vv and total Vp levels (excluding total Vp in oysters stored for 5 h) were not significantly different (P < 0.1) from levels in background oysters. Vv and total and pathogenic Vp levels were not significantly different (P > 0.1) from levels in background oysters after 14 days of resubmersion, regardless of dry storage time. These data demonstrate that oyster resubmersion after dry storage at elevated ambient temperatures allows vibrio levels to return to those of background control samples. These results can be used to help minimize the risk of Vv and Vp illnesses and to inform the oyster industry on the effectiveness of routine storing and resubmerging of aquaculture oysters.

  5. Genotyping of Staphylococcus aureus in bovine mastitis and correlation to phenotypic characteristics.

    PubMed

    Artursson, Karin; Söderlund, Robert; Liu, Lihong; Monecke, Stefan; Schelin, Jenny

    2016-09-25

    Reducing the prevalence of mastitis caused by Staphylococcus aureus (S. aureus) is essential to improve animal health and reduce economic losses for farmers. The clinical outcome of acute mastitis and risk of progression to persistent mastitis can, at least to some extent, be related to genetic variants of the strain causing the infection. In the present study we have used microarrays to investigate the presence of virulence genes in S. aureus isolates from dairy cows with acute clinical mastitis (n=70) and correlated the findings to other genotypic and phenotypic characteristics. Among the most commonly found virulence factors were genes encoding several hemolysin types, leukocidins D and lukM/lukF-P83, clumping factors A and B, fibrinogen binding protein and fibronectin-binding protein A. Some virulence factors e.g. fibronectin-binding protein B and Staphylococcus aureus surface protein G were less common. Genes coding for several staphylococcal enterotoxins and toxic shock syndrome toxin-1 (TSST-1) were commonly found, especially in one major pulsotype. No beta-lactamase genes were found in any common pulsotype, while present in some rare pulsotypes, indicated to be of human origin. Production of TSST-1, enterotoxins, hemolysins and beta-lactamase could all be positively correlated to presence of the corresponding genes. This study reveals a number of genotypic differences and similarities among common and rare pulsotypes of S. aureus from cases of mastitis in Sweden. The results could help the design of diagnostic tools to guide on-farm interventions according to the expected impact on udder health from a specific S. aureus genotype. PMID:27599942

  6. Genetic and phenotypic analysis of Vibrio cholerae non-O1, non-O139 isolated from German and Austrian patients.

    PubMed

    Schirmeister, F; Dieckmann, R; Bechlars, S; Bier, N; Faruque, S M; Strauch, E

    2014-05-01

    Vibrio cholerae belonging to the non-O1, non-O139 serogroups are present in the coastal waters of Germany and in some German and Austrian lakes. These bacteria can cause gastroenteritis and extraintestinal infections, and are transmitted through contaminated food and water. However, non-O1, non-O139 V. cholerae infections are rare in Germany. We studied 18 strains from German and Austrian patients with diarrhea or local infections for their virulence-associated genotype and phenotype to assess their potential for infectivity in anticipation of possible climatic changes that could enhance the transmission of these pathogens. The strains were examined for the presence of genes encoding cholera toxin and toxin-coregulated pilus (TCP), as well as other virulence-associated factors or markers, including hemolysins, repeats-in-toxin (RTX) toxins, Vibrio seventh pandemic islands VSP-1 and VSP-2, and the type III secretion system (TTSS). Phenotypic assays for hemolysin activity, serum resistance, and biofilm formation were also performed. A dendrogram generated by incorporating the results of these analyses revealed genetic differences of the strains correlating with their clinical origin. Non-O1, non-O139 strains from diarrheal patients possessed the TTSS and/or the multifunctional autoprocessing repeats-in-toxin (MARTX) toxin, which were not found in the strains from ear or wound infections. Routine matrix-assisted laser desorption/ionization (MALDI-TOF) mass spectrometry (MS) analysis of all strains provided reliable identification of the species but failed to differentiate between strains or clusters. The results of this study indicate the need for continued surveillance of V. cholerae non-O1, non-O139 in Germany, in view of the predicted increase in the prevalence of Vibrio spp. due to the rise in surface water temperatures.

  7. Capsule Expression and Genotypic Differences among Staphylococcus aureus Isolates from Patients with Chronic or Acute Osteomyelitis▿

    PubMed Central

    Lattar, Santiago M.; Tuchscherr, Lorena P. N.; Caccuri, Roberto L.; Centrón, Daniela; Becker, Karsten; Alonso, Claudio A.; Barberis, Claudia; Miranda, Graciela; Buzzola, Fernanda R.; von Eiff, Christof; Sordelli, Daniel O.

    2009-01-01

    There is ample evidence that Staphylococcus aureus capsular polysaccharide (CP) promotes virulence. Loss of capsule expression, however, may lead to S. aureus persistence in a chronically infected host. This study was conducted to determine the relative prevalence of nonencapsulated S. aureus in patients with chronic and acute osteomyelitis. Only 76/118 (64%) S. aureus isolates from patients with osteomyelitis expressed CP, whereas all 50 isolates from blood cultures of patients with infections other than osteoarticular infections expressed CP (P = 0.0001). A significantly higher prevalence of nonencapsulated S. aureus was found in patients with chronic osteomyelitis (53%) than in those with acute osteomyelitis (21%) (P = 0.0046). S. aureus isolates obtained from multiple specimens from five of six patients with chronic osteomyelitis exhibited phenotypic (expression of CP, α-hemolysin, β-hemolysin, slime, and the small-colony variant phenotype) and/or genotypic (pulsed-field gel electrophoresis and spa typing) differences. Nonencapsulated S. aureus was recovered from at least one specimen from each chronic osteomyelitis patient. Fourteen isolates obtained from two patients with acute osteomyelitis were indistinguishable from each other within each group, and all produced CP5. In conclusion, we demonstrated that nonencapsulated S. aureus is more frequently isolated from patients with chronic osteomyelitis than from those with acute osteomyelitis, suggesting that loss of CP expression may be advantageous to S. aureus during chronic infection. Our findings on multiple S. aureus isolates from individual patients allow us to suggest that selection of nonencapsulated S. aureus is likely to have occurred in the patient during long-term bone infection. PMID:19273557

  8. Vibrio parahaemolyticus Strains of Pandemic Serotypes Identified from Clinical and Environmental Samples from Jiangsu, China

    PubMed Central

    Li, Jingjiao; Xue, Feng; Yang, Zhenquan; Zhang, Xiaoping; Zeng, Dexin; Chao, Guoxiang; Jiang, Yuan; Li, Baoguang

    2016-01-01

    Vibrio parahaemolyticus has emerged as a major foodborne pathogen in China, Japan, Thailand, and other Asian countries. In this study, 72 strains of V. parahaemolyticus were isolated from clinical and environmental samples between 2006 and 2014 in Jiangsu, China. The serotypes and six virulence genes including thermostable direct hemolysin (TDR) and TDR-related hemolysin (TRH) genes were assessed among the isolates. Twenty five serotypes were identified and O3:K6 was one of the dominant serotypes. The genetic diversity was assessed by multilocus sequence typing (MLST) analysis, and 48 sequence types (STs) were found, suggesting this V. parahaemolyticus group is widely dispersed and undergoing rapid evolution. A total of 25 strains of pandemic serotypes such as O3:K6, O5:K17, and O1:KUT were identified. It is worth noting that the pandemic serotypes were not exclusively identified from clinical samples, rather, nine strains were also isolated from environmental samples; and some of these strains harbored several virulence genes, which may render those strains pathogenicity potential. Therefore, the emergence of these “environmental” pandemic V. parahaemolyticus strains may poses a new threat to the public health in China. Furthermore, six novel serotypes and 34 novel STs were identified among the 72 isolates, indicating that V. parahaemolyticus were widely distributed and fast evolving in the environment in Jiangsu, China. The findings of this study provide new insight into the phylogenic relationship between V. parahaemolyticus strains of pandemic serotypes from clinical and environmental sources and enhance the MLST database; and our proposed possible O- and K- antigen evolving paths of V. parahaemolyticus may help understand how the serotypes of this dispersed bacterial population evolve. PMID:27303379

  9. Prevalence, Molecular Characterization, and Antibiotic Susceptibility of Vibrio parahaemolyticus from Ready-to-Eat Foods in China.

    PubMed

    Xie, Tengfei; Xu, Xiaoke; Wu, Qingping; Zhang, Jumei; Cheng, Jianheng

    2016-01-01

    Vibrio parahaemolyticus is the leading cause of foodborne outbreaks, particularly outbreaks associated with consumption of fish and shellfish, and represents a major threat to human health worldwide. This bacterium harbors two main virulence factors: the thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH). Additionally, various serotypes have been identified. The extensive use of antibiotics is a contributing factor to the increasing incidence of antimicrobial-resistant V. parahaemolyticus. In the current study, we aimed to determine the incidence and features of V. parahaemolyticus in ready-to-eat (RTE) foods in China. We found 39 V. parahaemolyticus strains on Chinese RTE foods through investigation of 511 RTE foods samples from 24 cities in China. All isolates were analyzed for the presence of tdh and trh gene by PCR, serotyping was performed using multiplex PCR, antibiotic susceptibility analysis was carried out using the disk diffusion method, and molecular typing was performed using enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) typing and multilocus sequence typing (MLST). The results showed that none of the isolates were positive for tdh and trh. Most of the isolates (33.3%) were serotype O2. Antimicrobial susceptibility results indicated that most strains were resistant to streptomycin (89.7%), cefazolin (51.3%), and ampicillin (51.3%). The isolates were grouped into five clusters by ERIC-PCR and four clusters by MLST. We updated 10 novel loci and 33 sequence types (STs) in the MLST database. Thus, our findings demonstrated the presence of V. parahaemolyticus in Chinese RTE foods, provided insights into the dissemination of antibiotic-resistant strains, and improved our knowledge of methods of microbiological risk assessment in RTE foods. PMID:27148231

  10. Prevalence and antimicrobial susceptibility of Vibrio parahaemolyticus isolated from retail shrimps in Malaysia.

    PubMed

    Letchumanan, Vengadesh; Yin, Wai-Fong; Lee, Learn-Han; Chan, Kok-Gan

    2015-01-01

    Vibrio parahaemolyticus is a marine and estuarine bacterium that has been the leading cause of foodborne outbreaks which leads to a significant threat to human health worldwide. Consumption of seafood contaminated with V. parahaemolyticus causes acute gastroenteritis in individuals. The bacterium poses two main virulence factor including the thermostable direct hemolysin (tdh) which is a pore-forming protein that contributes to the invasiveness of the bacterium in humans and TDH-related hemolysin (trh), which plays a similar role as tdh in the disease pathogenesis. This study aimed to investigate the antimicrobial resistance V. parahaemolyticus strains in shrimps purchased from wetmarkets and supermarkets. The toxR-based PCR assay indicated that a total of 57.8% (185/320) isolates were positive for V. parahaemolyticus. Only 10% (19/185) toxR-positive isolate exhibit the trh gene and none of the isolates were tested positive for tdh. The MAR index was measured for 14 common antimicrobial agents. The results indicated 98% of the isolates were highly susceptible to imipenem, ampicillin sulbactam (96%), chloramphenicol (95%), trimethoprim-sulfamethoxazole (93%), gentamicin (85%), levofloxacin (83%), and tetracycline (82%). The chloramphenicol (catA2) and kanamycin (aphA-3) resistance genes were detected in the resistant V. parahaemolyticus isolates. Our results demonstrate that shrimps are contaminated with V. parahaemolyticus, some of which carry the trh-gene thus being potential to cause food borne illness. The occurrence of multidrug resistance strains in the environment could be an indication of excessive usage of antibiotics in agriculture and aquaculture fields. PMID:25688239

  11. Baicalein Inhibits Staphylococcus aureus Biofilm Formation and the Quorum Sensing System In Vitro.

    PubMed

    Chen, Yan; Liu, Tangjuan; Wang, Ke; Hou, Changchun; Cai, Shuangqi; Huang, Yingying; Du, Zhongye; Huang, Hong; Kong, Jinliang; Chen, Yiqiang

    2016-01-01

    Biofilm formed by Staphylococcus aureus significantly enhances antibiotic resistance by inhibiting the penetration of antibiotics, resulting in an increasingly serious situation. This study aimed to assess whether baicalein can prevent Staphylococcus aureus biofilm formation and whether it may have synergistic bactericidal effects with antibiotics in vitro. To do this, we used a clinically isolated strain of Staphylococcus aureus 17546 (t037) for biofilm formation. Virulence factors were detected following treatment with baicalein, and the molecular mechanism of its antibiofilm activity was studied. Plate counting, crystal violet staining, and fluorescence microscopy revealed that 32 μg/mL and 64 μg/mL baicalein clearly inhibited 3- and 7-day biofilm formation in vitro. Moreover, colony forming unit count, confocal laser scanning microscopy, and scanning electron microscopy showed that vancomycin (VCM) and baicalein generally enhanced destruction of biofilms, while VCM alone did not. Western blotting and real-time quantitative polymerase chain reaction analyses (RTQ-PCR) confirmed that baicalein treatment reduced staphylococcal enterotoxin A (SEA) and α-hemolysin (hla) levels. Most strikingly, real-time qualitative polymerase chain reaction data demonstrated that 32 μg/mL and 64 μg/mL baicalein downregulated the quorum-sensing system regulators agrA, RNAIII, and sarA, and gene expression of ica, but 16 μg/mL baicalein had no effect. In summary, baicalein inhibited Staphylococcus aureus biofilm formation, destroyed biofilms, increased the permeability of vancomycin, reduced the production of staphylococcal enterotoxin A and α-hemolysin, and inhibited the quorum sensing system. These results support baicalein as a novel drug candidate and an effective treatment strategy for Staphylococcus aureus biofilm-associated infections. PMID:27128436

  12. Phenotypic and genotypic detection of virulence factors of Staphylococcus aureus isolated from clinical and subclinical mastitis in cattle and water buffaloes from different farms of Sadat City in Egypt

    PubMed Central

    Elsayed, Mohamed Sabry; Mahmoud El-Bagoury, Abd Elrahman; Dawoud, Mai Abdallah

    2015-01-01

    Aim: To characterize Staphylococcus aureus from clinical and subclinical mastitis and identify virulence factors. Materials and Methods: Two hundred and two milk samples were collected, 143 from mastitic cattle and buffaloes 94 and 49, respectively, and 59 from apparently healthy cattle and buffaloes 35 and 24, respectively. Results: California mastitis test was applied and positive prevalence were 91.48% and 75.51% for cattle and buffalo with clinical mastitis and 37.14% and 45.83% for cattle and buffalo with subclinical mastitis. S. aureus was isolated from clinically mastitic cattle and buffaloes were 39.29% and 50%, respectively. While, from subclinical mastitic cattle and buffaloes were 80% and 72.73%, respectively. Hemolytic activity was assessed for S. aureus isolated from clinically and subclinical mastitic cases with prevalences of 100% and 56.25%, respectively. Thermo nuclease production from clinically and subclinical mastitic cases was 25% and 56.25%, respectively. Simplex polymerase chain reaction (PCR) conducted on S. aureus using 16S rRNA, clumping factor A, Panton valentine leukocidin, coagulase (Coa), alpha-hemolysin and beta-hemolysin those proved existence in 100%, 46.9%, 65.6%, 100%, 34.4%, and 43.75% of the isolates, respectively. While, multiplex PCR is utilized for detection of enterotoxins and proved that 12.5% was positive for enterotoxine Type D. Conclusions: It is concluded that simplex and multiplex PCR assays can be used as rapid and sensitive diagnostic tools to detect the presence of S. aureus and characterize its virulence factors that help in detection of severity of infection, distribution and stating preventive and control strategies. PMID:27047197

  13. Genotyping of Staphylococcus aureus in bovine mastitis and correlation to phenotypic characteristics.

    PubMed

    Artursson, Karin; Söderlund, Robert; Liu, Lihong; Monecke, Stefan; Schelin, Jenny

    2016-09-25

    Reducing the prevalence of mastitis caused by Staphylococcus aureus (S. aureus) is essential to improve animal health and reduce economic losses for farmers. The clinical outcome of acute mastitis and risk of progression to persistent mastitis can, at least to some extent, be related to genetic variants of the strain causing the infection. In the present study we have used microarrays to investigate the presence of virulence genes in S. aureus isolates from dairy cows with acute clinical mastitis (n=70) and correlated the findings to other genotypic and phenotypic characteristics. Among the most commonly found virulence factors were genes encoding several hemolysin types, leukocidins D and lukM/lukF-P83, clumping factors A and B, fibrinogen binding protein and fibronectin-binding protein A. Some virulence factors e.g. fibronectin-binding protein B and Staphylococcus aureus surface protein G were less common. Genes coding for several staphylococcal enterotoxins and toxic shock syndrome toxin-1 (TSST-1) were commonly found, especially in one major pulsotype. No beta-lactamase genes were found in any common pulsotype, while present in some rare pulsotypes, indicated to be of human origin. Production of TSST-1, enterotoxins, hemolysins and beta-lactamase could all be positively correlated to presence of the corresponding genes. This study reveals a number of genotypic differences and similarities among common and rare pulsotypes of S. aureus from cases of mastitis in Sweden. The results could help the design of diagnostic tools to guide on-farm interventions according to the expected impact on udder health from a specific S. aureus genotype.

  14. TTSS2-deficient hha mutant of Salmonella Typhimurium exhibits significant systemic attenuation in immunocompromised hosts

    PubMed Central

    Vishwakarma, Vikalp; Pati, Niladri Bhusan; Ray, Shilpa; Das, Susmita; Suar, Mrutyunjay

    2014-01-01

    Non-typhoidal Salmonella (NTS) infections are emerging as leading problem worldwide and the variations in host immune status append to the concern of NTS. Salmonella enterica serovar Typhimurium is one of the causative agents of NTS infections and has been extensively studied. The inactivation of Salmonella pathogenicity island 2 (SPI2) encoded type-III secretion system 2 (TTSS2) has been reported rendering the strain incapable for systemic dissemination to host sites and has also been proposed as live-attenuated vaccine. However, infections from TTSS2-deficient Salmonella have also been reported. In this study, mutant strain MT15 was developed by inactivation of the hemolysin expression modulating protein (hha) in TTSS2-deficient S. Typhimurium background. The MT15 strain showed significant level of attenuation in immune-deprived murine colitis model when tested in iNos−/−, IL10−/−, and CD40L−/− mice groups in C57BL/6 background. Further, the mutation in hha does not implicate any defect in bacterial colonization to the host gut. The long-term infection of developed mutant strain conferred protective immune responses to suitably immunized streptomycin pre-treated C57BL/6 mice. The immunization enhanced the CD4+ and CD8+ cell types involved in bacterial clearance. The serum IgG and luminal secretory IgA (sIgA) was also found to be elevated after the due course of infection. Additionally, the immunized C57BL/6 mice were protected from the subsequent lethal infection of Salmonella Typhimurium. Collectively, these findings implicate the involvement of hemolysin expression modulating protein (Hha) in establishment of bacterial infection. In light of the observed attenuation of the developed mutant strain, this study proposes the possible significance of SPI2-deficient hha mutant as an alternative live-attenuated vaccine strain for use against lethal Salmonella infections. PMID:24401482

  15. Anti-hemolytic, hemagglutination inhibition and bacterial membrane disruptive properties of selected herbal extracts attenuate virulence of Carbapenem Resistant Escherichia coli.

    PubMed

    Thakur, Pallavi; Chawla, Raman; Narula, Alka; Goel, Rajeev; Arora, Rajesh; Sharma, Rakesh Kumar

    2016-06-01

    Expression of a multitude of virulence factors by multi-drug resistant microbial strains, e.g., Carbapenem Resistant Escherichia coli (Family: Enterobacteriaceae; Class: Gammaproteobacteria), is responsible for resistance against beta-lactam antibiotics. Hemolysin production and induction of hemagglutination by bacterial surface receptors inflicts direct cytotoxicity by destroying host phagocytic and epithelial cells. We have previously reported that Berberis aristata, Camellia sinensis, Cyperus rotundus Holarrhena antidysenterica and Andrographis paniculata are promising herbal leads for targeting Carbapenem resistant Escherichia coli. These herbal leads were analyzed for their anti-hemolytic potential by employing spectrophotometric assay of hemoglobin liberation. Anti-hemagglutination potential of the extracts was assessed by employing qualitative assay of visible RBC aggregate formation. Camellia sinensis (PTRC-31911-A) exhibited anti-hemolytic potential of 73.97 ± 0.03%, followed by Holarrhena antidysenterica (PTRC-8111-A) i.e., 68.32 ± 0.05%, Berberis aristata (PTRC-2111-A) i.e., 60.26 ± 0.05% and Cyperus rotundus (PTRC-31811-A) i.e., 53.76 ± 0.03%. Comprehensive, visual analysis of hemagglutination inhibition revealed that only Berberis aristata (PTRC-2111-A) and Camellia sinensis (PTRC-31911-A) exhibited anti-hemagglutination activity. However, Andrographis paniculata (PTRC-11611-A) exhibited none of the inhibitory activities. Furthermore, the pair wise correlation analysis of the tested activities with quantitative phytochemical descriptors revealed that an increased content of alkaloid; flavonoids; polyphenols, and decreased content of saponins supported both the activities. Additionally, flow cytometry revealed that cell membrane structures of CRE were damaged by extracts of Berberis aristata (PTRC-2111-A) and Camellia sinensis (PTRC-31911-A) at their respective Minimum Inhibitory Concentrations, thereby confirming noteworthy antibacterial

  16. Clinical and molecular characteristics of infections with CO2-dependent small-colony variants of Staphylococcus aureus.

    PubMed

    Gómez-González, Carmen; Acosta, Joshi; Villa, Jennifer; Barrado, Laura; Sanz, Francisca; Orellana, M Angeles; Otero, Joaquín R; Chaves, Fernando

    2010-08-01

    Most Staphylococcus aureus small-colony variants (SCVs) are auxotrophs for menadione, hemin, or thymidine but rarely for CO(2). We conducted a prospective investigation of all clinical cases of CO(2)-dependent S. aureus during a 3-year period. We found 14 CO(2)-dependent isolates of S. aureus from 14 patients that fulfilled all requirements to be considered SCVs, 9 of which were methicillin resistant. The clinical presentations included four cases of catheter-related bacteremia, one complicated by endocarditis; two deep infections (mediastinitis and spondylodiscitis); four wound infections; two respiratory infections; and two cases of nasal colonization. Pulsed-field gel electrophoresis typing showed that the 14 isolates were distributed into 4 types corresponding to sequence types ST125-agr group II (agrII), ST30-agrIII, ST34-agrIII, and ST45-agrI. An array hybridization technique performed on the 14 CO(2)-dependent isolates and 20 S. aureus isolates with normal phenotype and representing the same sequence types showed that all possessed the enterotoxin gene cluster egc, as well as the genes for alpha-hemolysin and delta-hemolysin; biofilm genes icaA, icaC, and icaD; several microbial surface components recognizing adhesive matrix molecules (MSCRAMM) genes (clfA, clfB, ebh, eno, fib, ebpS, sdrC, and vw); and the isaB gene. Our study confirms the importance of CO(2)-dependent SCVs of S. aureus as significant pathogens. Clinical microbiologists should be aware of this kind of auxotrophy because recovery and identification are challenging and not routine. Further studies are necessary to determine the incidence of CO(2) auxotrophs of S. aureus, the factors that select these strains in the host, and the genetic basis of this type of auxotrophy.

  17. Vibrio parahaemolyticus Strains of Pandemic Serotypes Identified from Clinical and Environmental Samples from Jiangsu, China.

    PubMed

    Li, Jingjiao; Xue, Feng; Yang, Zhenquan; Zhang, Xiaoping; Zeng, Dexin; Chao, Guoxiang; Jiang, Yuan; Li, Baoguang

    2016-01-01

    Vibrio parahaemolyticus has emerged as a major foodborne pathogen in China, Japan, Thailand, and other Asian countries. In this study, 72 strains of V. parahaemolyticus were isolated from clinical and environmental samples between 2006 and 2014 in Jiangsu, China. The serotypes and six virulence genes including thermostable direct hemolysin (TDR) and TDR-related hemolysin (TRH) genes were assessed among the isolates. Twenty five serotypes were identified and O3:K6 was one of the dominant serotypes. The genetic diversity was assessed by multilocus sequence typing (MLST) analysis, and 48 sequence types (STs) were found, suggesting this V. parahaemolyticus group is widely dispersed and undergoing rapid evolution. A total of 25 strains of pandemic serotypes such as O3:K6, O5:K17, and O1:KUT were identified. It is worth noting that the pandemic serotypes were not exclusively identified from clinical samples, rather, nine strains were also isolated from environmental samples; and some of these strains harbored several virulence genes, which may render those strains pathogenicity potential. Therefore, the emergence of these "environmental" pandemic V. parahaemolyticus strains may poses a new threat to the public health in China. Furthermore, six novel serotypes and 34 novel STs were identified among the 72 isolates, indicating that V. parahaemolyticus were widely distributed and fast evolving in the environment in Jiangsu, China. The findings of this study provide new insight into the phylogenic relationship between V. parahaemolyticus strains of pandemic serotypes from clinical and environmental sources and enhance the MLST database; and our proposed possible O- and K- antigen evolving paths of V. parahaemolyticus may help understand how the serotypes of this dispersed bacterial population evolve. PMID:27303379

  18. Prevalence, Molecular Characterization, and Antibiotic Susceptibility of Vibrio parahaemolyticus from Ready-to-Eat Foods in China.

    PubMed

    Xie, Tengfei; Xu, Xiaoke; Wu, Qingping; Zhang, Jumei; Cheng, Jianheng

    2016-01-01

    Vibrio parahaemolyticus is the leading cause of foodborne outbreaks, particularly outbreaks associated with consumption of fish and shellfish, and represents a major threat to human health worldwide. This bacterium harbors two main virulence factors: the thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH). Additionally, various serotypes have been identified. The extensive use of antibiotics is a contributing factor to the increasing incidence of antimicrobial-resistant V. parahaemolyticus. In the current study, we aimed to determine the incidence and features of V. parahaemolyticus in ready-to-eat (RTE) foods in China. We found 39 V. parahaemolyticus strains on Chinese RTE foods through investigation of 511 RTE foods samples from 24 cities in China. All isolates were analyzed for the presence of tdh and trh gene by PCR, serotyping was performed using multiplex PCR, antibiotic susceptibility analysis was carried out using the disk diffusion method, and molecular typing was performed using enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) typing and multilocus sequence typing (MLST). The results showed that none of the isolates were positive for tdh and trh. Most of the isolates (33.3%) were serotype O2. Antimicrobial susceptibility results indicated that most strains were resistant to streptomycin (89.7%), cefazolin (51.3%), and ampicillin (51.3%). The isolates were grouped into five clusters by ERIC-PCR and four clusters by MLST. We updated 10 novel loci and 33 sequence types (STs) in the MLST database. Thus, our findings demonstrated the presence of V. parahaemolyticus in Chinese RTE foods, provided insights into the dissemination of antibiotic-resistant strains, and improved our knowledge of methods of microbiological risk assessment in RTE foods.

  19. Adaptive change inferred from genomic population analysis of the ST93 epidemic clone of community-associated methicillin-resistant Staphylococcus aureus.

    PubMed

    Stinear, Timothy P; Holt, Kathryn E; Chua, Kyra; Stepnell, Justin; Tuck, Kellie L; Coombs, Geoffrey; Harrison, Paul Francis; Seemann, Torsten; Howden, Benjamin P

    2014-02-01

    Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has emerged as a major public health problem around the world. In Australia, ST93-IV[2B] is the dominant CA-MRSA clone and displays significantly greater virulence than other S. aureus. Here, we have examined the evolution of ST93 via genomic analysis of 12 MSSA and 44 MRSA ST93 isolates, collected from around Australia over a 17-year period. Comparative analysis revealed a core genome of 2.6 Mb, sharing greater than 99.7% nucleotide identity. The accessory genome was 0.45 Mb and comprised additional mobile DNA elements, harboring resistance to erythromycin, trimethoprim, and tetracycline. Phylogenetic inference revealed a molecular clock and suggested that a single clone of methicillin susceptible, Panton-Valentine leukocidin (PVL) positive, ST93 S. aureus likely spread from North Western Australia in the early 1970s, acquiring methicillin resistance at least twice in the mid 1990s. We also explored associations between genotype and important MRSA phenotypes including oxacillin MIC and production of exotoxins (α-hemolysin [Hla], δ-hemolysin [Hld], PSMα3, and PVL). High-level expression of Hla is a signature feature of ST93 and reduced expression in eight isolates was readily explained by mutations in the agr locus. However, subtle but significant decreases in Hld were also noted over time that coincided with decreasing oxacillin resistance and were independent of agr mutations. The evolution of ST93 S. aureus is thus associated with a reduction in both exotoxin expression and oxacillin MIC, suggesting MRSA ST93 isolates are under pressure for adaptive change.

  20. Isolation and characterization of verocytotoxin-producing Escherichia coli O157 from slaughter pigs and poultry.

    PubMed

    Heuvelink, A E; Zwartkruis-Nahuis, J T; van den Biggelaar, F L; van Leeuwen, W J; de Boer, E

    1999-11-01

    Rectal contents and tonsils from Dutch slaughter pigs collected immediately after slaughter were examined for the presence of verocytotoxin (VT)-producing Escherichia coli (VTEC) of serogroup O157 (O157 VTEC). In addition, fresh fecal material from poultry layer flocks and turkey flocks collected on poultry farms was examined for the presence of O157 VTEC. E. coli O157 strains were isolated from two (1.4%) of 145 pigs. The strains were isolated from samples of rectal contents, all samples of tonsils being negative. While all 501 fecal samples from chicken flocks were found negative, E. coli O157 strains were isolated from six (1.3%) of 459 pooled fecal samples from turkey flocks. One of the porcine isolates and one of the turkey isolates contained the VT2 gene, the E. coli attaching-and-effacing gene, as well as the enterohemorrhagic E. coli hemolysin gene. Production of VT was confirmed by cytotoxicity tests on Vero cells. Based on these characteristics, the two stains were regarded as potentially pathogenic for humans. The porcine and the turkey isolate were further characterized as being of phage types 4 and 14, respectively. While biochemically typical of E. coli O157, the remaining six isolates were nonverocytotoxigenic and negative for both the E. coli attaching-and-effacing gene and the enterohemorrhagic E. coli hemolysin gene. All eight E. coli O157 isolates did not carry genes that encode E. coli heat-labile and heat-stable enterotoxins. It was concluded that pigs and poultry can be a source of O157 VTEC strains characteristic of those causing illness in man. The extent to which pigs and poultry play a role in the epidemiology of human O157 VTEC infection needs further research. PMID:10573393

  1. Characterization of toxigenic vibrios isolated from the freshwater environment of Hiroshima, Japan.

    PubMed Central

    Venkateswaran, K; Kiiyukia, C; Takaki, M; Nakano, H; Matsuda, H; Kawakami, H; Hashimoto, H

    1989-01-01

    The occurrence and characterization of toxigenic vibrios in surface water and sediment samples of the fresh water environment of the Ohta River were studied. The membrane filter, pad preenrichment technique, followed by the placement of membranes onto thiosulfate citrate-bile salt-sucrose agar, was used for the enumeration of total vibrios. Qualitative examination of pathogenic vibrios was also attempted. In addition, a survey was conducted to determine the incidence of Clostridium botulinum in sediment samples of the Ohta River and the Hiroshima coast. In the identification of 361 strains, 12 species of Vibrio and two species of Listonella were observed. Non-01 Vibrio cholerae was prevalent among the members of the genus Vibrio. Vibrio parahaemolyticus (serotype 04:K34), isolated in fresh water, is significant and suggests that some still unknown conditions promote the survival of these organisms in fresh water. An estimated 132 strains were hemolytic by a simple agar method, and further characterization revealed that 82% of the hemolytic vibrios (107 strains) produced various toxins. About 71% (93 strains) elaborated cytotoxin, 55% (72 strains) produced hemolysin, and 44% (58 strains) responded for both cytotoxin and hemolysin in the crude toxin extracts. All the non-01 V. cholerae showed cytotoxic activity, and the virulent strains of Vibrio fluvialis and Vibrio spp. showed cytotonic responses in RK-13 cells. Of 36 sediment samples tested, 10 harbored C. botulinum spores (28%) and were isolated invariably in all the regions of the Hiroshima coast and in the Ohta River, except the upper region of the Ohta River. PMID:2690736

  2. Cytotoxic Potential of Bacillus cereus Strains ATCC 11778 and 14579 Against Human Lung Epithelial Cells Under Microaerobic Growth Conditions

    PubMed Central

    Kilcullen, Kathleen; Teunis, Allison; Popova, Taissia G.; Popov, Serguei G.

    2016-01-01

    Bacillus cereus, a food poisoning bacterium closely related to Bacillus anthracis, secretes a multitude of virulence factors including enterotoxins, hemolysins, and phospholipases. However, the majority of the in vitro experiments evaluating the cytotoxic potential of B. cereus were carried out in the conditions of aeration, and the impact of the oxygen limitation in conditions encountered by the microbe in natural environment such as gastrointestinal tract remains poorly understood. This research reports comparative analysis of ATCC strains 11778 (BC1) and 14579 (BC2) in aerobic and microaerobic (static) cultures with regard to their toxicity for human lung epithelial cells. We showed that BC1 increased its toxicity upon oxygen limitation while BC2 was highly cytotoxic in both growth conditions. The combined effect of the pore-forming, cholesterol-dependent hemolysin, cereolysin O (CLO), and metabolic product(s) such as succinate produced in microaerobic conditions provided substantial contribution to the toxicity of BC1 but not BC2 which relied mainly on other toxins. This mechanism is shared between CB1 and B. anthracis. It involves the permeabilization of the cell membrane which facilitates transport of toxic bacterial metabolites into the cell. The toxicity of BC1 was potentiated in the presence of bovine serum albumin which appeared to serve as reservoir for bacteria-derived nitric oxide participating in the downstream production of reactive oxidizing species with the properties of peroxynitrite. In agreement with this the BC1 cultures demonstrated the increased oxidation of the indicator dye Amplex Red catalyzed by peroxidase as well as the increased toxicity in the presence of externally added ascorbic acid. PMID:26870026

  3. Virulence and antimicrobial resistance of common urinary bacteria from asymptomatic students of Niger Delta University, Amassoma, Bayelsa State, Nigeria

    PubMed Central

    Onanuga, Adebola; Selekere, Tamaradobra Laurretta

    2016-01-01

    Background: Asymptomatic bacteriuria frequently occurs among all ages with the possibility of developing into urinary tract infections, and the antimicrobial resistance patterns of the etiologic organisms are essential for appropriate therapy. Thus, we investigated the virulence and antimicrobial resistance patterns of common urinary bacteria in asymptomatic students of Niger Delta University, Amassoma, Bayelsa State, Nigeria in a cross-sectional study. Materials and Methods: Clean catch mid-stream early morning urine samples collected from 200 asymptomatic University students of aged ranges 15–30 years were cultured, screened and common bacteria were identified using standard microbiological procedures. The isolates were screened for hemolysin production and their susceptibility to antibiotics was determined using standard disc assay method. Results: A total prevalence rate of 52.0% significant bacteriuria was detected and it was significantly higher among the female with a weak association (χ2 = 6.01, phi = 0.173, P = 0.014). The Klebsiella pneumoniae and Staphylococcus aureus isolates were most frequently encountered among the isolated bacteria and 18 (12.7%) of all the bacterial isolates produced hemolysins. All the bacterial isolates exhibited 50–100% resistance to the tested beta-lactam antibiotics, tetracycline and co-trimoxazole. The isolated bacteria were 85-100% multi-drug resistant. However, most of the isolates were generally susceptible to gentamicin and ofloxacin. The phenotypic detection of extended-spectrum beta-lactamases was 9 (9.6%) among the tested Gram-negative bacterial isolates. Conclusions: The observed high proportions of multidrug resistant urinary bacteria among asymptomatic University students call for the need of greater control of antibiotic use in this study area. PMID:26957865

  4. Characterization of three Bacillus cereus strains involved in a major outbreak of food poisoning after consumption of fermented black beans (Douchi) in Yunan, China.

    PubMed

    Zhou, Guoping; Bester, Kai; Liao, Bin; Yang, Zushun; Jiang, Rongrong; Hendriksen, Niels Bohse

    2014-10-01

    Three Bacillus cereus strains isolated from an outbreak of food poisoning caused by the consumption of fermented black beans (douchi) containing B. cereus is described. The outbreak involved 139 persons who had nausea, vomiting, and diarrhea. The strains were isolated from vomit and the unprepared douchi. Two of the strains produced the emetic toxin cereulide, as evidenced by polymerase chain reaction analysis for the presence of the nonribosomal synthetase cluster responsible for the synthesis of cereulide and by chemical analysis by high-performance liquid chromatography-mass spectrometry. These two strains belong to genetic group III of B. cereus, and multiple locus sequence typing revealed that the type was ST26, as a major part of B. cereus emetic strains. One of these strains produced significantly more cereulide at 37°C than the type cereulide producer (F4810/72), and it was also able to produce the toxin at 40°C and 42°C. The third strain belongs to genetic group IV, and it is a new multiple locus sequence type closely related to strains that are cytotoxic and enterotoxigenic. It possesses genes for hemolysin BL, nonhemolytic enterotoxin, and cytotoxin K2; however, it varies from the majority of strains possessing genes for hemolysin BL by not being hemolytic. Thus, two B. cereus strains producing the emetic toxin cereulide and a strain producing enterotoxins might have been involved in this food-poisoning incident caused by the consumption of a natural fermented food. The ability of one of the strains to produce cereulide at ≥37°C makes it possible that it is produced in the human gut in addition to occurring in the food.

  5. Healthcare- and Community-Associated Methicillin-Resistant Staphylococcus aureus (MRSA) and Fatal Pneumonia with Pediatric Deaths in Krasnoyarsk, Siberian Russia: Unique MRSA's Multiple Virulence Factors, Genome, and Stepwise Evolution.

    PubMed

    Khokhlova, Olga E; Hung, Wei-Chun; Wan, Tsai-Wen; Iwao, Yasuhisa; Takano, Tomomi; Higuchi, Wataru; Yachenko, Svetlana V; Teplyakova, Olga V; Kamshilova, Vera V; Kotlovsky, Yuri V; Nishiyama, Akihito; Reva, Ivan V; Sidorenko, Sergey V; Peryanova, Olga V; Reva, Galina V; Teng, Lee-Jene; Salmina, Alla B; Yamamoto, Tatsuo

    2015-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a common multidrug-resistant (MDR) pathogen. We herein discussed MRSA and its infections in Krasnoyarsk, Siberian Russia between 2007 and 2011. The incidence of MRSA in 3,662 subjects was 22.0% and 2.9% for healthcare- and community-associated MRSA (HA- and CA-MRSA), respectively. The 15-day mortality rates for MRSA hospital- and community-acquired pneumonia (HAP and CAP) were 6.5% and 50%, respectively. MRSA CAP cases included pediatric deaths; of the MRSA pneumonia episodes available, ≥27.3% were associated with bacteremia. Most cases of HA-MRSA examined exhibited ST239/spa3(t037)/SCCmecIII.1.1.2 (designated as ST239Kras), while all CA-MRSA cases examined were ST8/spa1(t008)/SCCmecIV.3.1.1(IVc) (designated as ST8Kras). ST239Kras and ST8Kras strongly expressed cytolytic peptide (phenol-soluble modulin α, PSMα; and δ-hemolysin, Hld) genes, similar to CA-MRSA. ST239Kras pneumonia may have been attributed to a unique set of multiple virulence factors (MVFs): toxic shock syndrome toxin-1 (TSST-1), elevated PSMα/Hld expression, α-hemolysin, the staphylococcal enterotoxin SEK/SEQ, the immune evasion factor SCIN/SAK, and collagen adhesin. Regarding ST8Kras, SEA was included in MVFs, some of which were common to ST239Kras. The ST239Kras (strain OC3) genome contained: a completely unique phage, φSa7-like (W), with no att repetition; S. aureus pathogenicity island SaPI2R, the first TSST-1 gene-positive (tst+) SaPI in the ST239 lineage; and a super copy of IS256 (≥22 copies/genome). ST239Kras carried the Brazilian SCCmecIII.1.1.2 and United Kingdom-type tst. ST239Kras and ST8Kras were MDR, with the same levofloxacin resistance mutations; small, but transmissible chloramphenicol resistance plasmids spread widely enough to not be ignored. These results suggest that novel MDR and MVF+ HA- and CA-MRSA (ST239Kras and ST8Kras) emerged in Siberian Russia (Krasnoyarsk) associated with fatal pneumonia, and also with ST

  6. Characterization and Comparison of 2 Distinct Epidemic Community-Associated Methicillin-Resistant Staphylococcus aureus Clones of ST59 Lineage

    PubMed Central

    Chen, Chih-Jung; Unger, Clemens; Hoffmann, Wolfgang; Lindsay, Jodi A.; Huang, Yhu-Chering; Götz, Friedrich

    2013-01-01

    Sequence type (ST) 59 is an epidemic lineage of community-associated (CA) methicillin-resistant Staphylococcus aureus (MRSA) isolates. Taiwanese CA-MRSA isolates belong to ST59 and can be grouped into 2 distinct clones, a virulent Taiwan clone and a commensal Asian-Pacific clone. The Taiwan clone carries the Panton–Valentine leukocidin (PVL) genes and the staphylococcal chromosomal cassette mec (SCCmec) VT, and is frequently isolated from patients with severe disease. The Asian-Pacific clone is PVL-negative, carries SCCmec IV, and a frequent colonizer of healthy children. Isolates of both clones were characterized by their ability to adhere to respiratory A549 cells, cytotoxicity to human neutrophils, and nasal colonization of a murine and murine sepsis models. Genome variation was determined by polymerase chain reaction of selected virulence factors and by multi-strain whole genome microarray. Additionally, the expression of selected factors was compared between the 2 clones. The Taiwan clone showed a much higher cytotoxicity to the human neutrophils and caused more severe septic infections with a high mortality rate in the murine model. The clones were indistinguishable in their adhesion to A549 cells and persistence of murine nasal colonization. The microarray data revealed that the Taiwan clone had lost the ø3-prophage that integrates into the β-hemolysin gene and includes staphylokinase- and enterotoxin P-encoding genes, but had retained the genes for human immune evasion, scn and chps. Production of the virulence factors did not differ significantly in the 2 clonal groups, although more α-toxin was expressed in Taiwan clone isolates from pneumonia patients. In conclusion, the Taiwan CA-MRSA clone was distinguished by enhanced virulence in both humans and an animal infection model. The evolutionary acquisition of PVL, the higher expression of α-toxin, and possibly the loss of a large portion of the β-hemolysin-converting prophage likely contribute to

  7. Genomic Features of Environmental and Clinical Vibrio parahaemolyticus Isolates Lacking Recognized Virulence Factors Are Dissimilar.

    PubMed

    Ronholm, J; Petronella, N; Chew Leung, C; Pightling, A W; Banerjee, S K

    2015-12-04

    Vibrio parahaemolyticus is a bacterial pathogen that can cause illness after the consumption or handling of contaminated seafood. The primary virulence factors associated with V. parahaemolyticus illness are thermostable direct hemolysin (TDH) and Tdh-related hemolysin (TRH). However, clinical strains lacking tdh and trh have recently been isolated, and these clinical isolates are poorly understood. To help understand the emergence of clinical tdh- and trh-negative isolates, a genomic approach was used to comprehensively compare 4 clinical tdh- and trh-negative isolates with 16 environmental tdh- and trh-negative isolates and 34 clinical isolates positive for tdh or trh, or both, with the objective of identifying genomic features that are unique to clinical tdh- and trh-negative isolates. The prevalence of pathogenicity islands (PAIs) common to clinical isolates was thoroughly examined in each of the clinical tdh- and trh-negative isolates. The tdh PAI was not present in any clinical or environmental tdh- and trh-negative isolates. The trh PAI was not present in any environmental isolates; however, in clinical tdh- and trh-negative isolate 10-4238, the majority of the trh PAI including a partial trh1 gene was present, which resulted in reclassification of this isolate as a tdh-negative and trh-positive isolate. In the other clinical tdh- and trh-negative isolates, neither the trh gene nor the trh PAI was present. We identified 862 genes in clinical tdh- and trh-negative isolates but not in environmental tdh- and trh-negative isolates. Many of these genes are highly homologous to genes found in common enteric bacteria and included genes encoding a number of chemotaxis proteins and a novel putative type VI secretion system (T6SS) effector and immunity protein (T6SS1). The availability of genome sequences from clinical V. parahaemolyticus tdh- and trh-negative isolates and the comparative analysis may help provide an understanding of how this pathotype is able to

  8. Response of Staphylococcus Aureus to a Spaceflight Analogue

    NASA Technical Reports Server (NTRS)

    Castro, S. L.; Ott, C. M.

    2010-01-01

    The decreased gravity of the spaceflight environment creates quiescent, low fluid shear conditions. This environment can impart considerable effects on the physiology of microorganisms as well as their interactions with potential hosts. Using the rotating wall vessel (RWV), as a spaceflight analogue, the consequence of low fluid shear culture on microbial pathogenesis has provided a better understanding of the risks to the astronaut crew from infectious microorganisms. While the outcome of low fluid shear culture has been investigated for several bacterial pathogens, little has been done to understand how this environmental factor affects Staphylococcus aureus. S. aureus is an opportunistic human pathogen which presents a high level of infection risk to the crew, as it has been isolated from both the space shuttle and International Space Station. Given that approximately forty percent of the population are carriers of the bacteria, eradication of this organism from in flight environments is impractical. These reasons have lead to us to assess the response of S. aureus to a reduced fluid shear environment. Culture in the RWV demonstrated that S. aureus grown under the low-shear condition had lower cell concentrations after 10 hours when compared to the control culture. Furthermore, the low-shear cultured bacteria displayed a reduction in carotenoid production, pigments responsible for their yellow/gold coloration. When exposed to various environmental stressors, post low-shear culture, a decrease in the ability to survive oxidative assault was observed compared to control cultures. The low fluid shear environment also resulted in a decrease in hemolysin secretion, a staphylococcal toxin responsible for red blood cell lysis. When challenged by the immune components present in human whole blood, low-shear cultured S. aureus demonstrated significantly reduced survival rates as compared to the control culture. Assays to determine the duration of these alterations

  9. Use of porcine vaginal tissue ex-vivo to model environmental effects on vaginal mucosa to toxic shock syndrome toxin-1

    SciTech Connect

    Davis, Catherine C.; Baccam, Mekhine; Mantz, Mary J.; Osborn, Thomas W.; Hill, Donna R.; Squier, Christopher A.

    2014-01-15

    Menstrual toxic shock syndrome (mTSS) is a rare, recognizable, and treatable disease that has been associated with tampon use epidemiologically. It involves a confluence of microbial risk factors (Staphylococcus aureus strains that produce the superantigen—TSST-1), as well as environmental characteristics of the vaginal ecosystem during menstruation and host susceptibility factors. This paper describes a series of experiments using the well-characterized model of porcine vaginal mucosa ex-vivo to assess the effect of these factors associated with tampon use on the permeability of the mucosa. The flux of radiolabeled TSST-1 and tritiated water ({sup 3}H{sub 2}O) through porcine vaginal mucosa was determined at various temperatures, after mechanical disruption of the epithelial surface by tape stripping, after treatment with surfactants or other compounds, and in the presence of microbial virulence factors. Elevated temperatures (42, 47 and 52 °C) did not significantly increase flux of {sup 3}H{sub 2}O. Stripping of the epithelial layers significantly increased the flux of labeled toxin in a dose-dependent manner. Addition of benzalkonium chloride (0.1 and 0.5%) and glycerol (4%) significantly increased the flux of {sup 3}H{sub 2}O but sodium lauryl sulfate at any concentration tested did not. The flux of the labeled toxin was significantly increased in the presence of benzalkonium chloride but not Pluronic® L92 and Tween 20 and significantly increased with addition of α-hemolysin but not endotoxin. These results show that the permeability of porcine vagina ex-vivo to labeled toxin or water can be used to evaluate changes to the vaginal environment and modifications in tampon materials, and thus aid in risk assessment. - Highlights: • Model assessed local effects of tampon use on vaginal mucosa. • Risks were evaluated using two tracers to assess permeability in an ex vivo model. • Mechanical damage to the epithelial surface increased tracer penetration.

  10. The toxR Gene of Vibrio (Listonella) anguillarum Controls Expression of the Major Outer Membrane Proteins but Not Virulence in a Natural Host Model

    PubMed Central

    Okuda, Jun; Nakai, Toshihiro; Chang, Park Se; Oh, Takanori; Nishino, Takeshi; Koitabashi, Tsutomu; Nishibuchi, Mitsuaki

    2001-01-01

    To examine the hypothesis that the ancestral role of the toxR gene in the family Vibrionaceae is control of the expression of outer membrane protein (OMP)-encoding genes for adaptation to environmental change, we investigated the role of the toxR gene in Vibrio anguillarum, an important fish pathogen. The toxR gene of V. angullarum (Va-toxR) was cloned from strain PT-87050 isolated from diseased ayu (Plecoglossus altivelis), and the sequence was analyzed. The toxR sequence was 63 to 51% identical to those reported for other species of the family Vibrionaceae. Distribution of the Va-toxR gene sequence in V. anguillarum strains of various serotypes was confirmed by using DNA probe and PCR methods. An isogenic toxR mutant of V. anguillarum PT-24, isolated from diseased ayu, was constructed by using an allelic exchange method. The wild-type strain and the toxR mutant did not differ in the ability to produce a protease(s) and a hemolysin(s) or in pathogenicity for ayu when examined by the intramuscular injection and immersion methods. A 35-kDa major OMP was not produced by the toxR mutant. However, a 46-kDa OMP was hardly detected in the wild-type strain but was produced as the major OMP by the toxR mutant. For the toxR mutant, the MICs of two β-lactam antibiotics were higher and the minimum bactericidal concentration of sodium dodecyl sulfate was lower than for the wild-type strain. Analysis of the N-terminal amino acid sequences of the 35- and 46-kDa OMPs indicated that these proteins are the porin-like OMPs and are related to the toxR-regulated major OMPs of the family Vibrionaceae. The results indicate that the toxR gene is not involved in virulence expression in V. anguillarum PT-24 and that toxR regulation of major OMPs is universal in the family Vibrionaceae. These results support the hypothesis that the ancestral role of the toxR gene is regulation of OMP gene expression and that only in some Vibrio species has ToxR been appropriated for the regulation of a

  11. Peritoneal Dialysis-Related Peritonitis Due to Staphylococcus aureus: A Single-Center Experience over 15 Years

    PubMed Central

    Barretti, Pasqual; Moraes, Taíse M. C.; Camargo, Carlos H.; Caramori, Jacqueline C. T.; Mondelli, Alessandro L.; Montelli, Augusto C.; da Cunha, Maria de Lourdes R. S.

    2012-01-01

    Peritonitis caused by Staphylococcus aureus is a serious complication of peritoneal dialysis (PD), which is associated with poor outcome and high PD failure rates. We reviewed the records of 62 S. aureus peritonitis episodes that occurred between 1996 and 2010 in the dialysis unit of a single university hospital and evaluated the host and bacterial factors influencing peritonitis outcome. Peritonitis incidence was calculated for three subsequent 5-year periods and compared using a Poisson regression model. The production of biofilm, enzymes, and toxins was evaluated. Oxacillin resistance was evaluated based on minimum inhibitory concentration and presence of the mecA gene. Logistic regression was used for the analysis of demographic, clinical, and microbiological factors influencing peritonitis outcome. Resolution and death rates were compared with 117 contemporary coagulase-negative staphylococcus (CoNS) episodes. The incidence of S. aureus peritonitis declined significantly over time from 0.13 in 1996–2000 to 0.04 episodes/patient/year in 2006–2010 (p = 0.03). The oxacillin resistance rate was 11.3%. Toxin and enzyme production was expressive, except for enterotoxin D. Biofilm production was positive in 88.7% of strains. The presence of the mecA gene was associated with a higher frequency of fever and abdominal pain. The logistic regression model showed that diabetes mellitus (p = 0.009) and β-hemolysin production (p = 0.006) were independent predictors of non-resolution of infection. The probability of resolution was higher among patients aged 41 to 60 years than among those >60 years (p = 0.02). A trend to higher death rate was observed for S. aureus episodes (9.7%) compared to CoNS episodes (2.5%), (p = 0.08), whereas resolution rates were similar. Despite the decline in incidence, S. aureus peritonitis remains a serious complication of PD that is associated with a high death rate. The outcome of this infection is negatively influenced

  12. Healthcare- and Community-Associated Methicillin-Resistant Staphylococcus aureus (MRSA) and Fatal Pneumonia with Pediatric Deaths in Krasnoyarsk, Siberian Russia: Unique MRSA's Multiple Virulence Factors, Genome, and Stepwise Evolution.

    PubMed

    Khokhlova, Olga E; Hung, Wei-Chun; Wan, Tsai-Wen; Iwao, Yasuhisa; Takano, Tomomi; Higuchi, Wataru; Yachenko, Svetlana V; Teplyakova, Olga V; Kamshilova, Vera V; Kotlovsky, Yuri V; Nishiyama, Akihito; Reva, Ivan V; Sidorenko, Sergey V; Peryanova, Olga V; Reva, Galina V; Teng, Lee-Jene; Salmina, Alla B; Yamamoto, Tatsuo

    2015-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a common multidrug-resistant (MDR) pathogen. We herein discussed MRSA and its infections in Krasnoyarsk, Siberian Russia between 2007 and 2011. The incidence of MRSA in 3,662 subjects was 22.0% and 2.9% for healthcare- and community-associated MRSA (HA- and CA-MRSA), respectively. The 15-day mortality rates for MRSA hospital- and community-acquired pneumonia (HAP and CAP) were 6.5% and 50%, respectively. MRSA CAP cases included pediatric deaths; of the MRSA pneumonia episodes available, ≥27.3% were associated with bacteremia. Most cases of HA-MRSA examined exhibited ST239/spa3(t037)/SCCmecIII.1.1.2 (designated as ST239Kras), while all CA-MRSA cases examined were ST8/spa1(t008)/SCCmecIV.3.1.1(IVc) (designated as ST8Kras). ST239Kras and ST8Kras strongly expressed cytolytic peptide (phenol-soluble modulin α, PSMα; and δ-hemolysin, Hld) genes, similar to CA-MRSA. ST239Kras pneumonia may have been attributed to a unique set of multiple virulence factors (MVFs): toxic shock syndrome toxin-1 (TSST-1), elevated PSMα/Hld expression, α-hemolysin, the staphylococcal enterotoxin SEK/SEQ, the immune evasion factor SCIN/SAK, and collagen adhesin. Regarding ST8Kras, SEA was included in MVFs, some of which were common to ST239Kras. The ST239Kras (strain OC3) genome contained: a completely unique phage, φSa7-like (W), with no att repetition; S. aureus pathogenicity island SaPI2R, the first TSST-1 gene-positive (tst+) SaPI in the ST239 lineage; and a super copy of IS256 (≥22 copies/genome). ST239Kras carried the Brazilian SCCmecIII.1.1.2 and United Kingdom-type tst. ST239Kras and ST8Kras were MDR, with the same levofloxacin resistance mutations; small, but transmissible chloramphenicol resistance plasmids spread widely enough to not be ignored. These results suggest that novel MDR and MVF+ HA- and CA-MRSA (ST239Kras and ST8Kras) emerged in Siberian Russia (Krasnoyarsk) associated with fatal pneumonia, and also with ST

  13. Healthcare- and Community-Associated Methicillin-Resistant Staphylococcus aureus (MRSA) and Fatal Pneumonia with Pediatric Deaths in Krasnoyarsk, Siberian Russia: Unique MRSA's Multiple Virulence Factors, Genome, and Stepwise Evolution

    PubMed Central

    Khokhlova, Olga E.; Hung, Wei-Chun; Wan, Tsai-Wen; Iwao, Yasuhisa; Takano, Tomomi; Higuchi, Wataru; Yachenko, Svetlana V.; Teplyakova, Olga V.; Kamshilova, Vera V.; Kotlovsky, Yuri V.; Nishiyama, Akihito; Reva, Ivan V.; Sidorenko, Sergey V.; Peryanova, Olga V.; Reva, Galina V.; Teng, Lee-Jene; Salmina, Alla B.; Yamamoto, Tatsuo

    2015-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a common multidrug-resistant (MDR) pathogen. We herein discussed MRSA and its infections in Krasnoyarsk, Siberian Russia between 2007 and 2011. The incidence of MRSA in 3,662 subjects was 22.0% and 2.9% for healthcare- and community-associated MRSA (HA- and CA-MRSA), respectively. The 15-day mortality rates for MRSA hospital- and community-acquired pneumonia (HAP and CAP) were 6.5% and 50%, respectively. MRSA CAP cases included pediatric deaths; of the MRSA pneumonia episodes available, ≥27.3% were associated with bacteremia. Most cases of HA-MRSA examined exhibited ST239/spa3(t037)/SCCmecIII.1.1.2 (designated as ST239Kras), while all CA-MRSA cases examined were ST8/spa1(t008)/SCCmecIV.3.1.1(IVc) (designated as ST8Kras). ST239Kras and ST8Kras strongly expressed cytolytic peptide (phenol-soluble modulin α, PSMα; and δ-hemolysin, Hld) genes, similar to CA-MRSA. ST239Kras pneumonia may have been attributed to a unique set of multiple virulence factors (MVFs): toxic shock syndrome toxin-1 (TSST-1), elevated PSMα/Hld expression, α-hemolysin, the staphylococcal enterotoxin SEK/SEQ, the immune evasion factor SCIN/SAK, and collagen adhesin. Regarding ST8Kras, SEA was included in MVFs, some of which were common to ST239Kras. The ST239Kras (strain OC3) genome contained: a completely unique phage, φSa7-like (W), with no att repetition; S. aureus pathogenicity island SaPI2R, the first TSST-1 gene-positive (tst+) SaPI in the ST239 lineage; and a super copy of IS256 (≥22 copies/genome). ST239Kras carried the Brazilian SCCmecIII.1.1.2 and United Kingdom-type tst. ST239Kras and ST8Kras were MDR, with the same levofloxacin resistance mutations; small, but transmissible chloramphenicol resistance plasmids spread widely enough to not be ignored. These results suggest that novel MDR and MVF+ HA- and CA-MRSA (ST239Kras and ST8Kras) emerged in Siberian Russia (Krasnoyarsk) associated with fatal pneumonia, and also with ST

  14. Vibrio vulnificus and Vibrio parahaemolyticus in U.S. retail shell oysters: a national survey from June 1998 to July 1999.

    PubMed

    Cook, David W; Oleary, Paul; Hunsucker, Jeff C; Sloan, Edna M; Bowers, John C; Blodgett, Robert J; Depaola, Angelo

    2002-01-01

    From June 1998 to July 1999, 370 lots of oysters in the shell were sampled at 275 different establishments (71%, restaurants or oyster bars; 27%, retail seafood markets: and 2%, wholesale seafood markets) in coastal and inland markets throughout the United States. The oysters were harvested from the Gulf (49%). Pacific (14%), Mid-Atlantic (18%), and North Atlantic (11%) Coasts of the United States and from Canada (8%). Densities of Vibrio vulnificus and Vibrio parahaemolyticus were determined using a modification of the most probable number (MPN) techniques described in the Food and Drug Administration's Bacteriological Analytical Manual. DNA probes and enzyme immunoassay were used to identify suspect isolates and to determine the presence of the thermostable direct hemolysin gene associated with pathogenicity of V. parahaemolyticus. Densities of both V. vulnifcus and V. parahaemolyticus in market oysters from all harvest regions followed a seasonal distribution, with highest densities in the summer. Highest densities of both organisms were observed in oysters harvested from the Gulf Coast, where densities often exceeded 10,000 MPN/g. The majority (78%) of lots harvested in the North Atlantic, Pacific, and Canadian Coasts had V. vulnificus densities below the detectable level of 0.2 MPN/g; none exceeded 100 MPN/g. V. parahaemolyticus densities were greater than those of V. vulnificus in lots from these same areas, with some lots exceeding 1,000 MPN/g for V. parahaemolyticus. Some lots from the Mid-Atlantic states exceeded 10,000 MPN/g for both V. vulnificus and V. parahaemolyicus. Overall, there was a significant correlation between V. vulificus and V. parahaemolyticus densities (r = 0.72, n = 202, P < 0.0001), but neither density correlated with salinity. Storage time significantly affected the V. vulnificus (10% decrease per day) and V. parahaemolyticus (7% decrease per day) densities in market oysters. The thermostable direct hemolysin gene associated with V

  15. Effects of Fuzhuan Brick-Tea Water Extract on Mice Infected with E. coli O157:H7.

    PubMed

    Wang, Yuanliang; Xu, Aiqing; Liu, Ping; Li, Zongjun

    2015-07-01

    Fuzhuan brick-tea extract (FBTE) affects the physiology of mice infected with Escherichia coli O157:H7. For 10 consecutive days, 0.05, 0.5, and 1.0 g/mL FBTE was administered intragastrically to three groups of infected Kunming mice, and changes in immunological function, hematology, and histopathology were examined. The results revealed upregulation of platelets, total protein, and albumin along with downregulation of serum triglycerides, aspartate aminotransferase, creatinine, and urea nitrogen in FBTE-treated mice. Histological sections of stomach, kidney, duodenum, ileum, and colon suggested that infected mucous membranes could be rehabilitated by low- and high-dose FBTE and that inflammation was alleviated. Similarly, increased thymic function in mice treated with middle- and high-dose FBTE led to elevated serum hemolysin antibody titer and increased CD4+ and CD8+ T cells, as indicated by CD4+ and CD8+ expression on intestinal mucosa. Monocyte and macrophage function was improved by three FBTE dosages tested. Colonic microbiota analysis by denaturing gradient gel electrophoresis (DGGE) revealed characteristic bands in infected mice treated with middle- and high-dose FBTE and increased species diversity in Lactobacillus, Bacteroides, and Clostridium cluster IV. These results suggest that FBTE may protect kidney and liver of mice infected with E. coli O157:H7, improve immune function, and regulate the colonic microbiota. PMID:26140539

  16. Discrimination of biological and chemical threat simulants in residue mixtures on multiple substrates.

    PubMed

    Gottfried, Jennifer L

    2011-07-01

    The potential of laser-induced breakdown spectroscopy (LIBS) to discriminate biological and chemical threat simulant residues prepared on multiple substrates and in the presence of interferents has been explored. The simulant samples tested include Bacillus atrophaeus spores, Escherichia coli, MS-2 bacteriophage, α-hemolysin from Staphylococcus aureus, 2-chloroethyl ethyl sulfide, and dimethyl methylphosphonate. The residue samples were prepared on polycarbonate, stainless steel and aluminum foil substrates by Battelle Eastern Science and Technology Center. LIBS spectra were collected by Battelle on a portable LIBS instrument developed by A3 Technologies. This paper presents the chemometric analysis of the LIBS spectra using partial least-squares discriminant analysis (PLS-DA). The performance of PLS-DA models developed based on the full LIBS spectra, and selected emission intensities and ratios have been compared. The full-spectra models generally provided better classification results based on the inclusion of substrate emission features; however, the intensity/ratio models were able to correctly identify more types of simulant residues in the presence of interferents. The fusion of the two types of PLS-DA models resulted in a significant improvement in classification performance for models built using multiple substrates. In addition to identifying the major components of residue mixtures, minor components such as growth media and solvents can be identified with an appropriately designed PLS-DA model. PMID:21331489

  17. Schisandra polysaccharide evokes immunomodulatory activity through TLR 4-mediated activation of macrophages.

    PubMed

    Zhao, Ting; Feng, Yun; Li, Jing; Mao, Riwen; Zou, Ye; Feng, Weiwei; Zheng, Daheng; Wang, Wei; Chen, Yao; Yang, Liuqing; Wu, Xiangyang

    2014-04-01

    Schisandra chinensis (Turcz.) Baill has been used in traditional Chinese medicine for centuries. Previous studies have shown that Schisandra polysaccharide (SCPP11) has robust antitumor activity in vivo. In this study, the immunomodulatory activity and mechanisms of action of SCPP11 were investigated further to reveal its mechanism of action against tumors. Results showed that SCPP11 increased the thymus and spleen indices, pinocytic activity of peritoneal macrophages, and hemolysin formation in CTX-induced immunosuppressed mice. Moreover, SCPP11 significantly increased immunoglobulin levels, cytokines levels in vivo and induced RAW264.7 cells to secrete cytokines in vitro. RAW264.7 cells pretreated with SCPP11 significantly inhibited the proliferation of HepG-2 cells. In addition, SCPP11 promoted both the expression of iNOS protein and of iNOS and TNF-α mRNA. TLR-4 is a possible receptor for SCPP11-mediated macrophage activation. Therefore, the data suggest that SCPP11 exerted its antitumor activity by improving immune system functions through TLR-4-mediated up-regulation of NO and TNF-α.

  18. Toxin genes profiles and toxin production ability of Bacillus cereus isolated from clinical and food samples.

    PubMed

    Kim, Jung-Beom; Kim, Jai-Moung; Cho, Seung-Hak; Oh, Hyuk-Soo; Choi, Na Jung; Oh, Deog-Hwan

    2011-01-01

    Bacillus cereus can cause diarrheal and emetic type of food poisoning but little study has been done on the main toxins of food poisoning caused by B. cereus in Korea. The objective of this study is to characterize the toxin gene profiles and toxin-producing ability of 120 B. cereus isolates from clinical and food samples in Korea. The detection rate of nheABC, hblCDA, entFM, and cytK enterotoxin gene among all B. cereus strains was 94.2, 90.0, 65.8, and 52.5%, respectively. The ces gene encoding emetic toxin was not detected in all strains. Bacillus cereus strains carried at least 1 of the 8 enterotoxin genes were classified into 12 groups according to the presence or absence of 8 virulence genes. The 3 major patterns, I (nheABC, hblCDA, entFM, and cytK gene), II (nheABC, hblCDA and entFM gene), and VI (nheABC and hblCDA gene), accounted for 79.2% of all strains (95 out of 120 B. cereus isolates). Non-hemolytic enterotoxin (NHE) and hemolysin BL (HBL) enterotoxins were produced by 107 and 100 strains, respectively. Our finding revealed that NHE and HBL enterotoxins encoded by nhe and hbl genes were the major toxins among B. cereus tested in this study and enterotoxic type of B. cereus was predominant in Korea.

  19. Genetic analysis of functions involved in adhesion of Pseudomonas putida to seeds.

    PubMed

    Espinosa-Urgel, M; Salido, A; Ramos, J L

    2000-05-01

    Many agricultural uses of bacteria require the establishment of efficient bacterial populations in the rhizosphere, for which colonization of plant seeds often constitutes a critical first step. Pseudomonas putida KT2440 is a strain that colonizes the rhizosphere of a number of agronomically important plants at high population densities. To identify the functions involved in initial seed colonization by P. putida KT2440, we subjected this strain to transposon mutagenesis and screened for mutants defective in attachment to corn seeds. Eight different mutants were isolated and characterized. While all of them showed reduced attachment to seeds, only two had strong defects in their adhesion to abiotic surfaces (glass and different plastics). Sequences of the loci affected in all eight mutants were obtained. None of the isolated genes had previously been described in P. putida, although four of them showed clear similarities with genes of known functions in other organisms. They corresponded to putative surface and membrane proteins, including a calcium-binding protein, a hemolysin, a peptide transporter, and a potential multidrug efflux pump. One other showed limited similarities with surface proteins, while the remaining three presented no obvious similarities with known genes, indicating that this study has disclosed novel functions.

  20. Chemical Composition and Immunomodulatory Activity of Mycelia of the Hairy Bracket Mushroom, Trametes hirsuta (Higher Basidiomycetes).

    PubMed

    Ma, Rongxia; Yang, Rongling; Liu, Xueming; Chen, Zhiyi; Yang, Chunying; Wang, Siyuan

    2015-01-01

    Trametes hirsuta is a medicinal mushroom that produces laccase. Its mycelium is a by-product when this species is used for laccase production. Aiming to develop its potential medicinal value, we investigated the chemical composition and immunomodulatory activity of T. hirsuta mycelia (THM). Dried THM contained 26.06% protein, 1.15% fat, 57.87% carbohydrates, and 5.47% ash. Sixteen free amino acids (2.63% total content) and 6 5'-nucleotides (adenosine 5'-monophosphate, cytidine 5'-monophosphate, guanosine 5'-monophosphate, uridine 5'-monophosphate, xanthosine 5'-monophosphate, and inosine 5'-monophosphate) constituting 0.275% were detected. Dominant sugars and polyols were fructose (2.47%), mannitol (2.03%), and glucose (1.8%); trehalose and arabinose contents were less than 0.10%. Evaluation of immunomodulatory activity in mice showed that THM could improve macrophage phagocytic function and serum hemolysin concentrations, but only the low-dose group significantly enhanced the natural killer cell activity and increased the spleen index, and only the middle-dose group remarkably increased the thymus index. Therefore, T. hirsuta mycelia could enhance immune function in mice and have immunomodulatory activity.

  1. The controversial nature of the Weissella genus: technological and functional aspects versus whole genome analysis-based pathogenic potential for their application in food and health

    PubMed Central

    Abriouel, Hikmate; Lerma, Leyre Lavilla; Casado Muñoz, María del Carmen; Montoro, Beatriz Pérez; Kabisch, Jan; Pichner, Rohtraud; Cho, Gyu-Sung; Neve, Horst; Fusco, Vincenzina; Franz, Charles M. A. P.; Gálvez, Antonio; Benomar, Nabil

    2015-01-01

    Despite the use of several Weissella (W.) strains for biotechnological and probiotic purposes, certain species of this genus were found to act as opportunistic pathogens, while strains of W. ceti were recognized to be pathogenic for farmed rainbow trout. Herein, we investigated the pathogenic potential of weissellas based on in silico analyses of the 13 whole genome sequences available to date in the NCBI database. Our screening allowed us to find several virulence determinants such as collagen adhesins, aggregation substances, mucus-binding proteins, and hemolysins in some species. Moreover, we detected several antibiotic resistance-encoding genes, whose presence could increase the potential pathogenicity of some strains, but should not be regarded as an excluding trait for beneficial weissellas, as long as these genes are not present on mobile genetic elements. Thus, selection of weissellas intended to be used as starters or for biotechnological or probiotic purposes should be investigated regarding their safety aspects on a strain to strain basis, preferably also by genome sequencing, since nucleotide sequence heterogeneity in virulence and antibiotic resistance genes makes PCR-based screening unreliable for safety assessments. In this sense, the application of W. confusa and W. cibaria strains as starter cultures or as probiotics should be approached with caution, by carefully selecting strains that lack pathogenic potential. PMID:26579103

  2. Fabrication of solid-state nanopores with single-nanometre precision.

    PubMed

    Storm, A J; Chen, J H; Ling, X S; Zandbergen, H W; Dekker, C

    2003-08-01

    Single nanometre-sized pores (nanopores) embedded in an insulating membrane are an exciting new class of nanosensors for rapid electrical detection and characterization of biomolecules. Notable examples include alpha-hemolysin protein nanopores in lipid membranes and solid-state nanopores in Si3N4. Here we report a new technique for fabricating silicon oxide nanopores with single-nanometre precision and direct visual feedback, using state-of-the-art silicon technology and transmission electron microscopy. First, a pore of 20 nm is opened in a silicon membrane by using electron-beam lithography and anisotropic etching. After thermal oxidation, the pore can be reduced to a single-nanometre when it is exposed to a high-energy electron beam. This fluidizes the silicon oxide leading to a shrinking of the small hole due to surface tension. When the electron beam is switched off, the material quenches and retains its shape. This technique dramatically increases the level of control in the fabrication of a wide range of nanodevices.

  3. Re-emergence of Vibrio tubiashii in bivalve shellfish aquaculture: severity, environmental drivers, geographic extent and management.

    PubMed

    Elston, Ralph A; Hasegawa, Hiroaki; Humphrey, Karen L; Polyak, Ildiko K; Häse, Claudia C

    2008-11-20

    During 2006 and 2007, we documented the re-emergence of severe episodes of vibriosis caused by Vibrio tubiashii in shellfish hatcheries on the west coast of North America. Lost larval and juvenile production included 3 previously undescribed hosts, Pacific (Crassostrea gigas) and Kumamoto (C. sikamea) oysters and geoduck clams Panope abrupta, with a 2007 decline in larval oyster production of approximately 59% in one hatchery. Losses of larval and juvenile bivalves were linked to V. tubiashii blooms in the coastal environment, which were associated with the apparent mixing of unusually warm surface seawater and intermittently upwelled cooler, nutrient- and Vibrio spp.- enriched seawater. The ocean temperature elevation anomaly in 2007 was not clearly linked to an El Niño event, as was a similar episode in 1998. Concentrations of the dominant shellfish-pathogenic vibrios were as high as 1.6 x 10(5) cfu ml(-1) in the cold, upwelled water. The bacteria possessed the genes coding for a protease and hemolysin described for V. tubiashii, and pathogenic isolates secreted these peptides. Lesions resulting from a classic invasive disease and a toxigenic noninvasive disease occurred in oyster and geoduck clam larvae. Management and prevention require reduction of incoming concentrations of the bacteria, reduction of contamination in water and air supplies and in stock chemical solutions, removal of bacterial toxins, and interruption of the cycle of bacterial amplification in the hatchery and in microalgal food supplies.

  4. Mannheimia haemolytica and Its Leukotoxin Cause Macrophage Extracellular Trap Formation by Bovine Macrophages

    PubMed Central

    Aulik, Nicole A.; Hellenbrand, Katrina M.

    2012-01-01

    Human and bovine neutrophils release neutrophil extracellular traps (NETs), which are protein-studded DNA matrices capable of extracellular trapping and killing of pathogens. Recently, we reported that bovine neutrophils release NETs in response to the important respiratory pathogen Mannheimia haemolytica and its leukotoxin (LKT). Here, we demonstrate macrophage extracellular trap (MET) formation by bovine monocyte-derived macrophages exposed to M. haemolytica or its LKT. Both native fully active LKT and noncytolytic pro-LKT (produced by an lktC mutant of M. haemolytica) stimulated MET formation. Confocal and scanning electron microscopy revealed a network of DNA fibrils with colocalized histones in extracellular traps released from bovine macrophages. Formation of METs required NADPH oxidase activity, as previously demonstrated for NET formation. METs formed in response to LKT trapped and killed a portion of the M. haemolytica cells. Bovine alveolar macrophages, but not peripheral blood monocytes, also formed METs in response to M. haemolytica cells. MET formation was not restricted to bovine macrophages. We also observed MET formation by the mouse macrophage cell line RAW 264.7 and by human THP-1 cell-derived macrophages, in response to Escherichia coli hemolysin. The latter is a member of the repeats-in-toxin (RTX) toxin family related to the M. haemolytica leukotoxin. This study demonstrates that macrophages, like neutrophils, can form extracellular traps in response to bacterial pathogens and their exotoxins. PMID:22354029

  5. Channel-tunnels: outer membrane components of type I secretion systems and multidrug efflux pumps of Gram-negative bacteria.

    PubMed

    Andersen, C

    2003-01-01

    For translocation across the cell envelope of Gram-negative bacteria, substances have to overcome two permeability barriers, the inner and outer membrane. Channel-tunnels are outer membrane proteins, which are central to two distinct export systems: the type I secretion system exporting proteins such as toxins or proteases, and efflux pumps discharging antibiotics, dyes, or heavy metals and thus mediating drug resistance. Protein secretion is driven by an inner membrane ATP-binding cassette (ABC) transporter while drug efflux occurs via an inner membrane proton antiporter. Both inner membrane transporters are associated with a periplasmic accessory protein that recruits an outer membrane channel-tunnel to form a functional export complex. Prototypes of these export systems are the hemolysin secretion system and the AcrAB/TolC drug efflux pump of Escherichia coli, which both employ TolC as an outer membrane component. Its remarkable conduit-like structure, protruding 100 A into the periplasmic space, reveals how both systems are capable of transporting substrates across both membranes directly from the cytosol into the external environment. Proteins of the channel-tunnel family are widespread within Gram-negative bacteria. Their involvement in drug resistance and in secretion of pathogenic factors makes them an interesting system for further studies. Understanding the mechanism of the different export apparatus could help to develop new drugs, which block the efflux pumps or the secretion system.

  6. Chemical Composition and Antipathogenic Activity of Artemisia annua Essential Oil from Romania.

    PubMed

    Marinas, Ioana C; Oprea, Eliza; Chifiriuc, Mariana Carmen; Badea, Irinel Adriana; Buleandra, Mihaela; Lazar, Veronica

    2015-10-01

    The essential oil extracted by hydrodistillation from Romanian Artemisia annua aerial parts was characterized by GC/MS analysis, which allowed the identification of 94.64% of the total oil composition. The main components were camphor (17.74%), α-pinene (9.66%), germacrene D (7.55%), 1,8-cineole (7.24%), trans-β-caryophyllene (7.02%), and artemisia ketone (6.26%). The antimicrobial activity of this essential oil was evaluated by determining the following parameters: minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), minimal fungicidal concentration (MFC), and minimal biofilm eradication concentration (MBEC). Moreover, the soluble virulence factors were quantified with different biochemical substrates incorporated in the culture media. The reference and resistant, clinical strains proved to be susceptible to the A. annua oil, with MICs ranging from 0.51 to 16.33 mg/ml. The tested essential oil also showed good antibiofilm activity, inhibiting both the initial stage of the microbial cell adhesion to the inert substratum and the preformed mature biofilm. When used at subinhibitory concentrations, the essential oil proved to inhibit the phenotypic expression of five soluble virulence factors (hemolysins, gelatinase, DNase, lipases, and lecithinases). Briefly, the present results showed that the A. annua essential oil contained antimicrobial compounds with selective activity on Gram-positive and Gram-negative bacterial strains as well as on yeast strains and which also interfere with the expression of cell-associated and soluble virulence factors.

  7. In silico analysis for identifying potential vaccine candidates against Staphylococcus aureus

    PubMed Central

    Sedighian, Hamid

    2015-01-01

    Purpose Staphylococcus aureus is one of the most important causes of nosocomial and community-acquired infections. The increasing incidence of multiple antibiotic-resistant S. aureus strains and the emergence of vancomycin resistant S. aureus strains have placed renewed interest on alternative means of prevention and control of infection. S. aureus produces a variety of virulence factors, so a multi-subunit vaccine will be more successful for preventing S. aureus infections than a mono-subunit vaccine. Materials and Methods We selected three important virulence factors of S. aureus, clumping factor A (ClfA), iron-regulated surface determinant (IsdB), and gamma hemolysin (Hlg) that are potential candidates for vaccine development. We designed synthetic genes encoding the clfA, isdB, and hlg and used bioinformatics tools to predict structure of the synthetic construct and its stabilities. VaxiJen analysis of the protein showed a high antigenicity. Linear and conformational B-cell epitopes were identified. Results The proteins encoded by these genes were useful as vaccine candidates against S. aureus infections. Conclusion In silico tools are highly suited to study, design, and evaluate vaccine strategies. PMID:25649548

  8. SarA based novel therapeutic candidate against Staphylococcus aureus associated with vascular graft infections.

    PubMed

    Arya, Rekha; Ravikumar, R; Santhosh, R S; Princy, S Adline

    2015-01-01

    Staphylococcus aureus is a common pathogen seen in prosthetic vascular graft, leading to high morbidity and mortality. The virulence genes for severity of infections are under the control of global regulators. Staphylococcal accessory regulator A (SarA) a known master controller of biofilm formation is an attractive target for the drug development. A structure based screening of lead compounds was employed for the identification of novel small molecule inhibitors targeted to interact to the DNA binding domain of the transcriptional activator, SarA and hinder its response over the control of genes that up-regulate the phenotype, biofilm. The top-hit SarA selective inhibitor, 4-[(2,4-diflurobenzyl)amino] cyclohexanol (SarABI) was further validated in-vitro for its efficacy. The SarABI was found to have MBIC50value of 200 μg/ml and also down-regulated the expression of the RNA effector, (RNAIII), Hemolysin (hld), and fibronectin-binding protein (fnbA). The anti-adherence property of SarABI on S. aureus invasion to the host epithelial cell lines (Hep-2) was examined where no significant attachment of S. aureus was observed. The SarABI inhibits the colonization of MDR S. aureus in animal model experiment significantly cohere to the molecular docking studies and in vitro experiments. So, we propose that the SarABI could be a novel substitute to overcome a higher degree of MDR S. aureus colonization on vascular graft. PMID:26074884

  9. Necrotizing fasciitis due to Vibrio cholerae non-O1/non-O139 after exposure to Austrian bathing sites.

    PubMed

    Hirk, Sonja; Huhulescu, Steliana; Allerberger, Franz; Lepuschitz, Sarah; Rehak, Sonja; Weil, Sandra; Gschwandtner, Elisabeth; Hermann, Michael; Neuhold, Stephanie; Zoufaly, Alexander; Indra, Alexander

    2016-02-01

    We report on two cases of necrotizing fasciitis of the lower leg due to nontoxigenic Vibrio cholerae (V. cholerae). A 73-year-old woman (case 1) and an 80-year-old man (case 2) were hospitalized with symptoms of necrotizing fasciitis on July 18 and August 15, 2015, respectively. In both cases, symptoms started the day after swimming in local ponds. Swabs gained intraoperatively and a blood culture from the male patient, yielded V. cholerae non-O1/non-O139, negative for cholera toxin gene ctx and positive for hemolysin genes hlyA and hlyB. Water samples taken from pond A on August 17, 2015 (32 days after exposure of case 1) and from pond B on August 20, 2015 (7 days after exposure of case 2) yielded non-O1/non-O139 V. cholerae in most-probable numbers of > 11,000 per 100 ml each. The occurrence of two cases of necrotizing fasciitis within a 1 month period related to two Austrian non-saline bathing waters, previously not known to harbor V. cholerae, is probably linked to the prevailing extreme weather conditions (heat wave, drought) this summer in Austria. While case 1 was discharged in good clinical condition after 73 days, case 2 died after four months of hospitalization. Public health authorities are challenged to assess the effects of long-term climate change on pathogen growth and survival in continental bodies of fresh water. PMID:26825075

  10. Nitrosylation: an adverse factor in Uremic Hemolytic Syndrome. Antitoxin effect of Ziziphus mistol Griseb.

    PubMed

    Virginia, Aiassa; Claudia, Albrecht; Soledad, Bustos Pamela; Gabriela, Ortega; Jorge, Eraso Alberto; Albesa, Inés

    2013-06-01

    Toxins of Escherichia coli (STEC) causing Uremic Hemolytic Syndrome (UHS) generate oxidative stress in human blood with more production of nitric oxide (NO) than reactive oxygen species (ROS). Shiga toxin (Stx) together with the hemolysin (Hly) increased lipid oxidation, as evaluated by malondialdehyde MDA and oxidation of proteins. The addition of Ziziphus mistol Griseb extracts decreased NO, ROS, MDA and simultaneously caused an increase in the degradation of oxidized proteins to advanced oxidation protein products (AOPPs) in controls and samples with toxins. Furthermore, the nitrosylated proteins/AOPP ratio was reduced, due to the increase of AOPP. Z. mistol Griseb extracts exhibited a high proportion of polyphenols and flavonoids, with evident correlation with ferrous reduction antioxidant potential (FRAP). The plasma of eight children with UHS showed oxidative stress and NO stimulus, comparable to the effect of toxins during the assays in vitro. UHS children presented high levels of nitrosylated proteins respect to control children of similar age. Although the degradation of oxidized proteins to AOPP rose in UHS children, the nitrosylated proteins/AOPP rate increased as a consequence of the elevated nitrosative stress observed in these patients. PMID:23454150

  11. Bacillus cereus can attack the cell membranes of the alga Chara corallina by means of HlyII.

    PubMed

    Kataev, Anatoly A; Andreeva-Kovalevskaya, Zhanna I; Solonin, Alexander S; Ternovsky, Vadim I

    2012-05-01

    We studied the influence of Bacillus cereus bacteria on cells of the freshwater alga Chara corallina. These bacteria and recombinant Bacillus subtilis strains are capable of producing the secreted toxin HlyII, which changes the electrophysiological parameters of the algal electrically excitable plasma membrane by forming pores. Cooperative incubation of bacterial cells, which carry active hlyII gene, and Chara corallina cells caused a decrease in the resting potential (V(m)) and plasma membrane resistance (R(m)) of algal cells. The efficiency of each strain was commensurable with its ability to produce HlyII. Purified hemolysin II caused a similar effect on V(m) and R(m) of intact and perfused cells. This protein changed the kinetics and magnitude of transient voltage-dependent calcium and calcium-activated chloride currents owing to the formation of additional Ca(2+)-permeable pores in algal cell membrane. Occurrence of the cellulose cell wall with pores 2.1 to 4.6nm in diameter suggests that HlyII molecules reach the plasma membrane surface strictly as monomers.

  12. Virulence potential and genetic diversity of Aeromonas caviae, Aeromonas veronii, and Aeromonas hydrophila clinical isolates from Mexico and Spain: a comparative study.

    PubMed

    Aguilera-Arreola, Ma Guadalupe; Hernández-Rodríguez, César; Zúñiga, Gerardo; Figueras, María José; Garduño, Rafael A; Castro-Escarpulli, Graciela

    2007-07-01

    A comparative study of 109 Aeromonas clinical isolates belonging to the 3 species most frequently isolated from patients with diarrhea in Mexico and Spain was performed to investigate the distribution of 3 prominent toxin genes and the gene encoding flagellin of lateral flagella; 4 well-established virulence factors in the genus Aeromonas. The aerolysin-hemolysin toxin genes were the most prevalent, being present in 89% of the total isolates. The ast toxin gene was conspicuously absent from the Aeromonas caviae and Aeromonas veronii groups but was present in 91% of the Aeromonas hydrophila isolates. Both the alt toxin gene and the lafA flagellin gene also had a low incidence in A. caviae and A. veronii. Differences in the prevalence of alt and lafA were observed between isolates from Mexico and Spain, confirming genus heterogeneity according to geographic location. Carriage of multiple toxin genes was primarily restricted to A. hydrophila isolates, suggesting that A. caviae and A. veronii isolates circulating in Mexico and Spain possess a limited array of virulence genes. Enterobacterial repetitive intergenetic consensus - polymerase chain reaction showed that the Aeromonas populations sampled lack dominant clones and were genetically heterogeneous, with A. caviae being the most diverse species. Further surveys of virulence determinants in genetically heterogeneous populations of Aeromonas isolates circulating worldwide are required to enhance the understanding of their capacity to cause disease.

  13. Use of zebrafish to probe the divergent virulence potentials and toxin requirements of extraintestinal pathogenic Escherichia coli.

    PubMed

    Wiles, Travis J; Bower, Jean M; Redd, Michael J; Mulvey, Matthew A

    2009-12-01

    Extraintestinal pathogenic E. coli (ExPEC) cause an array of diseases, including sepsis, neonatal meningitis, and urinary tract infections. Many putative virulence factors that might modulate ExPEC pathogenesis have been identified through sequencing efforts, epidemiology, and gene expression profiling, but few of these genes have been assigned clearly defined functional roles during infection. Using zebrafish embryos as surrogate hosts, we have developed a model system with the ability to resolve diverse virulence phenotypes and niche-specific restrictions among closely related ExPEC isolates during either localized or systemic infections. In side-by-side comparisons of prototypic ExPEC isolates, we observed an unexpectedly high degree of phenotypic diversity that is not readily apparent using more traditional animal hosts. In particular, the capacity of different ExPEC isolates to persist and multiply within the zebrafish host and cause disease was shown to be variably dependent upon two secreted toxins, alpha-hemolysin and cytotoxic necrotizing factor. Both of these toxins appear to function primarily in the neutralization of phagocytes, which are recruited in high numbers to sites of infection where they act as an essential host defense against ExPEC as well as less virulent E. coli strains. These results establish zebrafish as a valuable tool for the elucidation and functional analysis of both ExPEC virulence factors and host defense mechanisms.

  14. MOLECULAR CHARACTERIZATION OF VIRULENCE AND ANTIMICROBIAL SUSCEPTIBILITY PROFILES OF UROPATHOGENIC ESCHERICHIA COLI FROM PATIENTS IN A TERTIARY HOSPITAL, SOUTHERN THAILAND.

    PubMed

    Themphachanal, Monchanok; Kongpheng, Suttiporn; Rattanachuay, Pattamarat; Khianngam, Saowapar; Singkhamanan, Kamonnut; Sukhumungoon, Pharanai

    2015-11-01

    Among uropathogens, uropathogenic Escherichia coli (UPEC) is the most common cause of urinary tract infection (UTI) worldwide, but clinical aspects due to this bacterial species is not fully understood in southern Thailand. Two hundred fifty-four UPEC isolates from patients admitted to Maharaj Nakhon Si Thammarat Hospital, southern Thailand were examined for crucial virulence genes, showing that 33.5% contained at least one of the virulence, genes tested. Genes encoding P fimbria, cytotoxic necrotizing factor-1 and α-hemolysin constituted the majority (15.8%) carried by UPEC isolates. Phylogenetic group classification revealed that 57.5% of UPEC belonged to group D. Antimicrobial susceptibility tests showed that 70.5% and 65.1% of the isolates were resistant to ciprofloxacin and norfloxacin, respectively. Moreover, 50.0% of UPEC were capable of producing extended spectrum beta-lactamases. These findings should be of benefit for more appropriate treatment of UTI patients in this region of Thailand. Keywords: uropathogenic Escherichia coli, antibiotics resistance, cnfl, hlyA, pap, Thailand PMID:26867360

  15. Bacillus cereus, a Volatile Human Pathogen

    PubMed Central

    Bottone, Edward J.

    2010-01-01

    Summary: Bacillus cereus is a Gram-positive aerobic or facultatively anaerobic, motile, spore-forming, rod-shaped bacterium that is widely distributed environmentally. While B. cereus is associated mainly with food poisoning, it is being increasingly reported to be a cause of serious and potentially fatal non-gastrointestinal-tract infections. The pathogenicity of B. cereus, whether intestinal or nonintestinal, is intimately associated with the production of tissue-destructive exoenzymes. Among these secreted toxins are four hemolysins, three distinct phospholipases, an emesis-inducing toxin, and proteases. The major hurdle in evaluating B. cereus when isolated from a clinical specimen is overcoming its stigma as an insignificant contaminant. Outside its notoriety in association with food poisoning and severe eye infections, this bacterium has been incriminated in a multitude of other clinical conditions such as anthrax-like progressive pneumonia, fulminant sepsis, and devastating central nervous system infections, particularly in immunosuppressed individuals, intravenous drug abusers, and neonates. Its role in nosocomial acquired bacteremia and wound infections in postsurgical patients has also been well defined, especially when intravascular devices such as catheters are inserted. Primary cutaneous infections mimicking clostridial gas gangrene induced subsequent to trauma have also been well documented. B. cereus produces a potent β-lactamase conferring marked resistance to β-lactam antibiotics. Antimicrobials noted to be effective in the empirical management of a B. cereus infection while awaiting antimicrobial susceptibility results for the isolate include ciprofloxacin and vancomycin. PMID:20375358

  16. Exploring simvastatin, an antihyperlipidemic drug, as a potential topical antibacterial agent.

    PubMed

    Thangamani, Shankar; Mohammad, Haroon; Abushahba, Mostafa F N; Hamed, Maha I; Sobreira, Tiago J P; Hedrick, Victoria E; Paul, Lake N; Seleem, Mohamed N

    2015-01-01

    The rapid rise of bacterial resistance to traditional antibiotics combined with the decline in discovery of novel antibacterial agents has created a global public health crisis. Repurposing existing drugs presents an alternative strategy to potentially expedite the discovery of new antimicrobial drugs. The present study demonstrates that simvastatin, an antihyperlipidemic drug exhibited broad-spectrum antibacterial activity against important Gram-positive (including methicillin-resistant Staphylococcus aureus (MRSA)) and Gram-negative pathogens (once the barrier imposed by the outer membrane was permeabilized). Proteomics and macromolecular synthesis analyses revealed that simvastatin inhibits multiple biosynthetic pathways and cellular processes in bacteria, including selective interference of bacterial protein synthesis. This property appears to assist in simvastatin's ability to suppress production of key MRSA toxins (α-hemolysin and Panton-Valentine leucocidin) that impair healing of infected skin wounds. A murine MRSA skin infection experiment confirmed that simvastatin significantly reduces the bacterial burden and inflammatory cytokines in the infected wounds. Additionally, simvastatin exhibits excellent anti-biofilm activity against established staphylococcal biofilms and demonstrates the ability to be combined with topical antimicrobials currently used to treat MRSA skin infections. Collectively the present study lays the foundation for further investigation of repurposing simvastatin as a topical antibacterial agent to treat skin infections. PMID:26553420

  17. Molecular Characterization and Antimicrobial Resistance Profile of Methicillin-Resistant Staphylococcus aureus in Retail Chicken.

    PubMed

    Sallam, Khalid Ibrahim; Abd-Elghany, Samir Mohammed; Elhadidy, Mohamed; Tamura, Tomohiro

    2015-10-01

    The emergence of livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) in food-producing animals is of increasing interest, raising questions about the presence of MRSA in food of animal origin and potential sources of transmission to humans via the food chain. In this study, the prevalence, molecular characterization, virulence factors, and antimicrobial susceptibility patterns of MRSA isolates from 200 retail raw chicken samples in Egypt were determined. MRSA was detected by positive amplification of the mecA gene in 38% (76 of 200) of chicken samples analyzed. This represents a potential public health threat in Egypt, as this contamination rate seems to be the highest among other studies reported worldwide. Furthermore, genes encoding α-hemolysin (hla) and staphylococcal enterotoxins (sea, seb, and sec) were detected in all of the 288 MRSA isolates. Nonetheless, none of the strains tested carried tst, the gene encoding toxic shock syndrome toxin 1. Antimicrobial resistance of MRSA isolates was most frequently detected against penicillin (93.4%), ampicillin (88.9%), and cloxacillin (83.3%). These results suggest that retail chicken might be a significant potential source for transmission of multidrug-resistant and toxigenic S. aureus in Egypt. This underlines the need for stricter hygienic measures in chicken production in Egypt to minimize the risk of transmission of these strains to consumers. To the best of our knowledge, this is the first study that reports the isolation and molecular characterization of MRSA in retail chicken samples in Egypt.

  18. The catecholamine stress hormones norepinephrine and dopamine increase the virulence of pathogenic Vibrio anguillarum and Vibrio campbellii.

    PubMed

    Pande, Gde Sasmita J; Suong, Nguyen Thao; Bossier, Peter; Defoirdt, Tom

    2014-12-01

    Obtaining a better understanding of mechanisms involved in bacterial infections is of paramount importance for the development of novel agents to control disease caused by (antibiotic resistant) pathogens in aquaculture. In this study, we investigated the impact of catecholamine stress hormones on growth and virulence factor production of pathogenic vibrios (i.e. two Vibrio campbellii strains and two Vibrio anguillarum strains). Both norepinephrine and dopamine (at 100 μM) significantly induced growth in media containing serum. The compounds also increased swimming motility of the tested strains, whereas they had no effect on caseinase, chitinase, and hemolysin activities. Further, antagonists for eukaryotic catecholamine receptors were able to neutralize some of the effects of the catecholamines. Indeed, the dopaminergic receptor antagonist chlorpromazine neutralized the effect of dopamine, and the α-adrenergic receptor antagonists phentolamine and phenoxybenzamine neutralized the effect of norepinephrine, whereas the β-adrenergic receptor antagonist propranolol had limited to no effect. Finally, pretreatment of pathogenic V. campbellii with catecholamines significantly increased its virulence toward giant freshwater prawn larvae. However, the impact of catecholamine receptor antagonists on in vivo virulence was less clear-cut when compared to the in vitro experiments. In summary, our results show that—similar to enteric pathogens—catecholamines also increase the virulence of vibrios that are pathogenic to aquatic organisms by increasing motility and growth in media containing serum.

  19. Purification and characterization of a new Bacillus thuringiensis bacteriocin active against Listeria monocytogenes, Bacillus cereus and Agrobacterium tumefaciens.

    PubMed

    Kamoun, Fakher; Fguira, Ines Ben; Hassen, Najeh Belguith Ben; Mejdoub, Hafedh; Lereclus, Didier; Jaoua, Samir

    2011-09-01

    This study reports on the identification, characterization and purification of a new bacteriocin, named Bacthuricin F103, from a Bacillus thuringiensis strain BUPM103. Bacthuricin F103 production began in the early exponential phase and reached a maximum in the middle of the same phase. Two chromatographic methods based on high performance liquid chromatography and fast protein liquid chromatography systems were used to purify Bacthuricin F103. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that this bacteriocin had a molecular weight of approximately 11 kDa. It also showed a wide range of thermostability of up to 80 °C for 60 min and a broad spectrum of antimicrobial activity over a pH range of 3.0-10.0. This bacteriocin was noted, and for the first time, to exhibit potent antimicrobial activity against Agrobacterium subsp. strains, the major causal agents of crown gall disease in tomato and vineyard crops, and against several challenging organisms in food, such as Listeria monocytogenes and Bacillus cereus. Complete killing with immediate impact on cells was observed within a short period of time. The sequence obtained for Bacthuricin F103 by direct N-terminal sequencing shared considerable homology with hemolysin. Bacthuricin F103 was noted to act through the depletion of intracellular ions, which suggest that the cell membrane was a possible target to Bacthuricin F103. PMID:21487734

  20. Indole and 7-benzyloxyindole attenuate the virulence of Staphylococcus aureus.

    PubMed

    Lee, Jin-Hyung; Cho, Hyun Seob; Kim, Younghoon; Kim, Jung-Ae; Banskota, Suhrid; Cho, Moo Hwan; Lee, Jintae

    2013-05-01

    Human pathogens can readily develop drug resistance due to the long-term use of antibiotics that mostly inhibit bacterial growth. Unlike antibiotics, antivirulence compounds diminish bacterial virulence without affecting cell viability and thus, may not lead to drug resistance. Staphylococcus aureus is a major agent of nosocomial infections and produces diverse virulence factors, such as the yellow carotenoid staphyloxanthin, which promotes resistance to reactive oxygen species (ROS) and the host immune system. To identify novel antivirulence compounds, bacterial signal indole present in animal gut and diverse indole derivatives were investigated with respect to reducing staphyloxanthin production and the hemolytic activity of S. aureus. Treatment with indole or its derivative 7-benzyloxyindole (7BOI) caused S. aureus to become colorless and inhibited its hemolytic ability without affecting bacterial growth. As a result, S. aureus was more easily killed by hydrogen peroxide (H₂O₂) and by human whole blood in the presence of indole or 7BOI. In addition, 7BOI attenuated S. aureus virulence in an in vivo model of nematode Caenorhabditis elegans, which is readily infected and killed by S. aureus. Transcriptional analyses showed that both indole and 7BOI repressed the expressions of several virulence genes such as α-hemolysin gene hla, enterotoxin seb, and the protease genes splA and sspA and modulated the expressions of the important regulatory genes agrA and sarA. These findings show that indole derivatives are potential candidates for use in antivirulence strategies against persistent S. aureus infection. PMID:23318836

  1. Streptococcus pyogenes CAMP factor attenuates phagocytic activity of RAW 264.7 cells.

    PubMed

    Kurosawa, Mie; Oda, Masataka; Domon, Hisanori; Saitoh, Issei; Hayasaki, Haruaki; Terao, Yutaka

    2016-02-01

    Streptococcus pyogenes produces molecules that inhibit the function of human immune system, thus allowing the pathogen to grow and spread in tissues. It is known that S. pyogenes CAMP factor increases erythrocytosis induced by Staphylococcus aureus β-hemolysin. However, the effects of CAMP factor for immune cells are unclear. In this study, we investigated the effects of CAMP factor to macrophages. Western blotting analysis demonstrated that all examined strains expressed CAMP factor protein. In the presence of calcium or magnesium ion, CAMP factor was significantly released in the supernatant. In addition, both culture supernatant from S. pyogenes strain SSI-9 and recombinant CAMP factor dose-dependently induced vacuolation in RAW 264.7 cells, but the culture supernatant from Δcfa isogenic mutant strain did not. CAMP factor formed oligomers in RAW 264.7 cells in a time-dependent manner. CAMP factor suppressed cell proliferation via G2 phase cell cycle arrest without inducing cell death. Furthermore, CAMP factor reduced the uptake of S. pyogenes and phagocytic activity indicator by RAW 264.7 cells. These results suggest that CAMP factor works as a macrophage dysfunction factor. Therefore, we conclude that CAMP factor allows S. pyogenes to escape the host immune system, and contribute to the spread of streptococcal infection.

  2. Mutations in the bvgA gene of Bordetella pertussis that differentially affect regulation of virulence determinants.

    PubMed Central

    Stibitz, S

    1994-01-01

    By using chemical mutagenesis and genetic mapping, a search was undertaken for previously undescribed genes which may be involved in different regulatory mechanisms governing different virulence factors of Bordetella pertussis. Previous studies have shown that the fha locus encoding filamentous hemagglutinin is regulated directly by the bvgAS two component system, while regulation of ptx encoding pertussis toxin is less direct or occurs by a different mechanism. With a strain containing gene fusions to each of these regulated loci, screening was done for mutations which were defective for ptx expression but maintained normal or nearly normal levels of fha expression. Two mutations which had such a phenotype and were also deficient in adenylate cyclase toxin/hemolysin expression were found and characterized more fully. Both were found to affect residues in the C-terminal portion of the BvgA response regulator protein, a domain which shares sequence similarity with a family of regulatory proteins including FixJ, UhpA, MalT, RcsA, RcsB, and LuxR. The residues affected are within a region which, by extension from studies on the LuxR protein, may be involved in transcriptional activation. Images PMID:8083156

  3. Calcium-chelating alizarin and other anthraquinones inhibit biofilm formation and the hemolytic activity of Staphylococcus aureus

    PubMed Central

    Lee, Jin-Hyung; Kim, Yong-Guy; Yong Ryu, Shi; Lee, Jintae

    2016-01-01

    Staphylococcal biofilms are problematic and play a critical role in the persistence of chronic infections because of their abilities to tolerate antimicrobial agents. Thus, the inhibitions of biofilm formation and/or toxin production are viewed as alternative means of controlling Staphylococcus aureus infections. Here, the antibiofilm activities of 560 purified phytochemicals were examined. Alizarin at 10 μg/ml was found to efficiently inhibit biofilm formation by three S. aureus strains and a Staphylococcus epidermidis strain. In addition, two other anthraquinones purpurin and quinalizarin were found to have antibiofilm activity. Binding of Ca2+ by alizarin decreased S. aureus biofilm formation and a calcium-specific chelating agent suppressed the effect of calcium. These three anthraquinones also markedly inhibited the hemolytic activity of S. aureus, and in-line with their antibiofilm activities, increased cell aggregation. A chemical structure-activity relationship study revealed that two hydroxyl units at the C-1 and C-2 positions of anthraquinone play important roles in antibiofilm and anti-hemolytic activities. Transcriptional analyses showed that alizarin repressed the α-hemolysin hla gene, biofilm-related genes (psmα, rbf, and spa), and modulated the expressions of cid/lrg genes (the holin/antiholin system). These findings suggest anthraquinones, especially alizarin, are potentially useful for controlling biofilm formation and the virulence of S. aureus. PMID:26763935

  4. Functional distinctness in the exoproteomes of marine S ynechococcus

    PubMed Central

    Armengaud, Jean; Guerin, Philippe; Scanlan, David J.

    2015-01-01

    Summary The exported protein fraction of an organism may reflect its life strategy and, ultimately, the way it is perceived by the outside world. Bioinformatic prediction of the exported pan‐proteome of P rochlorococcus and S ynechococcus lineages demonstrated that (i) this fraction of the encoded proteome had a much higher incidence of lineage‐specific proteins than the cytosolic fraction (57% and 73% homologue incidence respectively) and (ii) exported proteins are largely uncharacterized to date (54%) compared with proteins from the cytosolic fraction (35%). This suggests that the genomic and functional diversity of these organisms lies largely in the diverse pool of novel functions these organisms export to/through their membranes playing a key role in community diversification, e.g. for niche partitioning or evading predation. Experimental exoproteome analysis of marine S ynechococcus showed transport systems for inorganic nutrients, an interesting array of strain‐specific exoproteins involved in mutualistic or hostile interactions (i.e. hemolysins, pilins, adhesins), and exoenzymes with a potential mixotrophic goal (i.e. exoproteases and chitinases). We also show how these organisms can remodel their exoproteome, i.e. by increasing the repertoire of interaction proteins when grown in the presence of a heterotroph or decrease exposure to prey when grown in the dark. Finally, our data indicate that heterotrophic bacteria can feed on the exoproteome of S ynechococcus. PMID:25727668

  5. Comparative analysis of quantitative reverse transcription real-time PCR and commercial enzyme imunoassays for detection of enterotoxigenic Bacillus thuringiensis isolates.

    PubMed

    Kaminska, Paulina S; Yernazarova, Aliya; Murawska, Emilia; Swiecicki, Jakub; Fiedoruk, Krzysztof; Bideshi, Dennis K; Swiecicka, Izabela

    2014-08-01

    Entomopathogenic Bacillus thuringiensis is closely related to Bacillus cereus, a human pathogen known to cause emesis and diarrhea. Standard detection methods do not distinguish these bacilli. Hemolysin BL (hbl) and non-hemolytic enterotoxin (nhe) genes that encode, respectively, HBL and NHE enterotoxins, are known to be harbored in both bacterial species, suggesting that differentiation of these bacilli is clinically and epidemiologically relevant. In this study the reliability of quantitative reverse transcription real-time PCR (qRT-PCR) and enzyme immunoassays (EIAs) in detecting hbl and nhe transcripts and corresponding toxins in environmental B. thuringiensis isolates was assessed. At least one enterotoxin gene was present in each isolate, and nhe or hbl genes were found in 85% and 55% of the strains, respectively. Based on statistical analyses, both BCET-RPLA and Duopath detected HBL at similar levels, and TECRA and Duopath can be used interchangeably for the detection of NHE, although TECRA has significantly lower sensitivity than Duopath. Thus, as potential enterotoxic B. thuringiensis strains occur in the natural environment, and EIA results may not correspond with the presence of enterotoxin genes and their expression, we suggest that reliable interpretation will be significantly enhanced by including qRT-PCR to support inferences based on EIAs.

  6. Bacillus cereus and Bacillus thuringiensis spores in Korean rice: prevalence and toxin production as affected by production area and degree of milling.

    PubMed

    Kim, Booyoung; Bang, Jihyun; Kim, Hoikyung; Kim, Yoonsook; Kim, Byeong-Sam; Beuchat, Larry R; Ryu, Jee-Hoon

    2014-09-01

    We determined the prevalence of and toxin production by Bacillus cereus and Bacillus thuringiensis in Korean rice as affected by production area and degree of milling. Rough rice was collected from 64 farms in 22 agricultural areas and polished to produce brown and white rice. In total, rice samples were broadly contaminated with B. cereus spores, with no effect of production area. The prevalence and counts of B. cereus spores declined as milling progressed. Frequencies of hemolysin BL (HBL) production by isolates were significantly (P ≤ 0.01) reduced as milling progressed. This pattern corresponded with the presence of genes encoding the diarrheal enterotoxins. The frequency of B. cereus isolates positive for hblC, hblD, or nheB genes decreased as milling progressed. Because most B. cereus isolates from rice samples contained six enterotoxin genes, we concluded that B. cereus in rice produced in Korea is predominantly of the diarrheagenic type. The prevalence of B. thuringiensis in rice was significantly lower than that of B. cereus and not correlated with production area. All B. thuringiensis isolates were of the diarrheagenic type. This study provides information useful for predicting safety risks associated with B. cereus and B. thuringiensis in rough and processed Korean rice.

  7. Distribution of Toxin Genes and Enterotoxins in Bacillus thuringiensis Isolated from Microbial Insecticide Products.

    PubMed

    Cho, Seung-Hak; Kang, Suk-Ho; Lee, Yea-Eun; Kim, Sung-Jo; Yoo, Young-Bin; Bak, Yeong-Seok; Kim, Jung-Beom

    2015-12-28

    Bacillus thuringiensis microbial insecticide products have been applied worldwide. Although a few cases of B. thuringiensis foodborne illness have been reported, little is known about the toxigenic properties of B. thuringiensis isolates. The aims of this study were to estimate the pathogenic potential of B. thuringiensis selected from microbial insecticide products, based on its possession of toxin genes and production of enterotoxins. Fifty-two B. thuringiensis strains selected from four kinds of microbial insecticide products were analyzed. PCR assay for detection of toxin genes and immunoassay for detection of enterotoxins were performed. The hemolysin BL complex as a major enterotoxin was produced by 17 (32.7%), whereas the nonhemolytic enterotoxin complex was detected in 1 (1.9%) of 52 B. thuringiensis strains. However, cytK, entFM, and ces genes were not detected in any of the tested B. thuringiensis strains. The potential risk of food poisoning by B. thuringiensis along with concerns over B. thuringiensis microbial insecticide products has gained attention recently. Thus, microbial insecticide products based on B. thuringiensis should be carefully controlled.

  8. Preparation, characterization, and immunogenicity of conjugate vaccines directed against Actinobacillus pleuropneumoniae virulence determinants.

    PubMed Central

    Byrd, W; Kadis, S

    1992-01-01

    Conjugate vaccines were prepared in an attempt to protect pigs against swine pleuropneumonia induced by Actinobacillus pleuropneumoniae (SPAP). Two subunit conjugates were prepared by coupling the A. pleuropneumoniae 4074 serotype 1 capsular polysaccharide (CP) to the hemolysin protein (HP) and the lipopolysaccharide (LPS) to the HP. Adipic acid dihydrazide was used as a spacer to facilitate the conjugation in a carbodiimide-mediated reaction. The CP and the LPS were found to be covalently coupled to the HP in the conjugates as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detergent gel chromatography analyses. Following a booster vaccination, pigs exhibited significantly high (P less than 0.05) immunoglobulin G antibodies against CP, LPS, and HP. The anti-CP and anti-LPS immunoglobulin G antibodies were found to function as opsonins in the phagocytosis of A. pleuropneumoniae by polymorphonuclear leukocytes, whereas antibodies to the HP neutralized the cytotoxic effect of the HP on polymorphonuclear leukocytes. No killing of A. pleuropneumoniae was observed when the effects of the antibodies were tested in the presence of complement. Thus, polysaccharide-protein A. pleuropneumoniae conjugates elicit significant antibody responses against each component of each conjugate, which could be instrumental in protecting swine against SPAP. Images PMID:1639471

  9. SarA based novel therapeutic candidate against Staphylococcus aureus associated with vascular graft infections

    PubMed Central

    Arya, Rekha; Ravikumar, R.; Santhosh, R. S.; Princy, S. Adline

    2015-01-01

    Staphylococcus aureus is a common pathogen seen in prosthetic vascular graft, leading to high morbidity and mortality. The virulence genes for severity of infections are under the control of global regulators. Staphylococcal accessory regulator A (SarA) a known master controller of biofilm formation is an attractive target for the drug development. A structure based screening of lead compounds was employed for the identification of novel small molecule inhibitors targeted to interact to the DNA binding domain of the transcriptional activator, SarA and hinder its response over the control of genes that up-regulate the phenotype, biofilm. The top-hit SarA selective inhibitor, 4-[(2,4-diflurobenzyl)amino] cyclohexanol (SarABI) was further validated in-vitro for its efficacy. The SarABI was found to have MBIC50value of 200 μg/ml and also down-regulated the expression of the RNA effector, (RNAIII), Hemolysin (hld), and fibronectin-binding protein (fnbA). The anti-adherence property of SarABI on S. aureus invasion to the host epithelial cell lines (Hep-2) was examined where no significant attachment of S. aureus was observed. The SarABI inhibits the colonization of MDR S. aureus in animal model experiment significantly cohere to the molecular docking studies and in vitro experiments. So, we propose that the SarABI could be a novel substitute to overcome a higher degree of MDR S. aureus colonization on vascular graft. PMID:26074884

  10. Characterization of Virulence Properties of Aeromonas veronii Isolated from Diseased Gibel Carp (Carassius gibelio).

    PubMed

    Sun, Jingjing; Zhang, Xiaojun; Gao, Xiaojian; Jiang, Qun; Wen, Yi; Lin, Li

    2016-01-01

    Aeromonas veronii is a kind of opportunistic pathogen to fish and humans, significantly impending aquaculture production. Recently, we isolated two A. veronii strains, named GYC1 and GYC2, from diseased Gibel carp (Carassius gibelio) in China. Based on gyrB (DNA gyrase B subunit) genes of GYC1 and GYC2, the constructed phylogenetic tree showed that the two strains were clustered with A. veronii. Sixteen virulence genes related to the pathogenicity of Aeromonas spp. were subjected to PCR assay. The genes of ompAI, ompAII, lafA, act, aer, fla, gcaT and acg were detected in the two strains, while genes of hly, ahp, lip, ast and alt were not detected. Additionally, genes eprCAI, ela and exu were only detected in the strain GYC1. Furthermore, the results of extracellular enzyme analysis revealed that the two isolates can produce hemolysin, caseinase, esterase, amylase and lecithinase, which were closely related to the pathogenicity of the two strains. However, the results showed that there was no gelatinase activity in either strain. According to the antibiotic resistant assay, the two strains were sensitive to cephalosporins and aminoglycosides, while they were resistant to penicillins and quinolones. Through this study, the virulence characteristics, including virulence genes and extracellular enzymes, the pathogenicity of A. veronii was clarified, enhancing the understanding about this pathogenic bacterium and providing the theoretical basis in disease control. PMID:27043558

  11. Characterization of Virulence Properties of Aeromonas veronii Isolated from Diseased Gibel Carp (Carassius gibelio)

    PubMed Central

    Sun, Jingjing; Zhang, Xiaojun; Gao, Xiaojian; Jiang, Qun; Wen, Yi; Lin, Li

    2016-01-01

    Aeromonas veronii is a kind of opportunistic pathogen to fish and humans, significantly impending aquaculture production. Recently, we isolated two A. veronii strains, named GYC1 and GYC2, from diseased Gibel carp (Carassius gibelio) in China. Based on gyrB (DNA gyrase B subunit) genes of GYC1 and GYC2, the constructed phylogenetic tree showed that the two strains were clustered with A. veronii. Sixteen virulence genes related to the pathogenicity of Aeromonas spp. were subjected to PCR assay. The genes of ompAI, ompAII, lafA, act, aer, fla, gcaT and acg were detected in the two strains, while genes of hly, ahp, lip, ast and alt were not detected. Additionally, genes eprCAI, ela and exu were only detected in the strain GYC1. Furthermore, the results of extracellular enzyme analysis revealed that the two isolates can produce hemolysin, caseinase, esterase, amylase and lecithinase, which were closely related to the pathogenicity of the two strains. However, the results showed that there was no gelatinase activity in either strain. According to the antibiotic resistant assay, the two strains were sensitive to cephalosporins and aminoglycosides, while they were resistant to penicillins and quinolones. Through this study, the virulence characteristics, including virulence genes and extracellular enzymes, the pathogenicity of A. veronii was clarified, enhancing the understanding about this pathogenic bacterium and providing the theoretical basis in disease control. PMID:27043558

  12. Identification of Vibrio harveyi as a causative bacterium for a tail rot disease of sea bream Sparus aurata from research hatchery in Malta.

    PubMed

    Haldar, S; Maharajan, A; Chatterjee, S; Hunter, S A; Chowdhury, N; Hinenoya, A; Asakura, M; Yamasaki, S

    2010-10-20

    A bacterial disease was reported from gilthead sea bream (Sparus aurata) within a hatchery environment in Malta. Symptoms included complete erosion of tail, infection in the eye, mucous secretion and frequent mortality. A total of 540 strains were initially isolated in marine agar from different infected body parts and culture water sources. Subsequently 100 isolates were randomly selected, identified biochemically and all were found to be Vibrio harveyi-related organisms; finally from 100 isolates a total of 13 numbers were randomly selected and accurately identified as V. harveyi by 16S rRNA gene sequencing and species-specific PCR. Ribotyping of these strains with HindIII revealed total of six clusters. In vivo challenge study with representative isolates from each cluster proved two clusters each were highly pathogenic, moderately pathogenic and non-pathogenic. All 13 isolates were positive for hemolysin gene, a potential virulence factor. Further analysis revealed probably a single copy of this gene was encoded in all isolates, although not in the same locus in the genome. Although V. harveyi was reported to be an important pathogen for many aquatic organisms, to our knowledge this might be the first report of disease caused by V. harveyi and their systematic study in the sea bream hatchery from Malta.

  13. Statin-conferred enhanced cellular resistance against bacterial pore-forming toxins in airway epithelial cells.

    PubMed

    Statt, Sarah; Ruan, Jhen-Wei; Hung, Li-Yin; Chang, Ching-Yun; Huang, Chih-Ting; Lim, Jae Hyang; Li, Jian-Dong; Wu, Reen; Kao, Cheng-Yuan

    2015-11-01

    Statins are widely used to prevent cardiovascular disease. In addition to their inhibitory effects on cholesterol synthesis, statins have beneficial effects in patients with sepsis and pneumonia, although molecular mechanisms have mostly remained unclear. Using human airway epithelial cells as a proper in vitro model, we show that prior exposure to physiological nanomolar serum concentrations of simvastatin (ranging from 10-1,000 nM) confers significant cellular resistance to the cytotoxicity of pneumolysin, a pore-forming toxin and the main virulence factor of Streptococcus pneumoniae. This protection could be demonstrated with a different statin, pravastatin, or on a different toxin, α-hemolysin. Furthermore, through the use of gene silencing, pharmacological inhibitors, immunofluorescence microscopy, and biochemical and metabolic rescue approaches, we demonstrate that the mechanism of protection conferred by simvastatin at physiological nanomolar concentrations could be different from the canonical mevalonate pathways seen in most other mechanistic studies conducted with statins at micromolar levels. All of these data are integrated into a protein synthesis-dependent, calcium-dependent model showing the interconnected pathways used by statins in airway epithelial cells to elicit an increased resistance to pore-forming toxins. This research fills large gaps in our understanding of how statins may confer host cellular protection against bacterial infections in the context of airway epithelial cells without the confounding effect from the presence of immune cells. In addition, our discovery could be potentially developed into a host-centric strategy for the adjuvant treatment of pore-forming toxin associated bacterial infections.

  14. Distinct commensals induce interleukin-1β via NLRP3 inflammasome in inflammatory monocytes to promote intestinal inflammation in response to injury

    PubMed Central

    Seo, Sang-Uk; Kamada, Nobuhiko; Muñoz-Planillo, Raúl; Kim, Yun-Gi; Kim, Donghyun; Koizumi, Yukiko; Hasegawa, Mizuho; Himpsl, Stephanie D.; Browne, Hilary P.; Lawley, Trevor D.; Mobley, Harry L. T.; Inohara, Naohiro; Núñez, Gabriel

    2015-01-01

    SUMMARY The microbiota stimulate inflammation, but the signaling pathways and the members of the microbiota involved remain poorly understood. We found that the microbiota induce interleukin-1β (IL-1β) release upon intestinal injury and this is mediated via the NLRP3 inflammasome. Enterobacteriaceae and in particular the pathobiont Proteus mirabilis, induced robust IL-1β release that was comparable to that induced by the pathogen Salmonella. Upon epithelial injury, production of IL-1β in the intestine was largely mediated by intestinal Ly6Chigh monocytes, required chemokine receptor CCR2 and was abolished by deletion of IL-1β in CCR2+ blood monocytes. Furthermore, colonization with P. mirabilis promoted intestinal inflammation upon intestinal injury via the production of hemolysin which required NLRP3 and IL-1 receptor signaling in vivo. Thus, upon intestinal injury, selective members of the microbiota stimulate newly recruited monocytes to induce NLRP3-dependent IL-1β release which promotes inflammation in the intestine. PMID:25862092

  15. Role of wild birds as carriers of multi-drug resistant Escherichia coli and Escherichia vulneris

    PubMed Central

    Shobrak, Mohammed Y.; Abo-Amer, Aly E.

    2014-01-01

    Emergence and distribution of multi-drug resistant (MDR) bacteria in environments pose a risk to human and animal health. A total of 82 isolates of Escherichia spp. were recovered from cloacal swabs of migrating and non-migrating wild birds. All bacterial isolates were identified and characterized morphologically and biochemically. 72% and 50% of isolates recovered from non-migrating and migrating birds, respectively, showed positive congo red dye binding (a virulence factor). Also, hemolysin production (a virulence factor) was showed in 8% of isolates recovered from non-migrating birds and 75% of isolates recovered from migrating birds. All isolates recovered from non-migrating birds were found resistant to Oxacillin while all isolates recovered from migrating birds demonstrated resistance to Oxacillin, Chloramphenicol, Oxytetracycline and Lincomycin. Some bacterial isolates recovered from non-migrating birds and migrating birds exhibited MDR phenotype. The MDR isolates were further characterized by API 20E and 16S rRNA as E. coli and E. vulneris. MDR Escherichia isolates contain ~1–5 plasmids of high-molecular weights. Accordingly, wild birds could create a potential threat to human and animal health by transmitting MDR bacteria to water streams and other environmental sources through their faecal residues, and to remote regions by migration. PMID:25763023

  16. Stable Free-Standing Lipid Bilayer Membranes in Norland Optical Adhesive 81 Microchannels.

    PubMed

    Marin, Victor; Kieffer, Roland; Padmos, Raymond; Aubin-Tam, Marie-Eve

    2016-08-01

    We report a simple, cost-effective, and reproducible method to form free-standing lipid bilayer membranes in microdevices made with Norland Optical Adhesive 81 (NOA81). Surface treatment with either alkylsilane or fluoroalkylsilane enables the self-assembly of stable 1,2-diphytanoyl-sn-glycero-3-phosphocholine 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC) membranes. Capacitance measurements are used to characterize the lipid bilayer and to follow its formation in real-time. With current recordings, we detect the insertion of single α-hemolysin pores into the bilayer membrane, demonstrating the possibility of using this device for single-channel electrophysiology sensing applications. Optical transparency of the device and vertical position of the lipid bilayer with respect to the microscope focal plane allows easy integration with other single-molecule techniques, such as optical tweezers. Therefore, this method to form long-lived lipid bilayers finds a wide range of applications, from sensing measurements to biophysical studies of lipid bilayers and associated proteins.

  17. Crystal Structure of Hcp from Acinetobacter baumannii: A Component of the Type VI Secretion System

    PubMed Central

    Ruiz, Federico M.; Santillana, Elena; Spínola-Amilibia, Mercedes; Torreira, Eva; Culebras, Esther; Romero, Antonio

    2015-01-01

    The type VI secretion system (T6SS) is a bacterial macromolecular machine widely distributed in Gram-negative bacteria, which transports effector proteins into eukaryotic host cells or other bacteria. Membrane complexes and a central tubular structure, which resembles the tail of contractile bacteriophages, compose the T6SS. One of the proteins forming this tube is the hemolysin co-regulated protein (Hcp), which acts as virulence factor, as transporter of effectors and as a chaperone. In this study, we present the structure of Hcp from Acinetobacter baumannii, together with functional and oligomerization studies. The structure of this protein exhibits a tight β barrel formed by two β sheets and flanked at one side by a short α-helix. Six Hcp molecules associate to form a donut-shaped hexamer, as observed in both the crystal structure and solution. These results emphasize the importance of this oligomerization state in this family of proteins, despite the low similarity of sequence among them. The structure presented in this study is the first one for a protein forming part of a functional T6SS from A. baumannii. These results will help us to understand the mechanism and function of this secretion system in this opportunistic nosocomial pathogen. PMID:26079269

  18. The agr Inhibitors Solonamide B and Analogues Alter Immune Responses to Staphylococccus aureus but Do Not Exhibit Adverse Effects on Immune Cell Functions.

    PubMed

    Baldry, Mara; Kitir, Betül; Frøkiær, Hanne; Christensen, Simon B; Taverne, Nico; Meijerink, Marjolein; Franzyk, Henrik; Olsen, Christian A; Wells, Jerry M; Ingmer, Hanne

    2016-01-01

    Staphylococcus aureus infections are becoming increasingly difficult to treat due to antibiotic resistance with the community-associated methicillin-resistant S. aureus (CA-MRSA) strains such as USA300 being of particular concern. The inhibition of bacterial virulence has been proposed as an alternative approach to treat multi-drug resistant pathogens. One interesting anti-virulence target is the agr quorum-sensing system, which regulates virulence of CA-MRSA in response to agr-encoded autoinducing peptides. Agr regulation confines exotoxin production to the stationary growth phase with concomitant repression of surface-expressed adhesins. Solonamide B, a non-ribosomal depsipeptide of marine bacterial origin, was recently identified as a putative anti-virulence compound that markedly reduced expression of α-hemolysin and phenol-soluble modulins. To further strengthen solonamide anti-virulence candidacy, we report the chemical synthesis of solonamide analogues, investigation of structure-function relationships, and assessment of their potential to modulate immune cell functions. We found that structural differences between solonamide analogues confer significant differences in interference with agr, while immune cell activity and integrity is generally not affected. Furthermore, treatment of S. aureus with selected solonamides was found to only marginally influence the interaction with fibronectin and biofilm formation, thus addressing the concern that application of compounds inducing an agr-negative state may have adverse interactions with host factors in favor of host colonization. PMID:26731096

  19. The clinical significance of anti-H in an individual with the Oh (Bombay) phenotype.

    PubMed

    Davey, R J; Tourault, M A; Holland, P V

    1978-01-01

    To evaluate the clinical significance of anti-H present in individuals with the Oh (Bombay) phenotype, red blood cell 51chromium survival studies and related serological tests were undertaken in an Oh (Bombay) individual. A small sample of group O donor red blood cells was labeled with 51chromium and infused into the patient. The T 1/2 of the infused cells was six minutes, with two percent of the cells surviving at 24 hours. A similar study using the patient's own labeled red blood cells demonstrated 100 per cent survival at 24 hours. Initial laboratory studies indicated that the anti-H was active in saline at 4, 22 and 37 C and by the indirect antiglobulin test. Analysis of the antibody in both preand posttransfusion specimens showed it to have both IgM and IgG components. The anti-H titer at 37 C rose from 1:4 prior to the infusion of the O cells to 1:32 one week postinfusion, and a partial hemolysin appeared. Saliva inhibition studies demonstrated that the antibody was neutralizable prior to the group O exposure but was not neutralizable one week post exposure. We conclude that the anti-H present in this individual rapidly destroyed infused group O red blood cells. Individuals with the Oh (Bombay) phenotype should be transfused only with Oh (Bombay) blood.

  20. Mn(II) Oxidation Is Catalyzed by Heme Peroxidases in “Aurantimonas manganoxydans” Strain SI85-9A1 and Erythrobacter sp. Strain SD-21▿

    PubMed Central

    Anderson, C. R.; Johnson, H. A.; Caputo, N.; Davis, R. E.; Torpey, J. W.; Tebo, B. M.

    2009-01-01

    A new type of manganese-oxidizing enzyme has been identified in two alphaproteobacteria, “Aurantimonas manganoxydans” strain SI85-9A1 and Erythrobacter sp. strain SD-21. These proteins were identified by tandem mass spectrometry of manganese-oxidizing bands visualized by native polyacrylamide gel electrophoresis in-gel activity assays and fast protein liquid chromatography-purified proteins. Proteins of both alphaproteobacteria contain animal heme peroxidase and hemolysin-type calcium binding domains, with the 350-kDa active Mn-oxidizing protein of A. manganoxydans containing stainable heme. The addition of both Ca2+ ions and H2O2 to the enriched protein from Aurantimonas increased manganese oxidation activity 5.9-fold, and the highest activity recorded was 700 μM min−1 mg−1. Mn(II) is oxidized to Mn(IV) via an Mn(III) intermediate, which is consistent with known manganese peroxidase activity in fungi. The Mn-oxidizing protein in Erythrobacter sp. strain SD-21 is 225 kDa and contains only one peroxidase domain with strong homology to the first 2,000 amino acids of the peroxidase protein from A. manganoxydans. The heme peroxidase has tentatively been named MopA (manganese-oxidizing peroxidase) and sheds new light on the molecular mechanism of Mn oxidation in prokaryotes. PMID:19411418

  1. Identification of a novel insertion sequence element in Streptococcus agalactiae. bspeller@imib.rwth-aachen.de.

    PubMed

    Spellerberg, B; Martin, S; Franken, C; Berner, R; Lütticken, R

    2000-01-01

    Gain and loss of bacterial pathogenicity is often associated with mobile genetic elements. A novel insertion sequence (IS) element designated ISSa4 was identified in Streptococcus agalactiae (group B streptococci). The 963bp IS element is flanked by 25bp perfect inverted repeats and led to the duplication of a 9bp target sequence at the insertion site. ISSa4 contains one open reading frame coding for a putative transposase of 287 aa and exhibits closest similarities to insertion elements of the IS982 family which has previously not been identified in streptococci. Analysis of different S. agalactiae strains showed that the copy number of ISSa4 in S. agalactiae varies significantly between strains. The S. agalactiae strain with the highest copy number of ISSa4 was nonhemolytic and harbored one copy inserted in cylB, which encodes the membrane-spanning domain of the putative hemolysin transporter (Spellerberg et al., 1999. Identification of genetic determinants for the hemolytic activity of Streptococcus agalactiae by ISS1 transposition. J. Bacteriol. 181, 3212-3219). Determination of the distribution of ISSa4 in different S. agalactiae strains revealed that ISSa4 could be detected only in strains isolated after 1996, which might indicate a recent acquisition of this novel insertion element by S. agalactiae.

  2. Genomic Diversity of Escherichia Isolates from Diverse Habitats

    PubMed Central

    Yoder-Himes, Deborah R.; Tiedje, James M.; Konstantinidis, Konstantinos T.

    2012-01-01

    Our understanding of the Escherichia genus is heavily biased toward pathogenic or commensal isolates from human or animal hosts. Recent studies have recovered Escherichia isolates that persist, and even grow, outside these hosts. Although the environmental isolates are typically phylogenetically distinct, they are highly related to and phenotypically indistinguishable from their human counterparts, including for the coliform test. To gain insights into the genomic diversity of Escherichia isolates from diverse habitats, including freshwater, soil, animal, and human sources, we carried out comparative DNA-DNA hybridizations using a multi-genome E. coli DNA microarray. The microarray was validated based on hybridizations with selected strains whose genome sequences were available and used to assess the frequency of microarray false positive and negative signals. Our results showed that human fecal isolates share two sets of genes (n>90) that are rarely found among environmental isolates, including genes presumably important for evading host immune mechanisms (e.g., a multi-drug transporter for acids and antimicrobials) and adhering to epithelial cells (e.g., hemolysin E and fimbrial-like adhesin protein). These results imply that environmental isolates are characterized by decreased ability to colonize host cells relative to human isolates. Our study also provides gene markers that can distinguish human isolates from those of warm-blooded animal and environmental origins, and thus can be used to more reliably assess fecal contamination in natural ecosystems. PMID:23056556

  3. Modulation of host immune defenses by Aeromonas and Yersinia species: convergence on toxins secreted by various secretion systems

    PubMed Central

    Rosenzweig, Jason A.; Chopra, Ashok K.

    2013-01-01

    Like other pathogenic bacteria, Yersinia and Aeromonas species have been continuously co-evolving with their respective hosts. Although the former is a bonafide human pathogen, the latter has gained notararity as an emerging disease-causing agent. In response to immune cell challenges, bacterial pathogens have developed diverse mechanism(s) enabling their survival, and, at times, dominance over various host immune defense systems. The bacterial type three secretion system (T3SS) is evolutionarily derived from flagellar subunits and serves as a vehicle by which microbes can directly inject/translocate anti-host factors/effector proteins into targeted host immune cells. A large number of Gram-negative bacterial pathogens possess a T3SS empowering them to disrupt host cell signaling, actin cytoskeleton re-arrangements, and even to induce host-cell apoptotic and pyroptotic pathways. All pathogenic yersiniae and most Aeromonas species possess a T3SS, but they also possess T2- and T6-secreted toxins/effector proteins. This review will focus on the mechanisms by which the T3SS effectors Yersinia outer membrane protein J (YopJ) and an Aeromonas hydrophila AexU protein, isolated from the diarrheal isolate SSU, mollify host immune system defenses. Additionally, the mechanisms that are associated with host cell apoptosis/pyroptosis by Aeromonas T2SS secreted Act, a cytotoxic enterotoxin, and Hemolysin co-regulated protein (Hcp), an A. hydrophila T6SS effector, will also be discussed. PMID:24199174

  4. [Resistance to antimicrobial agents, hemolytic activity and plasmids in Aeromonas species].

    PubMed

    Morita, K; Watanabe, N; Kanamori, M

    1990-06-01

    A total of 174 Aeromonas isolates consisting of 100 strains from patients with diarrhea being mainly overseas travellers nd healthy subjects, and 74 strains from environmental sources including foods, fish, fresh water, sea water and river soil collected in the area of Tokyo Metropolis and Kanagawa Prefecture was examined for the antimicrobial resistance, presence of plasmids and hemolytic activity. Almost all the isolates (99.4%) were resistant to aminobenzyl penicillin. The isolation frequency of chloramphenicol- or tetracycline-resistant strain was low. Most environmental isolates of A. hydrophila were resistant to multiple antimicrobial agents. Thirty-seven percent of environmental isolates and 39% of human fecal ones carried plasmids. In environmental isolates, seven A. hydrophila and three A. sobria strains carried 63- to 150-kilobase pair (kb) conjugative R plasmids. Two A. hydrophila strains from both the healthy subject and domestic case with diarrhea carried 58- to 90-kb conjugative R plasmids, respectively. None of the isolates from the feces of overseas traveller's diarrhea carried the plasmid. Irrespective of the sources. A. hydrophila showed the highest hemolytic activity among three Aeromonas species. Eighty percent or more of A. hydrophila isolates were of hemolysin positive. The hemolytic titer of A. hydrophila strains from human feces was higher than that of the strains from environmental sources. PMID:2401817

  5. Cell envelope of Bordetella pertussis: immunological and biochemical analyses and characterization of a major outer membrane porin protein

    SciTech Connect

    Armstrong, S.K.

    1986-01-01

    Surface molecules of Bordetella pertussis which may be important in metabolism, pathogenesis, and immunity to whooping cough were examined using cell fractionation and /sup 125/I cell surface labeling. Antigenic envelope proteins were examined by immunofluorescence microscopy and Western blotting procedures using monoclonal antibodies and convalescent sera. A surface protein with a high M/sub r/, missing in a mutant lacking the filamentous hemagglutinin, was identified in virulent Bordetella pertussis but was absent in virulent B. pertussis strains. At least three envelope proteins were found only in virulent B. pertussis strains and were absent or diminished in avirulent and most phenotypically modulated strains. Transposon-induced mutants unable to produce hemolysin, dermonecrotic toxin, pertussis toxin, and filamentous hemagglutinin also lacked these three envelope proteins, confirming that virulence-associated envelope proteins were genetically regulated with other virulence-associated traits. Two dimensional gel electrophoresis revealed at least five heat modifiable proteins which migrated as higher or lower M/sub r/ moieties if solubilized at 25/sup 0/C instead of 100/sup 0/C.

  6. Enterococcus faecalis from Food, Clinical Specimens, and Oral Sites: Prevalence of Virulence Factors in Association with Biofilm Formation

    PubMed Central

    Anderson, Annette C.; Jonas, Daniel; Huber, Ingrid; Karygianni, Lamprini; Wölber, Johan; Hellwig, Elmar; Arweiler, Nicole; Vach, Kirstin; Wittmer, Annette; Al-Ahmad, Ali

    2016-01-01

    Enterococci have gained significance as the cause of nosocomial infections; they occur as food contaminants and have also been linked to dental diseases. E. faecalis has a great potential to spread virulence as well as antibiotic resistance genes via horizontal gene transfer. The integration of food-borne enterococci into the oral biofilm in-vivo has been observed. Therefore, we investigated the virulence determinants and antibiotic resistance of 97 E. faecalis isolates from the oral cavity, food, and clinical specimens. In addition, phenotypic expression of gelatinase and cytolysin were tested, in-vitro biofilm formation was quantified and isolates were compared for strain relatedness via pulsed field gel electrophoresis (PFGE). Each isolate was found to possess two or more virulence genes, most frequently gelE, efaA, and asa1. Notably, plaque/saliva isolates possessed the highest abundance of virulence genes, the highest levels of phenotypic gelatinase and hemolysin activity and concurrently a high ability to form biofilm. The presence of asa1 was associated with biofilm formation. The biofilm formation capacity of clinical and plaque/saliva isolates was considerably higher than that of food isolates and they also showed similar antibiotic resistance patterns. These results indicate that the oral cavity can constitute a reservoir for virulent E. faecalis strains possessing antibiotic resistance traits and at the same time distinct biofilm formation capabilities facilitating exchange of genetic material. PMID:26793174

  7. Roles of Hcp family proteins in the pathogenesis of the porcine extraintestinal pathogenic Escherichia coli type VI secretion system

    PubMed Central

    Peng, Ying; Wang, Xiangru; Shou, Jin; Zong, Bingbing; Zhang, Yanyan; Tan, Jia; Chen, Jing; Hu, Linlin; Zhu, Yongwei; Chen, Huanchun; Tan, Chen

    2016-01-01

    Hcp (hemolysin-coregulated protein) is considered a vital component of the functional T6SS (Type VI Secretion System), which is a newly discovered secretion system. Our laboratory has previously sequenced the whole genome of porcine extraintestinal pathogenic E. coli (ExPEC) strain PCN033, and identified an integrated T6SS encoding three different hcp family genes. In this study, we first identified a functional T6SS in porcine ExPEC strain PCN033, and demonstrated that the Hcp family proteins were involved in bacterial competition and the interactions with other cells. Interestingly, the three Hcp proteins had different functions. Hcp2 functioned predominantly in bacterial competition; all three proteins were involved in the colonization of mice; and Hcp1 and Hcp3 were predominantly contributed to bacterial-eukaryotic cell interactions. We showed an active T6SS in porcine ExPEC strain PCN033, and the Hcp family proteins had different functions in their interaction with other bacteria or host cells. PMID:27229766

  8. Staphylococcus aureus Formyl-Methionyl Transferase Mutants Demonstrate Reduced Virulence Factor Production and Pathogenicity

    PubMed Central

    Lewandowski, Thomas; Huang, Jianzhong; Fan, Frank; Rogers, Shannon; Gentry, Daniel; Holland, Reannon; DeMarsh, Peter; Zalacain, Magdalena

    2013-01-01

    Inhibitors of peptide deformylase (PDF) represent a new class of antibacterial agents with a novel mechanism of action. Mutations that inactivate formyl methionyl transferase (FMT), the enzyme that formylates initiator methionyl-tRNA, lead to an alternative initiation of protein synthesis that does not require deformylation and are the predominant cause of resistance to PDF inhibitors in Staphylococcus aureus. Here, we report that loss-of-function mutations in FMT impart pleiotropic effects that include a reduced growth rate, a nonhemolytic phenotype, and a drastic reduction in production of multiple extracellular proteins, including key virulence factors, such as α-hemolysin and Panton-Valentine leukocidin (PVL), that have been associated with S. aureus pathogenicity. Consequently, S. aureus FMT mutants are greatly attenuated in neutropenic and nonneutropenic murine pyelonephritis infection models and show very high survival rates compared with wild-type S. aureus. These newly discovered effects on extracellular virulence factor production demonstrate that FMT-null mutants have a more severe fitness cost than previously anticipated, leading to a substantial loss of pathogenicity and a restricted ability to produce an invasive infection. PMID:23571548

  9. Listeria monocytogenes, a food-borne pathogen.

    PubMed Central

    Farber, J M; Peterkin, P I

    1991-01-01

    The gram-positive bacterium Listeria monocytogenes is an ubiquitous, intracellular pathogen which has been implicated within the past decade as the causative organism in several outbreaks of foodborne disease. Listeriosis, with a mortality rate of about 24%, is found mainly among pregnant women, their fetuses, and immunocompromised persons, with symptoms of abortion, neonatal death, septicemia, and meningitis. Epidemiological investigations can make use of strain-typing procedures such as DNA restriction enzyme analysis or electrophoretic enzyme typing. The organism has a multifactorial virulence system, with the thiol-activated hemolysin, listeriolysin O, being identified as playing a crucial role in the organism's ability to multiply within host phagocytic cells and to spread from cell to cell. The organism occurs widely in food, with the highest incidences being found in meat, poultry, and seafood products. Improved methods for detecting and enumerating the organism in foodstuffs are now available, including those based on the use of monoclonal antibodies, DNA probes, or the polymerase chain reaction. As knowledge of the molecular and applied biology of L. monocytogenes increases, progress can be made in the prevention and control of human infection. PMID:1943998

  10. IVIG-mediated protection against necrotizing pneumonia caused by MRSA.

    PubMed

    Diep, Binh An; Le, Vien T M; Badiou, Cedric; Le, Hoan N; Pinheiro, Marcos Gabriel; Duong, Au H; Wang, Xing; Dip, Etyene Castro; Aguiar-Alves, Fábio; Basuino, Li; Marbach, Helene; Mai, Thuy T; Sarda, Marie N; Kajikawa, Osamu; Matute-Bello, Gustavo; Tkaczyk, Christine; Rasigade, Jean-Philippe; Sellman, Bret R; Chambers, Henry F; Lina, Gerard

    2016-09-21

    New therapeutic approaches are urgently needed to improve survival outcomes for patients with necrotizing pneumonia caused by Staphylococcus aureus One such approach is adjunctive treatment with intravenous immunoglobulin (IVIG), but clinical practice guidelines offer conflicting recommendations. In a preclinical rabbit model, prophylaxis with IVIG conferred protection against necrotizing pneumonia caused by five different epidemic strains of community-associated methicillin-resistant S. aureus (MRSA) as well as a widespread strain of hospital-associated MRSA. Treatment with IVIG, either alone or in combination with vancomycin or linezolid, improved survival outcomes in this rabbit model. Two specific IVIG antibodies that neutralized the toxic effects of α-hemolysin (Hla) and Panton-Valentine leukocidin (PVL) conferred protection against necrotizing pneumonia in the rabbit model. This mechanism of action of IVIG was uncovered by analyzing loss-of-function mutant bacterial strains containing deletions in 17 genes encoding staphylococcal exotoxins, which revealed only Hla and PVL as having an impact on necrotizing pneumonia. These results demonstrate the potential clinical utility of IVIG in the treatment of severe pneumonia induced by S. aureus. PMID:27655850

  11. Comparsion of selected growth media for culturing Serratia marcescens, Aeromonas sp., and Pseudomonas aeruginosa as pathogens of adult Stomoxys calcitrans (Diptera: Muscidae).

    PubMed

    Lysyk, T J; Kalischuk-Tymensen, L D; Selinger, L B

    2002-01-01

    Stable flies, Stomoxys calcitrans (L.), were orally infected with Aeromonas sp., Pseudomonas aeruginosa (Schroeter), and Serratia marcescens Bizio that were cultured on egg-yolk media, nutrient broth, and fly egg media. Aeromonas and Serratia caused mortality when the bacteria were originally grown on egg-yolk medium. Pseudomonas was equally lethal regardless of the media on which it was cultured. A wild isolate of Aeromonas caused greater death than an isolate that had been passed through host flies and had been reisolated from killed flies. Mortality increased with bacterial dose for all species. Aeromonas and Serratia caused mortality within several days after ingestion, whereas Pseudomonas caused a gradual increase in mortality 3-7 d after ingestion. The pathologic activity of Aeromonas and Serratia required extracellular products produced when cells were grown in egg yolk medium. Aeromonas required both supernatant and cells from egg yolk medium, wereas Serratia required supernatant from egg yolk medium and cells from either nutrient broth or egg yolk medium. Mortality due to ingestion of Aeromonas was correlated with the presence of enzymes that cause alpha- and beta-hemolysis, while mortality following ingestion of Serratia was associated with alpha-hemolysins, elastases, and chitinases.

  12. Separate inputs modulate phosphorylation-dependent and -independent type VI secretion activation

    PubMed Central

    Silverman, Julie M.; Austin, Laura S.; Hsu, FoSheng; Hicks, Kevin G.; Hood, Rachel D.; Mougous, Joseph D.

    2013-01-01

    Summary Productive intercellular delivery of cargo by secretory systems requires exquisite temporal and spatial choreography. Our laboratory has demonstrated that the hemolysin co-regulated secretion island I (HSI-I)-encoded type VI secretion system (H1-T6SS) of Pseudomonas aeruginosa transfers effector proteins to other bacterial cells. The activity of these effectors requires cell contact-dependent delivery by the secretion apparatus, and thus their export is highly repressed under planktonic growth conditions. Here we define regulatory pathways that orchestrate efficient secretion by this system. We identified a T6S-associated protein, TagF, as a posttranslational repressor of the H1-T6SS. Strains activated by TagF derepression or stimulation of a previously identified threonine phosphorylation pathway (TPP) share the property of secretory ATPase recruitment to the T6S apparatus, yet display different effector output levels and genetic requirements for their export. We also found that the pathways respond to distinct stimuli; we identified surface growth as a physiological cue that activates the H1-T6SS exclusively through the TPP. Coordination of posttranslational triggering with cell contact-promoting growth conditions provides a mechanism for the T6SS to avoid wasteful release of effectors. PMID:22017253

  13. Effect of negative pressure on growth, secretion and biofilm formation of Staphylococcus aureus.

    PubMed

    Li, Tongtong; Wang, Guoqi; Yin, Peng; Li, Zhirui; Zhang, Licheng; Liu, Jianheng; Li, Ming; Zhang, Lihai; Han, Li; Tang, Peifu

    2015-10-01

    Negative pressure wound therapy (NPWT) has gained popularity in the management of contaminated wounds as an effective physical therapy, although its influence on the bacteria in the wounds remains unclear. In this study, we attempted to explore the effect of negative pressure conditions on Staphylococcus aureus, the most frequently isolated pathogen during wound infection. S. aureus was cultured in Luria-Bertani medium at subatmospheric pressure of -125 mmHg for 24 h, with the bacteria grown at ambient pressure as the control. The application of negative pressure was found to slow down the growth rate and inhibit biofilm development of S. aureus, which was confirmed by static biofilm assays. Furthermore, decreases in the total amount of virulence factors and biofilm components were observed, including α-hemolysin, extracellular adherence protein, polysaccharide intercellular adhesin and extracellular DNA. With quantitative RT-PCR analysis, we also revealed a significant inhibition in the transcription of virulence and regulatory genes related to wound infections and bacterial biofilms. Together, these findings indicated that negative pressure could inhibit the growth, virulence and biofilm formation of S. aureus. A topical subatmospheric pressure condition, such as NPWT, may be a potential antivirulence and antibiofilm strategy in the field of wound care. PMID:26272011

  14. Molecular Characterization of Nonhemolytic and Nonpigmented Group B Streptococci Responsible for Human Invasive Infections

    PubMed Central

    Six, Anne; Firon, Arnaud; Plainvert, Céline; Caplain, Camille; Touak, Gérald; Dmytruk, Nicolas; Longo, Magalie; Letourneur, Franck; Fouet, Agnès; Trieu-Cuot, Patrick

    2015-01-01

    Group B Streptococcus (GBS) is a common commensal bacterium in adults, but is also the leading cause of invasive bacterial infections in neonates in developed countries. The β-hemolysin/cytolysin (β-h/c), which is always associated with the production of an orange-to-red pigment, is a major virulence factor that is also used for GBS diagnosis. A collection of 1,776 independent clinical GBS strains isolated in France between 2006 and 2013 was evaluated on specific medium for β-h/c activity and pigment production. The genomic sequences of nonhemolytic and nonpigmented (NH/NP) strains were analyzed to identify the molecular basis of this phenotype. Gene deletions or complementations were carried out to confirm the genotype-phenotype association. Sixty-three GBS strains (3.5%) were NH/NP, and 47 of these (74.6%) originated from invasive infections, including bacteremia and meningitis, in neonates or adults. The mutations are localized predominantly in the cyl operon, encoding the β-h/c pigment biosynthetic pathway and, in the abx1 gene, encoding a CovSR regulator partner. In conclusion, although usually associated with GBS virulence, β-h/c pigment production is not absolutely required to cause human invasive infections. Caution should therefore be taken in the use of hemolysis and pigmentation as criteria for GBS diagnosis in routine clinical laboratory settings. PMID:26491182

  15. Role of wild birds as carriers of multi-drug resistant Escherichia coli and Escherichia vulneris.

    PubMed

    Shobrak, Mohammed Y; Abo-Amer, Aly E

    2014-01-01

    Emergence and distribution of multi-drug resistant (MDR) bacteria in environments pose a risk to human and animal health. A total of 82 isolates of Escherichia spp. were recovered from cloacal swabs of migrating and non-migrating wild birds. All bacterial isolates were identified and characterized morphologically and biochemically. 72% and 50% of isolates recovered from non-migrating and migrating birds, respectively, showed positive congo red dye binding (a virulence factor). Also, hemolysin production (a virulence factor) was showed in 8% of isolates recovered from non-migrating birds and 75% of isolates recovered from migrating birds. All isolates recovered from non-migrating birds were found resistant to Oxacillin while all isolates recovered from migrating birds demonstrated resistance to Oxacillin, Chloramphenicol, Oxytetracycline and Lincomycin. Some bacterial isolates recovered from non-migrating birds and migrating birds exhibited MDR phenotype. The MDR isolates were further characterized by API 20E and 16S rRNA as E. coli and E. vulneris. MDR Escherichia isolates contain ~1-5 plasmids of high-molecular weights. Accordingly, wild birds could create a potential threat to human and animal health by transmitting MDR bacteria to water streams and other environmental sources through their faecal residues, and to remote regions by migration.

  16. The influence of environmental bacteria in freshwater stingray wound-healing.

    PubMed

    Domingos, Marta O; Franzolin, Márcia R; dos Anjos, Marina Tavares; Franzolin, Thais M P; Barbosa Albes, Rosely Cabette; de Andrade, Gabrielle Ribeiro; Lopes, Rossivan J L; Barbaro, Katia C

    2011-08-01

    Invasion by bacteria can influence the course of healing of wounds acquired in aquatic environment. In this study, the bacteria present in Potamotrygon motoro stingray mucus and in the Alto Paraná river water were identified, and their ability to induce tissue injury and resist antibiotics was determined. Biochemical identification analysis showed that 97% of all bacterial isolates were Gram negative, Aeromonas spp., Enterobacter cloacae and Citrobacter freundii being the species most prevalent. Gelatinase and caseinase were produced by Aeromonas hydrophila, Aeromonas sobria and Pseudomonas aeruginosa strains. Erythrocyte hemolysis assay showed that A. sobria, A. hydrophila and to a lesser extent, other Gram-negative bacteria produced hemolysin. It was also observed that molecules released in culture by these bacteria were toxic to human epithelial cells. Antibiogram results showed that 68% of all bacterial isolates were resistant to at least one type of antibiotic, mainly B-lactams. Finally, it was demonstrated that although P. motoro venom was toxic to epithelial cells it did not influence bacterial proliferation. In summary, the results obtained in this work indicate that during the accident, the mucus of P. motoro and the environmental water may transfer into the wound pathogenic multi-resistant bacteria with the potential to cause severe secondary infections. PMID:21635911

  17. The TolC Homologue of Brucella suis Is Involved in Resistance to Antimicrobial Compounds and Virulence▿

    PubMed Central

    Posadas, Diana M.; Martín, Fernando A.; Sabio y García, Julia V.; Spera, Juan M.; Delpino, M. Victoria; Baldi, Pablo; Campos, Eleonora; Cravero, Silvio L.; Zorreguieta, Angeles

    2007-01-01

    Brucella spp., like other pathogens, must cope with the environment of diverse host niches during the infection process. In doing this, pathogens evolved different type of transport systems to help them survive and disseminate within the host. Members of the TolC family have been shown to be involved in the export of chemically diverse molecules ranging from large protein toxins to small toxic compounds. The role of proteins from the TolC family in Brucella and other α-2-proteobacteria has been explored little. The gene encoding the unique member of the TolC family from Brucella suis (BepC) was cloned and expressed in an Escherichia coli mutant disrupted in the gene encoding TolC, which has the peculiarity of being involved in diverse transport functions. BepC fully complemented the resistance to drugs such as chloramphenicol and acriflavine but was incapable of restoring hemolysin secretion in the tolC mutant of E. coli. An insertional mutation in the bepC gene strongly affected the resistance phenotype of B. suis to bile salts and toxic chemicals such as ethidium bromide and rhodamine and significantly decreased the resistance to antibiotics such as erythromycin, ampicillin, tetracycline, and norfloxacin. Moreover, the B. suis bepC mutant was attenuated in the mouse model of infection. Taken together, these results suggest that BepC-dependent efflux processes of toxic compounds contribute to B. suis survival inside the host. PMID:17088356

  18. Ciprofloxacin and Trimethoprim Cause Phage Induction and Virulence Modulation in Staphylococcus aureus

    PubMed Central

    Goerke, Christiane; Köller, Johanna; Wolz, Christiane

    2006-01-01

    In Staphylococcus aureus strains of human origin, phages which integrate into the chromosomal gene coding for β-hemolysin (hlb) are widely distributed. Most of them encode accessory virulence determinants such as staphylokinase (sak) or enterotoxins. Here, we analyzed the effects of ciprofloxacin and trimethoprim on phage induction and expression of phage-encoded virulence factors by using isolates from patients with cystic fibrosis for which the induction of hlb-converting phages was demonstrated in vivo (C. Goerke, S. Matias y Papenberg, S. Dasbach, K. Dietz, R. Ziebach, B. C. Kahl, and C. Wolz, J. Infect. Dis. 189:724-734, 2004) as well as a φ13 lysogen of phage-cured strain 8325-4. Treatment of lysogens with subinhibitory concentrations of either antibiotic resulted in (i) delysogenization of strains resembling the isolates picked up after chronic lung infection and (ii) replication of phages in the bacterial host in a dose-dependent manner. Ciprofloxacin treatment resulted in enhanced recA transcription, indicating involvement of the SOS response in phage mobilization. Induction of φ13 was linked to elevated expression of the phage-encoded virulence gene sak, chiefly due to the activation of latent phage promoters. In summary, we could show the induction of hlb-converting phages and a subsequent virulence modulation of the host bacterium by ciprofloxacin and trimethoprim. PMID:16377683

  19. Storable droplet interface lipid bilayers for cell-free ion channel studies.

    PubMed

    Jung, Sung-Ho; Choi, Sangbaek; Kim, Young-Rok; Jeon, Tae-Joon

    2012-01-01

    An artificially created lipid bilayer is an important platform in studying ion channels and engineered biosensor applications. However, a lipid bilayer created using conventional techniques is fragile and short-lived, and the measurement of ion channels requires expertise and laborious procedures, precluding practical applications. Here, we demonstrate a storable droplet lipid bilayer precursor frozen with ion channels, resulting in a droplet interface bilayer upon thawing. A small vial with an aqueous droplet in organic solution was flash frozen in -80 °C methanol immediately after an aqueous droplet was introduced into the organic solution and gravity draws the droplet down to the interface upon thawing. A lipid bilayer created along the interface using this method had giga-ohm resistance and typical specific capacitance values. The noise level of this system is favorably comparable to the conventional system. The subsequent incorporation of ion channels, alpha-hemolysin and gramicidin A, showed typical conductance values consistent with those in previous literatures. This novel system to create a lipid bilayer as a whole can be automated from its manufacture to use and indefinitely stored when frozen. As a result, ion channel measurements can be carried out in any place, increasing the accessibility of ion channel studies as well as a number of applications, such as biosensors, ion channel drug screening, and biophysical studies. PMID:21909672

  20. Discovery of Antivirulence Agents against Methicillin-Resistant Staphylococcus aureus

    PubMed Central

    Khodaverdian, Varandt; Pesho, Michelle; Truitt, Barbara; Bollinger, Lucy; Patel, Parita; Nithianantham, Stanley; Yu, Guanping; Delaney, Elizabeth; Jankowsky, Eckhard

    2013-01-01

    Antivirulence agents inhibit the production of disease-causing virulence factors but are neither bacteriostatic nor bactericidal. Antivirulence agents against methicillin-resistant Staphylococcus aureus (MRSA) strain USA300, the most widespread community-associated MRSA strain in the United States, were discovered by virtual screening against the response regulator AgrA, which acts as a transcription factor for the expression of several of the most prominent S. aureus toxins and virulence factors involved in pathogenesis. Virtual screening was followed by similarity searches in the databases of commercial vendors. The small-molecule compounds discovered inhibit the production of the toxins alpha-hemolysin and phenol-soluble modulin α in a dose-dependent manner without inhibiting bacterial growth. These antivirulence agents are small-molecule biaryl compounds in which the aromatic rings either are fused or are separated by a short linker. One of these compounds is the FDA-approved nonsteroidal anti-inflammatory drug diflunisal. This represents a new use for an old drug. Antivirulence agents might be useful in prophylaxis and as adjuvants in antibiotic therapy for MRSA infections. PMID:23689713

  1. Effect of licorice extract on cell viability, biofilm formation and exotoxin production by Staphylococcus aureus.

    PubMed

    Rohinishree, Yadahalli Shrihari; Negi, Pradeep Singh

    2016-02-01

    Staphylococcus aureus is one of the most significant clinical pathogen, as it causes infections to humans and animals. Even though several antibiotics and other treatments have been used to control S. aureus infections and intoxication, bacterium is able to adapt, survive and produces exotoxins. Licorice (Glycyrrhiza glabra L.) has been used traditionally in various medicinal (antimicrobial) preparations, and Glycyrrhizic acid (GA) is the major active constituents present in it. In the present investigation the effect of licorice extract on methicillin susceptible S. aureus (FRI 722) and methicillin resistant S. aureus (ATCC 43300) growth and toxin production was studied. The MIC of licorice extract was found to be 0.25 and 2.5 mg GA ml(-1) against S. aureus FRI 722 and S. aureus ATCC 43300, respectively. Inhibition of biofilm formation was observed even at very low concentration (25 μg GA ml(-1)). Gradual decrease in expression and production of exotoxins such as α and β hemolysins and enterotoxin B was observed with the increasing concentrations of licorice extract, however, suboptimal concentration induced the expression of some of the virulence genes. This study indicated efficacy of licorice extract in controlling growth and pathogenicity of both methicillin susceptible and methicillin resistant S. aureus, however, the mechanisms of survival and toxin production at suboptimal concentration needs further study. PMID:27162389

  2. High-Resolution Size-Discrimination of Single Nonionic Synthetic Polymers with a Highly Charged Biological Nanopore.

    PubMed

    Baaken, Gerhard; Halimeh, Ibrahim; Bacri, Laurent; Pelta, Juan; Oukhaled, Abdelghani; Behrends, Jan C

    2015-06-23

    Electrophysiological studies of the interaction of polymers with pores formed by bacterial toxins (1) provide a window on single molecule interaction with proteins in real time, (2) report on the behavior of macromolecules in confinement, and (3) enable label-free single molecule sensing. Using pores formed by the staphylococcal toxin α-hemolysin (aHL), a particularly pertinent observation was that, under high salt conditions (3-4 M KCl), the current through the pore is blocked for periods of hundreds of microseconds to milliseconds by poly(ethylene glycol) (PEG) oligomers (degree of polymerization approximately 10-60). Notably, this block showed monomeric sensitivity on the degree of polymerization of individual oligomers, allowing the construction of size or mass spectra from the residual current values. Here, we show that the current through the pore formed by aerolysin (AeL) from Aeromonas hydrophila is also blocked by PEG but with drastic differences in the voltage-dependence of the interaction. In contrast to aHL, AeL strongly binds PEG at high transmembrane voltages. This fact, which is likely related to AeL's highly charged pore wall, allows discrimination of polymer sizes with particularly high resolution. Multiple applications are now conceivable with this pore to screen various nonionic or charged polymers. PMID:26028280

  3. Human case of bacteremia due to Streptococcus suis serotype 5 in Japan: The first report and literature review.

    PubMed

    Taniyama, Daisuke; Sakurai, Mayu; Sakai, Tetsuya; Kikuchi, Takahide; Takahashi, Takashi

    2016-01-01

    Streptococcus suis is a zoonotic pathogen that can be transferred from pigs to humans. The serotypes 2 and 14 are prevalent among patients with S. suis infections, while other serotypes (i.e., 1, 4, 5, 16, and 24) have been detected in rare human cases. To the best of our knowledge, the present patient handling with raw pork is the first human case of uncomplicated bacteremia due to S. suis serotype 5 in Japan. We confirmed the new sequence type 752 of this isolate. Virulence-associated gene profiling was performed; both sly (encoding the hemolysin suilysin) and mrp (encoding a muramidase-released protein) were detected without amplification of epf (encoding the extracellular factor). Our polymerase chain reaction-based results indicated that this isolate possessed both tet(O), the tetracycline-resistance determinant, and erm(B), the macrolide/lincosamide-resistance determinant. In addition, we provide the review of literature concerning clinical and microbiological features of four human cases of infection due to S. suis serotype 5. Clinicians should be aware of this microorganism when examining and treating patients with fever, who are handling raw pork or having close contact with infected pigs even if they are immunocompetent. PMID:27689023

  4. Methacrylate Polymer Scaffolding Enhances the Stability of Suspended Lipid Bilayers for Ion Channel Recordings and Biosensor Development

    PubMed Central

    Bright, Leonard K.; Baker, Christopher A.; Bränström, Robert; Saavedra, S. Scott; Aspinwall, Craig A.

    2016-01-01

    Black lipid membranes (BLMs) provide a synthetic environment that facilitates measurement of ion channel activity in diverse analytical platforms. The limited electrical, mechanical and temporal stabilities of BLMs pose a significant challenge to development of highly stable measurement platforms. Here, ethylene glycol dimethacrylate (EGDMA) and butyl methacrylate (BMA) were partitioned into BLMs and photopolymerized to create a cross-linked polymer scaffold in the bilayer lamella that dramatically improved BLM stability. The commercially available methacrylate monomers provide a simple, low cost, and broadly accessible approach for preparing highly stabilized BLMs useful for ion channel analytical platforms. When prepared on silane-modified glass microapertures, the resulting polymer scaffold-stabilized (PSS)-BLMs exhibited significantly improved lifetimes of 23 ± 9 to 40 ± 14 h and > 10-fold increase in mechanical stability, with breakdown potentials > 2000 mV attainable, depending on surface modification and polymer cross-link density. Additionally, the polymer scaffold exerted minimal perturbations to membrane electrical integrity as indicated by mean conductance measurements. When gramicidin A and α-hemolysin were reconstituted into PSS-BLMs, the ion channels retained function comparable to conventional BLMs. This approach is a key advance in the formation of stabilized BLMs and should be amenable to a wide range of receptor and ion channel functionalized platforms. PMID:26925461

  5. Screening ion-channel ligand interactions with passive pumping in a microfluidic bilayer lipid membrane chip

    PubMed Central

    Saha, Shimul C.; Powl, Andrew M.; Wallace, B. A.; de Planque, Maurits R. R.; Morgan, Hywel

    2015-01-01

    We describe a scalable artificial bilayer lipid membrane platform for rapid electrophysiological screening of ion channels and transporters. A passive pumping method is used to flow microliter volumes of ligand solution across a suspended bilayer within a microfluidic chip. Bilayers are stable at flow rates up to ∼0.5 μl/min. Phospholipid bilayers are formed across a photolithographically defined aperture made in a dry film resist within the microfluidic chip. Bilayers are stable for many days and the low shunt capacitance of the thin film support gives low-noise high-quality single ion channel recording. Dose-dependent transient blocking of α-hemolysin with β-cyclodextrin (β-CD) and polyethylene glycol is demonstrated and dose-dependent blocking studies of the KcsA potassium channel with tetraethylammonium show the potential for determining IC50 values. The assays are fast (30 min for a complete IC50 curve) and simple and require very small amounts of compounds (100 μg in 15 μl). The technology can be scaled so that multiple bilayers can be addressed, providing a screening platform for ion channels, transporters, and nanopores. PMID:25610515

  6. Polymerized Planar Suspended Lipid Bilayers for Single Ion Channel Recordings: Comparison of Several Dienoyl Lipids

    PubMed Central

    Heitz, Benjamin A.; Xu, Juhua; Jones, Ian W.; Keogh, John P.; Comi, Troy J.; Hall, Henry K.; Aspinwall, Craig A.; Saavedra, S. Scott

    2011-01-01

    The stabilization of suspended planar lipid membranes, or black lipid membranes (BLMs), through polymerization of mono- and bis-functionalized dienoyl lipids was investigated. Electrical properties, including capacitance, conductance, and dielectric breakdown voltage, were determined for BLMs composed of mono-DenPC, bis-DenPC, mono-SorbPC, and bis-SorbPC both prior to and following photopolymerization, with diphytanoyl phosphocholine (DPhPC) serving as a control. Poly(lipid) BLMs exhibited significantly longer lifetimes and increased the stability to air-water transfers. BLM stability followed the order: bis-DenPC > mono-DenPC ≈ mono-SorbPC > bis-SorbPC. The conductance of bis-SorbPC BLMs was significantly higher than that of the other lipids, which is attributed to a high density of hydrophilic pores, resulting in relatively unstable membranes. The use of poly(lipid) BLMs as matrices for supporting the activity of an ion channel protein (IC) was explored using α – hemolysin (α-HL), a model IC. Characteristic i-V plots of α-HL were maintained following photopolymerization of bis-DenPC, mono-DenPC, and mono-SorbPC, demonstrating the utility of these materials for preparing more durable BLMs for single channel recordings of reconstituted ICs. PMID:21226498

  7. Detection of single ion channel activity with carbon nanotubes

    PubMed Central

    Zhou, Weiwei; Wang, Yung Yu; Lim, Tae-Sun; Pham, Ted; Jain, Dheeraj; Burke, Peter J.

    2015-01-01

    Many processes in life are based on ion currents and membrane voltages controlled by a sophisticated and diverse family of membrane proteins (ion channels), which are comparable in size to the most advanced nanoelectronic components currently under development. Here we demonstrate an electrical assay of individual ion channel activity by measuring the dynamic opening and closing of the ion channel nanopores using single-walled carbon nanotubes (SWNTs). Two canonical dynamic ion channels (gramicidin A (gA) and alamethicin) and one static biological nanopore (α-hemolysin (α-HL)) were successfully incorporated into supported lipid bilayers (SLBs, an artificial cell membrane), which in turn were interfaced to the carbon nanotubes through a variety of polymer-cushion surface functionalization schemes. The ion channel current directly charges the quantum capacitance of a single nanotube in a network of purified semiconducting nanotubes. This work forms the foundation for a scalable, massively parallel architecture of 1d nanoelectronic devices interrogating electrophysiology at the single ion channel level. PMID:25778101

  8. Photolithographic fabrication of microapertures with well-defined, three-dimensional geometries for suspended lipid membrane studies

    PubMed Central

    Baker, Christopher A.; Bright, Leonard K.; Aspinwall, Craig A.

    2013-01-01

    Robust and high-density biosensors incorporating suspended lipid membranes require microfabricated apertures that can be readily integrated into complex analysis systems. Apertures with well-defined, three-dimensional geometries enable the formation of suspended lipid membranes, and facilitate reduced aperture size compared to vertical-walled apertures. Unfortunately, existing methods of producing apertures with well-defined, three-dimensional geometries are based on complex and expensive fabrication procedures, some of which yield apertures in excessively fragile thin-film materials. Here, we describe a microfabrication method utilizing incline and rotate lithography that achieves sloped-wall microapertures in SU-8 polymer substrates with precision control of aperture diameter, substrate thickness, and wall angle. This approach is simple, low cost, and readily scaled up to allow highly reproducible parallel fabrication. The effect of incident angle of UV exposure and the size of photomask features on aperture geometry were investigated, yielding aperture diameters as small as 7 µm, and aperture wall angles ranging from 8° to 36° measured from the normal axis. Black lipid membranes were suspended across the apertures and showed normalized conductance values of 0.02 to 0.05 pS µm−2 and break down voltages of 400 to 600 mV. The functionality of the resulting sloped-wall microapertures was validated via measurement of reconstituted α-hemolysin activity and the voltage-gated channel activity of alamethicin. PMID:23987300

  9. Exploring simvastatin, an antihyperlipidemic drug, as a potential topical antibacterial agent

    PubMed Central

    Thangamani, Shankar; Mohammad, Haroon; Abushahba, Mostafa F. N.; Hamed, Maha I.; Sobreira, Tiago J. P.; Hedrick, Victoria E.; Paul, Lake N.; Seleem, Mohamed N.

    2015-01-01

    The rapid rise of bacterial resistance to traditional antibiotics combined with the decline in discovery of novel antibacterial agents has created a global public health crisis. Repurposing existing drugs presents an alternative strategy to potentially expedite the discovery of new antimicrobial drugs. The present study demonstrates that simvastatin, an antihyperlipidemic drug exhibited broad-spectrum antibacterial activity against important Gram-positive (including methicillin-resistant Staphylococcus aureus (MRSA)) and Gram-negative pathogens (once the barrier imposed by the outer membrane was permeabilized). Proteomics and macromolecular synthesis analyses revealed that simvastatin inhibits multiple biosynthetic pathways and cellular processes in bacteria, including selective interference of bacterial protein synthesis. This property appears to assist in simvastatin’s ability to suppress production of key MRSA toxins (α-hemolysin and Panton-Valentine leucocidin) that impair healing of infected skin wounds. A murine MRSA skin infection experiment confirmed that simvastatin significantly reduces the bacterial burden and inflammatory cytokines in the infected wounds. Additionally, simvastatin exhibits excellent anti-biofilm activity against established staphylococcal biofilms and demonstrates the ability to be combined with topical antimicrobials currently used to treat MRSA skin infections. Collectively the present study lays the foundation for further investigation of repurposing simvastatin as a topical antibacterial agent to treat skin infections. PMID:26553420

  10. The genome sequence of the facultative intracellular pathogen Brucella melitensis.

    PubMed

    DelVecchio, Vito G; Kapatral, Vinayak; Redkar, Rajendra J; Patra, Guy; Mujer, Cesar; Los, Tamara; Ivanova, Natalia; Anderson, Iain; Bhattacharyya, Anamitra; Lykidis, Athanasios; Reznik, Gary; Jablonski, Lynn; Larsen, Niels; D'Souza, Mark; Bernal, Axel; Mazur, Mikhail; Goltsman, Eugene; Selkov, Eugene; Elzer, Philip H; Hagius, Sue; O'Callaghan, David; Letesson, Jean-Jacques; Haselkorn, Robert; Kyrpides, Nikos; Overbeek, Ross

    2002-01-01

    Brucella melitensis is a facultative intracellular bacterial pathogen that causes abortion in goats and sheep and Malta fever in humans. The genome of B. melitensis strain 16M was sequenced and found to contain 3,294,935 bp distributed over two circular chromosomes of 2,117,144 bp and 1,177,787 bp encoding 3,197 ORFs. By using the bioinformatics suite ERGO, 2,487 (78%) ORFs were assigned functions. The origins of replication of the two chromosomes are similar to those of other alpha-proteobacteria. Housekeeping genes, including those involved in DNA replication, transcription, translation, core metabolism, and cell wall biosynthesis, are distributed on both chromosomes. Type I, II, and III secretion systems are absent, but genes encoding sec-dependent, sec-independent, and flagella-specific type III, type IV, and type V secretion systems as well as adhesins, invasins, and hemolysins were identified. Several features of the B. melitensis genome are similar to those of the symbiotic Sinorhizobium meliloti.

  11. Occurrence of Natural Bacillus thuringiensis Contaminants and Residues of Bacillus thuringiensis-Based Insecticides on Fresh Fruits and Vegetables

    PubMed Central

    Frederiksen, Kristine; Rosenquist, Hanne; Jørgensen, Kirsten; Wilcks, Andrea

    2006-01-01

    A total of 128 Bacillus cereus-like strains isolated from fresh fruits and vegetables for sale in retail shops in Denmark were characterized. Of these strains, 39% (50/128) were classified as Bacillus thuringiensis on the basis of their content of cry genes determined by PCR or crystal proteins visualized by microscopy. Random amplified polymorphic DNA analysis and plasmid profiling indicated that 23 of the 50 B. thuringiensis strains were of the same subtype as B. thuringiensis strains used as commercial bioinsecticides. Fourteen isolates were indistinguishable from B. thuringiensis subsp. kurstaki HD1 present in the products Dipel, Biobit, and Foray, and nine isolates grouped with B. thuringiensis subsp. aizawai present in Turex. The commercial strains were primarily isolated from samples of tomatoes, cucumbers, and peppers. A multiplex PCR method was developed to simultaneously detect all three genes in the enterotoxin hemolysin BL (HBL) and the nonhemolytic enterotoxin (NHE), respectively. This revealed that the frequency of these enterotoxin genes was higher among the strains indistinguishable from the commercial strains than among the other B. thuringiensis and B. cereus-like strains isolated from fruits and vegetables. The same was seen for a third enterotoxin, CytK. In conclusion, the present study strongly indicates that residues of B. thuringiensis-based insecticides can be found on fresh fruits and vegetables and that these are potentially enterotoxigenic. PMID:16672488

  12. FlhF Is Required for Swarming Motility and Full Pathogenicity of Bacillus cereus

    PubMed Central

    Mazzantini, Diletta; Celandroni, Francesco; Salvetti, Sara; Gueye, Sokhna A.; Lupetti, Antonella; Senesi, Sonia; Ghelardi, Emilia

    2016-01-01

    Besides sporulation, Bacillus cereus can undergo a differentiation process in which short swimmer cells become elongated and hyperflagellated swarmer cells that favor migration of the bacterial community on a surface. The functionally enigmatic flagellar protein FlhF, which is the third paralog of the signal recognition particle (SRP) GTPases Ffh and FtsY, is required for swarming in many bacteria. Previous data showed that FlhF is involved in the control of the number and positioning of flagella in B. cereus. In this study, in silico analysis of B. cereus FlhF revealed that this protein presents conserved domains that are typical of SRPs in many organisms and a peculiar N-terminal basic domain. By proteomic analysis, a significant effect of FlhF depletion on the amount of secreted proteins was found with some proteins increased (e.g., B component of the non-hemolytic enterotoxin, cereolysin O, enolase) and others reduced (e.g., flagellin, L2 component of hemolysin BL, bacillolysin, sphingomyelinase, PC-PLC, PI-PLC, cytotoxin K) in the extracellular proteome of a ΔflhF mutant. Deprivation of FlhF also resulted in significant attenuation in the pathogenicity of this strain in an experimental model of infection in Galleria mellonella larvae. Our work highlights the multifunctional role of FlhF in B. cereus, being this protein involved in bacterial flagellation, swarming, protein secretion, and pathogenicity. PMID:27807433

  13. Assessment of adhesion, invasion and cytotoxicity potential of oral Staphylococcus aureus strains.

    PubMed

    Merghni, Abderrahmen; Ben Nejma, Mouna; Helali, Imen; Hentati, Hajer; Bongiovanni, Antonino; Lafont, Frank; Aouni, Mahjoub; Mastouri, Maha

    2015-09-01

    The oral cavity is regarded as a relevant site for Staphylococcus aureus colonization. However, characterization of virulence mechanisms of oral S. aureus remains to be uncovered. In this study, twenty one S. aureus strains isolated from the oral cavity of Tunisian patients were screened for adherence, invasion and cytotoxicity against HeLa cells. In addition, the presence of adhesins (icaA, icaD, can, fnbA and fnbB) and α-hemolysin (hla) genes in each strain was achieved by polymerase chain reaction (PCR). Our finding revealed that oral S. aureus strains were able to adhere and invade epithelial cells, with variable degrees (P < 0.05). Moreover they exhibited either low (23.8%) or moderate (76.2%) cytotoxic effects. In addition 76.2% of strains were icaA and icaD positive and 90.5% harbor both the fnbA and the fnbB gene. While the cna gene was detected in 12 strains (57.2%). Furthermore, the hla gene encoding the α-toxin was found in 52.4% of the isolates. All these virulence factors give to S. aureus the right qualities to become a redoubtable pathogen associated to oral infections.

  14. In vivo immunological activity of carboxymethylated-sulfated (1→3)-β-D-glucan from sclerotium of Poria cocos.

    PubMed

    Wang, Haili; Mukerabigwi, Jean Felix; Zhang, Yuannian; Han, Lin; Jiayinaguli, Telieke; Wang, Qing; Liu, Lina; Cao, Yu; Sun, Renqiang; Huang, Xueying

    2015-08-01

    β-Glucans are one of the polysaccharides known as biological response modifiers extracted from the sclerotium of Poria cocos which has been used for several decades as Traditional Chinese Medicine. Due to its ability to activate immune system, it can be applied in chemotherapy after being chemically modified. In this study, sulfated (1→3)-β-D-glucan (S-P), carboxymethyl (1→3)-β-D-glucan (CMP), and carboxymethylated-sulfated (1→3)-β-D-glucan (S-CMP), which are (1→3)-β-D-glucan derivatives were synthesized. The current study was aimed to investigate in vivo potential immunological activity of S-CMP in mice. In addition, mice were separately treated with S-P, CMP and S-CMP to evaluate the relationship between single and multiple functional groups. Interestingly, S-CMP exhibited the best in vivo immunological activities and the highest inhibition rate against the implanted HepG2 tumor in BALB/c mice, with significant increase in serum hemolysin antibody titer, spleen antibody production as well as delayed type hypersensitivity compared with S-P and CMP. Furthermore, it was assumed that simultaneous introduction of carboxymethyl and sulfate groups also had great potential effect on antioxidant activity, as substantial decrease in malondialdehyde (MDA) content was remarked. Therefore, it may suggest that S-CMP has better immunological and anti-tumor effects on mice in vivo.

  15. Identification and Characterization of Vancomycin-resistant Enterococcus species Frequently Isolated from Laboratory Mice

    PubMed Central

    Yamanaka, Hitoki; Takagi, Toshikazu; Ohsawa, Makiko; Yamamoto, Naoto; Kubo, Noriaki; Takemoto, Takahira; Sasano, Shoko; Masuyama, Ritsuko; Ohsawa, Kazutaka

    2014-01-01

    To determine the prevalence of drug resistant bacteria colonizing laboratory mice, we isolated and characterized vancomycin-resistant Enterococcus species (VRE) from commercially available mice. A total of 24 VRE isolates were obtained from 19 of 21 mouse strains supplied by 4 commercial breeding companies. Of these, 19 isolates of E. gallinarum and 5 isolates of E. casseliflavus possessing the vanC1 and vanC2/3 genes intrinsically, exhibited intermediate resistance to vancomycin respectively. In addition, these isolates also exhibited diverse resistant patterns to erythromycin, tetracycline, and ciprofloxacin, whereas the use of antibiotics had not been undertaken in mouse strains tested in this study. Although 6 virulence-associated genes (ace, asa, cylA, efaA, esp, and gelE) and secretion of gelatinase and hemolysin were not detected in all isolates, 23 of 24 isolates including the isolates of E. casselifalvus secreted ATP into culture supernatants. Since secretion of ATP by bacteria resident in the intestinal tract modulates the local immune responses, the prevalence of ATP-secreting VRE in mice therefore needs to be considered in animal experiments that alter the gut microflora by use of antibiotics. PMID:25077759

  16. Membrane-on-a-chip: microstructured silicon/silicon-dioxide chips for high-throughput screening of membrane transport and viral membrane fusion.

    PubMed

    Kusters, Ilja; van Oijen, Antoine M; Driessen, Arnold J M

    2014-04-22

    Screening of transport processes across biological membranes is hindered by the challenge to establish fragile supported lipid bilayers and the difficulty to determine at which side of the membrane reactants reside. Here, we present a method for the generation of suspended lipid bilayers with physiological relevant lipid compositions on microstructured Si/SiO2 chips that allow for high-throughput screening of both membrane transport and viral membrane fusion. Simultaneous observation of hundreds of single-membrane channels yields statistical information revealing population heterogeneities of the pore assembly and conductance of the bacterial toxin α-hemolysin (αHL). The influence of lipid composition and ionic strength on αHL pore formation was investigated at the single-channel level, resolving features of the pore-assembly pathway. Pore formation is inhibited by a specific antibody, demonstrating the applicability of the platform for drug screening of bacterial toxins and cell-penetrating agents. Furthermore, fusion of H3N2 influenza viruses with suspended lipid bilayers can be observed directly using a specialized chip architecture. The presented micropore arrays are compatible with fluorescence readout from below using an air objective, thus allowing high-throughput screening of membrane transport in multiwell formats in analogy to plate readers.

  17. Single Molecule Investigation of Ag+ Interactions with Single Cytosine-, Methylcytosine- and Hydroxymethylcytosine-Cytosine Mismatches in a Nanopore

    PubMed Central

    Wang, Yong; Luan, Bin-Quan; Yang, Zhiyu; Zhang, Xinyue; Ritzo, Brandon; Gates, Kent; Gu, Li-Qun

    2014-01-01

    Both cytosine-Ag-cytosine interactions and cytosine modifications in a DNA duplex have attracted great interest for research. Cytosine (C) modifications such as methylcytosine (mC) and hydroxymethylcytosine (hmC) are associated with tumorigenesis. However, a method for directly discriminating C, mC and hmC bases without labeling, modification and amplification is still missing. Additionally, the nature of coordination of Ag+ with cytosine-cytosine (C-C) mismatches is not clearly understood. Utilizing the alpha-hemolysin nanopore, we show that in the presence of Ag+, duplex stability is most increased for the cytosine-cytosine (C-C) pair, followed by the cytosine-methylcytosine (C-mC) pair, and the cytosine-hydroxymethylcytosine (C-hmC) pair, which has no observable Ag+ induced stabilization. Molecular dynamics simulations reveal that the hydrogen-bond-mediated paring of a C-C mismatch results in a binding site for Ag+. Cytosine modifications (such as mC and hmC) disrupted the hydrogen bond, resulting in disruption of the Ag+ binding site. Our experimental method provides a novel platform to study the metal ion-DNA interactions and could also serve as a direct detection method for nucleobase modifications. PMID:25103463

  18. Isolation, antibiogram and pathogenicity of Salmonella spp. recovered from slaughtered food animals in Nagpur region of Central India

    PubMed Central

    Kalambhe, D. G.; Zade, N. N.; Chaudhari, S. P.; Shinde, S. V.; Khan, W.; Patil, A. R.

    2016-01-01

    Aim: To determine the prevalence, antibiogram and pathogenicity of Salmonella spp. in the common food animals slaughtered for consumption purpose at government approved slaughter houses located in and around Nagpur region during a period of 2010-2012. Materials and Methods: A total of 400 samples comprising 50 each of blood and meat from each slaughtered male cattle, buffaloes, pigs and goats were collected. Isolation was done by pre-enrichment in buffered peptone water and enrichment in Rappaport-Vassiliadis broth with subsequent selective plating onto xylose lysine deoxycholate agar. Presumptive Salmonella colonies were biochemically confirmed and analyzed for pathogenicity by hemolysin production and Congo red dye binding assay (CRDA). An antibiotic sensitivity test was performed to assess the antibiotic resistance pattern of the isolates. Results: A total of 10 isolates of Salmonella spp. from meat (3 from cattle, 1 from buffaloes and 6 from pigs) with an overall prevalence of 5% among food animals was recorded. No isolation was reported from any blood samples. Pathogenicity assays revealed 100% and 80% positivity for CRDA and hemolytic activity, respectively. Antimicrobial sensitivity test showed multi-drug resistance. The overall resistance of 50% was noted for trimethoprim followed by ampicillin (20%). A maximum sensitivity (80%) was reported to gentamycin followed by 40% each to ampicillin and trimethoprim, 30% to amikacin and 10% to kanamycin. Conclusion: The presence of multidrug resistant and potentially pathogenic Salmonella spp. in slaughtered food animals in Nagpur region can be a matter of concern for public health. PMID:27051204

  19. Protective potency of clove oil and its transcriptional down-regulation of Aeromonas sobria virulence genes in African catfish (Clarias gariepinus L.).

    PubMed

    Abd El-Hamid, M I; Abd El-Aziz, N K; Ali, H A

    2016-01-01

    Disease episodes of fish caused by Aeromonas species are moved to the top list of limiting problems worldwide. The present study was planned to verify the in vitro antibacterial activities as well as the in vivo potential values of clove oil and ciprofloxacin against Aeromonas sobria in African catfish (Clarias gariepinus). The in vitro phenotypic virulence activities and the successful amplification of aerolysin and hemolysin genes in the precisely identified A. sobria strain were predictive for its virulence. In the in vivo assay, virulence of A. sobria strain was fully demonstrated based on constituent mRNA expression profile of tested virulence genes and typical septicemia associated with high mortalities of infected fish. Apparent lower mortality rates were correlated well with both decrescent bacterial burden and significant down-regulated transcripts of representative genes in the treated groups with clove oil, followed by ciprofloxacin as a prophylactic use for 15 days (P < 0.0001); however, the essential oil apart from ciprofloxacin significantly enhanced different hematological parameters (P < 0.05). In addition, administration of antibiotic may be considered as a pronounced stress factor in the fish even when it used in the prophylactic dose. In conclusion, medicinal plants-derived essential oils provide a virtually safer alternative to chemotherapeutics on fish, consumers and ecosystems. PMID:27609474

  20. Channel-forming bacterial toxins in biosensing and macromolecule delivery.

    PubMed

    Gurnev, Philip A; Nestorovich, Ekaterina M

    2014-08-21

    To intoxicate cells, pore-forming bacterial toxins are evolved to allow for the transmembrane traffic of different substrates, ranging from small inorganic ions to cell-specific polypeptides. Recent developments in single-channel electrical recordings, X-ray crystallography, protein engineering, and computational methods have generated a large body of knowledge about the basic principles of channel-mediated molecular transport. These discoveries provide a robust framework for expansion of the described principles and methods toward use of biological nanopores in the growing field of nanobiotechnology. This article, written for a special volume on "Intracellular Traffic and Transport of Bacterial Protein Toxins", reviews the current state of applications of pore-forming bacterial toxins in small- and macromolecule-sensing, targeted cancer therapy, and drug delivery. We discuss the electrophysiological studies that explore molecular details of channel-facilitated protein and polymer transport across cellular membranes using both natural and foreign substrates. The review focuses on the structurally and functionally different bacterial toxins: gramicidin A of Bacillus brevis, α-hemolysin of Staphylococcus aureus, and binary toxin of Bacillus anthracis, which have found their "second life" in a variety of developing medical and technological applications.

  1. Channel formation by RTX-toxins of pathogenic bacteria: Basis of their biological activity.

    PubMed

    Benz, Roland

    2016-03-01

    The pore-forming cytolysins of the RTX-toxin (Repeats in ToXin) family are a relatively small fraction of a steadily increasing family of proteins that contain several functionally important glycine-rich and aspartate containing nonapeptide repeats. These cytolysins produced by a variety of Gram-negative bacteria form ion-permeable channels in erythrocytes and other eukaryotic cells. Hemolytic and cytolytic RTX-toxins represent pathogenicity factors of the toxin-producing bacteria and are very often important key factors in pathogenesis of the bacteria. Channel formation by RTX-toxins lead to the dissipation of ionic gradients and membrane potential across the cytoplasmic membrane of target cells, which results in cell death. Here we discuss channel formation and channel properties of some of the best known RTX-toxins, such as α-hemolysin (HlyA) of Escherichia coli and the uropathogenic EHEC strains, the adenylate cyclase toxin (ACT, CyaA) of Bordetella pertussis and the RTX-toxins (ApxI, ApxII and ApxIII) produced by different strains of Actinobacillus pleuropneumoniae. The channels formed by these RTX-toxins in lipid bilayers share some common properties such as cation selectivity and voltage-dependence. Furthermore the channels are transient and show frequent switching between different ion-conducting states. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.

  2. Toxin Profile, Biofilm Formation, and Molecular Characterization of Emetic Toxin-Producing Bacillus cereus Group Isolates from Human Stools.

    PubMed

    Oh, Su Kyung; Chang, Hyun-Joo; Choi, Sung-Wook; Ok, Gyeongsik; Lee, Nari

    2015-11-01

    Emetic toxin-producing Bacillus cereus group species are an important problem, because the staple food for Korean is grains such as rice. In this study, we determined the prevalence (24 of 129 isolates) of emetic B. cereus in 36,745 stool samples from sporadic food-poisoning cases in Korea between 2007 and 2008. The toxin gene profile, toxin production, and biofilm-forming ability of the emetic B. cereus isolates were investigated. Repetitive element sequence polymorphism polymerase chain reaction fingerprints (rep-PCR) were also used to assess the intraspecific biodiversity of these isolates. Emetic B. cereus was present in 0.07% of the sporadic food-poisoning cases. The 24 emetic isolates identified all carried the nheABC and entFM genes and produced NHE enterotoxin. However, they did not have hemolysin BL toxin or related genes. A relationship between biofilm formation and toxin production was not observed in this study. The rep-PCR fingerprints of the B. cereus isolates were not influenced by the presence of toxin genes, or biofilm-forming ability. The rep-PCR assay discriminated emetic B. cereus isolates from nonemetic isolates, even if this assay did not perfectly discriminate these isolates. Further study on emetic isolates possessing a high degree of diversity may be necessary to evaluate the performance of the subtyping assay to discriminate emetic and nonemetic B. cereus isolates and could provide a more accurate indication of the risk from B. cereus strains.

  3. Virulence properties of extended spectrum β-lactamase-producing Klebsiella species in meat samples.

    PubMed

    Gundogan, Neslihan; Citak, Sumru; Yalcin, Emel

    2011-04-01

    The present study was carried out to identify virulence properties (siderophores, serum resistance, and hemolysin) and antibiotic resistance in extended spectrum β-lactamase (ESBL)-producing Klebsiella isolates from 60 calf and chicken meat samples purchased from various supermarkets in Ankara, Turkey. Of the 45 Klebsiella isolates, 24 (53%) were identified as K. oxytoca and 21 (47%) were identified as K. pneumoniae. A high proportion of Klebsiella isolates had virulence factors such as hemolytic activity (67%), siderophore production (44%), and serum resistance (38%). The double-disk synergy test was used to determine ESBL production. ESBL production was detected in 13 (29%) of the 45 Klebsiella isolates. Resistance to 14 antimicrobials was tested in all Klebsiella isolates by the disk diffusion method. All isolates were resistant to two or more antimicrobial agents. All ESBL-producing Klebsiella isolates were highly resistant to cephalosporins and monobactams. Our findings indicate that meat and its products represent potential hazardous sources of multidrug-resistant and virulent Klebsiella species.

  4. Toxigenic clostridia.

    PubMed Central

    Hatheway, C L

    1990-01-01

    Toxigenic clostridia belonging to 13 recognized species are discussed in this review. Each species or group of organisms is, in general, introduced by presenting the historical aspects of its discovery by early investigators of human and animal diseases. The diseases caused by each species or group are described and usually discussed in relation to the toxins involved in the pathology. Morphological and physiological characteristics of the organisms are described. Finally, the toxins produced by each organism are listed, with a presentation of their biological activities and physical and biochemical characteristics. The complete amino acid sequences for some are known, and some of the genes have been cloned. The term toxin is used loosely to include the various antigenic protein products of these organisms with biological and serological activities which have served as distinguishing characteristics for differentiation and classification. Some of these factors are not truly toxic and have no known role in pathogenicity. Some of the interesting factors common to more than one species or group are the following: neurotoxins, lethal toxins, lecithinases, oxygen-labile hemolysins, binary toxins, and ADP-ribosyltransferases. Problems in bacterial nomenclature and designation of biologically active factors are noted. PMID:2404569

  5. Attenuated Virulence and Genomic Reductive Evolution in the Entomopathogenic Bacterial Symbiont Species, Xenorhabdus poinarii

    PubMed Central

    Ogier, Jean-Claude; Pagès, Sylvie; Bisch, Gaëlle; Chiapello, Hélène; Médigue, Claudine; Rouy, Zoé; Teyssier, Corinne; Vincent, Stéphanie; Tailliez, Patrick; Givaudan, Alain; Gaudriault, Sophie

    2014-01-01

    Bacteria of the genus Xenorhabdus are symbionts of soil entomopathogenic nematodes of the genus Steinernema. This symbiotic association constitutes an insecticidal complex active against a wide range of insect pests. Unlike other Xenorhabdus species, Xenorhabdus poinarii is avirulent when injected into insects in the absence of its nematode host. We sequenced the genome of the X. poinarii strain G6 and the closely related but virulent X. doucetiae strain FRM16. G6 had a smaller genome (500–700 kb smaller) than virulent Xenorhabdus strains and lacked genes encoding potential virulence factors (hemolysins, type 5 secretion systems, enzymes involved in the synthesis of secondary metabolites, and toxin–antitoxin systems). The genomes of all the X. poinarii strains analyzed here had a similar small size. We did not observe the accumulation of pseudogenes, insertion sequences or decrease in coding density usually seen as a sign of genomic erosion driven by genetic drift in host-adapted bacteria. Instead, genome reduction of X. poinarii seems to have been mediated by the excision of genomic blocks from the flexible genome, as reported for the genomes of attenuated free pathogenic bacteria and some facultative mutualistic bacteria growing exclusively within hosts. This evolutionary pathway probably reflects the adaptation of X. poinarii to specific host. PMID:24904010

  6. A functional VipA-VipB interaction is required for the type VI secretion system activity of Vibrio cholerae O1 strain A1552

    PubMed Central

    2013-01-01

    Background Many Gram-negative bacteria rely on a type VI secretion system (T6SS) to infect eukaryotic cells or to compete against other microbes. Common to these systems is the presence of two conserved proteins, in Vibrio cholerae denoted VipA and VipB, which have been shown to interact in many clinically relevant pathogens. In this study, mutagenesis of a defined region within the VipA protein was used to identify residues important for VipB binding in V. cholerae O1 strain A1552. Results A dramatically diminished interaction was shown to correlate with a decrease in VipB stability and a loss of hemolysin co-regulated protein (Hcp) secretion and rendered the bacterium unable to compete with Escherichia coli in a competition assay. Conclusions This confirms the biological relevance of the VipA-VipB interaction, which is essential for the T6SS activity of many important human pathogens. PMID:23642157

  7. Nanoscale Bioengineering Solutions for Space Exploration the Nanopore Sequencer

    NASA Technical Reports Server (NTRS)

    Ioana, Cozmuta; Viktor, Stoic

    2005-01-01

    Characterization of biological systems at the molecular level and extraction of essential information for nano-engineering design to guide the nano-fabrication of solid-state sensors and molecular identification devices is a computational challenge. The alpha hemolysin protein ion channel is used as a model system for structural analysis of nucleic acids like DNA. Applied voltage draws a DNA strand and surrounding ionic solution through the biological nanopore. The subunits in the DNA strand block ion flow by differing amounts. Atomistic scale simulations are employed using NASA supercomputers to study DNA translocation. with the aim to enhance single DNA subunit identification. Compared to protein channels, solid-state nanopores offer a better temporal control of the translocation of DNA and the possibility to easily tune its chemistry to increase the signal resolution. Potential applications for NASA missions, besides real-time genome sequencing include astronaut health, life detection and decoding of various genomes. http://phenomrph.arc.nasa.gov/index.php

  8. Genome of Herbaspirillum seropedicae Strain SmR1, a Specialized Diazotrophic Endophyte of Tropical Grasses

    PubMed Central

    Pedrosa, Fábio O.; Monteiro, Rose Adele; Wassem, Roseli; Cruz, Leonardo M.; Ayub, Ricardo A.; Colauto, Nelson B.; Fernandez, Maria Aparecida; Fungaro, Maria Helena P.; Grisard, Edmundo C.; Hungria, Mariangela; Madeira, Humberto M. F.; Nodari, Rubens O.; Osaku, Clarice A.; Petzl-Erler, Maria Luiza; Terenzi, Hernán; Vieira, Luiz G. E.; Steffens, Maria Berenice R.; Weiss, Vinicius A.; Pereira, Luiz F. P.; Almeida, Marina I. M.; Alves, Lysangela R.; Marin, Anelis; Araujo, Luiza Maria; Balsanelli, Eduardo; Baura, Valter A.; Chubatsu, Leda S.; Faoro, Helisson; Favetti, Augusto; Friedermann, Geraldo; Glienke, Chirlei; Karp, Susan; Kava-Cordeiro, Vanessa; Raittz, Roberto T.; Ramos, Humberto J. O.; Ribeiro, Enilze Maria S. F.; Rigo, Liu Un; Rocha, Saul N.; Schwab, Stefan; Silva, Anilda G.; Souza, Eliel M.; Tadra-Sfeir, Michelle Z.; Torres, Rodrigo A.; Dabul, Audrei N. G.; Soares, Maria Albertina M.; Gasques, Luciano S.; Gimenes, Ciela C. T.; Valle, Juliana S.; Ciferri, Ricardo R.; Correa, Luiz C.; Murace, Norma K.; Pamphile, João A.; Patussi, Eliana Valéria; Prioli, Alberto J.; Prioli, Sonia Maria A.; Rocha, Carmem Lúcia M. S. C.; Arantes, Olívia Márcia N.; Furlaneto, Márcia Cristina; Godoy, Leandro P.; Oliveira, Carlos E. C.; Satori, Daniele; Vilas-Boas, Laurival A.; Watanabe, Maria Angélica E.; Dambros, Bibiana Paula; Guerra, Miguel P.; Mathioni, Sandra Marisa; Santos, Karine Louise; Steindel, Mario; Vernal, Javier; Barcellos, Fernando G.; Campo, Rubens J.; Chueire, Ligia Maria O.; Nicolás, Marisa Fabiana; Pereira-Ferrari, Lilian; da Conceição Silva, José L.; Gioppo, Nereida M. R.; Margarido, Vladimir P.; Menck-Soares, Maria Amélia; Pinto, Fabiana Gisele S.; Simão, Rita de Cássia G.; Takahashi, Elizabete K.; Yates, Marshall G.; Souza, Emanuel M.

    2011-01-01

    The molecular mechanisms of plant recognition, colonization, and nutrient exchange between diazotrophic endophytes and plants are scarcely known. Herbaspirillum seropedicae is an endophytic bacterium capable of colonizing intercellular spaces of grasses such as rice and sugar cane. The genome of H. seropedicae strain SmR1 was sequenced and annotated by The Paraná State Genome Programme—GENOPAR. The genome is composed of a circular chromosome of 5,513,887 bp and contains a total of 4,804 genes. The genome sequence revealed that H. seropedicae is a highly versatile microorganism with capacity to metabolize a wide range of carbon and nitrogen sources and with possession of four distinct terminal oxidases. The genome contains a multitude of protein secretion systems, including type I, type II, type III, type V, and type VI secretion systems, and type IV pili, suggesting a high potential to interact with host plants. H. seropedicae is able to synthesize indole acetic acid as reflected by the four IAA biosynthetic pathways present. A gene coding for ACC deaminase, which may be involved in modulating the associated plant ethylene-signaling pathway, is also present. Genes for hemagglutinins/hemolysins/adhesins were found and may play a role in plant cell surface adhesion. These features may endow H. seropedicae with the ability to establish an endophytic life-style in a large number of plant species. PMID:21589895

  9. Comparison of the White-Nose Syndrome Agent Pseudogymnoascus destructans to Cave-Dwelling Relatives Suggests Reduced Saprotrophic Enzyme Activity

    PubMed Central

    Reynolds, Hannah T.; Barton, Hazel A.

    2014-01-01

    White-nose Syndrome (WNS) is an emerging infectious mycosis that has impacted multiple species of North American bats since its initial discovery in 2006, yet the physiology of the causal agent, the psychrophilic fungus Pseudogymnoascus destructans ( = Geomyces destructans), is not well understood. We investigated the ability of P. destructans to secrete enzymes that could permit environmental growth or affect pathogenesis and compared enzyme activity across several Pseudogymnoascus species isolated from both hibernating bats and cave sediments. We found that P. destructans produced enzymes that could be beneficial in either a pathogenic or saprotrophic context, such as lipases, hemolysins, and urease, as well as chitinase and cellulases, which could aid in saprotrophic growth. The WNS pathogen showed significantly lower activity for urease and endoglucanase compared to con-generic species (Pseudogymnoascus), which may indicate a shift in selective pressure to the detriment of P. destructans’ saprotrophic ability. Based on the positive function of multiple saprotrophic enzymes, the causal agent of White-nose Syndrome shows potential for environmental growth on a variety of substrates found in caves, albeit at a reduced level compared to environmental strains. Our data suggest that if P. destructans emerged as an opportunistic infection from an environmental source, co-evolution with its host may have led to a reduced capacity for saprotrophic growth. PMID:24466096

  10. Hcp2, a Secreted Protein of the Phytopathogen Pseudomonas syringae pv. Tomato DC3000, Is Required for Fitness for Competition against Bacteria and Yeasts

    PubMed Central

    Mosorin, Hanna; Dorati, Federico; Wu, Ru-Fen; Roine, Elina; Taira, Suvi; Nissinen, Riitta; Mattinen, Laura; Jackson, Robert; Pirhonen, Minna

    2012-01-01

    When analyzing the secretome of the plant pathogen Pseudomonas syringae pv. tomato DC3000, we identified hemolysin-coregulated protein (Hcp) as one of the secreted proteins. Hcp is assumed to be an extracellular component of the type VI secretion system (T6SS). Two copies of hcp genes are present in the P. syringae pv. tomato DC3000 genome, hcp1 (PSPTO_2539) and hcp2 (PSPTO_5435). We studied the expression patterns of the hcp genes and tested the fitness of hcp knockout mutants in host plant colonization and in intermicrobial competition. We found that the hcp2 gene is expressed most actively at the stationary growth phase and that the Hcp2 protein is secreted via the T6SS and appears in the culture medium as covalently linked dimers. Expression of hcp2 is not induced in planta and does not contribute to virulence in or colonization of tomato or Arabidopsis plants. Instead, hcp2 is required for survival in competition with enterobacteria and yeasts, and its function is associated with the suppression of the growth of these competitors. This is the first report on bacterial T6SS-associated genes functioning in competition with yeast. Our results suggest that the T6SS of P. syringae may play an important role in bacterial fitness, allowing this plant pathogen to survive under conditions where it has to compete with other microorganisms for resources. PMID:22753062

  11. Crystal Structure and Self-Interaction of the Type VI Secretion Tail-Tube Protein from Enteroaggregative Escherichia coli

    PubMed Central

    Douzi, Badreddine; Spinelli, Silvia; Blangy, Stéphanie; Roussel, Alain; Durand, Eric; Brunet, Yannick R.; Cascales, Eric; Cambillau, Christian

    2014-01-01

    The type VI secretion system (T6SS) is a widespread machine used by bacteria to control their environment and kill or disable bacterial species or eukaryotes through toxin injection. The T6SS comprises a central tube formed of stacked hexamers of hemolysin co-regulated proteins (Hcp) and terminated by a trimeric valine-glycine repeat protein G (VgrG) component, the cell puncturing device. A contractile tail sheath, formed by the TssB and TssC proteins, surrounds this tube. This syringe-like machine has been compared to an inverted phage, as both Hcp and VgrG share structural homology with tail components of Caudovirales. Here we solved the crystal structure of a tryptophan-substituted double mutant of Hcp1 from enteroaggregative Escherichia coli and compared it to the structures of other Hcps. Interestingly, we observed that the purified Hcp native protein is unable to form tubes in vitro. To better understand the rationale for observation, we measured the affinity of Hcp1 hexamers with themselves by surface plasmon resonance. The intra-hexamer interaction is weak, with a KD value of 7.2 µM. However, by engineering double cysteine mutants at defined positions, tubes of Hcp1 gathering up to 15 stacked hexamers formed in oxidative conditions. These results, together with those available in the literature regarding TssB and TssC, suggest that assembly of the T6SS tube differs significantly from that of Sipho- or Myoviridae. PMID:24551044

  12. Arrayed lipid bilayer chambers allow single-molecule analysis of membrane transporter activity.

    PubMed

    Watanabe, Rikiya; Soga, Naoki; Fujita, Daishi; Tabata, Kazuhito V; Yamauchi, Lisa; Hyeon Kim, Soo; Asanuma, Daisuke; Kamiya, Mako; Urano, Yasuteru; Suga, Hiroaki; Noji, Hiroyuki

    2014-07-24

    Nano- to micron-size reaction chamber arrays (femtolitre chamber arrays) have facilitated the development of sensitive and quantitative biological assays, such as single-molecule enzymatic assays, digital PCR and digital ELISA. However, the versatility of femtolitre chamber arrays is limited to reactions that occur in aqueous solutions. Here we report an arrayed lipid bilayer chamber system (ALBiC) that contains sub-million femtolitre chambers, each sealed with a stable 4-μm-diameter lipid bilayer membrane. When reconstituted with a limiting amount of the membrane transporter proteins α-hemolysin or F0F1-ATP synthase, the chambers within the ALBiC exhibit stochastic and quantized transporting activities. This demonstrates that the single-molecule analysis of passive and active membrane transport is achievable with the ALBiC system. This new platform broadens the versatility of femtolitre chamber arrays and paves the way for novel applications aimed at furthering our mechanistic understanding of membrane proteins' function.

  13. The pathogenesis, detection, and prevention of Vibrio parahaemolyticus

    PubMed Central

    Wang, Rongzhi; Zhong, Yanfang; Gu, Xiaosong; Yuan, Jun; Saeed, Abdullah F.; Wang, Shihua

    2015-01-01

    Vibrio parahaemolyticus, a Gram-negative motile bacterium that inhabits marine and estuarine environments throughout the world, is a major food-borne pathogen that causes life-threatening diseases in humans after the consumption of raw or undercooked seafood. The global occurrence of V. parahaemolyticus accentuates the importance of investigating its virulence factors and their effects on the human host. This review describes the virulence factors of V. parahaemolyticus reported to date, including hemolysin, urease, two type III secretion systems and two type VI secretion systems, which both cause both cytotoxicity in cultured cells and enterotoxicity in animal models. We describe various types of detection methods, based on virulence factors, that are used for quantitative detection of V. parahaemolyticus in seafood. We also discuss some useful preventive measures and therapeutic strategies for the diseases mediated by V. parahaemolyticus, which can reduce, to some extent, the damage to humans and aquatic animals attributable to V. parahaemolyticus. This review extends our understanding of the pathogenic mechanisms of V. parahaemolyticus mediated by virulence factors and the diseases it causes in its human host. It should provide new insights for the diagnosis, treatment, and prevention of V. parahaemolyticus infection. PMID:25798132

  14. Bacterial Pore-Forming Cytolysins Induce Neuronal Damage in a Rat Model of Neonatal Meningitis

    PubMed Central

    Reiß, Anja; Braun, Johann S.; Jäger, Katja; Freyer, Dorette; Laube, Gregor; Bührer, Christoph; Felderhoff-Müser, Ursula; Stadelmann, Christine; Nizet, Victor

    2011-01-01

    Background. Group B Streptococcus (GBS) and Streptococcus pneumoniae (SP) are leading causes of bacterial meningitis in neonates and children. Each pathogen produces a pore-forming cytolytic toxin, β-hemolysin/cytolysin (β-h/c) by GBS and pneumolysin by SP. The aim of this study was to understand the role of these pore-forming cytotoxins, in particular of the GBS β-h/c, as potential neurotoxins in experimental neonatal meningitis. Methods. Meningitis was induced in 7- and 11-day-old rats by intracisternal injection of wild type (WT) GBS or SP and compared with isogenic β-h/c- or pneumolysin-deficient mutants, or a double mutant of SP deficient in pneumolysin and hydrogen peroxide production. Results. GBS β-h/c and SP pneumolysin contributed to neuronal damage, worsened clinical outcome and weight loss, but had no influence on the early kinetics of leukocyte influx and bacterial growth in the cerebrospinal fluid. In vitro, β-h/c-induced neuronal apoptosis occurred independently of caspase-activation and was not preventable by the broad spectrum caspase-inhibitor z-VAD-fmk. Conclusions. These data suggest that both cytolytic toxins, the GBS β-h/c and SP pneumolysin, contribute to neuronal damage in meningitis and extend the concept of a key role for bacterial pore-forming cytolysins in the pathogenesis and sequelae of neonatal meningitis. PMID:21186256

  15. Prevalence of Virulence Factors and Drug Resistance in Clinical Isolates of Enterococci: A Study from North India

    PubMed Central

    Banerjee, Tuhina; Anupurba, Shampa

    2015-01-01

    Along with emergence of multidrug resistance, presence of several virulence factors in enterococci is an emerging concept. This study was undertaken to determine the prevalence of various virulence factors phenotypically and genotypically in enterococci and study their association with multidrug resistance. A total of 310 enterococcal isolates were studied, comprising 155 E. faecium and 155 E. faecalis. Antimicrobial susceptibility testing was done by disc diffusion and agar dilution method. Hemolysin, gelatinase, biofilm production, and haemagglutination were detected phenotypically and presence of virulence genes, namely, asa1, gelE, cylA, esp, and hyl, was detected by multiplex PCR. Of the total, 47.41% isolates were high level gentamicin resistant (HLGRE) and 7.09% were vancomycin resistant (VRE). All the virulence traits studied were found in varying proportions, with majority in E. faecalis (p > 0.05). Strong biofilm producers possessed either asa1 or gelE gene. gelE silent gene was detected in 41.37% (12/29). However, increase in resistance was associated with significant decrease in expression or acquisition of virulence genes. Further, acquisition of vancomycin resistance was the significant factor responsible for the loss of virulence traits. Though it is presumed that increased drug resistance correlates with increased virulence, acquisition of vancomycin resistance might be responsible for reduced expression of virulence traits to meet the “biological cost” relating to VRE. PMID:26366302

  16. Discrimination of biological and chemical threat simulants in residue mixtures on multiple substrates.

    PubMed

    Gottfried, Jennifer L

    2011-07-01

    The potential of laser-induced breakdown spectroscopy (LIBS) to discriminate biological and chemical threat simulant residues prepared on multiple substrates and in the presence of interferents has been explored. The simulant samples tested include Bacillus atrophaeus spores, Escherichia coli, MS-2 bacteriophage, α-hemolysin from Staphylococcus aureus, 2-chloroethyl ethyl sulfide, and dimethyl methylphosphonate. The residue samples were prepared on polycarbonate, stainless steel and aluminum foil substrates by Battelle Eastern Science and Technology Center. LIBS spectra were collected by Battelle on a portable LIBS instrument developed by A3 Technologies. This paper presents the chemometric analysis of the LIBS spectra using partial least-squares discriminant analysis (PLS-DA). The performance of PLS-DA models developed based on the full LIBS spectra, and selected emission intensities and ratios have been compared. The full-spectra models generally provided better classification results based on the inclusion of substrate emission features; however, the intensity/ratio models were able to correctly identify more types of simulant residues in the presence of interferents. The fusion of the two types of PLS-DA models resulted in a significant improvement in classification performance for models built using multiple substrates. In addition to identifying the major components of residue mixtures, minor components such as growth media and solvents can be identified with an appropriately designed PLS-DA model.

  17. MOLECULAR CHARACTERIZATION OF VIRULENCE AND ANTIMICROBIAL SUSCEPTIBILITY PROFILES OF UROPATHOGENIC ESCHERICHIA COLI FROM PATIENTS IN A TERTIARY HOSPITAL, SOUTHERN THAILAND.

    PubMed

    Themphachanal, Monchanok; Kongpheng, Suttiporn; Rattanachuay, Pattamarat; Khianngam, Saowapar; Singkhamanan, Kamonnut; Sukhumungoon, Pharanai

    2015-11-01

    Among uropathogens, uropathogenic Escherichia coli (UPEC) is the most common cause of urinary tract infection (UTI) worldwide, but clinical aspects due to this bacterial species is not fully understood in southern Thailand. Two hundred fifty-four UPEC isolates from patients admitted to Maharaj Nakhon Si Thammarat Hospital, southern Thailand were examined for crucial virulence genes, showing that 33.5% contained at least one of the virulence, genes tested. Genes encoding P fimbria, cytotoxic necrotizing factor-1 and α-hemolysin constituted the majority (15.8%) carried by UPEC isolates. Phylogenetic group classification revealed that 57.5% of UPEC belonged to group D. Antimicrobial susceptibility tests showed that 70.5% and 65.1% of the isolates were resistant to ciprofloxacin and norfloxacin, respectively. Moreover, 50.0% of UPEC were capable of producing extended spectrum beta-lactamases. These findings should be of benefit for more appropriate treatment of UTI patients in this region of Thailand. Keywords: uropathogenic Escherichia coli, antibiotics resistance, cnfl, hlyA, pap, Thailand

  18. Virulence potential and genetic diversity of Aeromonas caviae, Aeromonas veronii, and Aeromonas hydrophila clinical isolates from Mexico and Spain: a comparative study.

    PubMed

    Aguilera-Arreola, Ma Guadalupe; Hernández-Rodríguez, César; Zúñiga, Gerardo; Figueras, María José; Garduño, Rafael A; Castro-Escarpulli, Graciela

    2007-07-01

    A comparative study of 109 Aeromonas clinical isolates belonging to the 3 species most frequently isolated from patients with diarrhea in Mexico and Spain was performed to investigate the distribution of 3 prominent toxin genes and the gene encoding flagellin of lateral flagella; 4 well-established virulence factors in the genus Aeromonas. The aerolysin-hemolysin toxin genes were the most prevalent, being present in 89% of the total isolates. The ast toxin gene was conspicuously absent from the Aeromonas caviae and Aeromonas veronii groups but was present in 91% of the Aeromonas hydrophila isolates. Both the alt toxin gene and the lafA flagellin gene also had a low incidence in A. caviae and A. veronii. Differences in the prevalence of alt and lafA were observed between isolates from Mexico and Spain, confirming genus heterogeneity according to geographic location. Carriage of multiple toxin genes was primarily restricted to A. hydrophila isolates, suggesting that A. caviae and A. veronii isolates circulating in Mexico and Spain possess a limited array of virulence genes. Enterobacterial repetitive intergenetic consensus - polymerase chain reaction showed that the Aeromonas populations sampled lack dominant clones and were genetically heterogeneous, with A. caviae being the most diverse species. Further surveys of virulence determinants in genetically heterogeneous populations of Aeromonas isolates circulating worldwide are required to enhance the understanding of their capacity to cause disease. PMID:17898843

  19. Construction of a chromosome map for the phage group II Staphylococcus aureus Ps55.

    PubMed Central

    Bannantine, J P; Pattee, P A

    1996-01-01

    The genome size and a partial physical and genetic map have been defined for the phage group II Staphylococcus aureus Ps55. The genome size was estimated to be 2,771 kb by pulsed-field gel electrophoresis (PFGE) using the restriction enzymes SmaI, CspI, and SgrAI. The Ps55 chromosome map was constructed by transduction of auxotrophic and cryptic transposon insertions, with known genetic and physical locations in S. aureus NCTC 8325, into the Ps55 background. PFGE and DNA hybridization analysis were used to detect the location of the transposon in Ps55. Ps55 restriction fragments were then ordered on the basis of genetic conservation between the two strains. Cloned DNA probes containing the lactose operon (lac) and genes encoding staphylococcal protein A (spa), gamma hemolysin (hlg), and coagulase (coa) were also located on the map by PFGE and hybridization analysis. This methodology enabled a direct comparison of chromosomal organization between NCTC 8325 and Ps55 strains. The chromosome size, gene order, and some of the restriction sites are conserved between the two phage group strains. PMID:8955305

  20. Acidity-Mediated, Electrostatic Tuning of Asymmetrically Charged Peptides Interactions with Protein Nanopores.

    PubMed

    Asandei, Alina; Chinappi, Mauro; Kang, Hee-Kyoung; Seo, Chang Ho; Mereuta, Loredana; Park, Yoonkyung; Luchian, Tudor

    2015-08-01

    Despite success in probing chemical reactions and dynamics of macromolecules on submillisecond time and nanometer length scales, a major impasse faced by nanopore technology is the need to cheaply and controllably modulate macromolecule capture and trafficking across the nanopore. We demonstrate herein that tunable charge separation engineered at the both ends of a macromolecule very efficiently modulates the dynamics of macromolecules capture and traffic through a nanometer-size pore. In the proof-of-principle approach, we employed a 36 amino acids long peptide containing at the N- and C-termini uniform patches of glutamic acids and arginines, flanking a central segment of asparagines, and we studied its capture by the α-hemolysin (α-HL) and the mean residence time inside the pore in the presence of a pH gradient across the protein. We propose a solution to effectively control the dynamics of peptide interaction with the nanopore, with both association and dissociation reaction rates of peptide-α-HL interactions spanning orders of magnitude depending upon solution acidity on the peptide addition side and the transmembrane electric potential, while preserving the amplitude of the blockade current signature. PMID:26144534

  1. Use of Zebrafish to Probe the Divergent Virulence Potentials and Toxin Requirements of Extraintestinal Pathogenic Escherichia coli

    PubMed Central

    Wiles, Travis J.; Bower, Jean M.; Redd, Michael J.; Mulvey, Matthew A.

    2009-01-01

    Extraintestinal pathogenic E. coli (ExPEC) cause an array of diseases, including sepsis, neonatal meningitis, and urinary tract infections. Many putative virulence factors that might modulate ExPEC pathogenesis have been identified through sequencing efforts, epidemiology, and gene expression profiling, but few of these genes have been assigned clearly defined functional roles during infection. Using zebrafish embryos as surrogate hosts, we have developed a model system with the ability to resolve diverse virulence phenotypes and niche-specific restrictions among closely related ExPEC isolates during either localized or systemic infections. In side-by-side comparisons of prototypic ExPEC isolates, we observed an unexpectedly high degree of phenotypic diversity that is not readily apparent using more traditional animal hosts. In particular, the capacity of different ExPEC isolates to persist and multiply within the zebrafish host and cause disease was shown to be variably dependent upon two secreted toxins, α-hemolysin and cytotoxic necrotizing factor. Both of these toxins appear to function primarily in the neutralization of phagocytes, which are recruited in high numbers to sites of infection where they act as an essential host defense against ExPEC as well as less virulent E. coli strains. These results establish zebrafish as a valuable tool for the elucidation and functional analysis of both ExPEC virulence factors and host defense mechanisms. PMID:20019794

  2. Origins and Virulence Mechanisms of Uropathogenic Escherichia coli

    PubMed Central

    Wiles, Travis J.; Kulesus, Richard R.; Mulvey, Matthew A.

    2008-01-01

    Strains of uropathogenic E. coli (UPEC) are the primary cause of urinary tract infections, including both cystitis and pyelonephritis. These bacteria have evolved a multitude of virulence factors and strategies that facilitate bacterial growth and persistence within the adverse settings of the host urinary tract. Expression of adhesive organelles like type 1 and P pili allow UPEC to bind and invade host cells and tissues within the urinary tract while expression of iron chelating factors (siderophores) enable UPEC to pilfer host iron stores. Deployment of an array of toxins, including hemolysin and cytotoxic necrotizing factor 1, provide UPEC with the means to inflict extensive tissue damage, facilitating bacterial dissemination as well as releasing host nutrients and disabling immune effector cells. These toxins also have the capacity to modulate, in more subtle ways, host signaling pathways affecting myriad processes, including inflammatory responses, host cell survival, and cytoskeletal dynamics. Here, we discuss the mechanisms by which these and other virulence factors promote UPEC survival and growth within the urinary tract. Comparisons are also made between UPEC and other strains of extraintestinal pathogenic E. coli that, although closely related to UPEC, are distinct in their abilities to colonize the host and cause disease. PMID:18482721

  3. The Draft Genome Sequence of the Yersinia entomophaga Entomopathogenic Type Strain MH96T

    PubMed Central

    Hurst, Mark R. H.; Beattie, Amy; Altermann, Eric; Moraga, Roger M.; Harper, Lincoln A.; Calder, Joanne; Laugraud, Aurelie

    2016-01-01

    Here we report the draft genome of Yersinia entomophaga type strain MH96T. The genome shows 93.8% nucleotide sequence identity to that of Yersinia nurmii type strain APN3a-cT, and comprises a single chromosome of approximately 4,275,531 bp. In silico analysis identified that, in addition to the previously documented Y. entomophaga Yen-TC gene cluster, the genome encodes a diverse array of toxins, including two type III secretion systems, and five rhs-associated gene clusters. As well as these multicomponent systems, several orthologs of known insect toxins, such as VIP2 toxin and the binary toxin PirAB, and distant orthologs of some mammalian toxins, including repeats-in-toxin, a cytolethal distending toxin, hemolysin-like genes and an adenylate cyclase were identified. The genome also contains a large number of hypothetical proteins and orthologs of known effector proteins, such as LopT, as well as genes encoding a wide range of proteolytic determinants, including metalloproteases and pathogen fitness determinants, such as genes involved in iron metabolism. The bioinformatic data derived from the current in silico analysis, along with previous information on the pathobiology of Y. entomophaga against its insect hosts, suggests that a number of these virulence systems are required for survival in the hemocoel and incapacitation of the insect host. PMID:27187466

  4. Campylobacter jejuni Increases Flagellar Expression and Adhesion of Noninvasive Escherichia coli: Effects on Enterocytic Toll-Like Receptor 4 and CXCL-8 Expression

    PubMed Central

    Reti, Kristen L.; Tymensen, Lisa D.; Davis, Shevaun P.; Amrein, Matthias W.

    2015-01-01

    Campylobacter jejuni is the most common cause of bacterium-induced gastroenteritis, and while typically self-limiting, C. jejuni infections are associated with postinfectious intestinal disorders, including flares in patients with inflammatory bowel disease and postinfectious irritable bowel syndrome (PI-IBS), via mechanisms that remain obscure. Based on the hypothesis that acute campylobacteriosis may cause pathogenic microbiota dysbiosis, we investigated whether C. jejuni may activate dormant virulence genes in noninvasive Escherichia coli and examined the epithelial pathophysiological consequences of these alterations. Microarray and quantitative real-time PCR analyses revealed that E. coli adhesin, flagellum, and hemolysin gene expression were increased when E. coli was exposed to C. jejuni-conditioned medium. Increased development of bacterial flagella upon exposure to live C. jejuni or C. jejuni-conditioned medium was observed under transmission electron microscopy. Atomic force microscopy demonstrated that the forces of bacterial adhesion to colonic T84 enterocytes, and the work required to rupture this adhesion, were significantly increased in E. coli exposed to C. jejuni-conditioned media. Finally, C. jejuni-modified E. coli disrupted TLR4 gene expression and induced proinflammatory CXCL-8 gene expression in colonic enterocytes. Together, these data suggest that exposure to live C. jejuni, and/or to its secretory-excretory products, may activate latent virulence genes in noninvasive E. coli and that these alterations may directly trigger proinflammatory signaling in intestinal epithelia. These observations shed new light on mechanisms that may contribute, at least in part, to postcampylobacteriosis inflammatory disorders. PMID:26371123

  5. Detecting and genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays.

    PubMed

    Call, D R; Brockman, F J; Chandler, D P

    2001-07-20

    Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification. The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFU ml(-1) (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass-based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products; the system is amenable to automation.

  6. Slowing down single-molecule trafficking through a protein nanopore reveals intermediates for peptide translocation

    NASA Astrophysics Data System (ADS)

    Mereuta, Loredana; Roy, Mahua; Asandei, Alina; Lee, Jong Kook; Park, Yoonkyung; Andricioaei, Ioan; Luchian, Tudor

    2014-01-01

    The microscopic details of how peptides translocate one at a time through nanopores are crucial determinants for transport through membrane pores and important in developing nano-technologies. To date, the translocation process has been too fast relative to the resolution of the single molecule techniques that sought to detect its milestones. Using pH-tuned single-molecule electrophysiology and molecular dynamics simulations, we demonstrate how peptide passage through the α-hemolysin protein can be sufficiently slowed down to observe intermediate single-peptide sub-states associated to distinct structural milestones along the pore, and how to control residence time, direction and the sequence of spatio-temporal state-to-state dynamics of a single peptide. Molecular dynamics simulations of peptide translocation reveal the time- dependent ordering of intermediate structures of the translocating peptide inside the pore at atomic resolution. Calculations of the expected current ratios of the different pore-blocking microstates and their time sequencing are in accord with the recorded current traces.

  7. Crystal Structure of the Monomeric Porin OmpG

    SciTech Connect

    Subbarao,G.; van den Berg, B.

    2006-01-01

    The outer membrane (OM) of Gram-negative bacteria contains a large number of channel proteins that mediate the uptake of ions and nutrients necessary for growth and functioning of the cell. An important group of OM channel proteins are the porins, which mediate the non-specific, diffusion-based passage of small (<600 Da) polar molecules. All porins of Gram-negative bacteria that have been crystallized to date form stable trimers, with each monomer composed of a 16-stranded {beta}-barrel with a relatively narrow central pore. In contrast, the OmpG porin is unique, as it appears to function as a monomer. We have determined the X-ray crystal structure of OmpG from Escherichia coli to a resolution of 2.3 Angstroms. The structure shows a 14-stranded {beta}{beta}-barrel with a relatively simple architecture. Due to the absence of loops that fold back into the channel, OmpG has a large ({approx}13 Angstroms) central pore that is considerably wider than those of other E. coli porins, and very similar in size to that of the toxin a-hemolysin. The architecture of the channel, together with previous biochemical and other data, suggests that OmpG may form a non-specific channel for the transport of larger oligosaccharides. The structure of OmpG provides the starting point for engineering studies aiming to generate selective channels and for the development of biosensors.

  8. Imaging of bacterial multicellular behaviour in biofilms in liquid by atmospheric scanning electron microscopy

    PubMed Central

    Sugimoto, Shinya; Okuda, Ken-ichi; Miyakawa, Reina; Sato, Mari; Arita-Morioka, Ken-ichi; Chiba, Akio; Yamanaka, Kunitoshi; Ogura, Teru; Mizunoe, Yoshimitsu; Sato, Chikara

    2016-01-01

    Biofilms are complex communities of microbes that attach to biotic or abiotic surfaces causing chronic infectious diseases. Within a biofilm, microbes are embedded in a self-produced soft extracellular matrix (ECM), which protects them from the host immune system and antibiotics. The nanoscale visualisation of delicate biofilms in liquid is challenging. Here, we develop atmospheric scanning electron microscopy (ASEM) to visualise Gram-positive and -negative bacterial biofilms immersed in aqueous solution. Biofilms cultured on electron-transparent film were directly imaged from below using the inverted SEM, allowing the formation of the region near the substrate to be studied at high resolution. We visualised intercellular nanostructures and the exocytosis of membrane vesicles, and linked the latter to the trafficking of cargos, including cytoplasmic proteins and the toxins hemolysin and coagulase. A thick dendritic nanotube network was observed between microbes, suggesting multicellular communication in biofilms. A universal immuno-labelling system was developed for biofilms and tested on various examples, including S. aureus biofilms. In the ECM, fine DNA and protein networks were visualised and the precise distribution of protein complexes was determined (e.g., straight curli, flagella, and excreted cytoplasmic molecular chaperones). Our observations provide structural insights into bacteria-substratum interactions, biofilm development and the internal microbe community. PMID:27180609

  9. Phosphate regulation of gene expression in Vibrio parahaemolyticus.

    PubMed Central

    McCarter, L L; Silverman, M

    1987-01-01

    The synthesis of a major outer membrane protein, OmpP, in Vibrio parahaemolyticus was induced by growth in media deficient in phosphate. The gene, ompP, encoding this protein was cloned. Synthesis of OmpP in Escherichia coli was regulated by the availability of phosphate, and this control required the function of pho regulatory genes of E. coli. Analysis of gene fusion strains constructed by mutagenesis with transposon mini-Mulux revealed that ompP was transcriptionally regulated in V. parahaemolyticus. Impaired growth of a strain with an ompP defect was observed in media which contained large linear polyphosphates as the phosphate source. This and other evidence suggested that OmpP functions as a porin channel for the entry of phosphate into the cell. A number of other proteins or activities were induced by phosphate limitation including hemolysin, phospholipase C, and phosphatase activities. A regulatory locus controlling expression of phosphate-regulated genes was identified and cloned. This regulatory locus cloned from V. parahaemolyticus was shown to complement E. coli strains with defects in pho regulatory genes. Images PMID:3038839

  10. Modelling the Bioelectronic Interface in Engineered Tethered Membranes: From Biosensing to Electroporation.

    PubMed

    Hoiles, William; Krishnamurthy, Vikram; Cornell, Bruce

    2015-06-01

    This paper studies the construction and predictive models of three novel measurement platforms: (i) a Pore Formation Measurement Platform (PFMP) for detecting the presence of pore forming proteins and peptides, (ii) the Ion Channel Switch (ICS) biosensor for detecting the presence of analyte molecules in a fluid chamber, and (iii) an Electroporation Measurement Platform (EMP) that provides reliable measurements of the electroporation phenomenon. Common to all three measurement platforms is that they are comprised of an engineered tethered membrane that is formed via a rapid solvent exchange technique allowing the platform to have a lifetime of several months. The membrane is tethered to a gold electrode bioelectronic interface that includes an ionic reservoir separating the membrane and gold surface, allowing the membrane to mimic the physiological response of natural cell membranes. The electrical response of the PFMP, ICS, and EMP are predicted using continuum theories for electrodiffusive flow coupled with boundary conditions for modelling chemical reactions and electrical double layers present at the bioelectronic interface. Experimental measurements are used to validate the predictive accuracy of the dynamic models. These include using the PFMP for measuring the pore formation dynamics of the antimicrobial peptide PGLa and the protein toxin Staphylococcal α-Hemolysin; the ICS biosensor for measuring nano-molar concentrations of streptavidin, ferritin, thyroid stimulating hormone (TSH), and human chorionic gonadotropin (pregnancy hormone hCG); and the EMP for measuring electroporation of membranes with different tethering densities, and membrane compositions.

  11. Genome-Wide Analysis of Oceanimonas sp. GK1 Isolated from Gavkhouni Wetland (Iran) Demonstrates Presence of Genes for Virulence and Pathogenicity

    PubMed Central

    Parsa Yeganeh, Laleh; Azarbaijani, Reza; Mousavi, Hossein; Shahzadeh Fazeli, Seyed Abolhassan; Amoozgar, Mohammad Ali; Salekdeh, Ghasem Hosseini

    2015-01-01

    Objective The bacterium Oceanimonas sp. (O. sp.) GK1 is a member of the Aeromonadaceae family and its genome represents several virulence genes involved in fish and human pathogenicity. In this original research study we aimed to identify and characterize the putative virulence factors and pathogenicity of this halotolerant marine bacterium using genome wide analysis. Materials and Methods The genome data of O. sp. GK1 was obtained from NCBI. Comparative genomic study was done using MetaCyc database. Results Whole genome data analysis of the O. sp. GK1 revealed that the bacterium possesses some important virulence genes (e.g. ZOT, RTX toxin, thermostable hemolysin, lateral flagella and type IV pili) which have been implicated in adhesion and biofilm formation and infection in some other pathogenic bacteria. Conclusion This is the first report of the putative pathogenicity of O. sp.GK1. The genome wide analysis of the bacterium demonstrates the presence of virulence genes causing infectious diseases in many warmand cold-blooded animals. PMID:26464816

  12. Identification of a two-component fatty acid kinase responsible for host fatty acid incorporation by Staphylococcus aureus

    PubMed Central

    Parsons, Joshua B.; Broussard, Tyler C.; Bose, Jeffrey L.; Rosch, Jason W.; Jackson, Pamela; Subramanian, Chitra; Rock, Charles O.

    2014-01-01

    Extracellular fatty acid incorporation into the phospholipids of Staphylococcus aureus occurs via fatty acid phosphorylation. We show that fatty acid kinase (Fak) is composed of two dissociable protein subunits encoded by separate genes. FakA provides the ATP binding domain and interacts with two distinct FakB proteins to produce acyl-phosphate. The FakBs are fatty acid binding proteins that exchange bound fatty acid/acyl-phosphate with fatty acid/acyl-phosphate presented in detergent micelles or liposomes. The ΔfakA and ΔfakB1 ΔfakB2 strains were unable to incorporate extracellular fatty acids into phospholipid. FakB1 selectively bound saturated fatty acids whereas FakB2 preferred unsaturated fatty acids. Affymetrix array showed a global perturbation in the expression of virulence genes in the ΔfakA strain. The severe deficiency in α-hemolysin protein secretion in ΔfakA and ΔfakB1 ΔfakB2 mutants coupled with quantitative mRNA measurements showed that fatty acid kinase activity was required to support virulence factor transcription. These data reveal the function of two conserved gene families, their essential role in the incorporation of host fatty acids by Gram-positive pathogens, and connects fatty acid kinase to the regulation of virulence factor transcription in S. aureus. PMID:25002480

  13. Chemical Composition and Antipathogenic Activity of Artemisia annua Essential Oil from Romania.

    PubMed

    Marinas, Ioana C; Oprea, Eliza; Chifiriuc, Mariana Carmen; Badea, Irinel Adriana; Buleandra, Mihaela; Lazar, Veronica

    2015-10-01

    The essential oil extracted by hydrodistillation from Romanian Artemisia annua aerial parts was characterized by GC/MS analysis, which allowed the identification of 94.64% of the total oil composition. The main components were camphor (17.74%), α-pinene (9.66%), germacrene D (7.55%), 1,8-cineole (7.24%), trans-β-caryophyllene (7.02%), and artemisia ketone (6.26%). The antimicrobial activity of this essential oil was evaluated by determining the following parameters: minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), minimal fungicidal concentration (MFC), and minimal biofilm eradication concentration (MBEC). Moreover, the soluble virulence factors were quantified with different biochemical substrates incorporated in the culture media. The reference and resistant, clinical strains proved to be susceptible to the A. annua oil, with MICs ranging from 0.51 to 16.33 mg/ml. The tested essential oil also showed good antibiofilm activity, inhibiting both the initial stage of the microbial cell adhesion to the inert substratum and the preformed mature biofilm. When used at subinhibitory concentrations, the essential oil proved to inhibit the phenotypic expression of five soluble virulence factors (hemolysins, gelatinase, DNase, lipases, and lecithinases). Briefly, the present results showed that the A. annua essential oil contained antimicrobial compounds with selective activity on Gram-positive and Gram-negative bacterial strains as well as on yeast strains and which also interfere with the expression of cell-associated and soluble virulence factors. PMID:26460560

  14. Antistaphylococcal and biofilm inhibitory activities of gallic, caffeic, and chlorogenic acids.

    PubMed

    Luís, Ângelo; Silva, Filomena; Sousa, Sónia; Duarte, Ana Paula; Domingues, Fernanda

    2014-01-01

    Staphylococcus aureus is a Gram-positive pathogen which is able to form biofilms, exhibiting a more pronounced resistance to antibiotics and disinfectants. The hurdles posed in eradicating biofilms have driven the search for new compounds able to fight these structures. Phenolic compounds constitute one of the most numerous and ubiquitous group of plant secondary metabolites with many biological activities. The aim of the present work was to study the potential antimicrobial and antibiofilm properties of gallic, caffeic, and chlorogenic acids against S. aureus as well to elucidate its mechanism of action. It was concluded that the phenolic acids studied in this work have antistaphylococcal properties. For instance, gallic acid is able to influence the adhesion properties of S. aureus. The phenolic acids tested were also able to inhibit the production of α-hemolysin by this microorganism, with the exception of chlorogenic acid. Regarding its mechanism of action, caffeic acid interferes with the stability of the cell membrane and with the metabolic activity of the cells of S. aureus. PMID:24228999

  15. ABC transporters: bacterial exporters.

    PubMed Central

    Fath, M J; Kolter, R

    1993-01-01

    The ABC transporters (also called traffic ATPases) make up a large superfamily of proteins which share a common function and a common ATP-binding domain. ABC transporters are classified into three major groups: bacterial importers (the periplasmic permeases), eukaryotic transporters, and bacterial exporters. We present a comprehensive review of the bacterial ABC exporter group, which currently includes over 40 systems. The bacterial ABC exporter systems are functionally subdivided on the basis of the type of substrate that each translocates. We describe three main groups: protein exporters, peptide exporters, and systems that transport nonprotein substrates. Prototype exporters from each group are described in detail to illustrate our current understanding of this protein family. The prototype systems include the alpha-hemolysin, colicin V, and capsular polysaccharide exporters from Escherichia coli, the protease exporter from Erwinia chrysanthemi, and the glucan exporters from Agrobacterium tumefaciens and Rhizobium meliloti. Phylogenetic analysis of the ATP-binding domains from 29 bacterial ABC exporters indicates that the bacterial ABC exporters can be divided into two primary branches. One branch contains the transport systems where the ATP-binding domain and the membrane-spanning domain are present on the same polypeptide, and the other branch contains the systems where these domains are found on separate polypeptides. Differences in substrate specificity do not correlate with evolutionary relatedness. A complete survey of the known and putative bacterial ABC exporters is included at the end of the review. PMID:8302219

  16. Sea cucumber (Codonopsis pilosula) oligopeptides: immunomodulatory effects based on stimulating Th cells, cytokine secretion and antibody production.

    PubMed

    He, Li-Xia; Zhang, Zhao-Feng; Sun, Bin; Chen, Qi-He; Liu, Rui; Ren, Jin-Wei; Wang, Jun-Bo; Li, Yong

    2016-02-01

    This study aimed to investigate the immunomodulating activity of small molecule oligopeptides from sea cucumber (Codonopsis pilosula) (SOP) in mice. Seven assays were performed to determine the immunomodulatory effects, including splenic lymphocyte proliferation and delayed-type hypersensitivity assays (cell-mediated immunity), IgM antibody response of spleen to sheep red blood cells (SRBC) and serum hemolysin level assays (humoral immunity), the carbon clearance assay and the phagocytic capacity of peritoneal cavity phagocytes assay (macrophage phagocytosis), and the NK cell activity assay. Spleen T lymphocyte subpopulations, multiplex sandwich immunoassays of serum cytokine and immunoglobulin levels and enzyme-linked immunosorbent assays for small intestinal secretory immunoglobulin were performed to study the mechanism by which SOP affects the immune system. We found that SOP could improve immune functions in mice, which may be due to the enhancement of the functions of cell-mediated immunity, humoral immunity, macrophage phagocytosis and NK cell activity. From the cellular and molecular assays, we postulated that the immunomodulatory effects are most likely attributed to the stimulation of Th cells, cytokine secretion and antibody production. PMID:26838796

  17. Semisynthetic protein nanoreactor for single-molecule chemistry

    PubMed Central

    Lee, Joongoo; Bayley, Hagan

    2015-01-01

    The covalent chemistry of individual reactants bound within a protein pore can be monitored by observing the ionic current flow through the pore, which acts as a nanoreactor responding to bond-making and bond-breaking events. In the present work, we incorporated an unnatural amino acid into the α-hemolysin (αHL) pore by using solid-phase peptide synthesis to make the central segment of the polypeptide chain, which forms the transmembrane β-barrel of the assembled heptamer. The full-length αHL monomer was obtained by native chemical ligation of the central synthetic peptide to flanking recombinant polypeptides. αHL pores with one semisynthetic subunit were then used as nanoreactors for single-molecule chemistry. By introducing an amino acid with a terminal alkyne group, we were able to visualize click chemistry at the single-molecule level, which revealed a long-lived (4.5-s) reaction intermediate. Additional side chains might be introduced in a similar fashion, thereby greatly expanding the range of single-molecule covalent chemistry that can be investigated by the nanoreactor approach. PMID:26504203

  18. Staphylococcus epidermidis and Staphylococcus haemolyticus: Molecular Detection of Cytotoxin and Enterotoxin Genes

    PubMed Central

    Pinheiro, Luiza; Ivo Brito, Carla; de Oliveira, Adilson; Yoshida Faccioli Martins, Patrícia; Cataneli Pereira, Valéria; Ribeiro de Souza da Cunha, Maria de Lourdes

    2015-01-01

    Although opportunistic pathogens, coagulase-negative staphylococci (CoNS), including Staphylococcus epidermidis and Staphylococcus haemolyticus, have long been regarded as avirulent organisms. The role of toxins in the development of infections caused by CoNS is still controversial. The objective of this study was to characterize the presence of enterotoxin and cytotoxin genes in S. epidermidis and S. haemolyticus isolates obtained from blood cultures. Cytotoxin genes were detected by PCR using novel species-specific primers. Among the 85 S. epidermidis and 84 S. haemolyticus isolates, 95.3% and 79.8%, respectively, carried at least one enterotoxin gene. The most frequent enterotoxin genes were sea (53.3%), seg (64.5%) and sei (67.5%). The seg gene was positively associated with S. epidermidis (p = 0.02), and this species was more toxigenic than S. haemolyticus. The hla/yidD gene was detected in 92.9% of S. epidermidis and the hla gene in 91.7% of S. haemolyticus isolates; hlb was detected in 92.9% of the S. epidermidis isolates and hld in 95.3%. Nosocomial Staphylococcus epidermidis and S. haemolyticus isolates exhibited a high toxigenic potential, mainly containing the non-classical enterotoxin genes seg and sei. The previously unreported detection of hla/yidD and hlb in S. epidermidis and S. haemolyticus using species-specific primers showed that these hemolysin genes differ between CoNS species and that they are highly frequent in blood culture isolates. PMID:26389954

  19. CD4 T Cell Antigens from Staphylococcus aureus Newman Strain Identified following Immunization with Heat-Killed Bacteria

    PubMed Central

    Lawrence, Paulraj K.; Rokbi, Bachra; Arnaud-Barbe, Nadège; Sutten, Eric L.; Norimine, Junzo; Lahmers, Kevin K.

    2012-01-01

    Staphylococcus aureus is a commensal bacterium associated with the skin and mucosal surfaces of humans and animals that can also cause chronic infection. The emergence of antibiotic-resistant strains such as methicillin-resistant S. aureus (MRSA) and strains causing chronic intramammary infections (IMI) in cows results in severe human and livestock infections. Conventional approaches to vaccine development have yielded only a few noneffective vaccines against MRSA or IMI strains, so there is a need for improved vaccine development. CD4 T lymphocytes are required for promoting gamma interferon (IFN-γ) mediated immunoglobulin isotype switching in B lymphocytes to produce high-affinity IgG antibodies and IFN-γ-mediated phagocyte activation for an effective resolution of bacterial infection. However, the lack of known CD4 T cell antigens from S. aureus has made it difficult to design effective vaccines. The goal of this study was to identify S. aureus proteins recognized by immune CD4 T cells. Using a reverse genetics approach, 43 antigens were selected from the S. aureus Newman strain. These included lipoproteins, proteases, transcription regulators, an alkaline shock protein, conserved-domain proteins, hemolysins, fibrinogen-binding protein, staphylokinase, exotoxin, enterotoxin, sortase, and protein A. Screening of expressed proteins for recall T cell responses in outbred, immune calves identified 13 proteins that share over 80% sequence identity among MRSA or IMI strains. These may be useful for inclusion in a broadly protective multiantigen vaccine against MRSA or IMI. PMID:22323557

  20. Clonal analysis of Escherichia coli serotype O6 strains from urinary tract infections.

    PubMed

    Zingler, G; Ott, M; Blum, G; Falkenhagen, U; Naumann, G; Sokolowska-Köhler, W; Hacker, J

    1992-04-01

    A total of 36 Escherichia coli urinary tract isolates (UTI) of serotype O6, with different combinations of capsule (K) and flagellin (H) antigens, were analysed according to the outer membrane pattern (OMP), serum resistance properties, mannose-resistant hemagglutination using various types of erythrocytes, and also for the genetic presence and the expression of P-fimbriae, S fimbriae/F1C fimbriae, Type 1 fimbriae, aerobactin and hemolysin. Twenty selected strains were further analysed by pulsed field gel electrophoresis (PFGE), elaborating genomic profiles by XbaI cleavage and subsequent Southern hybridization to virulence-associated DNA probes. It could be shown that O6 UTI isolates represent a highly heterogeneous group of strains according to the occurrence and combination of these traits. Relatedness on the genetic and the phenotypic level was found for some of the strains exhibiting the same O:K:H:F serotype. DNA long-range mapping further indicated some interesting features, according to the copy number and the genomic linkage of virulence genes.

  1. Distribution of uropathogenic virulence factors among Escherichia coli strains isolated from dogs and cats.

    PubMed

    Yuri, K; Nakata, K; Katae, H; Yamamoto, S; Hasegawa, A

    1998-03-01

    A variety of virulence factors (VFs) such as type 1 fimbriae, pilus associated with pyelonephritis, S fimbriae, afimbrial adhesin, alpha-hemolysin, aerobactin and cytotoxic necrotizing factor 1 are associated with uropathogenic Escherichia coli. In this study, 80 uropathogenic E. coli strains in 50 dogs and 30 cats suffering from UTI. In addition, 60 E. coli strains were isolated from fecal samples from 30 each of healthy dogs and cats. The distribution of VFs of uropathogenic E. coli strains isolated from dogs and cats suffering from urinary tract infections (UTI) were examined by the colony hybridization test with seven DNA probes specific for VFs, and the results were compared with those obtained in the studies on strains from humans with UTI. In uropathogenic E. coli strains isolated from dogs and cats suffering from UTI, VFs were detected as frequently as in the strains isolated from humans with UTI. Although less frequently, genes encoding these VFs especially pap, sfa, hly, and cnf 1 genes were also associated with E. coli strains isolated from feces of healthy cats, in contrast to the distribution pattern of uropathogenic E. coli observed in humans. Furthermore, all VFs except pil were significantly more frequently detected in strains isolated from urine of animals with cystitis than in those isolated from feces of healthy humans. These results indicate that VFs of E. coli contribute to the pathogenesis of UTI in dogs and cats.

  2. Clonal differentiation of uropathogenic Escherichia coli isolates of serotype O6:K5 by fimbrial antigen typing and DNA long-range mapping techniques.

    PubMed

    Zingler, G; Blum, G; Falkenhagen, U; Orskov, I; Orskov, F; Hacker, J; Ott, M

    1993-03-01

    Escherichia coli isolates of serotype O6:K5 are the most common causative agents of cystitis and pyelonephritis in adults. To answer the question, as to whether strains of this particular serotype represent one special clonal group, out of a collection of 34 serotype O6:K5 isolates [Zingler et al. (1990) Zentralbl. Bakteriol Mikrobiol Hyg [A] 274:372-381] 15 strains were selected and analyzed in detail. The flagellar (H) antigen and the outer membrane protein (OMP) pattern were determined. Further serum resistance properties and the genetic presence and expression of other virulence factors, including hemolysin, aerobactin, P fimbriae, S/F1C fimbriae and type 1 fimbriae was evaluated. In addition the XbaI-macrorestriction pattern of ten representative isolates was elaborated and the fimbrial (F) antigen type of the P fimbriae was determined, to obtain the complete O:K:H:F pattern. These analyses could clearly show that the O6:K5 isolates do not represent one clonal group. The XbaI-macrorestriction profiles were heterogeneous and marked differences in the hybridization patterns, using virulence-associated gene probes in Southern hybridization of long-range-separated genomic DNA, were observed among the strains. However, some of strains showed similarities in the genomic profiles, arguing for clonal groupings among the O6:K5 isolates. Interestingly the strains grouped together exhibited the same fimbrial F type that many indicate a coincidence of this phenotypic trait with clonality.

  3. Phenotypic Analysis Reveals that the 2010 Haiti Cholera Epidemic Is Linked to a Hypervirulent Strain.

    PubMed

    Satchell, Karla J F; Jones, Christopher J; Wong, Jennifer; Queen, Jessica; Agarwal, Shivani; Yildiz, Fitnat H

    2016-09-01

    Vibrio cholerae O1 El Tor strains have been responsible for pandemic cholera since 1961. These strains have evolved over time, spreading globally in three separate waves. Wave 3 is caused by altered El Tor (AET) variant strains, which include the strain with the signature ctxB7 allele that was introduced in 2010 into Haiti, where it caused a devastating epidemic. In this study, we used phenotypic analysis to compare an early isolate from the Haiti epidemic to wave 1 El Tor isolates commonly used for research. It is demonstrated that the Haiti isolate has increased production of cholera toxin (CT) and hemolysin, increased motility, and a reduced ability to form biofilms. This strain also outcompetes common wave 1 El Tor isolates for colonization of infant mice, indicating that it has increased virulence. Monitoring of CT production and motility in additional wave 3 isolates revealed that this phenotypic variation likely evolved over time rather than in a single genetic event. Analysis of available whole-genome sequences and phylogenetic analyses suggested that increased virulence arose from positive selection for mutations found in known and putative regulatory genes, including hns and vieA, diguanylate cyclase genes, and genes belonging to the lysR and gntR regulatory families. Overall, the studies presented here revealed that V. cholerae virulence potential can evolve and that the currently prevalent wave 3 AET strains are both phenotypically distinct from and more virulent than many El Tor isolates. PMID:27297393

  4. Preparation, characterization, and immunogenicity of conjugate vaccines directed against Actinobacillus pleuropneumoniae virulence determinants.

    PubMed

    Byrd, W; Kadis, S

    1992-08-01

    Conjugate vaccines were prepared in an attempt to protect pigs against swine pleuropneumonia induced by Actinobacillus pleuropneumoniae (SPAP). Two subunit conjugates were prepared by coupling the A. pleuropneumoniae 4074 serotype 1 capsular polysaccharide (CP) to the hemolysin protein (HP) and the lipopolysaccharide (LPS) to the HP. Adipic acid dihydrazide was used as a spacer to facilitate the conjugation in a carbodiimide-mediated reaction. The CP and the LPS were found to be covalently coupled to the HP in the conjugates as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detergent gel chromatography analyses. Following a booster vaccination, pigs exhibited significantly high (P less than 0.05) immunoglobulin G antibodies against CP, LPS, and HP. The anti-CP and anti-LPS immunoglobulin G antibodies were found to function as opsonins in the phagocytosis of A. pleuropneumoniae by polymorphonuclear leukocytes, whereas antibodies to the HP neutralized the cytotoxic effect of the HP on polymorphonuclear leukocytes. No killing of A. pleuropneumoniae was observed when the effects of the antibodies were tested in the presence of complement. Thus, polysaccharide-protein A. pleuropneumoniae conjugates elicit significant antibody responses against each component of each conjugate, which could be instrumental in protecting swine against SPAP. PMID:1639471

  5. Characterisation of Aeromonas spp. isolated from frozen fish intended for human consumption in Mexico.

    PubMed

    Castro-Escarpulli, G; Figueras, M J; Aguilera-Arreola, G; Soler, L; Fernández-Rendón, E; Aparicio, G O; Guarro, J; Chacón, M R

    2003-07-15

    A total of 82 strains of presumptive Aeromonas spp. were identified biochemically and genetically (16S rDNA-RFLP). The strains were isolated from 250 samples of frozen fish (Tilapia, Oreochromis niloticus niloticus) purchased in local markets in Mexico City. In the present study, we detected the presence of several genes encoding for putative virulence factors and phenotypic activities that may play an important role in bacterial infection. In addition, we studied the antimicrobial patterns of those strains. Molecular identification demonstrated that the prevalent species in frozen fish were Aeromonas salmonicida (67.5%) and Aeromonas bestiarum (20.9%), accounting for 88.3% of the isolates, while the other strains belonged to the species Aeromonas veronii (5.2%), Aeromonas encheleia (3.9%) and Aeromonas hydrophila (2.6%). Detection by polymerase chain reaction (PCR) of genes encoding putative virulence factors common in Aeromonas, such as aerolysin/hemolysin, lipases including the glycerophospholipid-cholesterol acyltransferase (GCAT), serine protease and DNases, revealed that they were all common in these strains. Our results showed that first generation quinolones and second and third generation cephalosporins were the drugs with the best antimicrobial effect against Aeromonas spp. In Mexico, there have been few studies on Aeromonas and its putative virulence factors. The present work therefore highlights an important incidence of Aeromonas spp., with virulence potential and antimicrobial resistance, isolated from frozen fish intended for human consumption in Mexico City. PMID:12781953

  6. Channel-Forming Bacterial Toxins in Biosensing and Macromolecule Delivery

    PubMed Central

    Gurnev, Philip A.; Nestorovich, Ekaterina M.

    2014-01-01

    To intoxicate cells, pore-forming bacterial toxins are evolved to allow for the transmembrane traffic of different substrates, ranging from small inorganic ions to cell-specific polypeptides. Recent developments in single-channel electrical recordings, X-ray crystallography, protein engineering, and computational methods have generated a large body of knowledge about the basic principles of channel-mediated molecular transport. These discoveries provide a robust framework for expansion of the described principles and methods toward use of biological nanopores in the growing field of nanobiotechnology. This article, written for a special volume on “Intracellular Traffic and Transport of Bacterial Protein Toxins”, reviews the current state of applications of pore-forming bacterial toxins in small- and macromolecule-sensing, targeted cancer therapy, and drug delivery. We discuss the electrophysiological studies that explore molecular details of channel-facilitated protein and polymer transport across cellular membranes using both natural and foreign substrates. The review focuses on the structurally and functionally different bacterial toxins: gramicidin A of Bacillus brevis, α-hemolysin of Staphylococcus aureus, and binary toxin of Bacillus anthracis, which have found their “second life” in a variety of developing medical and technological applications. PMID:25153255

  7. Local Epidermal Growth Factor Receptor Signaling Mediates the Systemic Pathogenic Effects of Staphylococcus aureus Toxic Shock Syndrome

    PubMed Central

    Gillman, Aaron N.; Stach, Christopher S.; Schlievert, Patrick M.; Peterson, Marnie L.

    2016-01-01

    Secreted factors of Staphylococcus aureus can activate host signaling from the epidermal growth factor receptor (EGFR). The superantigen toxic shock syndrome toxin-1 (TSST-1) contributes to mucosal cytokine production through a disintegrin and metalloproteinase (ADAM)-mediated shedding of EGFR ligands and subsequent EGFR activation. The secreted hemolysin, α-toxin, can also induce EGFR signaling and directly interacts with ADAM10, a sheddase of EGFR ligands. The current work explores the role of EGFR signaling in menstrual toxic shock syndrome (mTSS), a disease mediated by TSST-1. The data presented show that TSST-1 and α-toxin induce ADAM- and EGFR-dependent cytokine production from human vaginal epithelial cells. TSST-1 and α-toxin also induce cytokine production from an ex vivo porcine vaginal mucosa (PVM) model. EGFR signaling is responsible for the majority of IL-8 production from PVM in response to secreted toxins and live S. aureus. Finally, data are presented demonstrating that inhibition of EGFR signaling with the EGFR-specific tyrosine kinase inhibitor AG1478 significantly increases survival in a rabbit model of mTSS. These data indicate that EGFR signaling is critical for progression of an S. aureus exotoxin-mediated disease and may represent an attractive host target for therapeutics. PMID:27414801

  8. Secreted biofilm factors adversely affect cellular wound healing responses in vitro.

    PubMed

    Jeffery Marano, Robert; Jane Wallace, Hilary; Wijeratne, Dulharie; William Fear, Mark; San Wong, Hui; O'Handley, Ryan

    2015-08-17

    Although most chronic wounds possess an underlying pathology, infectious agents also contribute. In many instances, pathogens exist as biofilms forming clusters surrounded by a secreted extracellular substance. We hypothesized that compounds secreted by biofilm bacteria may inhibit normal wound healing events including cell proliferation and migration. Conditioned media from two common bacterial species associated with chronic skin wounds and chronic tympanic membrane perforations, Staphylococcus aureus and Pseudomonas aeruginosa, were evaluated for their capacity to affect keratinocyte proliferation and migration. Additionally, proteomic analysis was performed to identify proteins within the biofilm conditioned media that may contribute to these observed effects. Biofilm conditioned media from both species inhibited proliferation in human tympanic membrane derived keratinocytes, whereas only biofilm conditioned media from S. aureus inhibited migration. Human epidermal keratinocytes were found to be more sensitive to the effects of the conditioned media resulting in high levels of cell death. Heat treatment and microfiltration suggested that S. aureus activity was due to a protein, while P. aeruginosa activity was more likely due to a small molecule. Proteomic analysis identified several proteins with putative links to delayed wound healing. These include alpha hemolysin, alcohol dehydrogenase, fructose-bisphosphate aldolase, lactate dehydrogenase and epidermal cell differentiation inhibitor.

  9. Targeting Multidrug-resistant Staphylococci with an anti-rpoA Peptide Nucleic Acid Conjugated to the HIV-1 TAT Cell Penetrating Peptide.

    PubMed

    Abushahba, Mostafa Fn; Mohammad, Haroon; Seleem, Mohamed N

    2016-01-01

    Staphylococcus aureus infections present a serious challenge to healthcare practitioners due to the emergence of resistance to numerous conventional antibiotics. Due to their unique mode of action, peptide nucleic acids are novel alternatives to traditional antibiotics to tackle the issue of bacterial multidrug resistance. In this study, we designed a peptide nucleic acid covalently conjugated to the HIV-TAT cell penetrating peptide (GRKKKRRQRRRYK) in order to target the RNA polymerase α subunit gene (rpoA) required for bacterial genes transcription. We explored the antimicrobial activity of the anti-rpoA construct (peptide nucleic acid-TAT) against methicillin-resistant S. aureus, vancomycin-intermediate S. aureus, vancomycin-resistant S. aureus, linezolid-resistant S. aureus, and methicillin-resistant S. epidermidis in pure culture, infected mammalian cell culture, and in an in vivo Caenorhabditis elegans infection model. The anti-rpoA construct led to a concentration-dependent inhibition of bacterial growth (at micromolar concentrations) in vitro and in both infected cell culture and in vivo in C. elegans. Moreover, rpoA gene silencing resulted in suppression of its message as well as reduced expression of two important methicillin-resistant S. aureus USA300 toxins (α-hemolysin and Panton-Valentine leukocidin). This study confirms that rpoA gene is a potential target for development of novel antisense therapeutics to treat infections caused by methicillin-resistant S. aureus. PMID:27434684

  10. Secreted biofilm factors adversely affect cellular wound healing responses in vitro.

    PubMed

    Jeffery Marano, Robert; Jane Wallace, Hilary; Wijeratne, Dulharie; William Fear, Mark; San Wong, Hui; O'Handley, Ryan

    2015-01-01

    Although most chronic wounds possess an underlying pathology, infectious agents also contribute. In many instances, pathogens exist as biofilms forming clusters surrounded by a secreted extracellular substance. We hypothesized that compounds secreted by biofilm bacteria may inhibit normal wound healing events including cell proliferation and migration. Conditioned media from two common bacterial species associated with chronic skin wounds and chronic tympanic membrane perforations, Staphylococcus aureus and Pseudomonas aeruginosa, were evaluated for their capacity to affect keratinocyte proliferation and migration. Additionally, proteomic analysis was performed to identify proteins within the biofilm conditioned media that may contribute to these observed effects. Biofilm conditioned media from both species inhibited proliferation in human tympanic membrane derived keratinocytes, whereas only biofilm conditioned media from S. aureus inhibited migration. Human epidermal keratinocytes were found to be more sensitive to the effects of the conditioned media resulting in high levels of cell death. Heat treatment and microfiltration suggested that S. aureus activity was due to a protein, while P. aeruginosa activity was more likely due to a small molecule. Proteomic analysis identified several proteins with putative links to delayed wound healing. These include alpha hemolysin, alcohol dehydrogenase, fructose-bisphosphate aldolase, lactate dehydrogenase and epidermal cell differentiation inhibitor. PMID:26278131

  11. Virulence and antimicrobial susceptibility of clinical and environmental strains of Aeromonas spp. from northeastern Brazil.

    PubMed

    Castelo-Branco, Débora de Souza Collares Maia; Guedes, Glaucia Morgana de Melo; Brilhante, Raimunda Sâmia Nogueira; Rocha, Marcos Fábio Gadelha; Sidrim, José Júlio Costa; Moreira, José Luciano Bezerra; Cordeiro, Rossana de Aguiar; Sales, Jamille Alencar; Riello, Giovanna Barbosa; de Alencar, Lucas Pereira; Paiva, Manoel de Araújo Neto; Vasconcelos, David Caldas; de Menezes, Isis Sousa Bezerra; de Ponte, Yago Brito; Sampaio, Célia Maria de Souza; Monteiro, André Jalles; Bandeira, Tereza de Jesus Pinheiro Gomes

    2015-08-01

    The aims of the present study were to isolate and identify clinical and environmental strains of Aeromonas spp. by means of biochemical tests and the automated method VITEK 2 and to investigate the presence of the virulence genes cytotoxic enterotoxin (act), hemolysin (asa-1), and type III secretion system (ascV), and also the in vitro antimicrobial susceptibility of the strains. From the clinical isolates, 19 Aeromonas hydrophila, 3 Aeromonas veronii bv. sobria, and 1 Aeromonas caviae were identified, while from the environmental strains, 11 A. hydrophila, 22 A. veronii bv. sobria, 1 A. veronii bv. veronii, and 1 A. caviae were recovered. The gene act was detected in 69.5% of clinical isolates, asa-1 in 8.6%, and ascV in 34.7%. In the environmental strains, the detection rates were 51.4%, 45.7%, and 54.2% for the genes act, asa-1, and ascV, respectively. Resistance to amoxicillin-clavulanate and piperacillin-tazobactam was observed in 15 and 3 clinical strains, respectively, and resistance to ceftazidime, meropenem, imipenem, ciprofloxacin, and trimethoprim-sulfamethoxazole was observed in 1 strain for each drug. Resistance to amoxicillin-clavulanate and piperacillin-tazobactam was detected in 17 and 1 environmental strain, respectively. Higher resistance percentages were observed in clinical strains, but environmental strains also showed this phenomenon and presented a higher detection rate of virulence genes. Thus, it is important to monitor the antimicrobial susceptibility and pathogenic potential of the environmental isolates.

  12. Neonatal Sulfhemoglobinemia and Hemolytic Anemia Associated With Intestinal Morganella morganii.

    PubMed

    Murphy, Kiera; Ryan, Clodagh; Dempsey, Eugene M; O'Toole, Paul W; Ross, R Paul; Stanton, Catherine; Ryan, C Anthony

    2015-12-01

    Sulfhemoglobinemia is a rare disorder characterized by the presence of sulfhemoglobin in the blood. It is typically drug-induced and may cause hypoxia, end-organ damage, and death through oxygen deprivation. We present here a case of non-drug-induced sulfhemoglobinemia in a 7-day-old preterm infant complicated by hemolytic anemia. Microbiota compositional analysis of fecal samples to investigate the origin of hydrogen sulphide revealed the presence of Morganella morganii at a relative abundance of 38% of the total fecal microbiota at the time of diagnosis. M morganii was not detected in the fecal samples of 40 age-matched control preterm infants. M morganii is an opportunistic pathogen that can cause serious infection, particularly in immunocompromised hosts such as neonates. Strains of M morganii are capable of producing hydrogen sulphide, and virulence factors include the production of a diffusible α-hemolysin. The infant in this case survived intact through empirical oral and intravenous antibiotic therapy, probiotic administration, and red blood cell transfusions. This coincided with a reduction in the relative abundance of M morganii to 3%. Neonatologists should have a high index of suspicion for intestinal pathogens in cases of non-drug-induced sulfhemoglobinemia and consider empirical treatment of the intestinal microbiota in this potentially lethal condition.

  13. Novel Twin Streptolysin S-Like Peptides Encoded in the sag Operon Homologue of Beta-Hemolytic Streptococcus anginosus

    PubMed Central

    Tabata, Atsushi; Nakano, Kota; Ohkura, Kazuto; Tomoyasu, Toshifumi; Kikuchi, Ken; Whiley, Robert A.

    2013-01-01

    Streptococcus anginosus is a member of the anginosus group streptococci, which form part of the normal human oral flora. In contrast to the pyogenic group streptococci, our knowledge of the virulence factors of the anginosus group streptococci, including S. anginosus, is not sufficient to allow a clear understanding of the basis of their pathogenicity. Generally, hemolysins are thought to be important virulence factors in streptococcal infections. In the present study, a sag operon homologue was shown to be responsible for beta-hemolysis in S. anginosus strains by random gene knockout. Interestingly, contrary to pyogenic group streptococci, beta-hemolytic S. anginosus was shown to have two tandem sagA homologues, encoding streptolysin S (SLS)-like peptides, in the sag operon homologue. Gene deletion and complementation experiments revealed that both genes were functional, and these SLS-like peptides were essential for beta-hemolysis in beta-hemolytic S. anginosus. Furthermore, the amino acid sequence of these SLS-like peptides differed from that of the typical SLS of S. pyogenes, especially in their propeptide domain, and an amino acid residue indicated to be important for the cytolytic activity of SLS in S. pyogenes was deleted in both S. anginosus homologues. These data suggest that SLS-like peptides encoded by two sagA homologues in beta-hemolytic S. anginosus may be potential virulence factors with a different structure essential for hemolytic activity and/or the maturation process compared to the typical SLS present in pyogenic group streptococci. PMID:23292