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Sample records for rtx toxin gene

  1. Detection of RTX toxin genes in gram-negative bacteria with a set of specific probes.

    PubMed Central

    Kuhnert, P; Heyberger-Meyer, B; Burnens, A P; Nicolet, J; Frey, J

    1997-01-01

    The family of RTX (RTX representing repeats in the structural toxin) toxins is composed of several protein toxins with a characteristic nonapeptide glycine-rich repeat motif. Most of its members were shown to have cytolytic activity. By comparing the genetic relationships of the RTX toxin genes we established a set of 10 gene probes to be used for screening as-yet-unknown RTX toxin genes in bacterial species. The probes include parts of apxIA, apxIIA, and apxIIIA from Actinobacillus pleuropneumoniae, cyaA from Bordetella pertusis, frpA from Neisseria meningitidis, prtC from Erwinia chrysanthemi, hlyA and elyA from Escherichia coli, aaltA from Actinobacillus actinomycetemcomitans and lktA from Pasteurella haemolytica. A panel of pathogenic and nonpathogenic gram-negative bacteria were investigated for the presence of RTX toxin genes. The probes detected all known genes for RTX toxins. Moreover, we found potential RTX toxin genes in several pathogenic bacterial species for which no such toxins are known yet. This indicates that RTX or RTX-like toxins are widely distributed among pathogenic gram-negative bacteria. The probes generated by PCR and the hybridization method were optimized to allow broad-range screening for RTX toxin genes in one step. This included the binding of unlabelled probes to a nylon filter and subsequent hybridization of the filter with labelled genomic DNA of the strain to be tested. The method constitutes a powerful tool for the assessment of the potential pathogenicity of poorly characterized strains intended to be used in biotechnological applications. Moreover, it is useful for the detection of already-known or new RTX toxin genes in bacteria of medical importance. PMID:9172345

  2. The rtxA Toxin Gene of Kingella kingae: a Pertinent Target for Molecular Diagnosis of Osteoarticular Infections▿†

    PubMed Central

    Lehours, Philippe; Freydière, Anne-Marie; Richer, Olivier; Burucoa, Christophe; Boisset, Sandrine; Lanotte, Philippe; Prère, Marie Françoise; Ferroni, Agnès; Lafuente, Christine; Vandenesch, Francois; Mégraud, Francis; Ménard, Armelle

    2011-01-01

    Kingella kingae is an emerging osteoarticular pathogen in young children. Its isolation by traditional culture methods remains difficult, underscoring the need to implement other diagnostic methods for its detection and identification, such as nucleic acid amplification tests. Although the genome of this bacterium has not yet been sequenced, a toxin named RTX has been identified. The goal of this study was to develop sensitive, specific, and rapid molecular methods based on the rtxA toxin gene sequence to diagnose this infection. Two real-time PCR assays (SYBR green and TaqMan chemistries) targeting this gene are reported. Sensitivity and specificity were first evaluated successfully with 67 strains: 31 Kingella kingae isolates and 36 strains from other bacterial species. Then, 52 clinical specimens positive or negative by culture and/or PCR (16S rRNA and cpn60 genes) were tested with these assays. A nested PCR assay with subsequent sequencing was also developed to confirm the presence of Kingella kingae isolates in these clinical specimens. The results obtained demonstrate that these assays are accurate for the diagnosis of Kingella kingae infection. PMID:21248099

  3. Multilocus Sequence Typing and rtxA Toxin Gene Sequencing Analysis of Kingella kingae Isolates Demonstrates Genetic Diversity and International Clones

    PubMed Central

    Basmaci, Romain; Yagupsky, Pablo; Ilharreborde, Brice; Guyot, Kathleen; Porat, Nurith; Chomton, Marilyn; Thiberge, Jean-Michel; Mazda, Keyvan; Bingen, Edouard

    2012-01-01

    Background Kingella kingae, a normal component of the upper respiratory flora, is being increasingly recognized as an important invasive pathogen in young children. Genetic diversity of this species has not been studied. Methods We analyzed 103 strains from different countries and clinical origins by a new multilocus sequence-typing (MLST) schema. Putative virulence gene rtxA, encoding an RTX toxin, was also sequenced, and experimental virulence of representative strains was assessed in a juvenile-rat model. Results Thirty-six sequence-types (ST) and nine ST-complexes (STc) were detected. The main STc 6, 14 and 23 comprised 23, 17 and 20 strains respectively, and were internationally distributed. rtxA sequencing results were mostly congruent with MLST, and showed horizontal transfer events. Of interest, all members of the distantly related ST-6 (n = 22) and ST-5 (n = 4) harboured a 33 bp duplication or triplication in their rtxA sequence, suggesting that this genetic trait arose through selective advantage. The animal model revealed significant differences in virulence among strains of the species. Conclusion MLST analysis reveals international spread of ST-complexes and will help to decipher acquisition and evolution of virulence traits and diversity of pathogenicity among K. kingae strains, for which an experimental animal model is now available. PMID:22693588

  4. The RTX pore-forming toxin α-hemolysin of uropathogenic Escherichia coli: progress and perspectives.

    PubMed

    Wiles, Travis J; Mulvey, Matthew A

    2013-01-01

    Members of the RTX family of protein toxins are functionally conserved among an assortment of bacterial pathogens. By disrupting host cell integrity through their pore-forming and cytolytic activities, this class of toxins allows pathogens to effectively tamper with normal host cell processes, promoting pathogenesis. Here, we focus on the biology of RTX toxins by describing salient properties of a prototype member, α-hemolysin, which is often encoded by strains of uropathogenic Escherichia coli. It has long been appreciated that RTX toxins can have distinct effects on host cells aside from outright lysis. Recently, advances in modeling and analysis of host-pathogen interactions have led to novel findings concerning the consequences of pore formation during host-pathogen interactions. We discuss current progress on longstanding questions concerning cell specificity and pore formation, new areas of investigation that involve toxin-mediated perturbations of host cell signaling cascades and perspectives on the future of RTX toxin investigation.

  5. 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.

  6. Pore forming activity of the potent RTX-toxin produced by pediatric pathogen Kingella kingae: characterization and comparison to other RTX-family members*

    PubMed Central

    Bárcena-Uribarri, Iván; Benz, Roland; Winterhalter, Mathias; Zakharian, Eleonora; Balashova, Nataliya

    2015-01-01

    Pediatric septic arthritis in patients under age of four is frequently caused by the oral Gram-negative bacterium Kingella kingae. This organism may be responsible for a severe form of infective endocarditis in otherwise healthy children and adults. A major virulence factor of K. kingae is RtxA, a toxin that belongs to the RTX (Repeats-in-ToXin) group of secreted pore forming toxins. To understand the RtxA effects on host cell membranes, the toxin activity was studied using planar lipid bilayers. K. kingae strain PYKK081 and its isogenic RtxA-deficient strain, KKNB100, were tested for their ability to form pores in artificial membranes of asolectin/n-decane. RtxA, purified from PYKK081, was able to rapidly form pores with an apparent diameter of 1.9 nm as measured by the partition of nonelectrolytes in the pores. The RtxA channels are cation-selective and showed strong voltage-dependent gating. In contrast to supernatants of PYKK081, those of KKNB100 did not show any pore forming activity. We concluded that RtxA toxin is the only secreted protein from K. kingae forming large channels in host cell membranes where it induces cation flux leading to programmed cell death. Furthermore, our findings suggested that the planar lipid bilayer technique can effectively be used to test possible inhibitors of RTX toxin activity and to investigate the mechanism of the toxin binding to the membrane. PMID:25858109

  7. Pore forming activity of the potent RTX-toxin produced by pediatric pathogen Kingella kingae: Characterization and comparison to other RTX-family members.

    PubMed

    Bárcena-Uribarri, Iván; Benz, Roland; Winterhalter, Mathias; Zakharian, Eleonora; Balashova, Nataliya

    2015-07-01

    Pediatric septic arthritis in patients under age of four is frequently caused by the oral Gram-negative bacterium Kingella kingae. This organism may be responsible for a severe form of infective endocarditis in otherwise healthy children and adults. A major virulence factor of K. kingae is RtxA, a toxin that belongs to the RTX (Repeats-in-ToXin) group of secreted pore forming toxins. To understand the RtxA effects on host cell membranes, the toxin activity was studied using planar lipid bilayers. K. kingae strain PYKK081 and its isogenic RtxA-deficient strain, KKNB100, were tested for their ability to form pores in artificial membranes of asolectin/n-decane. RtxA, purified from PYKK081, was able to rapidly form pores with an apparent diameter of 1.9nm as measured by the partition of nonelectrolytes in the pores. The RtxA channels are cation-selective and showed strong voltage-dependent gating. In contrast to supernatants of PYKK081, those of KKNB100 did not show any pore forming activity. We concluded that RtxA toxin is the only secreted protein from K. kingae forming large channels in host cell membranes where it induces cation flux leading to programmed cell death. Furthermore, our findings suggested that the planar lipid bilayer technique can effectively be used to test possible inhibitors of RTX toxin activity and to investigate the mechanism of the toxin binding to the membrane.

  8. Characterization of PaxA and Its Operon: a Cohemolytic RTX Toxin Determinant from Pathogenic Pasteurella aerogenes

    PubMed Central

    Kuhnert, Peter; Heyberger-Meyer, Bénédicte; Nicolet, Jacques; Frey, Joachim

    2000-01-01

    Pasteurella aerogenes is known as a commensal bacterium or as an opportunistic pathogen, as well as a primary pathogen found to be involved in abortion cases of humans, swine, and other mammals. Using broad-range DNA probes for bacterial RTX toxin genes, we cloned and subsequently sequenced a new operon named paxCABD encoding the RTX toxin PaxA in P. aerogenes. The pax operon is organized analogous to the classical RTX operons containing the activator gene paxC upstream of the structural toxin gene paxA, which is followed by the secretion protein genes paxB and paxD. The highest sequence similarity of paxA with known RTX toxin genes is found with apxIIIA (82%). PaxA is structurally similar to ApxIIIA and also shows functional analogy to ApxIIIA, since it shows cohemolytic activity with the sphingomyelinase of Staphylococcus aureus, known as the CAMP effect, but is devoid of direct hemolytic activity. In addition, it shows to some extent immunological cross-reactions with ApxIIIA. P. aerogenes isolated from various specimens showed that the pax operon was present in about one-third of the strains. All of the pax-positive strains were specifically related to swine abortion cases or septicemia of newborn piglets. These strains were also shown to produce the PaxA toxin as determined by the CAMP phenomenon, whereas none of the pax-negative strains did. This indicated that the PaxA toxin is involved in the pathogenic potential of P. aerogenes. The examined P. aerogenes isolates were phylogenetically analyzed by 16S rRNA gene (rrs) sequencing in order to confirm their species. Only a small heterogeneity (<0.5%) was observed between the rrs genes of the strains originating from geographically distant farms and isolated at different times. PMID:10603361

  9. Structural analysis of the Actinobacillus pleuropneumoniae-RTX-toxin I (ApxI) operon.

    PubMed Central

    Jansen, R; Briaire, J; Kamp, E M; Gielkens, A L; Smits, M A

    1993-01-01

    Actinobacillus pleuropneumoniae-RTX-toxin I (ApxI), an important virulence factor, is secreted by serotypes 1, 5, 9, 10, and 11 of A. pleuropneumoniae. However, sequences homologous to the secretion genes apxIBD of the ApxI operon are present in all 12 serotypes except serotype 3. The purpose of this study was to determine and compare the structures of the ApxI operons of the 12 A. pleuropneumoniae serotypes. We focused on the nucleotide sequence comparison of the ApxI-coding genes, the structures of the ApxI operons, and the transcription of the ApxI operons. We determined the nucleotide sequences of the toxin-encoding apxICA genes of serotype 9 and found that the gene for the structural toxin, apxIA, was almost identical to the apxIA gene of serotype 1. The toxin-encoding genes of the other serotypes are also similar for the main part; nevertheless, two variants were identified, one in serotypes 1, 9, and 11 and one in serotypes 5 and 10. The two apxIA variants differ mainly within the distal 110 nucleotides. Structural analysis demonstrated that intact ApxI operons, consisting of the four contiguous genes apxICABD, are present in serotypes 1, 5, 9, 10, and 11. ApxI operons with a major deletion in the apxICA genes are present in serotypes 2, 4, 6, 7, 8, and 12. Serotype 3 does not contain ApxI operon sequences. We found that all ApxI operons are transcriptionally active despite the partial deletion of the operon in some serotypes. The implications of these data for the expression and secretion of ApxI and the other Apx-toxins, ApxII and ApxIII, as well as for the development of a subunit vaccine against A. pleuropneumoniae will be discussed. Images PMID:8359891

  10. Identification and characterization of an RTX toxin in the emerging pathogen Kingella kingae.

    PubMed

    Kehl-Fie, Thomas E; St Geme, Joseph W

    2007-01-01

    Kingella kingae is an emerging bacterial pathogen that is increasingly recognized as the causative agent of a variety of pediatric diseases, including septic arthritis and osteomyelitis. The pathogenesis of K. kingae disease is believed to begin with colonization of the upper respiratory tract. In the present study, we examined interactions between K. kingae and cultured respiratory epithelial cells and observed potent cytotoxicity, detected by both microscopy and lactic acid dehydrogenase (LDH) release assays. Experiments with synovial and macrophage cell lines revealed cytotoxicity for these cell types as well. Using mariner mutagenesis and a screen for loss of cytotoxicity, a genetic locus encoding an RTX toxin system was identified. Disruption of the K. kingae RTX locus resulted in a loss of cytotoxicity for respiratory epithelial, synovial, and macrophage cell lines. DNA sequence analysis demonstrated that the RTX locus is flanked by insertion elements and has a reduced G+C content compared to that of the whole genome. Two relatively less invasive Kingella species, K. oralis and K. denitrificans, were found to be noncytotoxic and to lack the RTX region, as determined by LDH release assays and Southern blotting. We concluded that K. kingae expresses an RTX toxin that has wide cellular specificity and was likely acquired horizontally. The possible roles for this toxin in the pathogenesis of K. kingae disease include breaching of the epithelial barrier and destruction of target tissues, such as synovium (joint lining).

  11. RTX Toxin Plays a Key Role in Kingella kingae Virulence in an Infant Rat Model

    PubMed Central

    Chang, Dennis W.; Nudell, Yoav A.; Lau, Jenny; Zakharian, Eleonora

    2014-01-01

    Kingella kingae is a human oral bacterium that can cause diseases of the skeletal system in children and infective endocarditis in children and adults. K. kingae produces a toxin of the RTX group, RtxA. To investigate the role of RtxA in disease pathogenesis in vivo, K. kingae strain PYKK081 and its isogenic RtxA-deficient strain KKNB100 were tested for their virulence and pathological consequences upon intraperitoneal injections in 7-day-postnatal (PN 7) rats. At the doses above 8.0 × 106 cells/animal, PYKK081 was able to cause a fatal illness, resulting in rapid weight loss, bacteremia, and abdominal necrotic lesion formation. Significant histopathology was observed in thymus, spleen, and bone marrow. Strain KKNB100 was less toxic to animals. Neither weight loss, bacteremia, nor histopathological changes were evident. Animals injected with KKNB100 exhibited a significantly elevated circulating white blood cell (WBC) count, whereas animals injected with PYKK081 had a WBC count that resembled that of the uninfected control. This observation parallels the subtleties associated with clinical presentation of K. kingae disease in humans and suggests that the toxin contributes to WBC depletion. Thus, our results demonstrate that RtxA is a key K. kingae virulence factor. Furthermore, our findings suggest that the PN 7 rat can serve as a useful model for understanding disease caused by K. kingae and for elucidating diagnostic parameters in human patients. PMID:24664507

  12. Vibrio vulnificus Biotype 3 Multifunctional Autoprocessing RTX Toxin Is an Adenylate Cyclase Toxin Essential for Virulence in Mice

    PubMed Central

    Ziolo, Kevin J.; Jeong, Hee-Gon; Kwak, Jayme S.; Yang, Shuangni; Lavker, Robert M.

    2014-01-01

    Vibrio vulnificus is an environmental organism that causes both food-borne and wound infections with high morbidity and mortality in humans. The annual incidence and global distribution of infections associated with this pathogen are increasing with climate change. In the late 1990s, an outbreak of tilapia-associated wound infections in Israel was linked to a previously unrecognized variant of V. vulnificus designated biotype 3. The sudden emergence and clonality of the outbreak suggest that this strain may be a true newly emergent pathogen with novel virulence properties compared to those of other V. vulnificus strains. In a subcutaneous infection model to mimic wound infection, the multifunctional autoprocessing RTX (MARTX) toxin of biotype 3 strains was shown to be an essential virulence factor contributing to highly inflammatory skin wounds with severe damage affecting every tissue layer. We conducted a sequencing-based analysis of the MARTX toxin and found that biotype 3 MARTX toxin has an effector domain structure distinct from that of either biotype 1 or biotype 2. Of the two new domains identified, a domain similar to Pseudomonas aeruginosa ExoY was shown to confer adenylate cyclase activity on the MARTX toxin. This is the first demonstration that the biotype 3 MARTX toxin is essential for virulence and that the ExoY-like MARTX effector domain is a catalytically active adenylate cyclase. PMID:24614656

  13. Vibrio vulnificus biotype 3 multifunctional autoprocessing RTX toxin is an adenylate cyclase toxin essential for virulence in mice.

    PubMed

    Ziolo, Kevin J; Jeong, Hee-Gon; Kwak, Jayme S; Yang, Shuangni; Lavker, Robert M; Satchell, Karla J F

    2014-05-01

    Vibrio vulnificus is an environmental organism that causes both food-borne and wound infections with high morbidity and mortality in humans. The annual incidence and global distribution of infections associated with this pathogen are increasing with climate change. In the late 1990s, an outbreak of tilapia-associated wound infections in Israel was linked to a previously unrecognized variant of V. vulnificus designated biotype 3. The sudden emergence and clonality of the outbreak suggest that this strain may be a true newly emergent pathogen with novel virulence properties compared to those of other V. vulnificus strains. In a subcutaneous infection model to mimic wound infection, the multifunctional autoprocessing RTX (MARTX) toxin of biotype 3 strains was shown to be an essential virulence factor contributing to highly inflammatory skin wounds with severe damage affecting every tissue layer. We conducted a sequencing-based analysis of the MARTX toxin and found that biotype 3 MARTX toxin has an effector domain structure distinct from that of either biotype 1 or biotype 2. Of the two new domains identified, a domain similar to Pseudomonas aeruginosa ExoY was shown to confer adenylate cyclase activity on the MARTX toxin. This is the first demonstration that the biotype 3 MARTX toxin is essential for virulence and that the ExoY-like MARTX effector domain is a catalytically active adenylate cyclase.

  14. Assessing the Exoproteome of Marine Bacteria, Lesson from a RTX-Toxin Abundantly Secreted by Phaeobacter Strain DSM 17395

    PubMed Central

    Durighello, Emie; Christie-Oleza, Joseph Alexander; Armengaud, Jean

    2014-01-01

    Bacteria from the Roseobacter clade are abundant in surface marine ecosystems as over 10% of bacterial cells in the open ocean and 20% in coastal waters belong to this group. In order to document how these marine bacteria interact with their environment, we analyzed the exoproteome of Phaeobacter strain DSM 17395. We grew the strain in marine medium, collected the exoproteome and catalogued its content with high-throughput nanoLC-MS/MS shotgun proteomics. The major component represented 60% of the total protein content but was refractory to either classical proteomic identification or proteogenomics. We de novo sequenced this abundant protein with high-resolution tandem mass spectra which turned out being the 53 kDa RTX-toxin ZP_02147451. It comprised a peptidase M10 serralysin domain. We explained its recalcitrance to trypsin proteolysis and proteomic identification by its unusual low number of basic residues. We found this is a conserved trait in RTX-toxins from Roseobacter strains which probably explains their persistence in the harsh conditions around bacteria. Comprehensive analysis of exoproteomes from environmental bacteria should take into account this proteolytic recalcitrance. PMID:24586966

  15. Virulence factor rtx in Legionella pneumophila, evidence suggesting it is a modular multifunctional protein

    PubMed Central

    D'Auria, Giuseppe; Jiménez, Núria; Peris-Bondia, Francesc; Pelaz, Carmen; Latorre, Amparo; Moya, Andrés

    2008-01-01

    Background The repeats in toxin (Rtx) are an important pathogenicity factor involved in host cells invasion of Legionella pneumophila and other pathogenic bacteria. Its role in escaping the host immune system and cytotoxic activity is well known. Its repeated motives and modularity make Rtx a multifunctional factor in pathogenicity. Results The comparative analysis of rtx gene among 6 strains of L. pneumophila showed modularity in their structures. Among compared genomes, the N-terminal region of the protein presents highly dissimilar repeats with functionally similar domains. On the contrary, the C-terminal region is maintained with a fashionable modular configuration, which gives support to its proposed role in adhesion and pore formation. Despite the variability of rtx among the considered strains, the flanking genes are maintained in synteny and similarity. Conclusion In contrast to the extracellular bacteria Vibrio cholerae, in which the rtx gene is highly conserved and flanking genes have lost synteny and similarity, the gene region coding for the Rtx toxin in the intracellular pathogen L. pneumophila shows a rapid evolution. Changes in the rtx could play a role in pathogenicity. The interplay of the Rtx toxin with host membranes might lead to the evolution of new variants that are able to escape host cell defences. PMID:18194518

  16. Inducible polymerization and two-dimensional assembly of the repeats-in-toxin (RTX) domain from the Pseudomonas aeruginosa alkaline protease.

    PubMed

    Zhang, Liang; Franks, Jonathon; Stolz, Donna B; Conway, James F; Thibodeau, Patrick H

    2014-10-21

    Self-assembling proteins represent potential scaffolds for the organization of enzymatic activities. The alkaline protease repeats-in-toxin (RTX) domain from Pseudomonas aeruginosa undergoes multiple structural transitions in the presence and absence of calcium, a native structural cofactor. In the absence of calcium, this domain is capable of spontaneous, ordered polymerization, producing amyloid-like fibrils and large two-dimensional protein sheets. This polymerization occurs under near-physiological conditions, is rapid, and can be controlled by regulating calcium in solution. Fusion of the RTX domain to a soluble protein results in the incorporation of engineered protein function into these macromolecular assemblies. Applications of this protein sequence in bacterial adherence and colonization and the generation of biomaterials are discussed.

  17. Differences in purinergic amplification of osmotic cell lysis by the pore-forming RTX toxins Bordetella pertussis CyaA and Actinobacillus pleuropneumoniae ApxIA: the role of pore size.

    PubMed

    Masin, Jiri; Fiser, Radovan; Linhartova, Irena; Osicka, Radim; Bumba, Ladislav; Hewlett, Erik L; Benz, Roland; Sebo, Peter

    2013-12-01

    A large subgroup of the repeat in toxin (RTX) family of leukotoxins of Gram-negative pathogens consists of pore-forming hemolysins. These can permeabilize mammalian erythrocytes (RBCs) and provoke their colloid osmotic lysis (hemolytic activity). Recently, ATP leakage through pannexin channels and P2X receptor-mediated opening of cellular calcium and potassium channels were implicated in cell permeabilization by pore-forming toxins. In the study described here, we examined the role played by purinergic signaling in the cytolytic action of two RTX toxins that form pores of different sizes. The cytolytic potency of ApxIA hemolysin of Actinobacillus pleuropneumoniae, which forms pores about 2.4 nm wide, was clearly reduced in the presence of P2X7 receptor antagonists or an ATP scavenger, such as pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), Brilliant Blue G, ATP oxidized sodium salt, or hexokinase. In contrast, antagonists of purinergic signaling had no impact on the hemolytic potency of the adenylate cyclase toxin-hemolysin (CyaA) of Bordetella pertussis, which forms pores of 0.6 to 0.8 nm in diameter. Moreover, the conductance of pores formed by ApxIA increased with the toxin concentration, while the conductance of the CyaA single pore units was constant at various toxin concentrations. However, the P2X7 receptor antagonist PPADS inhibited in a concentration-dependent manner the exacerbated hemolytic activity of a CyaA-ΔN489 construct (lacking 489 N-terminal residues of CyaA), which exhibited a strongly enhanced pore-forming propensity (>20-fold) and also formed severalfold larger conductance units in planar lipid bilayers than intact CyaA. These results point to a pore size threshold of purinergic amplification involvement in cell permeabilization by pore-forming RTX toxins.

  18. Cloning and Characterization of Two Bistructural S-Layer-RTX Proteins from Campylobacter rectus

    PubMed Central

    Braun, Martin; Kuhnert, Peter; Nicolet, Jacques; Burnens, André P.; Frey, Joachim

    1999-01-01

    Campylobacter rectus is an important periodontal pathogen in humans. A surface-layer (S-layer) protein and a cytotoxic activity have been characterized and are thought to be its major virulence factors. The cytotoxic activity was suggested to be due to a pore-forming protein toxin belonging to the RTX (repeats in the structural toxins) family. In the present work, two closely related genes, csxA and csxB (for C. rectus S-layer and RTX protein) were cloned from C. rectus and characterized. The Csx proteins appear to be bifunctional and possess two structurally different domains. The N-terminal part shows similarity with S-layer protein, especially SapA and SapB of C. fetus and Crs of C. rectus. The C-terminal part comprising most of CsxA and CsxB is a domain with 48 and 59 glycine-rich canonical nonapeptide repeats, respectively, arranged in three blocks. Purified recombinant Csx peptides bind Ca2+. These are characteristic traits of RTX toxin proteins. The S-layer and RTX domains of Csx are separated by a proline-rich stretch of 48 amino acids. All C. rectus isolates studied contained copies of either the csxA or csxB gene or both; csx genes were absent from all other Campylobacter and Helicobacter species examined. Serum of a patient with acute gingivitis showed a strong reaction to recombinant Csx protein on immunoblots. PMID:10198015

  19. Cloning and characterization of two bistructural S-layer-RTX proteins from Campylobacter rectus.

    PubMed

    Braun, M; Kuhnert, P; Nicolet, J; Burnens, A P; Frey, J

    1999-04-01

    Campylobacter rectus is an important periodontal pathogen in humans. A surface-layer (S-layer) protein and a cytotoxic activity have been characterized and are thought to be its major virulence factors. The cytotoxic activity was suggested to be due to a pore-forming protein toxin belonging to the RTX (repeats in the structural toxins) family. In the present work, two closely related genes, csxA and csxB (for C. rectus S-layer and RTX protein) were cloned from C. rectus and characterized. The Csx proteins appear to be bifunctional and possess two structurally different domains. The N-terminal part shows similarity with S-layer protein, especially SapA and SapB of C. fetus and Crs of C. rectus. The C-terminal part comprising most of CsxA and CsxB is a domain with 48 and 59 glycine-rich canonical nonapeptide repeats, respectively, arranged in three blocks. Purified recombinant Csx peptides bind Ca2+. These are characteristic traits of RTX toxin proteins. The S-layer and RTX domains of Csx are separated by a proline-rich stretch of 48 amino acids. All C. rectus isolates studied contained copies of either the csxA or csxB gene or both; csx genes were absent from all other Campylobacter and Helicobacter species examined. Serum of a patient with acute gingivitis showed a strong reaction to recombinant Csx protein on immunoblots.

  20. Nemertean Toxin Genes Revealed through Transcriptome Sequencing

    PubMed Central

    Whelan, Nathan V.; Kocot, Kevin M.; Santos, Scott R.; Halanych, Kenneth M.

    2014-01-01

    Nemerteans are one of few animal groups that have evolved the ability to utilize toxins for both defense and subduing prey, but little is known about specific nemertean toxins. In particular, no study has identified specific toxin genes even though peptide toxins are known from some nemertean species. Information about toxin genes is needed to better understand evolution of toxins across animals and possibly provide novel targets for pharmaceutical and industrial applications. We sequenced and annotated transcriptomes of two free-living and one commensal nemertean and annotated an additional six publicly available nemertean transcriptomes to identify putative toxin genes. Approximately 63–74% of predicted open reading frames in each transcriptome were annotated with gene names, and all species had similar percentages of transcripts annotated with each higher-level GO term. Every nemertean analyzed possessed genes with high sequence similarities to known animal toxins including those from stonefish, cephalopods, and sea anemones. One toxin-like gene found in all nemerteans analyzed had high sequence similarity to Plancitoxin-1, a DNase II hepatotoxin that may function well at low pH, which suggests that the acidic body walls of some nemerteans could work to enhance the efficacy of protein toxins. The highest number of toxin-like genes found in any one species was seven and the lowest was three. The diversity of toxin-like nemertean genes found here is greater than previously documented, and these animals are likely an ideal system for exploring toxin evolution and industrial applications of toxins. PMID:25432940

  1. Penicillium roqueforti PR toxin gene cluster characterization.

    PubMed

    Hidalgo, Pedro I; Poirier, Elisabeth; Ullán, Ricardo V; Piqueras, Justine; Meslet-Cladière, Laurence; Coton, Emmanuel; Coton, Monika

    2017-03-01

    PR toxin is a well-known isoprenoid mycotoxin almost solely produced by Penicillium roqueforti after growth on food or animal feed. This mycotoxin has been described as the most toxic produced by this species. In this study, an in silico analysis allowed identifying for the first time a 22.4-kb biosynthetic gene cluster involved in PR toxin biosynthesis in P. roqueforti. The pathway contains 11 open reading frames encoding for ten putative proteins including the major fungal terpene cyclase, aristolochene synthase, involved in the first farnesyl-diphosphate cyclization step as well as an oxidoreductase, an oxidase, two P450 monooxygenases, a transferase, and two dehydrogenase enzymes. Gene silencing was used to study three genes (ORF5, ORF6, and ORF8 encoding for an acetyltransferase and two P450 monooxygenases, respectively) and resulted in 20 to 40% PR toxin production reductions in all transformants proving the involvement of these genes and the corresponding enzyme activities in PR toxin biosynthesis. According to the considered silenced gene target, eremofortin A and B productions were also affected suggesting their involvement as biosynthetic intermediates in this pathway. A PR toxin biosynthesis pathway is proposed based on the most recent and available data.

  2. Small Molecule-Induced Allosteric Activation of the Vibrio Cholerae RTX Cysteine Protease Domain

    SciTech Connect

    Lupardus, P.J.; Shen, A.; Bogyo, M.; Garcia, K.C.

    2009-05-19

    Vibrio cholerae RTX (repeats in toxin) is an actin-disrupting toxin that is autoprocessed by an internal cysteine protease domain (CPD). The RTX CPD is efficiently activated by the eukaryote-specific small molecule inositol hexakisphosphate (InsP{sub 6}), and we present the 2.1 angstrom structure of the RTX CPD in complex with InsP{sub 6}. InsP{sub 6} binds to a conserved basic cleft that is distant from the protease active site. Biochemical and kinetic analyses of CPD mutants indicate that InsP{sub 6} binding induces an allosteric switch that leads to the autoprocessing and intracellular release of toxin-effector domains.

  3. Gene therapy and targeted toxins for glioma.

    PubMed

    Castro, Maria G; Candolfi, Marianela; Kroeger, Kurt; King, Gwendalyn D; Curtin, James F; Yagiz, Kader; Mineharu, Yohei; Assi, Hikmat; Wibowo, Mia; Ghulam Muhammad, A K M; Foulad, David; Puntel, Mariana; Lowenstein, Pedro R

    2011-06-01

    The most common primary brain tumor in adults is glioblastoma. These tumors are highly invasive and aggressive with a mean survival time of 15-18 months from diagnosis to death. Current treatment modalities are unable to significantly prolong survival in patients diagnosed with glioblastoma. As such, glioma is an attractive target for developing novel therapeutic approaches utilizing gene therapy. This review will examine the available preclinical models for glioma including xenographs, syngeneic and genetic models. Several promising therapeutic targets are currently being pursued in pre-clinical investigations. These targets will be reviewed by mechanism of action, i.e., conditional cytotoxic, targeted toxins, oncolytic viruses, tumor suppressors/oncogenes, and immune stimulatory approaches. Preclinical gene therapy paradigms aim to determine which strategies will provide rapid tumor regression and long-term protection from recurrence. While a wide range of potential targets are being investigated preclinically, only the most efficacious are further transitioned into clinical trial paradigms. Clinical trials reported to date are summarized including results from conditionally cytotoxic, targeted toxins, oncolytic viruses and oncogene targeting approaches. Clinical trial results have not been as robust as preclinical models predicted; this could be due to the limitations of the GBM models employed. Once this is addressed, and we develop effective gene therapies in models that better replicate the clinical scenario, gene therapy will provide a powerful approach to treat and manage brain tumors.

  4. Gene Therapy and Targeted Toxins for Glioma

    PubMed Central

    Castro, Maria G.; Candolfi, Marianela; Kroeger, Kurt; King, Gwendalyn D.; Curtin, James F.; Yagiz, Kader; Mineharu, Yohei; Assi, Hikmat; Wibowo, Mia; Muhammad, AKM Ghulam; Foulad, David; Puntel, Mariana; Lowenstein, Pedro R.

    2011-01-01

    The most common primary brain tumor in adults is glioblastoma. These tumors are highly invasive and aggressive with a mean survival time of nine to twelve months from diagnosis to death. Current treatment modalities are unable to significantly prolong survival in patients diagnosed with glioblastoma. As such, glioma is an attractive target for developing novel therapeutic approaches utilizing gene therapy. This review will examine the available preclinical models for glioma including xenographs, syngeneic and genetic models. Several promising therapeutic targets are currently being pursued in pre-clinical investigations. These targets will be reviewed by mechanism of action, i.e., conditional cytotoxic, targeted toxins, oncolytic viruses, tumor suppressors/oncogenes, and immune stimulatory approaches. Preclinical gene therapy paradigms aim to determine which strategies will provide rapid tumor regression and long-term protection from recurrence. While a wide range of potential targets are being investigated preclinically, only the most efficacious are further transitioned into clinical trial paradigms. Clinical trials reported to date are summarized including results from conditionally cytotoxic, targeted toxins, oncolytic viruses and oncogene targeting approaches. Clinical trial results have not been as robust as preclinical models predicted; this could be due to the limitations of the GBM models employed. Once this is addressed, and we develop effective gene therapies in models that better replicate the clinical scenario, gene therapy will provide a powerful approach to treat and manage brain tumors. PMID:21453286

  5. RTX proteins: a highly diverse family secreted by a common mechanism

    PubMed Central

    Linhartová, Irena; Bumba, Ladislav; Mašín, Jiří; Basler, Marek; Osička, Radim; Kamanová, Jana; Procházková, Kateřina; Adkins, Irena; Hejnová-Holubová, Jana; Sadílková, Lenka; Morová, Jana; Šebo, Peter

    2010-01-01

    Repeats-in-toxin (RTX) exoproteins of Gram-negative bacteria form a steadily growing family of proteins with diverse biological functions. Their common feature is the unique mode of export across the bacterial envelope via the type I secretion system and the characteristic, typically nonapeptide, glycine- and aspartate-rich repeats binding Ca2+ ions. In this review, we summarize the current state of knowledge on the organization of rtx loci and on the biological and biochemical activities of therein encoded proteins. Applying several types of bioinformatic screens on the steadily growing set of sequenced bacterial genomes, over 1000 RTX family members were detected, with the biological functions of most of them remaining to be characterized. Activities of the so far characterized RTX family members are then discussed and classified according to functional categories, ranging from the historically first characterized pore-forming RTX leukotoxins, through the large multifunctional enzymatic toxins, bacteriocins, nodulation proteins, surface layer proteins, up to secreted hydrolytic enzymes exhibiting metalloprotease or lipase activities of industrial interest. PMID:20528947

  6. Toxin-mediated gene regulatory mechanism in Staphylococcus aureus

    PubMed Central

    Joo, Hwang-Soo; Otto, Michael

    2016-01-01

    The dangerous human pathogen Staphylococcus aureus relies heavily on toxins to cause disease, but toxin production can put a strong burden on the bacteria’s energy balance. Thus, controlling the synthesis of proteins solely needed in times of toxin production represents a way for the bacteria to avoid wasting energy. One hypothetical manner to accomplish this sort of regulation is by gene regulatory functions of the toxins themselves. There have been several reports about gene regulation by toxins in S. aureus, but these were never verified on the molecular level. In our study published in MBio [Joo et al., 7(5). pii: e01579-16], we show that phenol-soluble modulins (PSMs), important peptide toxins of S. aureus, release a repressor from the promoter of the operon encoding the toxin export system, thereby enabling toxin secretion. This study describes the first molecular regulatory mechanism exerted by an S. aureus toxin, setting a paradigmatic example of how S. aureus toxins may influence cell functions to adjust them to times of toxin production.

  7. Molecular evolution of toxin genes in Elapidae snakes.

    PubMed

    Tamiya, Toru; Fujimi, Takahiko J

    2006-11-01

    The venom of the sea krait, Laticauda semifasciata, consists primarily of two toxic proteins, phospholipase A(2) (PLA(2)) and a three-finger-structure toxin. We have cloned both toxic protein genes, including the upstream region. PLA(2) genes contain three types of inserted sequences: an AG-rich region, a chicken repeat 1-like long interspersed nucleotide element sequence and an intron II 3' side repeat sequence. The molecular divergence of L. semifasciata PLA(2) genes was defined on the basis of the inserted sequences and their sequence homology. The length of intron I in the three-finger-structure toxin genes differs from species to species. The alignment analysis of intron I of the three-finger-structure toxin genes revealed that the intron I sequence of the ancestral gene comprised ten genetic regions. A hypothetical evolutionary process for the three-finger-structure toxin genes has also been developed.

  8. A Double Selection Approach to Achieve Specific Expression of Toxin Genes for Ovarian Cancer Gene Therapy

    DTIC Science & Technology

    2006-11-01

    specific expression of toxin genes for ovarian cancer gene therapy PRINCIPAL INVESTIGATOR: David T. Curiel, M.D., Ph.D. Gene Siegal...A double selection approach to achieve specific expression of toxin genes for ovarian cancer gene therapy 5b. GRANT NUMBER W81XWH-05-1-0035...cancer. This system should result in highly efficient and specific expression of toxin encoding genes in tumor cells, enabling these cells to be

  9. Cholera toxin structure, gene regulation and pathophysiological and immunological aspects.

    PubMed

    Sánchez, J; Holmgren, J

    2008-05-01

    Many notions regarding the function, structure and regulation of cholera toxin expression have remained essentially unaltered in the last 15 years. At the same time, recent findings have generated additional perspectives. For example, the cholera toxin genes are now known to be carried by a non-lytic bacteriophage, a previously unsuspected condition. Understanding of how the expression of cholera toxin genes is controlled by the bacterium at the molecular level has advanced significantly and relationships with cell-density-associated (quorum-sensing) responses have recently been discovered. Regarding the cell intoxication process, the mode of entry and intracellular transport of cholera toxin are becoming clearer. In the immunological field, the strong oral immunogenicity of the non-toxic B subunit of cholera toxin (CTB) has been exploited in the development of a now widely licensed oral cholera vaccine. Additionally, CTB has been shown to induce tolerance against co-administered (linked) foreign antigens in some autoimmune and allergic diseases.

  10. Identification and Characterization of Clostridium sordellii Toxin Gene Regulator

    PubMed Central

    Sirigi Reddy, Apoorva Reddy; Girinathan, Brintha Parasumanna; Zapotocny, Ryan

    2013-01-01

    Toxigenic Clostridium sordellii causes uncommon but highly lethal infections in humans and animals. Recently, an increased incidence of C. sordellii infections has been reported in women undergoing obstetric interventions. Pathogenic strains of C. sordellii produce numerous virulence factors, including sordellilysin, phospholipase, neuraminidase, and two large clostridial glucosylating toxins, TcsL and TcsH. Recent studies have demonstrated that TcsL toxin is an essential virulence factor for the pathogenicity of C. sordellii. In this study, we identified and characterized TcsR as the toxin gene (tcsL) regulator in C. sordellii. High-throughput sequencing of two C. sordellii strains revealed that tcsR lies within a genomic region that encodes TcsL, TcsH, and TcsE, a putative holin. By using ClosTron technology, we inactivated the tcsR gene in strain ATCC 9714. Toxin production and tcsL transcription were decreased in the tcsR mutant strain. However, the complemented tcsR mutant produced large amounts of toxins, similar to the parental strain. Expression of the Clostridium difficile toxin gene regulator tcdR also restored toxin production to the C. sordellii tcsR mutant, showing that these sigma factors are functionally interchangeable. PMID:23873908

  11. Discovery of a widely distributed toxin biosynthetic gene cluster

    PubMed Central

    Lee, Shaun W.; Mitchell, Douglas A.; Markley, Andrew L.; Hensler, Mary E.; Gonzalez, David; Wohlrab, Aaron; Dorrestein, Pieter C.; Nizet, Victor; Dixon, Jack E.

    2008-01-01

    Bacteriocins represent a large family of ribosomally produced peptide antibiotics. Here we describe the discovery of a widely conserved biosynthetic gene cluster for the synthesis of thiazole and oxazole heterocycles on ribosomally produced peptides. These clusters encode a toxin precursor and all necessary proteins for toxin maturation and export. Using the toxin precursor peptide and heterocycle-forming synthetase proteins from the human pathogen Streptococcus pyogenes, we demonstrate the in vitro reconstitution of streptolysin S activity. We provide evidence that the synthetase enzymes, as predicted from our bioinformatics analysis, introduce heterocycles onto precursor peptides, thereby providing molecular insight into the chemical structure of streptolysin S. Furthermore, our studies reveal that the synthetase exhibits relaxed substrate specificity and modifies toxin precursors from both related and distant species. Given our findings, it is likely that the discovery of similar peptidic toxins will rapidly expand to existing and emerging genomes. PMID:18375757

  12. [A promoter responsible for over-expression of cholera toxin B subunit in cholera toxin A subunit structure gene].

    PubMed

    Cao, C; Shi, C; Li, P; Ma, Q

    1997-01-01

    A promoter sequence, which promotes the transcription of cholera toxin B subunit gene, was found in cholera toxin A subunit structure gene. The transcription starts at the adenine Located at +833, that is 456bp upstream to the A of the initiation codon ATG of cholera toxin B gene. Under the control of the promoter, cholera toxin B subunit was over-expressed as high as 200 mg/L at an optimized culture condition. The chloramphenicol acetyl transferase gene and beta-galactosidase could also be efficiently expressed under the direction of the promoter. This promoter may be responsible for the 6 fold and 7 fold higher expression level of cholera toxin B subunit than cholera toxin A subunit in V. cholerae and Escheria coli respectively. The over-expression of CTB may be useful in preparing vaccine against cholera and facilitating the construction of peptide-bearing immunogenic hybrid proteins.

  13. Phylogenetic analysis and genetic diversity of 3' region of rtxA gene from geographically diverse strains of Moraxella bovis, Moraxella bovoculi and Moraxella ovis.

    PubMed

    Farias, Luana D'Avila; Maboni, Grazieli; Matter, Letícia Beatriz; Scherer, Charles Fernando Capinos; Libardoni, Felipe; de Vargas, Agueda Castagna

    2015-08-05

    The cytotoxin A (MbxA) is one of the main virulence factors of Moraxella bovis involved in the pathogenesis of infectious bovine keratoconjunctivitis (IBK). Moraxella ovis and Moraxella bovoculi, suspected to be associated with infectious keratitis in sheep and cattle respectively, also have a gene that encodes the cytotoxin A (movA and mbvA, respectively). The aim of this study was to determine the molecular sequence of the 3' region of the cytotoxin gene of Moraxella spp. strains isolated from clinical cases to establish phylogenetic and evolutionary comparisons. PCR amplification, nucleotide sequencing (nt) and amino acid (aa) sequence prediction were performed, followed by the sequences comparison, identity level calculation and selective pressure analysis. The phylogenetic reconstruction based on nt and aa sequences clearly differentiate M. bovis (n=15), M. bovoculi (n=11) and M. ovis (n=7) and their respective reference strains. An alignment of 843nt revealed high similarity within bacterial species (MbxA=99.9% nt and aa; MbvA=99.3% nt and 98.8% aa; MovA=99.5% nt and 99.3% aa). The similarity of partial sequences (nt 1807-2649) of MbxA in relation to MbvA and MovA ranged from 76.3 to 78.5%; similarity between MbvA and MovA ranged from 95.7 to 97.5%. A negative selection on mbvA and movA sequences was revealed by the molecular evolution analysis. The phylogenetic analysis of movA and mbvA allowed different strains of Moraxella spp. to be grouped according to the period of isolation. Sequence analysis of cytotoxin may provide insights into genetic and evolutionary relationships and into the genetic/molecular basis of Moraxella spp.

  14. Reporter gene assays for algal-derived toxins.

    PubMed

    Fairey, E R; Ramsdell, J S

    1999-01-01

    We have modified the cell-based directed cytotoxicity assay for sodium channel and calcium channel active phycotoxins using a c-fos-luciferase reporter gene construct. In this report we describe the conceptual basis to the development of reporter gene assays for algal-derived toxins and summarize both published and unpublished data using this method. N2A mouse neuroblastoma cells, which express voltage-dependent sodium channels, were stably transfected with the reporter gene c-fos-luc, which contains the firefly luciferase gene under the transcriptional regulation of the human c-fos response element. The characteristics of the N2A reporter gene assay were determined by dose response with brevetoxin and ciguatoxin. Brevetoxin-1 and ciguatoxin-1 induced c-fos-luc with an EC50 of 4.6 and 3.0 ng ml(-1), respectively. Saxitoxin caused a concentration-dependent inhibition of brevetoxin-1 induction of c-fos-luc with an EC50 of 3.5 ng ml(-1). GH4C1 rat pituitary cells, which lack voltage-dependent sodium channels but express voltage-dependent calcium channels, were also stably transfected with the c-fos-luc. GH4C1 cells expressing c-fos-luciferase were responsive to maitotoxin (1 ng ml(-1)) and a putative toxin produced by Pfiesteria piscicida. Although reporter gene assays are not designed to replace existing detection methods used to measure toxin activity in seafood, they do provide a valuable means to screen algal cultures for toxin activity, to conduct assay-guided fractionation and to characterize pharmacologic properties of algal toxins.

  15. Sequence Analysis of Toxin Gene-Bearing Corynebacterium diphtheriae Strains, Australia.

    PubMed

    Doyle, Christine J; Mazins, Adam; Graham, Rikki M A; Fang, Ning-Xia; Smith, Helen V; Jennison, Amy V

    2017-01-01

    By conducting a molecular characterization of Corynebacterium diphtheriae strains in Australia, we identified novel sequences, nonfunctional toxin genes, and 5 recent cases of toxigenic cutaneous diphtheria. These findings highlight the importance of extrapharyngeal infections for toxin gene-bearing (functional or not) and non-toxin gene-bearing C. diphtheriae strains. Continued surveillance is recommended.

  16. Arrangement of the Clostridium baratii F7 Toxin Gene Cluster with Identification of a σ Factor That Recognizes the Botulinum Toxin Gene Cluster Promoters

    SciTech Connect

    Dover, Nir; Barash, Jason R.; Burke, Julianne N.; Hill, Karen K.; Detter, John C.; Arnon, Stephen S.

    2014-05-22

    Botulinum neurotoxin (BoNT) is the most poisonous substances known and its eight toxin types (A to H) are distinguished by the inability of polyclonal antibodies that neutralize one toxin type to neutralize any of the other seven toxin types. Infant botulism, an intestinal toxemia orphan disease, is the most common form of human botulism in the United States. It results from swallowed spores of Clostridium botulinum (or rarely, neurotoxigenic Clostridium butyricum or Clostridium baratii) that germinate and temporarily colonize the lumen of the large intestine, where, as vegetative cells, they produce botulinum toxin. Botulinum neurotoxin is encoded by the bont gene that is part of a toxin gene cluster that includes several accessory genes. In this paper, we sequenced for the first time the complete botulinum neurotoxin gene cluster of nonproteolytic C. baratii type F7. Like the type E and the nonproteolytic type F6 botulinum toxin gene clusters, the C. baratii type F7 had an orfX toxin gene cluster that lacked the regulatory botR gene which is found in proteolytic C. botulinum strains and codes for an alternative σ factor. In the absence of botR, we identified a putative alternative regulatory gene located upstream of the C. baratii type F7 toxin gene cluster. This putative regulatory gene codes for a predicted σ factor that contains DNA-binding-domain homologues to the DNA-binding domains both of BotR and of other members of the TcdR-related group 5 of the σ70 family that are involved in the regulation of toxin gene expression in clostridia. We showed that this TcdR-related protein in association with RNA polymerase core enzyme specifically binds to the C. baratii type F7 botulinum toxin gene cluster promoters. Finally, this TcdR-related protein may therefore be involved in regulating the expression of the genes of the botulinum toxin gene cluster in neurotoxigenic C. baratii.

  17. Arrangement of the Clostridium baratii F7 toxin gene cluster with identification of a σ factor that recognizes the botulinum toxin gene cluster promoters.

    PubMed

    Dover, Nir; Barash, Jason R; Burke, Julianne N; Hill, Karen K; Detter, John C; Arnon, Stephen S

    2014-01-01

    Botulinum neurotoxin (BoNT) is the most poisonous substances known and its eight toxin types (A to H) are distinguished by the inability of polyclonal antibodies that neutralize one toxin type to neutralize any of the other seven toxin types. Infant botulism, an intestinal toxemia orphan disease, is the most common form of human botulism in the United States. It results from swallowed spores of Clostridium botulinum (or rarely, neurotoxigenic Clostridium butyricum or Clostridium baratii) that germinate and temporarily colonize the lumen of the large intestine, where, as vegetative cells, they produce botulinum toxin. Botulinum neurotoxin is encoded by the bont gene that is part of a toxin gene cluster that includes several accessory genes. We sequenced for the first time the complete botulinum neurotoxin gene cluster of nonproteolytic C. baratii type F7. Like the type E and the nonproteolytic type F6 botulinum toxin gene clusters, the C. baratii type F7 had an orfX toxin gene cluster that lacked the regulatory botR gene which is found in proteolytic C. botulinum strains and codes for an alternative σ factor. In the absence of botR, we identified a putative alternative regulatory gene located upstream of the C. baratii type F7 toxin gene cluster. This putative regulatory gene codes for a predicted σ factor that contains DNA-binding-domain homologues to the DNA-binding domains both of BotR and of other members of the TcdR-related group 5 of the σ70 family that are involved in the regulation of toxin gene expression in clostridia. We showed that this TcdR-related protein in association with RNA polymerase core enzyme specifically binds to the C. baratii type F7 botulinum toxin gene cluster promoters. This TcdR-related protein may therefore be involved in regulating the expression of the genes of the botulinum toxin gene cluster in neurotoxigenic C. baratii.

  18. Clonal Spread of a Clostridium difficile Strain with a Complete Set of Toxin A, Toxin B, and Binary Toxin Genes among Polish Patients with Clostridium difficile-Associated Diarrhea

    PubMed Central

    Pituch, Hanna; Kreft, Deborah; Obuch-Woszczatyński, Piotr; Wultańska, Dorota; Meisel-Mikołajczyk, Felicja; Łuczak, Mirosław; van Belkum, Alex

    2005-01-01

    Clinically relevant Clostridium difficile strains usually produce toxins A and B. Some C. difficile strains can produce an additional binary toxin. We report clonality among five strains carrying all toxin genes from Polish patients with C. difficile-associated diarrhea. In another strain, possible recombination between binary toxin genes is documented. PMID:15635019

  19. Presence of Putative Repeat-in-Toxin Gene tosA in Escherichia coli Predicts Successful Colonization of the Urinary Tract

    PubMed Central

    Vigil, Patrick D.; Stapleton, Ann E.; Johnson, James R.; Hooton, Thomas M.; Hodges, Andrew P.; He, Yongqun; Mobley, Harry L. T.

    2011-01-01

    ABSTRACT Uropathogenic Escherichia coli (UPEC) strains, which cause the majority of uncomplicated urinary tract infections (UTIs), carry a unique assortment of virulence or fitness genes. However, no single defining set of virulence or fitness genes has been found in all strains of UPEC, making the differentiation between UPEC and fecal commensal strains of E. coli difficult without the use of animal models of infection or phylogenetic grouping. In the present study, we consider three broad categories of virulence factors simultaneously to better define a combination of virulence factors that predicts success in the urinary tract. A total of 314 strains of E. coli, representing isolates from fecal samples, asymptomatic bacteriuria, complicated UTIs, and uncomplicated bladder and kidney infections, were assessed by multiplex PCR for the presence of 15 virulence or fitness genes encoding adhesins, toxins, and iron acquisition systems. The results confirm previous reports of gene prevalence among isolates from different clinical settings and identify several new patterns of gene associations. One gene, tosA, a putative repeat-in-toxin (RTX) homolog, is present in 11% of fecal strains but 25% of urinary isolates. Whereas tosA-positive strains carry an unusually high number (11.2) of the 15 virulence or fitness genes, tosA-negative strains have an average of only 5.4 virulence or fitness genes. The presence of tosA was predictive of successful colonization of a murine model of infection, even among fecal isolates, and can be used as a marker of pathogenic strains of UPEC within a distinct subset of the B2 lineage. PMID:21540363

  20. Characterization of Shiga toxin subtypes and virulence genes in porcine Shiga toxin-producing Escherichia coli

    DOE PAGES

    Baranzoni, Gian Marco; Fratamico, Pina M.; Gangiredla, Jayanthi; ...

    2016-04-21

    Similar to ruminants, swine have been shown to be a reservoir for Shiga toxin-producing Escherichia coli (STEC), and pork products have been linked with outbreaks associated with STEC O157 and O111:H-. STEC strains, isolated in a previous study from fecal samples of late-finisher pigs, belonged to a total of 56 serotypes, including O15:H27, O91:H14, and other serogroups previously associated with human illness. The isolates were tested by polymerase chain reaction (PCR) and a high-throughput real-time PCR system to determine the Shiga toxin (Stx) subtype and virulence-associated and putative virulence-associated genes they carried. Select STEC strains were further analyzed using amore » Minimal Signature E. coli Array Strip. As expected, stx2e (81%) was the most common Stx variant, followed by stx1a (14%), stx2d (3%), and stx1c (1%). The STEC serogroups that carried stx2d were O15:H27, O159:H16 and O159:H-. Similar to stx2a and stx2c, the stx2d variant is associated with development of hemorrhagic colitis and hemolytic uremic syndrome, and reports on the presence of this variant in STEC strains isolated from swine are lacking. Moreover, the genes encoding heat stable toxin (estIa) and enteroaggregative E. coli heat stable enterotoxin-1 (astA) were commonly found in 50 and 44% of isolates, respectively. The hemolysin genes, hlyA and ehxA, were both detected in 7% of the swine STEC strains. Although the eae gene was not found, other genes involved in host cell adhesion, including lpfAO113 and paa were detected in more than 50% of swine STEC strains, and a number of strains also carried iha, lpfAO26, lpfAO157, fedA, orfA, and orfB. Furthermore, the present work provides new insights on the distribution of virulence factors among swine STEC strains and shows that swine may carry Stx1a-, Stx2e-, or Stx2d-producing E. coli with virulence gene profiles associated with human infections.« less

  1. Characterization of Shiga Toxin Subtypes and Virulence Genes in Porcine Shiga Toxin-Producing Escherichia coli

    PubMed Central

    Baranzoni, Gian Marco; Fratamico, Pina M.; Gangiredla, Jayanthi; Patel, Isha; Bagi, Lori K.; Delannoy, Sabine; Fach, Patrick; Boccia, Federica; Anastasio, Aniello; Pepe, Tiziana

    2016-01-01

    Similar to ruminants, swine have been shown to be a reservoir for Shiga toxin-producing Escherichia coli (STEC), and pork products have been linked with outbreaks associated with STEC O157 and O111:H-. STEC strains, isolated in a previous study from fecal samples of late-finisher pigs, belonged to a total of 56 serotypes, including O15:H27, O91:H14, and other serogroups previously associated with human illness. The isolates were tested by polymerase chain reaction (PCR) and a high-throughput real-time PCR system to determine the Shiga toxin (Stx) subtype and virulence-associated and putative virulence-associated genes they carried. Select STEC strains were further analyzed using a Minimal Signature E. coli Array Strip. As expected, stx2e (81%) was the most common Stx variant, followed by stx1a (14%), stx2d (3%), and stx1c (1%). The STEC serogroups that carried stx2d were O15:H27, O159:H16 and O159:H-. Similar to stx2a and stx2c, the stx2d variant is associated with development of hemorrhagic colitis and hemolytic uremic syndrome, and reports on the presence of this variant in STEC strains isolated from swine are lacking. Moreover, the genes encoding heat stable toxin (estIa) and enteroaggregative E. coli heat stable enterotoxin-1 (astA) were commonly found in 50 and 44% of isolates, respectively. The hemolysin genes, hlyA and ehxA, were both detected in 7% of the swine STEC strains. Although the eae gene was not found, other genes involved in host cell adhesion, including lpfAO113 and paa were detected in more than 50% of swine STEC strains, and a number of strains also carried iha, lpfAO26, lpfAO157, fedA, orfA, and orfB. The present work provides new insights on the distribution of virulence factors among swine STEC strains and shows that swine may carry Stx1a-, Stx2e-, or Stx2d-producing E. coli with virulence gene profiles associated with human infections. PMID:27148249

  2. Arrangement of the Clostridium baratii F7 Toxin Gene Cluster with Identification of a σ Factor That Recognizes the Botulinum Toxin Gene Cluster Promoters

    DOE PAGES

    Dover, Nir; Barash, Jason R.; Burke, Julianne N.; ...

    2014-05-22

    Botulinum neurotoxin (BoNT) is the most poisonous substances known and its eight toxin types (A to H) are distinguished by the inability of polyclonal antibodies that neutralize one toxin type to neutralize any of the other seven toxin types. Infant botulism, an intestinal toxemia orphan disease, is the most common form of human botulism in the United States. It results from swallowed spores of Clostridium botulinum (or rarely, neurotoxigenic Clostridium butyricum or Clostridium baratii) that germinate and temporarily colonize the lumen of the large intestine, where, as vegetative cells, they produce botulinum toxin. Botulinum neurotoxin is encoded by the bontmore » gene that is part of a toxin gene cluster that includes several accessory genes. In this paper, we sequenced for the first time the complete botulinum neurotoxin gene cluster of nonproteolytic C. baratii type F7. Like the type E and the nonproteolytic type F6 botulinum toxin gene clusters, the C. baratii type F7 had an orfX toxin gene cluster that lacked the regulatory botR gene which is found in proteolytic C. botulinum strains and codes for an alternative σ factor. In the absence of botR, we identified a putative alternative regulatory gene located upstream of the C. baratii type F7 toxin gene cluster. This putative regulatory gene codes for a predicted σ factor that contains DNA-binding-domain homologues to the DNA-binding domains both of BotR and of other members of the TcdR-related group 5 of the σ70 family that are involved in the regulation of toxin gene expression in clostridia. We showed that this TcdR-related protein in association with RNA polymerase core enzyme specifically binds to the C. baratii type F7 botulinum toxin gene cluster promoters. Finally, this TcdR-related protein may therefore be involved in regulating the expression of the genes of the botulinum toxin gene cluster in neurotoxigenic C. baratii.« less

  3. Prediction of Toxin Genes from Chinese Yellow Catfish Based on Transcriptomic and Proteomic Sequencing

    PubMed Central

    Xie, Bing; Li, Xiaofeng; Lin, Zhilong; Ruan, Zhiqiang; Wang, Min; Liu, Jie; Tong, Ting; Li, Jia; Huang, Yu; Wen, Bo; Sun, Ying; Shi, Qiong

    2016-01-01

    Fish venom remains a virtually untapped resource. There are so few fish toxin sequences for reference, which increases the difficulty to study toxins from venomous fish and to develop efficient and fast methods to dig out toxin genes or proteins. Here, we utilized Chinese yellow catfish (Pelteobagrus fulvidraco) as our research object, since it is a representative species in Siluriformes with its venom glands embedded in the pectoral and dorsal fins. In this study, we set up an in-house toxin database and a novel toxin-discovering protocol to dig out precise toxin genes by combination of transcriptomic and proteomic sequencing. Finally, we obtained 15 putative toxin proteins distributed in five groups, namely Veficolin, Ink toxin, Adamalysin, Za2G and CRISP toxin. It seems that we have developed a novel bioinformatics method, through which we could identify toxin proteins with high confidence. Meanwhile, these toxins can also be useful for comparative studies in other fish and development of potential drugs. PMID:27089325

  4. Cross-protection against Vibrio cholerae infection by monoclonal antibodies against Vibrio vulnificus RtxA1/MARTXVv.

    PubMed

    Lee, Tae Hee; Cha, Sun-Shin; Lee, Chang-Seop; Rhee, Joon Haeng; Woo, Hye Ryun; Chung, Kyung Min

    2016-11-01

    Gram-negative Vibrio species secrete multifunctional autoprocessing repeats-in-toxin (MARTX) toxins associated with bacterial pathogenesis. Here, the cross-reactivity and cross-protectivity of mAbs against V. vulnificus RtxA1/MARTXVv was evaluated. Passive administration of any of these mAbs (21RA, 24RA, 46RA, 47RA and 50RA) provided strong protection against lethal V. cholerae infection. Interestingly, 24RA and 46RA, which map to the cysteine protease domain of V. cholerae MARTXVc , inhibited CPD autocleavage in vitro; this process is involved in V. cholerae pathogenesis. These results generate new insight into the development of broadly protective mAbs and/or vaccines against Vibrio species with MARTX toxins.

  5. Genetic diversity within Clostridium botulinum serotypes, botulinum neurotoxin gene clusters and toxin subtypes.

    PubMed

    Hill, Karen K; Smith, Theresa J

    2013-01-01

    Clostridium botulinum is a species of spore-forming anaerobic bacteria defined by the expression of any one or two of seven serologically distinct botulinum neurotoxins (BoNTs) designated BoNT/A-G. This Gram-positive bacterium was first identified in 1897 and since then the paralyzing and lethal effects of its toxin have resulted in the recognition of different forms of the intoxication known as food-borne, infant, or wound botulism. Early microbiological and biochemical characterization of C. botulinum isolates revealed that the bacteria within the species had different characteristics and expressed different toxin types. To organize the variable bacterial traits within the species, Group I-IV designations were created. Interestingly, it was observed that isolates within different Groups could express the same toxin type and conversely a single Group could express different toxin types. This discordant phylogeny between the toxin and the host bacteria indicated that horizontal gene transfer of the toxin was responsible for the variation observed within the species. The recent availability of multiple C. botulinum genomic sequences has offered the ability to bioinformatically analyze the locations of the bont genes, the composition of their toxin gene clusters, and the genes flanking these regions to understand their variation. Comparison of the genomic sequences representing multiple serotypes indicates that the bont genes are not in random locations. Instead the analyses revealed specific regions where the toxin genes occur within the genomes representing serotype A, B, C, E, and F C. botulinum strains and C. butyricum type E strains. The genomic analyses have provided evidence of horizontal gene transfer, site-specific insertion, and recombination events. These events have contributed to the variation observed among the neurotoxins, the toxin gene clusters and the bacteria that contain them, and has supported the historical microbiological, and biochemical

  6. Gene family encoding the major toxins of lethal Amanita mushrooms.

    PubMed

    Hallen, Heather E; Luo, Hong; Scott-Craig, John S; Walton, Jonathan D

    2007-11-27

    Amatoxins, the lethal constituents of poisonous mushrooms in the genus Amanita, are bicyclic octapeptides. Two genes in A. bisporigera, AMA1 and PHA1, directly encode alpha-amanitin, an amatoxin, and the related bicyclic heptapeptide phallacidin, a phallotoxin, indicating that these compounds are synthesized on ribosomes and not by nonribosomal peptide synthetases. alpha-Amanitin and phallacidin are synthesized as proproteins of 35 and 34 amino acids, respectively, from which they are predicted to be cleaved by a prolyl oligopeptidase. AMA1 and PHA1 are present in other toxic species of Amanita section Phalloidae but are absent from nontoxic species in other sections. The genomes of A. bisporigera and A. phalloides contain multiple sequences related to AMA1 and PHA1. The predicted protein products of this family of genes are characterized by a hypervariable "toxin" region capable of encoding a wide variety of peptides of 7-10 amino acids flanked by conserved sequences. Our results suggest that these fungi have a broad capacity to synthesize cyclic peptides on ribosomes.

  7. Characterization of shiga toxin subtypes and virulence genes in Porcine shiga toxin-producing Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Similar to ruminants, swine have been shown to be a reservoir for Shiga toxin-producing Escherichia coli (STEC), and pork products have been linked with outbreaks associated with STEC O157 and O111:H-. STEC strains, isolated in a previous study from fecal samples of late-finisher pigs, belonged to a...

  8. Detection of Clostridium perfringens alpha toxin gene in lambs by loop mediated isothermal amplification

    PubMed Central

    Radhika, B.; Kumar, N. Vinod; Sreenivasulu, D.

    2016-01-01

    Aim: The loop mediated isothermal amplification (LAMP) was standardized for rapid detection of Clostridium perfringens. Materials and Methods: A total of 120 fecal samples were collected from enterotoxemia suspected lambs were used for screening of C. perfringens cpa gene by LAMP. The specificity of the LAMP amplified products was tested by digesting with restriction enzyme XmnI for alpha toxin gene. Results: Out of 120 samples screened 112 (93.3%) samples were positive by both LAMP and polymerase chain reaction (PCR) for detection of cpa gene which indicated the equal sensitivity of both the tests. The enzyme produced single cut in 162 base pair amplified product of alpha toxin gene at 81 base pair resulting in a single band in gel electrophoresis. Conclusion: Both LAMP and PCR for detection of cpa gene indicated the equal sensitivity of both the tests. Standardization of LAMP reaction for amplification of epsilon and beta toxin genes will help to identify the C. perfringens toxin types from the clinical samples. The test could be a suitable alternative to the PCR in detection of toxin types without the help of sophisticated machinery like thermal cycler. Considering its simplicity in operation and high sensitivity, there is the potential use of this technique in clinical diagnosis and surveillance of infectious diseases. PMID:27051186

  9. Rapid detection of vip1-type genes from Bacillus cereus and characterization of a novel vip binary toxin gene.

    PubMed

    Yu, Xiumei; Liu, Tao; Liang, Xiaoxing; Tang, Changqing; Zhu, Jun; Wang, Shiquan; Li, Shuangcheng; Deng, Qiming; Wang, Linxia; Zheng, Aiping; Li, Ping

    2011-12-01

    A PCR-restriction fragment length polymorphism (PCR-RFLP) method for identifying vegetative insecticidal protein (vip) 1-type genes from Bacillus cereus was developed by designing specific primers based on the conserved regions of the genes to amplify vip1-type gene fragments. PCR products were digested with endonuclease AciI, and four known vip1-type genes were identified. Vip1Ac and vip1Aa-type genes appeared in 17 of 26 B. cereus strains. A novel vip1-type gene, vip1Ac1, was identified from B. cereus strain HL12. The vip1Ac1 and vip2Ae3 genes were co-expressed in Escherichia coli strain BL21 by vector pCOLADuet-1. The binary toxin showed activity only against Aphis gossypii (Homoptera), but not for Coleptera (Tenebrio molitor, Holotrichia oblita), Lepidoptera (Spodoptera exigua, Helicoverpa armigera, and Chilo suppressalis), Diptera (Culex quinquefasciatus). The LC(50) of this binary toxin for A. gossypii is 87.5 (34.2-145.3) ng mL(-1) . This is probably only the second report that Vip1 and Vip2 binary toxin shows toxicity against homopteran pests. The PCR-RFLP method developed could be very useful for identifying novel Vip1-Vip2-type binary toxins, and the novel binary toxins, Vip1Ac1 and Vip2Ae3, identified in this study may have applications in biological control of insects, thus avoiding potential problems of resistance.

  10. Contamination of refuges by Bacillus thuringiensis toxin genes from transgenic maize.

    PubMed

    Chilcutt, Charles F; Tabashnik, Bruce E

    2004-05-18

    Transgenic crops producing insecticidal toxins from Bacillus thuringiensis (Bt) are widely used to control pests, but their benefits will be lost if pests evolve resistance. The mandated high-dose/refuge strategy for delaying pest resistance requires planting refuges of toxin-free crops near Bt crops to promote survival of susceptible pests. We report that pollen-mediated gene flow up to 31 m from Bt maize caused low to moderate Bt toxin levels in kernels of non-Bt maize refuge plants. Immunoassays of non-Bt maize sampled from the field showed that the mean concentration of Bt toxin Cry1Ab in kernels and the percentage of kernels with Cry1Ab decreased with distance from Bt maize. The highest Bt toxin concentration in pooled kernels of non-Bt maize plants was 45% of the mean concentration in kernels from adjacent Bt maize plants. Most previous work on gene flow from transgenic crops has emphasized potential effects of transgene movement on wild relatives of crops, landraces, and organic plantings, whereas implications for pest resistance have been largely ignored. Variable Bt toxin production in seeds of refuge plants undermines the high-dose/refuge strategy and could accelerate pest resistance to Bt crops. Thus, guidelines should be revised to reduce gene flow between Bt crops and refuge plants.

  11. Molecular mechanism of acquisition of the cholera toxin genes.

    PubMed

    Das, Bhabatosh; Bischerour, Julien; Barre, Francois-Xavier

    2011-02-01

    One of the major pathogenic determinants of Vibrio cholerae, the cholera toxin, is encoded in the genome of a filamentous phage, CTXφ. CTXφ makes use of the chromosome dimer resolution system of V. cholerae to integrate its single stranded genome into one, the other, or both V. cholerae chromosomes. Here, we review current knowledge about this smart integration process.

  12. An attempt to identify the likely sources of Escherichia coli harboring toxin genes in rainwater tanks.

    PubMed

    Ahmed, W; Sidhu, J P S; Toze, S

    2012-05-01

    In this study, 200 Escherichia coli isolates from 22 rainwater tank samples in Southeast Queensland, Australia were tested for the presence of 10 toxin genes (i.e., stx(1), stx(2), hlyA, ehxA, LT1, ST1, cdtB, east1, cnf1, and cvaC) associated with intestinal and extraintestinal pathotypes. Among the 22 rainwater tanks tested, 5 (28%), 7 (32%), 7 (32%), and 1 (5%) tanks contained E. coli harboring ST1, east1, cdtB, and cvaC genes, respectively. Of the 200 E. coli isolates from the 22 tanks, 43 (22%) strains from 13 (59%) tanks were harboring toxin gene. An attempt was made to establish a link between bird and possum fecal contamination and the presence of these potential clinically significant E. coli strains harboring toxin genes in rainwater tanks. Among the 214 E. coli isolates tested from birds, 30 (14%), 11 (5%) and 18 (8%) strains contained east1, cdtB, and cvaC toxin genes, respectively. Similarly, among the 214 possum E. coli isolates, 74 (35%) contained only the east1 toxin gene. All E. coli strains from rainwater tanks, bird and possum fecal samples harboring toxin genes were biochemically fingerprinted. Biochemical phenotypes (BPTs) of 14 (33%) E. coli strains from 7 rainwater tanks and 9 (21%) E. coli strains from 6 rainwater tanks were identical to a number of BPTs of E. coli strains isolated from bird and possum feces suggesting that these animals may be the sources of these E. coli in rainwater tanks. as a precautionary measure, it is recommended that rainwater should be treated prior to drinking. In addition, proper maintenance of roof and gutter hygiene and elimination of overhanging tree branches and other structures where possible to prevent the movement of possums are highly recommended.

  13. Host–Pathogen Coevolution: The Selective Advantage of Bacillus thuringiensis Virulence and Its Cry Toxin Genes

    PubMed Central

    Papkou, Andrei; Laehnemann, David; Guenther, Patrick S.; Prahl, Swantje; Saebelfeld, Manja; Hollensteiner, Jacqueline; Liesegang, Heiko; Brzuszkiewicz, Elzbieta; Daniel, Rolf; Michiels, Nicolaas K.; Schulte, Rebecca D.; Kurtz, Joachim; Rosenstiel, Philip; Telschow, Arndt; Bornberg-Bauer, Erich; Schulenburg, Hinrich

    2015-01-01

    Reciprocal coevolution between host and pathogen is widely seen as a major driver of evolution and biological innovation. Yet, to date, the underlying genetic mechanisms and associated trait functions that are unique to rapid coevolutionary change are generally unknown. We here combined experimental evolution of the bacterial biocontrol agent Bacillus thuringiensis and its nematode host Caenorhabditis elegans with large-scale phenotyping, whole genome analysis, and functional genetics to demonstrate the selective benefit of pathogen virulence and the underlying toxin genes during the adaptation process. We show that: (i) high virulence was specifically favoured during pathogen–host coevolution rather than pathogen one-sided adaptation to a nonchanging host or to an environment without host; (ii) the pathogen genotype BT-679 with known nematocidal toxin genes and high virulence specifically swept to fixation in all of the independent replicate populations under coevolution but only some under one-sided adaptation; (iii) high virulence in the BT-679-dominated populations correlated with elevated copy numbers of the plasmid containing the nematocidal toxin genes; (iv) loss of virulence in a toxin-plasmid lacking BT-679 isolate was reconstituted by genetic reintroduction or external addition of the toxins. We conclude that sustained coevolution is distinct from unidirectional selection in shaping the pathogen's genome and life history characteristics. To our knowledge, this study is the first to characterize the pathogen genes involved in coevolutionary adaptation in an animal host–pathogen interaction system. PMID:26042786

  14. Distribution of toxin genes among different spa types and phage types of animal Staphylococcus aureus.

    PubMed

    Garbacz, Katarzyna; Piechowicz, Lidia; Mroczkowska, Aneta

    2015-09-01

    We analyzed distribution of toxin genes (sea-seo, eta, etb, tst, lukS/lukF-PV) among spa types and phage types of 39 Staphylococcus aureus (S. aureus) isolates from healthy and diseased animals. All isolates turned out to be mecA negative (MSSA). Nine spa types were identified: t144 and t723 (dogs), t084 (dogs and pigs), t5447 (cat), t1491 and t008 (pigs), t002, t127 and t3478 (poultry). Seven phage types were detected, enclosed within four phage groups: I (cat), II (dogs), III (pigs) and mixed group (dogs and pigs). Three poultry spa types proved to be non-typeable by phages. Toxin genes were detected in 33 out of the 39 animal isolates. Our analysis revealed that the incidence of some toxin genes in S. aureus is host specific. Canine isolates t144 of phage group II harbored exfoliative toxin gene (eta), and porcine isolates type t1491 representing phage group III showed enterotoxin A gene (sea). The enterotoxin gene cluster (egc1) and enterotoxin gene seh were found in non-typeable isolates from chicken and in one feline isolate type t5447.

  15. Characterization of Shiga toxin subtypes and virulence genes in porcine Shiga toxin-producing Escherichia coli

    SciTech Connect

    Baranzoni, Gian Marco; Fratamico, Pina M.; Gangiredla, Jayanthi; Patel, Isha; Bagi, Lori K.; Delannoy, Sabine; Fach, Patrick; Boccia, Federica; Anastasio, Aniello; Pepe, Tiziana

    2016-04-21

    Similar to ruminants, swine have been shown to be a reservoir for Shiga toxin-producing Escherichia coli (STEC), and pork products have been linked with outbreaks associated with STEC O157 and O111:H-. STEC strains, isolated in a previous study from fecal samples of late-finisher pigs, belonged to a total of 56 serotypes, including O15:H27, O91:H14, and other serogroups previously associated with human illness. The isolates were tested by polymerase chain reaction (PCR) and a high-throughput real-time PCR system to determine the Shiga toxin (Stx) subtype and virulence-associated and putative virulence-associated genes they carried. Select STEC strains were further analyzed using a Minimal Signature E. coli Array Strip. As expected, stx2e (81%) was the most common Stx variant, followed by stx1a (14%), stx2d (3%), and stx1c (1%). The STEC serogroups that carried stx2d were O15:H27, O159:H16 and O159:H-. Similar to stx2a and stx2c, the stx2d variant is associated with development of hemorrhagic colitis and hemolytic uremic syndrome, and reports on the presence of this variant in STEC strains isolated from swine are lacking. Moreover, the genes encoding heat stable toxin (estIa) and enteroaggregative E. coli heat stable enterotoxin-1 (astA) were commonly found in 50 and 44% of isolates, respectively. The hemolysin genes, hlyA and ehxA, were both detected in 7% of the swine STEC strains. Although the eae gene was not found, other genes involved in host cell adhesion, including lpfAO113 and paa were detected in more than 50% of swine STEC strains, and a number of strains also carried iha, lpfAO26, lpfAO157, fedA, orfA, and orfB. Furthermore, the present work provides new insights on the distribution of virulence factors among swine STEC strains and shows that swine may carry Stx1a-, Stx2e-, or Stx2d-producing E. coli with virulence gene profiles

  16. In situ detection of the Clostridium botulinum type C1 toxin gene in wetland sediments with a nested PCR assay

    USGS Publications Warehouse

    Williamson, J.L.; Rocke, T.E.; Aiken, Judd M.

    1999-01-01

    A nested PCR was developed for detection of the Clostridium botulinum type C1 toxin gene in sediments collected from wetlands where avian botulism outbreaks had or had not occurred. The C1 toxin gene was detected in 16 of 18 sites, demonstrating both the ubiquitous distribution of C. botulinum type C in wetland sediments and the sensitivity of the detection assay.

  17. Produce isolates of the Escherichia coli Ont:H52 serotype that carry both Shiga toxin 1 and stable toxin genes.

    PubMed

    Monday, Steven R; Keys, Christina; Hanson, Patricia; Shen, Yuelian; Whittam, Thomas S; Feng, Peter

    2006-04-01

    Produce isolates of the Escherichia coli Ont:H52 serotype carried Shiga toxin 1 and stable toxin genes but only expressed Stx1. These strains had pulsed-field gel electrophoresis profiles that were 90% homologous to clinical Ont:H52 strains that had identical phenotypes and genotypes. All Ont:H52 strains had identical single nucleotide polymorphism profiles that are suggestive of a unique clonal group.

  18. Expression of the yeast cpd1 gene in tobacco confers resistance to the fungal toxin cercosporin.

    PubMed

    Panagiotis, Madesis; Kritonas, Kalantidis; Irini, Nianiou Obeidat; Kiriaki, Chatzidimitriou; Nicolaos, Panopoulos; Athanasios, Tsaftaris

    2007-06-01

    Many phytopathogenic species of the fungus Cercospora produce cercosporin, a photoactivated perylenequinone toxin that belongs to a family of photosensitizers, which absorb light energy and produce extremely cytotoxic, reactive oxygen species. The cpd1 (cercosporin photosensitizer detoxification) gene of yeast (Saccharomyces cerevisiae), which encodes for a novel protein with significant similarity to the FAD-dependent pyridine nucleotide reductases, confers resistance to cercosporin when over-expressed in yeast. The aim of this work was to investigate the potential ability of cpd1 gene to confer resistance to cercosporin when expressed in tobacco plants (Nicotiana tabacum). Transgenic tobacco plants were produced using Agrobacterium tumefaciens, with cpd1 integrated as the gene of interest. We report here that expression of cpd1 gene in tobacco can mediate resistance to cercosporin. The involvement of cpd1 gene in the detoxification of the cercosporin reinforces previous observations, which suggested that resistance to cercosporin is mediated by a mechanism involving toxin reduction.

  19. One gene in diamondback moth confers resistance to four Bacillus thuringiensis toxins

    PubMed Central

    Tabashnik, Bruce E.; Liu, Yong-Biao; Finson, Naomi; Masson, Luke; Heckel, David G.

    1997-01-01

    Environmentally benign insecticides derived from the soil bacterium Bacillus thuringiensis (Bt) are the most widely used biopesticides, but their success will be short-lived if pests quickly adapt to them. The risk of evolution of resistance by pests has increased, because transgenic crops producing insecticidal proteins from Bt are being grown commercially. Efforts to delay resistance with two or more Bt toxins assume that independent mutations are required to counter each toxin. Moreover, it generally is assumed that resistance alleles are rare in susceptible populations. We tested these assumptions by conducting single-pair crosses with diamondback moth (Plutella xylostella), the first insect known to have evolved resistance to Bt in open field populations. An autosomal recessive gene conferred extremely high resistance to four Bt toxins (Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F). The finding that 21% of the individuals from a susceptible strain were heterozygous for the multiple-toxin resistance gene implies that the resistance allele frequency was 10 times higher than the most widely cited estimate of the upper limit for the initial frequency of resistance alleles in susceptible populations. These findings suggest that pests may evolve resistance to some groups of toxins much faster than previously expected. PMID:9050831

  20. Map-Based Cloning of the Fungal Toxin Sensitivity Gene Tsn1 in Wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The wheat Tsn1 gene on wheat chromosome arm 5BL confers sensitivity to the host-selective proteinaceous toxins Ptr ToxA and SnToxA produced by the pathogenic fungi Pyrenophora tritici-repentis and Stagonospora nodorum, respectively. Compatible Tsn1-ToxA interactions lead to extensive cell death and...

  1. Characterization of six type A strains of Clostridium botulinum that contain type B toxin gene sequences.

    PubMed

    Kirma, Nameer; Ferreira, Joseph L; Baumstark, Barbara R

    2004-02-16

    Six Clostridium botulinum isolates exhibiting type A toxicity as measured by the mouse bioassay were found to contain both type A and type B neurotoxin DNA sequences. The six strains were divided into three groups based on the DNA sequence of the type B neurotoxin gene. Members of each group exhibited 100% sequence identity over the 3876 bp type B toxin open reading frame. The type B toxin sequence of all groups differed at more than 60 positions when compared to the BGB control strain.

  2. Whole genome sequences of three Clade 3 Clostridium difficile strains carrying binary toxin genes in China.

    PubMed

    Chen, Rong; Feng, Yu; Wang, Xiaohui; Yang, Jingyu; Zhang, Xiaoxia; Lü, Xiaoju; Zong, Zhiyong

    2017-03-06

    Clostridium difficile consists of six clades but studies on Clade 3 are limited. Here, we report genome sequences of three Clade 3 C. difficile strains carrying genes encoding toxin A and B and the binary toxin. Isolates 103 and 133 (both of ST5) and isolate 106 (ST285) were recovered from three ICU patients. Whole genome sequencing using HiSeq 2500 revealed 4.1-Mb genomes with 28-29% GC content. There were ≥1,104 SNP between the isolates, suggesting they were not of a single clone. The toxin A and B gene-carrying pathogenicity locus (PaLoc) of the three isolates were identical and had the insertion of the transposon Tn6218. The genetic components of PaLoc among Clade 3 strains were the same with only a few nucleotide mutations and deletions/insertions, suggesting that the Tn6218 insertion might have occurred before the divergence within Clade 3. The binary toxin-genes carrying CDT locus (CdtLoc) of the three isolates were identical and were highly similar to those of other Clade 3 strains, but were more divergent from those of other clades. In conclusion, Clade 3 has an unusual clade-specific PaLoc characteristic of a Tn6218 insertion which appears to be the main feature to distinguish Clade 3 from other C. difficile.

  3. Whole genome sequences of three Clade 3 Clostridium difficile strains carrying binary toxin genes in China

    PubMed Central

    Chen, Rong; Feng, Yu; Wang, Xiaohui; Yang, Jingyu; Zhang, Xiaoxia; Lü, Xiaoju; Zong, Zhiyong

    2017-01-01

    Clostridium difficile consists of six clades but studies on Clade 3 are limited. Here, we report genome sequences of three Clade 3 C. difficile strains carrying genes encoding toxin A and B and the binary toxin. Isolates 103 and 133 (both of ST5) and isolate 106 (ST285) were recovered from three ICU patients. Whole genome sequencing using HiSeq 2500 revealed 4.1-Mb genomes with 28–29% GC content. There were ≥1,104 SNP between the isolates, suggesting they were not of a single clone. The toxin A and B gene-carrying pathogenicity locus (PaLoc) of the three isolates were identical and had the insertion of the transposon Tn6218. The genetic components of PaLoc among Clade 3 strains were the same with only a few nucleotide mutations and deletions/insertions, suggesting that the Tn6218 insertion might have occurred before the divergence within Clade 3. The binary toxin-genes carrying CDT locus (CdtLoc) of the three isolates were identical and were highly similar to those of other Clade 3 strains, but were more divergent from those of other clades. In conclusion, Clade 3 has an unusual clade-specific PaLoc characteristic of a Tn6218 insertion which appears to be the main feature to distinguish Clade 3 from other C. difficile. PMID:28262711

  4. Construction of "Toxin Complex" in a Mutant Serotype C Strain of Clostridium botulinum Harboring a Defective Neurotoxin Gene.

    PubMed

    Suzuki, Tomonori; Nagano, Thomas; Niwa, Koichi; Uchino, Masataka; Tomizawa, Motohiro; Sagane, Yoshimasa; Watanabe, Toshihiro

    2017-01-01

    A non-toxigenic mutant of the toxigenic serotype C Clostridium botulinum strain Stockholm (C-St), C-N71, does not produce the botulinum neurotoxin (BoNT). However, the original strain C-St produces botulinum toxin complex, in which BoNT is associated with non-toxic non-hemagglutinin (NTNHA) and three hemagglutinin proteins (HA-70, HA-33, and HA-17). Therefore, in this study, we aimed to elucidate the effects of bont gene knockout on the formation of the "toxin complex." Nucleotide sequence analysis revealed that a premature stop codon was introduced in the bont gene, whereas other genes were not affected by this mutation. Moreover, we successfully purified the "toxin complex" produced by C-N71. The "toxin complex" was identified as a mixture of NTNHA/HA-70/HA-17/HA-33 complexes with intact NTNHA or C-terminally truncated NTNHA, without BoNT. These results indicated that knockout of the bont gene does not affect the formation of the "toxin complex." Since the botulinum toxin complex has been shown to play an important role in oral toxin transport in the human and animal body, a non-neurotoxic "toxin complex" of C-N71 may be valuable for the development of an oral drug delivery system.

  5. Detection of Shiga toxin variants, virulence genes and the relationship to cytotoxicity in Shiga toxin-producing Escherichia coli (STEC) from domestic farm animals

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing Escherichia coli (STEC) can cause foodborne illnesses ranging from diarrhea to life-threating diseases such as hemorrhagic colitis, and hemolytic uremic syndrome in humans. In this study, we determined virulence genes, stx subtypes and we evaluated the cytotoxicity in STEC stra...

  6. Identification of Anthrax Toxin Genes in a Bacillus cereus Associated With An Illness Resembling Inhalation Anthrax

    DTIC Science & Technology

    2004-01-01

    Identification of anthrax toxin genes in a Bacillus cereus associated with an illness resembling inhalation anthrax Alex R. Hoffmaster*†, Jacques... Bacillus anthracis is the etiologic agent of anthrax, an acute fatal disease among mammals. It was thought to differ from Bacillus cereus , an...correlation of phenotypic characteristics and their genetic basis. Bacillus cereus and Bacillus anthracis are members of a closelyrelated phylogenetic

  7. Use of Redundant Exclusion PCR To Identify a Novel Bacillus thuringiensis Cry8 Toxin Gene from Pooled Genomic DNA

    PubMed Central

    Zhang, Fengjiao; Shu, Changlong; Crickmore, Neil; Li, Yanqiu; Song, Fuping; Liu, Chunqin

    2016-01-01

    ABSTRACT With the aim of optimizing the cloning of novel genes from a genomic pool containing many previously identified homologous genes, we designed a redundant exclusion PCR (RE-PCR) technique. In RE-PCR, a pair of generic amplification primers are combined with additional primers that are designed to specifically bind to redundant, unwanted genes that are a subset of those copied by the amplification primers. During RE-PCR, the specific primer blocks amplification of the full-length redundant gene. Using this method, we managed to clone a number of cry8 or cry9 toxin genes from a pool of Bacillus thuringiensis genomic DNA while excluding amplicons for cry9Da, cry9Ea, and cry9Eb. The method proved to be very efficient at increasing the number of rare genes in the resulting library. One such rare (and novel) cry8-like gene was expressed, and the encoded toxin was shown to be toxic to Anomala corpulenta. IMPORTANCE Protein toxins from the bacterium Bacillus thuringiensis are being increasingly used as biopesticides against a wide range of insect pests, yet the search for new or improved toxins is becoming more difficult, as traditional methods for gene discovery routinely isolate previously identified clones. This paper describes an approach that we have developed to increase the success rate for novel toxin gene identification through reducing or eliminating the cloning of previously characterized genes. PMID:27084017

  8. Diversification of a single ancestral gene into a successful toxin superfamily in highly venomous Australian funnel-web spiders

    PubMed Central

    2014-01-01

    Background Spiders have evolved pharmacologically complex venoms that serve to rapidly subdue prey and deter predators. The major toxic factors in most spider venoms are small, disulfide-rich peptides. While there is abundant evidence that snake venoms evolved by recruitment of genes encoding normal body proteins followed by extensive gene duplication accompanied by explosive structural and functional diversification, the evolutionary trajectory of spider-venom peptides is less clear. Results Here we present evidence of a spider-toxin superfamily encoding a high degree of sequence and functional diversity that has evolved via accelerated duplication and diversification of a single ancestral gene. The peptides within this toxin superfamily are translated as prepropeptides that are posttranslationally processed to yield the mature toxin. The N-terminal signal sequence, as well as the protease recognition site at the junction of the propeptide and mature toxin are conserved, whereas the remainder of the propeptide and mature toxin sequences are variable. All toxin transcripts within this superfamily exhibit a striking cysteine codon bias. We show that different pharmacological classes of toxins within this peptide superfamily evolved under different evolutionary selection pressures. Conclusions Overall, this study reinforces the hypothesis that spiders use a combinatorial peptide library strategy to evolve a complex cocktail of peptide toxins that target neuronal receptors and ion channels in prey and predators. We show that the ω-hexatoxins that target insect voltage-gated calcium channels evolved under the influence of positive Darwinian selection in an episodic fashion, whereas the κ-hexatoxins that target insect calcium-activated potassium channels appear to be under negative selection. A majority of the diversifying sites in the ω-hexatoxins are concentrated on the molecular surface of the toxins, thereby facilitating neofunctionalisation leading to new toxin

  9. Restriction and Recruitment—Gene Duplication and the Origin and Evolution of Snake Venom Toxins

    PubMed Central

    Hargreaves, Adam D.; Swain, Martin T.; Hegarty, Matthew J.; Logan, Darren W.; Mulley, John F.

    2014-01-01

    Snake venom has been hypothesized to have originated and diversified through a process that involves duplication of genes encoding body proteins with subsequent recruitment of the copy to the venom gland, where natural selection acts to develop or increase toxicity. However, gene duplication is known to be a rare event in vertebrate genomes, and the recruitment of duplicated genes to a novel expression domain (neofunctionalization) is an even rarer process that requires the evolution of novel combinations of transcription factor binding sites in upstream regulatory regions. Therefore, although this hypothesis concerning the evolution of snake venom is very unlikely and should be regarded with caution, it is nonetheless often assumed to be established fact, hindering research into the true origins of snake venom toxins. To critically evaluate this hypothesis, we have generated transcriptomic data for body tissues and salivary and venom glands from five species of venomous and nonvenomous reptiles. Our comparative transcriptomic analysis of these data reveals that snake venom does not evolve through the hypothesized process of duplication and recruitment of genes encoding body proteins. Indeed, our results show that many proposed venom toxins are in fact expressed in a wide variety of body tissues, including the salivary gland of nonvenomous reptiles and that these genes have therefore been restricted to the venom gland following duplication, not recruited. Thus, snake venom evolves through the duplication and subfunctionalization of genes encoding existing salivary proteins. These results highlight the danger of the elegant and intuitive “just-so story” in evolutionary biology. PMID:25079342

  10. Restriction and recruitment-gene duplication and the origin and evolution of snake venom toxins.

    PubMed

    Hargreaves, Adam D; Swain, Martin T; Hegarty, Matthew J; Logan, Darren W; Mulley, John F

    2014-08-01

    Snake venom has been hypothesized to have originated and diversified through a process that involves duplication of genes encoding body proteins with subsequent recruitment of the copy to the venom gland, where natural selection acts to develop or increase toxicity. However, gene duplication is known to be a rare event in vertebrate genomes, and the recruitment of duplicated genes to a novel expression domain (neofunctionalization) is an even rarer process that requires the evolution of novel combinations of transcription factor binding sites in upstream regulatory regions. Therefore, although this hypothesis concerning the evolution of snake venom is very unlikely and should be regarded with caution, it is nonetheless often assumed to be established fact, hindering research into the true origins of snake venom toxins. To critically evaluate this hypothesis, we have generated transcriptomic data for body tissues and salivary and venom glands from five species of venomous and nonvenomous reptiles. Our comparative transcriptomic analysis of these data reveals that snake venom does not evolve through the hypothesized process of duplication and recruitment of genes encoding body proteins. Indeed, our results show that many proposed venom toxins are in fact expressed in a wide variety of body tissues, including the salivary gland of nonvenomous reptiles and that these genes have therefore been restricted to the venom gland following duplication, not recruited. Thus, snake venom evolves through the duplication and subfunctionalization of genes encoding existing salivary proteins. These results highlight the danger of the elegant and intuitive "just-so story" in evolutionary biology.

  11. Mining rare and ubiquitous toxin genes from a large collection of Bacillus thuringiensis strains.

    PubMed

    Li, Ying; Shu, Changlong; Zhang, Xuewen; Crickmore, Neil; Liang, Gemei; Jiang, Xingfu; Liu, Rongmei; Song, Fuping; Zhang, Jie

    2014-10-01

    There has been considerable effort made in recent years for research groups and other organizations to build up large collections of strains of Bacillus thuringiensis in the search for genes encoding novel insecticidal toxins, or encoding novel metabolic pathways. Whilst next generation sequencing allows the detailed genetic characterization of a bacterial strain with relative ease it is still not practicable for large strain collections. In this work we assess the practicability of mining a mixture of genomic DNA from a two thousand strain collection for particular genes. Using PCR the collection was screened for both a rare (cry15) toxin gene as well as a more commonly found gene (vip3A). The method was successful in identifying both a cry15 gene and multiple examples of the vip3A gene family including a novel member of this family (vip3Aj). A number of variants of vip3Ag were cloned and expressed, and differences in toxicity observed despite extremely high sequence similarity.

  12. Toxic peptides and genes encoding toxin gamma of the Brazilian scorpions Tityus bahiensis and Tityus stigmurus.

    PubMed

    Becerril, B; Corona, M; Coronas, F I; Zamudio, F; Calderon-Aranda, E S; Fletcher, P L; Martin, B M; Possani, L D

    1996-02-01

    Seven toxic peptides from the venom of Tityus bahiensis and Tityus stigmurus was isolated and sequenced, five of them to completion. The most abundant peptide from each of these two species of scorpion was 95% identical with that of toxin gamma from the venom of Tityus serrulatus. They were consequently named gamma-b and gamma-st respectively. The genes encoding these new gamma-like peptides were cloned and sequenced by utilizing oligonucleotides synthesized according to known cDNA sequences of toxin gamma, and amplified by PCR on templates of DNA purified from both T. bahiensis and T. stigmurus. They contain an intron of approx. 470 bp. Possible mechanisms of processing and expressing these peptides are discussed, in view of the fact that glycine is the first residue of the N-terminal sequence of T. stigmurus, whereas lysine is the residue at position 1 of toxin gamma from T. serrulatus and T. bahiensis. In addition, chemical characterization of the less abundant toxic peptides showed the presence of at least four distinct families of peptides in all three species of the genus Tityus studied. There is a large degree of similarity among peptides from different venoms of the same family. By using specific horse and rabbit antisera, the venoms of T. bahiensis, T. serrulatus and T. stigmurus were compared. They showed an extended degree of cross-reactivity. Thus these three species of scorpion have similar toxic components, the genes of which are similarly organized, processed and expressed.

  13. Toxic peptides and genes encoding toxin gamma of the Brazilian scorpions Tityus bahiensis and Tityus stigmurus.

    PubMed Central

    Becerril, B; Corona, M; Coronas, F I; Zamudio, F; Calderon-Aranda, E S; Fletcher, P L; Martin, B M; Possani, L D

    1996-01-01

    Seven toxic peptides from the venom of Tityus bahiensis and Tityus stigmurus was isolated and sequenced, five of them to completion. The most abundant peptide from each of these two species of scorpion was 95% identical with that of toxin gamma from the venom of Tityus serrulatus. They were consequently named gamma-b and gamma-st respectively. The genes encoding these new gamma-like peptides were cloned and sequenced by utilizing oligonucleotides synthesized according to known cDNA sequences of toxin gamma, and amplified by PCR on templates of DNA purified from both T. bahiensis and T. stigmurus. They contain an intron of approx. 470 bp. Possible mechanisms of processing and expressing these peptides are discussed, in view of the fact that glycine is the first residue of the N-terminal sequence of T. stigmurus, whereas lysine is the residue at position 1 of toxin gamma from T. serrulatus and T. bahiensis. In addition, chemical characterization of the less abundant toxic peptides showed the presence of at least four distinct families of peptides in all three species of the genus Tityus studied. There is a large degree of similarity among peptides from different venoms of the same family. By using specific horse and rabbit antisera, the venoms of T. bahiensis, T. serrulatus and T. stigmurus were compared. They showed an extended degree of cross-reactivity. Thus these three species of scorpion have similar toxic components, the genes of which are similarly organized, processed and expressed. PMID:8611151

  14. The Deep Origin and Recent Loss of Venom Toxin Genes in Rattlesnakes

    PubMed Central

    Dowell, Noah L.; Giorgianni, Matt W.; Kassner, Victoria A.; Selegue, Jane E.; Sanchez, Elda E.; Carroll, Sean B.

    2016-01-01

    SUMMARY The genetic origin of novel traits is a central but challenging puzzle in evolutionary biology. Among snakes, phospholipase A2 (PLA2)-related toxins have evolved in different lineages to function as potent neurotoxins, myotoxins, or hemotoxins. Here, we traced the genomic origin and evolution of PLA2 toxins by examining PLA2 gene number, organization, and expression in both neurotoxic and non-neurotoxic rattlesnakes. We found that even though most North American rattlesnakes do not produce neurotoxins, the genes of a specialized heterodimeric neurotoxin predate the origin of rattlesnakes and were present in their last common ancestor (~22 mya). The neurotoxin genes were then deleted independently in the lineages leading to the Western Diamondback (Crotalus atrox) and Eastern Diamondback (C. adamanteus) rattlesnakes (~6 mya), while a PLA2 myotoxin gene retained in C. atrox was deleted from the neurotoxic Mojave rattlesnake (C. scutulatus; ~4 mya). The rapid evolution of PLA2 gene number appears to be due to transposon invasion that provided a template for non-allelic homologous recombination. PMID:27641771

  15. Characterization of Clostridium perfringens TpeL Toxin Gene Carriage, Production, Cytotoxic Contributions, and Trypsin Sensitivity

    PubMed Central

    Chen, Jianming

    2015-01-01

    Large clostridial toxins (LCTs) are produced by at least four pathogenic clostridial species, and several LCTs are proven pivotal virulence factors for both human and veterinary diseases. TpeL is a recently identified LCT produced by Clostridium perfringens that has received relatively limited study. In response, the current study surveyed carriage of the tpeL gene among different C. perfringens strains, detecting this toxin gene in some type A, B, and C strains but not in any type D or E strains. This study also determined that all tested strains maximally produce, and extracellularly release, TpeL at the late-log or early-stationary growth stage during in vitro culture, which is different from the maximal late-stationary-phase production reported previously for other LCTs and for TpeL production by C. perfringens strain JIR12688. In addition, the present study found that TpeL levels in culture supernatants can be repressed by either glucose or sucrose. It was also shown that, at natural production levels, TpeL is a significant contributor to the cytotoxic activity of supernatants from cultures of tpeL-positive strain CN3685. Lastly, this study identified TpeL, which presumably is produced in the intestines during diseases caused by TpeL-positive type B and C strains, as a toxin whose cytotoxicity decreases after treatment with trypsin; this finding may have pathophysiologic relevance by suggesting that, like beta toxin, TpeL contributes to type B and C infections in hosts with decreased trypsin levels due to disease, diet, or age. PMID:25824828

  16. Structure of the yellow sac spider Cheiracanthium punctorium genes provides clues to evolution of insecticidal two-domain knottin toxins.

    PubMed

    Sachkova, M Y; Slavokhotova, A A; Grishin, E V; Vassilevski, A A

    2014-08-01

    Yellow sac spiders (Cheiracanthium punctorium, family Miturgidae) are unique in terms of venom composition, because, as we show here, two-domain toxins have replaced the usual one-domain peptides as the major constituents. We report the structure of the two-domain Che. punctorium toxins (CpTx), along with the corresponding cDNA and genomic DNA sequences. At least three groups of insecticidal CpTx were identified, each consisting of several members. Unlike many cone snail and snake toxins, accelerated evolution is not typical of cptx genes, which instead appear to be under the pressure of purifying selection. Both CpTx modules present the inhibitor cystine knot (ICK), or knottin signature; however, the sequence similarity between the domains is low. Conversely, notable similarity was found between separate domains of CpTx and one-domain toxins from spiders of the Lycosidae family. The observed chimerism is a landmark of exon shuffling events, but in contrast to many families of multidomain protein genes no introns were found in the cptx genes. Considering the possible scenarios, we suggest that an early transcription-mediated fusion event between two related one-domain toxin genes led to the emergence of a primordial cptx-like sequence. We conclude that evolution of toxin variability in spiders appears to be quite different from other venomous animals.

  17. [Cloning of Clostridium perfringens alpha-toxin gene and extracellular expression in Escherichia coli].

    PubMed

    Inoue, Masaharu; Kikuchi, Maho; Komoriya, Tomoe; Watanabe, Kunitomo; Kouno, Hideki

    2007-01-01

    Clostridium perfringens (C. perfringens) is a Gram-positive bacterial pathogen that widely propagets in the soil and the gastrointestinal tract of human and animals. This bacteria causes food poisoning, gas gangrene and other various range of infectious diseases. But there is no standard diagnosis method of C. perfringens. In order to develop a new type of immunoassay for clinical purpose, we studied expression and extracellular secretion of recombinant alpha-toxin having enzyme activity in E. coli expression system. Cloning was carried out after PCR amplification from C. perfringens GAI 94074 which was clinical isolate. Three kinds of fragment were cloned using pET100/D-TOPO vector. These fragments coded for ribosome binding site, signal peptide, and alpha-toxin gene respectively. Recombinant pET100 plasmid transformed into TOP 10 cells and the obtained plasmids were transformed into BL21 (DE3) cells. Then, the transformants were induced expression with IPTG. In conclusion, we successfully cloned, expressed and exteracellular secreted C. perfringens alpha-toxin containing signal peptide. Biologically, the obtained recombinant protein was positive for phospholipase C activity.

  18. Identification of genes differentially expressed in the phytopathogenic fungus Cercospora nicotianae between cercosporin toxin-resistant and -susceptible strains.

    PubMed

    Herrero, Sonia; Amnuaykanjanasin, Alongkorn; Daub, Margaret E

    2007-10-01

    Plant pathogens from the genus Cercospora produce cercosporin, a photoactivated fungal toxin that generates toxic reactive oxygen species. Mechanisms governing toxin auto-resistance in Cercospora spp. are poorly understood. In this work, suppressive subtractive hybridization was used to identify genes differentially expressed between the cercosporin-resistant wild-type (WT) Cercospora nicotianae and a sensitive strain lacking a transcription factor (CRG1) that regulates resistance. Out of 338 sequences recovered, 185 unique expressed sequence tags (ESTs) were obtained and classified into functional categories. The majority of genes showed predicted expression differences, and 38.5% were differentially expressed at least twofold between the WT and mutant strain. ESTs were recovered with homology to genes involved in detoxification of noxious compounds, multidrug membrane transporters and antioxidant and polyketide biosynthetic enzymes as well as to ATPases and ATP synthases. The findings suggest that CRG1 regulates genes involved in pH responses in addition to those involved in toxin resistance and biosynthesis.

  19. Coexpression of chitinase and the cry11Aa1 toxin genes in Bacillus thuringiensis serovar israelensis.

    PubMed

    Sirichotpakorn, N; Rongnoparut, P; Choosang, K; Panbangred, W

    2001-10-01

    At the spore stage, a cloned chitinase gene was coexpressed with the regulatory gene p19 and the toxin gene cry11Aa1 in the hosts Bacillus thuringiensis serovar israelensis strains 4Q2-72 and c4Q2-72. The chitinase gene was derived from a high-chitinase producer, Bacillus licheniformis TP-1. Two transcriptional fusion plasmids between the p19 or p19-cry11Aa1 genes and the promoterless chitinase gene were constructed. In transcription order, the p16-19CHI construct contained the p19 gene together with the chitinase gene only while the p16-1968CHI construct contained p19 together with the toxin gene cry11Aa1 and the chitinase gene. The inserted sequences were regulated by a spore-specific promoter located upstream of p19. The recombinant chitinase of all transformed B. thuringiensis serovar israelensis strains was initially synthesized at low level at about 9 h of growth when a portion of the cells started to sporulate. It increased thereafter and reached maximum levels of 5.5, 4.9, and 4.7 mU/ml at 48 h, for strain 4Q2-72 transformed with p16-19CHI and p16-1968CHI and strain c4Q2-72 transformed with p16-19CHI, respectively. This activity was approximately 2 times higher than the maximum activity (2.7 mU/ml) of the parental strain, B. licheniformis TP-1. Although crude chitinase alone from B. thuringiensis serovar israelensis c4Q2-72 (p16-19CHI) at 4.5 mU/ml caused 40% mortality in second instar Aedes aegypti larvae, transformants containing the chitinase alone or in combination with cry11Aa1 resulted in lower toxicity to A. aegypti larvae than the untransformed 4Q2-72 host. For example the LC(50) for the transformed 4Q2-72 harboring the chitinase gene only (p16-19CHI) was 5.6 x 10(4) +/- 0.7 x 10(4) cells, 40 times higher than that of the untransformed host at 1.4 x 10(3) +/- 0.19 x 10(3). The lower toxicity correlated with poor sporulation in the transformants (i.e., 35 times lower than that in the untransformed host). However, the transformed 4Q2-72 strain

  20. The lethal toxin from Australian funnel-web spiders is encoded by an intronless gene.

    PubMed

    Pineda, Sandy Steffany; Wilson, David; Mattick, John S; King, Glenn F

    2012-01-01

    Australian funnel-web spiders are generally considered the most dangerous spiders in the world, with envenomations from the Sydney funnel-web spider Atrax robustus resulting in at least 14 human fatalities prior to the introduction of an effective anti-venom in 1980. The clinical envenomation syndrome resulting from bites by Australian funnel-web spiders is due to a single 42-residue peptide known as δ-hexatoxin. This peptide delays the inactivation of voltage-gated sodium channels, which results in spontaneous repetitive firing and prolongation of action potentials, thereby causing massive neurotransmitter release from both somatic and autonomic nerve endings. Here we show that δ-hexatoxin from the Australian funnel-web spider Hadronyche versuta is produced from an intronless gene that encodes a prepropeptide that is post-translationally processed to yield the mature toxin. A limited sampling of genes encoding unrelated venom peptides from this spider indicated that they are all intronless. Thus, in distinct contrast to cone snails and scorpions, whose toxin genes contain introns, spiders may have developed a quite different genetic strategy for evolving their venom peptidome.

  1. 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.

  2. The Lethal Toxin from Australian Funnel-Web Spiders Is Encoded by an Intronless Gene

    PubMed Central

    Pineda, Sandy Steffany; Wilson, David; Mattick, John S.; King, Glenn F.

    2012-01-01

    Australian funnel-web spiders are generally considered the most dangerous spiders in the world, with envenomations from the Sydney funnel-web spider Atrax robustus resulting in at least 14 human fatalities prior to the introduction of an effective anti-venom in 1980. The clinical envenomation syndrome resulting from bites by Australian funnel-web spiders is due to a single 42-residue peptide known as δ-hexatoxin. This peptide delays the inactivation of voltage-gated sodium channels, which results in spontaneous repetitive firing and prolongation of action potentials, thereby causing massive neurotransmitter release from both somatic and autonomic nerve endings. Here we show that δ-hexatoxin from the Australian funnel-web spider Hadronyche versuta is produced from an intronless gene that encodes a prepropeptide that is post-translationally processed to yield the mature toxin. A limited sampling of genes encoding unrelated venom peptides from this spider indicated that they are all intronless. Thus, in distinct contrast to cone snails and scorpions, whose toxin genes contain introns, spiders may have developed a quite different genetic strategy for evolving their venom peptidome. PMID:22928020

  3. The Repeat-In-Toxin Family Member TosA Mediates Adherence of Uropathogenic Escherichia coli and Survival during Bacteremia

    PubMed Central

    Vigil, Patrick D.; Wiles, Travis J.; Engstrom, Michael D.; Prasov, Lev; Mulvey, Matthew A.

    2012-01-01

    Uropathogenic Escherichia coli (UPEC) is responsible for the majority of uncomplicated urinary tract infections (UTI) and represents the most common bacterial infection in adults. UPEC utilizes a wide range of virulence factors to colonize the host, including the novel repeat-in-toxin (RTX) protein TosA, which is specifically expressed in the host urinary tract and contributes significantly to the virulence and survival of UPEC. tosA, found in strains within the B2 phylogenetic subgroup of E. coli, serves as a marker for strains that also contain a large number of well-characterized UPEC virulence factors. The presence of tosA in an E. coli isolate predicts successful colonization of the murine model of ascending UTI, regardless of the source of the isolate. Here, a detailed analysis of the function of tosA revealed that this gene is transcriptionally linked to genes encoding a conserved type 1 secretion system similar to other RTX family members. TosA localized to the cell surface and was found to mediate (i) adherence to host cells derived from the upper urinary tract and (ii) survival in disseminated infections and (iii) to enhance lethality during sepsis (as assessed in two different animal models of infection). An experimental vaccine, using purified TosA, protected vaccinated animals against urosepsis. From this work, it was concluded that TosA belongs to a novel group of RTX proteins that mediate adherence and host damage during UTI and urosepsis and could be a novel target for the development of therapeutics to treat ascending UTIs. PMID:22083710

  4. Role of peptidergic nerve terminals in the skin: reversal of thermal sensation by calcitonin gene-related peptide in TRPV1-depleted neuropathy.

    PubMed

    Hsieh, Yu-Lin; Lin, Chih-Lung; Chiang, Hao; Fu, Yaw-Syan; Lue, June-Horng; Hsieh, Sung-Tsang

    2012-01-01

    To investigate the contribution of peptidergic intraepidermal nerve fibers (IENFs) to nociceptive responses after depletion of the thermal-sensitive receptor, transient receptor potential vanilloid subtype 1 (TRPV1), we took advantage of a resiniferatoxin (RTX)-induced neuropathy which specifically affected small-diameter dorsal root ganglion (DRG) neurons and their corresponding nerve terminals in the skin. Thermal hypoalgesia (p<0.001) developed from RTX-treatment day 7 (RTXd7) and became normalized from RTXd56 to RTXd84. Substance P (SP)(+) and TRPV1(+) neurons were completely depleted (p = 0.0001 and p<0.0001, respectively), but RTX had a relatively minor effect on calcitonin gene-related peptide (CGRP)(+) neurons (p = 0.029). Accordingly, SP(+) (p<0.0001) and TRPV1(+) (p = 0.0008) IENFs were permanently depleted, but CGRP(+) IENFs (p = 0.012) were only transiently reduced and had recovered by RTXd84 (p = 0.83). The different effects of RTX on peptidergic neurons were attributed to the higher co-localization ratio of TRPV1/SP than of TRPV1/CGRP (p = 0.029). Thermal hypoalgesia (p = 0.0018) reappeared with an intraplantar injection of botulinum toxin type A (botox), and the temporal course of withdrawal latencies in the hot-plate test paralleled the innervation of CGRP(+) IENFs (p = 0.0003) and CGRP contents in skin (p = 0.01). In summary, this study demonstrated the preferential effects of RTX on depletion of SP(+) IENFs which caused thermal hypoalgesia. In contrast, the skin was reinnervated by CGRP(+) IENFs, which resulted in a normalization of nociceptive functions.

  5. SxtA and sxtG Gene Expression and Toxin Production in the Mediterranean Alexandrium minutum (Dinophyceae)

    PubMed Central

    Perini, Federico; Galluzzi, Luca; Dell’Aversano, Carmela; Dello Iacovo, Emma; Tartaglione, Luciana; Ricci, Fabio; Forino, Martino; Ciminiello, Patrizia; Penna, Antonella

    2014-01-01

    The dinoflagellate Alexandrium minutum is known for the production of potent neurotoxins affecting the health of human seafood consumers via paralytic shellfish poisoning (PSP). The aim of this study was to investigate the relationship between the toxin content and the expression level of the genes involved in paralytic shellfish toxin (PST) production. The algal cultures were grown both in standard f/2 medium and in phosphorus/nitrogen limitation. In our study, LC-HRMS analyses of PST profile and content in different Mediterranean A. minutum strains confirmed that this species was able to synthesize mainly the saxitoxin analogues Gonyautoxin-1 (GTX1) and Gonyautoxin-4 (GTX4). The average cellular toxin content varied among different strains, and between growth phases, highlighting a decreasing trend from exponential to stationary phase in all culture conditions tested. The absolute quantities of intracellular sxtA1 and sxtG mRNA were not correlated with the amount of intracellular toxins in the analysed A. minutum suggesting that the production of toxins may be regulated by post-transcriptional mechanisms and/or by the concerted actions of alternative genes belonging to the PST biosynthesis gene cluster. Therefore, it is likely that the sxtA1 and sxtG gene expression could not reflect the PST accumulation in the Mediterranean A. minutum populations under the examined standard and nutrient limiting conditions. PMID:25341029

  6. Expression of mosquito active toxin genes by a Colombian native strain of the gram-negative bacterium Asticcacaulis excentricus.

    PubMed

    Romero, M; Gil, F M; Orduz, S

    2001-02-01

    Mosquito control with biological insecticides, such as Bacillus sp. toxins, has been used widely in many countries. However, rapid sedimentation away from the mosquito larvae feeding zone causes a low residual effect. In order to overcome this problem, it has been proposed to clone the Bacillus toxin genes in aquatic bacteria which are able to live in the upper part of the water column. Two strains of Asticcacaulis excentricus were chosen to introduce the B. sphaericus binary toxin gene and B. thuringiensis subsp. medellin cry11Bb gene cloned in suitable vectors. In feeding experiments with these aquatic bacteria, it was shown that Culex quinquefasciatus, Aedes aegypti, and Anopheles albimanus larvae were able to survive on a diet based on this wild bacterium. A. excentricus recombinant strains were able to express both genes, but the recombinant strain expressing the B. sphaericus binary toxin was toxic to mosquito larvae. Crude protease A. excentricus extracts did not degrade the Cry11Bb toxin. The flotability studies indicated that the recombinant A. excentricus strains remained in the upper part of the water column longer than the wild type Bacillus strains.

  7. Transport and magnetic properties of RTX and related compounds

    NASA Astrophysics Data System (ADS)

    Goruganti, Venkateshwarlu

    Physical properties of RTX compounds (R = Rare earth, T = Transition metal and X = main group element from B, C or N group) compounds have been studied by means of electrical resistivity, heat capacity, dc magnetization and NMR. Searching for new magnetic materials is always an interesting topic from both a technological and basic research prospective; it is even more interesting when unusual magnetic phases are observed. Ternary intermetallic plumbides are interesting because of their unconventional magnetic ordering and variety of multiple magnetic transitions. Crystalline electric fields (CEF) also strongly effect the magnetic properties of these intermetallics. To understand the phase transitions, CEF effects, and magnetic interactions, a systematic study of the RNiPb, R 2Ni2Pb, R5NiPb3 and RCuGe systems were conducted. Among the results for NdNiPb a single antiferromagnetic transition was found at 3.5K, while the superconductivity found in some ingots of this material was shown not to correspond to a bulk behavior for this phase. Nd2Ni 2Pb was shown to have a canted zero field magnetic structure with a low temperature metamagnetic transition 3 T. In NdCuGe, a 3K AF transition was found along with a corresponding magnon contribution to the specific heat and magnetic and thermodynamic behavior from which the detailed CEF configuration was obtained. In a series of measurements on recently-synthesized R 5NiPb3 (R=Ce, Nd, Gd), for Ce5NiPb 3 a transition at 48 K was found, which was confirmed to be ferromagnetic character from field dependent heat capacity and Curie-Weiss susceptibility. Nd5NiPb3 exhibits two transitions, an antiferromagnetic transition at 42 K and an apparently weak ferromagnetic canting transition at 8 K. For Gd5NiPb3, a ferro- or ferrimagnetic transition was found at 68 K. For the Ce and Nd materials metamagnetism was also observed at low temperatures. In addition, very large metallic type gamma terms were found in the specific heat, as well as a

  8. A novel gene required for cercosporin toxin resistance in the fungus Cercospora nicotianae.

    PubMed

    Chung, K R; Jenns, A E; Ehrenshaft, M; Daub, M E

    1999-09-01

    Cercosporin, a photosensitizing perylenequinone toxin produced by the plant pathogenic Cercospora fungi, generates the highly toxic singlet oxygen (1O2) upon exposure to light. Cercosporin shows broad toxicity against a wide range of organisms, including bacteria, fungi, plants, and animals; however, Cercospora fungi are resistant to its effects. A novel gene, crg1 (cercosporin-resistance gene) was isolated from a wild-type strain of C. nicotianae by genetic complementation of a C. nicotianae mutant (CS10) which is cercosporin sensitive and down-regulated in cercosporin production. Sequence analysis indicated that crg1 encodes a putative protein of 550 amino acids with four putative transmembrane helical regions, however CRG1 shows no strong similarity to any other protein in sequence databases. Northern analysis identified two transcripts (4.5 and 2.6 kb) that are unaffected by the presence of light or cercosporin. Southern analysis demonstrated that crg1 is present in a single copy in the C. nicotianae genome and can be detected only in Cercospora species. Targeted disruption of crg1 resulted in mutants that, like CS10, are sensitive to cercosporin. However, unlike CS10, crg1 disruption mutants are not down-regulated in toxin production. Both CS10 and the crg1 disruption mutants are unaffected in their response to other 1O2-generating photosensitizers, suggesting that CRG1 functions specifically against cercosporin, rather than against 1O2.

  9. A satellite phage-encoded antirepressor induces repressor aggregation and cholera toxin gene transfer.

    PubMed

    Davis, Brigid M; Kimsey, Harvey H; Kane, Anne V; Waldor, Matthew K

    2002-08-15

    CTXphi is a filamentous bacteriophage whose genome encodes cholera toxin, the principal virulence factor of Vibrio cholerae. We have found that the CTXphi-related element RS1 is a satellite phage whose transmission depends upon proteins produced from a CTX prophage (its helper phage). However, unlike other satellite phages and satellite animal viruses, RS1 can aid the CTX prophage as well as exploit it, due to the RS1-encoded protein RstC. RstC, whose function previously was unknown, is an antirepressor that counteracts the activity of the phage repressor RstR. RstC promotes transcription of genes required for phage production and thereby promotes transmission of both RS1 and CTXphi. Antirepression by RstC also induces expression of the cholera toxin genes, ctxAB, and thus may contribute to the virulence of V.cholerae. In vitro, RstC binds directly to RstR, producing unusual, insoluble aggregates containing both proteins. In vivo, RstC and RstR are both found at the cell pole, where they again appear to form stable complexes. The sequestration/inactivation process induced by RstC resembles those induced by mutant polyglutamine-containing proteins implicated in human neurodegenerative disorders.

  10. Transfer of toxin genes to alternate bacterial hosts for mosquito control.

    PubMed

    Orduz, S; Restrepo, N; Patiño, M M; Rojas, W

    1995-01-01

    Mosquitoes are vector of serious human and animal diseases, such as malaria, dengue, yellow fever, among others. The use of biological control agents has provide an environmentally safe and highly specific alternative to the use of chemical insecticides in the control of vector borne diseases. Bacillus thuringiensis and B. sphaericus produce toxic proteins to mosquito larvae. Great progress has been made on the biochemical and molecular characterization of such proteins and the genes encoding them. Nevertheless, the low residuality of these biological insecticides is one of the major drawbacks. This article present some interesting aspects of the mosquito larvae feeding habits and review the attempts that have been made to genetically engineer microorganisms that while are used by mosquito larvae as a food source should express the Bacillus toxin genes in order to improve the residuality and stability in the mosquito breeding ponds.

  11. Effects of a natural toxin on life history and gene expression of Eisenia andrei.

    PubMed

    van Ommen Kloeke, A E Elaine; Gong, Ping; Ellers, Jacintha; Roelofs, Dick

    2014-02-01

    Earthworms perform key functions for a healthy soil ecosystem, such as bioturbation. The soil ecosystem can be challenged by natural toxins such as isothiocyanates (ITCs), produced by many commercial crops. Therefore, the effects of 2-phenylethyl ITC were investigated on the earthworm Eisenia andrei using an ecotoxicogenomics approach. Exposure to 2-phenylethyl ITC reduced both survival and reproduction of E. andrei in a dose-dependent manner (median effective concentration [EC50] = 556 nmol/g). Cross-species comparative genomic hybridization validated the applicability of an existing 4 × 44,000 Eisenia fetida microarray to E. andrei. Gene expression profiles revealed the importance of metallothionein (MT) as an early warning signal when E. andrei was exposed to low concentrations of 2-phenylethyl ITC. Alignment of these MT genes with the MT-2 gene of Lumbricus rubellus showed that at least 2 MT gene clusters are present in the Eisenia sp. genome. At high-exposure concentrations, gene expression was mainly affected by inhibiting chitinase activity, inducing an oxidative stress response, and stimulating energy metabolism. Furthermore, analysis by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway implied that the high concentration may have caused impaired light sensitivity, angiogenesis, olfactory perception, learning, and memory. Increased levels of ITCs may be found in the field in the near future. The results presented call for a careful investigation to quantify the risk of such compounds before allowing them to enter the soil on a large scale.

  12. Horizontal gene transfer of a bacterial insect toxin gene into the Epichloë fungal symbionts of grasses

    PubMed Central

    Ambrose, Karen V.; Koppenhöfer, Albrecht M.; Belanger, Faith C.

    2014-01-01

    Horizontal gene transfer is recognized as an important factor in genome evolution, particularly when the newly acquired gene confers a new capability to the recipient species. We identified a gene similar to the makes caterpillars floppy (mcf1 and mcf2) insect toxin genes in Photorhabdus, bacterial symbionts of nematodes, in the genomes of the Epichloë fungi, which are intercellular symbionts of grasses. Infection by Epichloë spp. often confers insect resistance to the grass hosts, largely due to the production of fungal alkaloids. A mcf-like gene is present in all of the Epichloë genome sequences currently available but in no other fungal genomes. This suggests the Epichloë genes were derived from a single lineage-specific HGT event. Molecular dating was used to estimate the time of the HGT event at between 7.2 and 58.8 million years ago. The mcf-like coding sequence from Epichloë typhina subsp. poae was cloned and expressed in Escherichia coli. E. coli cells expressing the Mcf protein were toxic to black cutworms (Agrotis ipsilon), whereas E. coli cells containing the vector only were non-toxic. These results suggest that the Epichloë mcf-like genes may be a component, in addition to the fungal alkaloids, of the insect resistance observed in Epichloë-infected grasses. PMID:24990771

  13. Escherichia coli O157:H7 survives within human macrophages: global gene expression profile and involvement of the Shiga toxins.

    PubMed

    Poirier, Katherine; Faucher, Sébastien P; Béland, Maxime; Brousseau, Roland; Gannon, Victor; Martin, Christine; Harel, Josée; Daigle, France

    2008-11-01

    Escherichia coli O157:H7 is an important food-borne pathogen that specifically binds to the follicle-associated epithelium in the intestine, which rapidly brings this bacterial pathogen in contact with underlying human macrophages. Very little information is available about the interaction between E. coli O157:H7 and human macrophages. We evaluated the uptake and survival of strain EDL933 during infection of human macrophages. Surprisingly, EDL933 survived and multiplied in human macrophages at 24 h postinfection. The global gene expression profile of this pathogen during macrophage infection was determined. Inside human macrophages, upregulation of E. coli O157:H7 genes carried on O islands (such as pagC, the genes for both of the Shiga toxins, and the two iron transport system operons fit and chu) was observed. Genes involved in acid resistance and in the SOS response were upregulated. However, genes of the locus of enterocyte effacement or genes involved in peroxide resistance were not differentially expressed. Many genes with putative or unknown functions were upregulated inside human macrophages and may be newly discovered virulence factors. As the Shiga toxin genes were upregulated in macrophages, survival and cytotoxicity assays were performed with isogenic Shiga toxin mutants. The initial uptake of Shiga toxins mutants was higher than that of the wild type; however, the survival rates were significantly lower at 24 h postinfection. Thus, Shiga toxins are implicated in the interaction between E. coli O157:H7 and human macrophages. Understanding the molecular mechanisms used by E. coli to survive within macrophages may help in the identification of targets for new therapeutic agents.

  14. Characterization of Toxin Genes and Antimicrobial Susceptibility of Staphylococcus aureus Isolates in Fishery Products in Iran

    PubMed Central

    Arfatahery, Noushin; Davoodabadi, Abolfazl; Abedimohtasab, Taranehpeimaneh

    2016-01-01

    Staphylococcus aureus is one of the most common causes of seafood-borne diseases worldwide, which are attributable to the contamination of food by preformed enterotoxins. In this study, a total of 206 (34.3%) Staphylococcus aureus strains were obtained from 600 fish and shrimp samples and were tested for their antimicrobial susceptibility. We assessed the prevalence of the genes responsible for the staphylococcal enterotoxins (SEA, SEB) and toxic shock syndrome toxin 1 (TSST-1) genes. The results indicated that 34% of aqua food samples were contaminated with S. aureus, and 23.8% of these isolates were mec-A-positive. Sixty-four percent of the strains isolated from contaminated seafood was enterotoxigenic S. aureus, and 28.2% of SEs were MRSA-positive. The most prevalent genotype was characterized by the presence of the sea gene (45.2%), followed by the seb gene (18.5%), and the tst gene encoding TSST-1 was found in eight strains (3.9%). Of the 206 S. aureus isolates, 189 strains (84.9%) were resistant to at least one antibiotic. Given the frequent outbreaks of enterotoxigenic MRSA, it is necessary to make revisions to mandatory programmes to facilitate improved hygiene practices during fishing, aquaculture, processing, and sales to prevent the contamination of fishery products in Iran. PMID:27694813

  15. Alterations in gene expression precede sarcopenia and osteopenia in botulinum toxin immobilized mice

    PubMed Central

    Vegger, J.B.; Brüel, A.; Dahlgaard, A.F.; Thomsen, J.S.

    2016-01-01

    Objectives: To investigate alteration of bone and muscle gene expression at different time points during 3 weeks of botulinum toxin (BTX) induced immobilization and how this correlate with conventional analysis of bone and muscle. Methods: Thirty-five 16-week-old female C57BL/6-mice were investigated; 15 were injected with BTX, 15 served as age-matched controls, and 5 as baseline. 5 BTX-injected and 5 control mice were euthanized after 1, 2, and 3 weeks. Analysis included RT-qPCR, dynamic bone histomorphometry, DEXA, µCT, mechanical testing, and muscle cell cross-sectional-area (CSA). Results: Genes related to osteoblasts were expressed at a lower level after 1 week, but not after 2 and 3 weeks of disuse. Moreover, genes related to osteoclasts were expressed at a higher level after 1 and 2 weeks of disuse, whereafter they approached the level of the controls. Genes related to muscle atrophy were upregulated 1 and 2 weeks after the BTX-injection, but not after 3 weeks. In contrast, deterioration of bone microstructure and strength, and reduction in muscle cell CSA were most evident after 3 weeks of disuse. Conclusions: Gene expression should be investigated during the first two weeks of immobilization, whereas changes in bone microstructure and muscle cell CSA are most prominent after 3 weeks of immobilization. PMID:27973388

  16. Bacillus subtilis HJ18-4 from traditional fermented soybean food inhibits Bacillus cereus growth and toxin-related genes.

    PubMed

    Eom, Jeong Seon; Lee, Sun Young; Choi, Hye Sun

    2014-11-01

    Bacillus subtilis HJ18-4 isolated from buckwheat sokseongjang, a traditional Korean fermented soybean food, exhibits broad-spectrum antimicrobial activity against foodborne pathogens, including Bacillus cereus. In this study, we investigated the antibacterial efficacy and regulation of toxin gene expression in B. cereus by B. subtilis HJ18-4. Expression of B. cereus toxin-related genes (groEL, nheA, nheC, and entFM) was downregulated by B. subtilis HJ18-4, which also exhibited strong antibacterial activity against B. cereus. We also found that water extracts of soy product fermented with B. subtilis HJ18-4 significantly inhibited the growth of B. cereus and toxin expression. These results indicate that B. subtilis HJ18-4 could be used as an antimicrobial agent to control B. cereus in the fermented soybean food industry. Our findings also provide an opportunity to develop an efficient biological control agent against B. cereus.

  17. Domain loss facilitates accelerated evolution and neofunctionalization of duplicate snake venom metalloproteinase toxin genes.

    PubMed

    Casewell, Nicholas R; Wagstaff, Simon C; Harrison, Robert A; Renjifo, Camila; Wüster, Wolfgang

    2011-09-01

    Gene duplication is a key mechanism for the adaptive evolution and neofunctionalization of gene families. Large multigene families often exhibit complex evolutionary histories as a result of frequent gene duplication acting in concordance with positive selection pressures. Alterations in the domain structure of genes, causing changes in the molecular scaffold of proteins, can also result in a complex evolutionary history and has been observed in functionally diverse multigene toxin families. Here, we investigate the role alterations in domain structure have on the tempo of evolution and neofunctionalization of multigene families using the snake venom metalloproteinases (SVMPs) as a model system. Our results reveal that the evolutionary history of viperid (Serpentes: Viperidae) SVMPs is repeatedly punctuated by domain loss, with the single loss of the cysteine-rich domain, facilitating the formation of P-II class SVMPs, occurring prior to the convergent loss of the disintegrin domain to form multiple P-I SVMP structures. Notably, the majority of phylogenetic branches where domain loss was inferred to have occurred exhibited highly significant evidence of positive selection in surface-exposed amino acid residues, resulting in the neofunctionalization of P-II and P-I SVMP classes. These results provide a valuable insight into the mechanisms by which complex gene families evolve and detail how the loss of domain structures can catalyze the accelerated evolution of novel gene paralogues. The ensuing generation of differing molecular scaffolds encoded by the same multigene family facilitates gene neofunctionalization while presenting an evolutionary advantage through the retention of multiple genes capable of encoding functionally distinct proteins.

  18. Characterization of foodborne Staphylococcus aureus isolates: association of toxin gene profile with genotype and food commodities in Shanghai, China

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Staphylococcus aureus is an important clinical and foodborne pathogen. Zoonotic risk of transmission to humans highlights the need to understand the ecology of S. aureus in various foods. We characterized the genetic diversity and the distribution of 25 toxin genes in 142 foodborne Staphylococcus au...

  19. Usefulness of Adjunctive Fecal Calprotectin and Serum Procalcitonin in Individuals Positive for Clostridium difficile Toxin Gene by PCR Assay

    PubMed Central

    Gheorghe, Romina; Eastmond, Jennifer; Miller, Mark A.

    2015-01-01

    In 54/64 subjects with nosocomial diarrhea, fecal calprotectin levels correlated with the results of stool samples tested for Clostridium difficile toxin gene by PCR. Fecal calprotectin levels can be used as an adjunctive measure to PCR to support the diagnosis of C. difficile infection. PMID:26354814

  20. A highly efficient molecular cloning platform that utilises a small bacterial toxin gene.

    PubMed

    Mok, Wendy W K; Li, Yingfu

    2013-04-15

    Molecular cloning technologies that have emerged in recent years are more efficient and simpler to use than traditional strategies, but many have the disadvantages of requiring multiple steps and expensive proprietary enzymes. We have engineered cloning vectors containing variants of IbsC, a 19-residue toxin from Escherichia coli K-12. These toxic peptides offer selectivity to minimise the background, labour, and cost associated with conventional molecular cloning. As demonstrated with the cloning of reporter genes, this "detox cloning" system consistently produced over 95 % positive clones. Purification steps between digestion and ligation are not necessary, and the total time between digestion and plating of transformants can be as little as three hours. Thus, these IbsC-based cloning vectors are as reliable and amenable to high-throughput cloning as commercially available systems, and have the advantage of being more time-efficient and cost-effective.

  1. Variability in the sxt Gene Clusters of PSP Toxin Producing Aphanizomenon gracile Strains from Norway, Spain, Germany and North America

    PubMed Central

    Cerasino, Leonardo; Hostyeva, Vladyslava; Cirés, Samuel

    2016-01-01

    Paralytic shellfish poisoning (PSP) toxin production has been detected worldwide in the cyanobacterial genera Anabaena, Lyngbya, Scytonema, Cuspidothrix and Aphanizomenon. In Europe Aphanizomenon gracile and Cuspidothrix issatschenkoi are the only known producers of PSP toxins and are found in Southwest and Central European freshwater bodies. In this study the PSP toxin producing Aphanizomenon sp. strain NIVA-CYA 851 was isolated from the Norwegian Lake Hillestadvannet. In a polyphasic approach NIVA-CYA 851 was morphologically and phylogenetically classified, and investigated for toxin production. The strain NIVA-CYA 851 was identified as A. gracile using 16S rRNA gene phylogeny and was confirmed to produce neosaxitoxin, saxitoxin and gonyautoxin 5 by LC-MS. The whole sxt gene clusters (circa 27.3 kb) of four A. gracile strains: NIVA-CYA 851 (Norway); NIVA-CYA 655 & NIVA-CYA 676 (Germany); and UAM 529 (Spain), all from latitudes between 40° and 59° North were sequenced and compared with the sxt gene cluster of reference strain A. gracile NH-5 from the USA. All five sxt gene clusters are highly conserved with similarities exceeding 99.4%, but they differ slightly in the number and presence of single nucleotide polymorphisms (SNPs) and insertions/deletions (In/Dels). Altogether 178 variable sites (44 SNPs and 4 In/Dels, comprising 134 nucleotides) were found in the sxt gene clusters of the Norwegian, German and Spanish strains compared to the reference strain. Thirty-nine SNPs were located in 16 of the 27 coding regions. The sxt gene clusters of NIVA-CYA 851, NIVA-CYA 655, NIVA-CYA 676 and UAM 529, were characterized by 15, 16, 19 and 23 SNPs respectively. Only the Norwegian strain NIVA-CYA 851 possessed an insertion of 126 base pairs (bp) in the noncoding area between the sxtA and sxtE genes and a deletion of 6 nucleotides in the sxtN gene. The sxtI gene showed the highest variability and is recommended as the best genetic marker for further phylogenetic studies

  2. Variability in the sxt Gene Clusters of PSP Toxin Producing Aphanizomenon gracile Strains from Norway, Spain, Germany and North America.

    PubMed

    Ballot, Andreas; Cerasino, Leonardo; Hostyeva, Vladyslava; Cirés, Samuel

    2016-01-01

    Paralytic shellfish poisoning (PSP) toxin production has been detected worldwide in the cyanobacterial genera Anabaena, Lyngbya, Scytonema, Cuspidothrix and Aphanizomenon. In Europe Aphanizomenon gracile and Cuspidothrix issatschenkoi are the only known producers of PSP toxins and are found in Southwest and Central European freshwater bodies. In this study the PSP toxin producing Aphanizomenon sp. strain NIVA-CYA 851 was isolated from the Norwegian Lake Hillestadvannet. In a polyphasic approach NIVA-CYA 851 was morphologically and phylogenetically classified, and investigated for toxin production. The strain NIVA-CYA 851 was identified as A. gracile using 16S rRNA gene phylogeny and was confirmed to produce neosaxitoxin, saxitoxin and gonyautoxin 5 by LC-MS. The whole sxt gene clusters (circa 27.3 kb) of four A. gracile strains: NIVA-CYA 851 (Norway); NIVA-CYA 655 & NIVA-CYA 676 (Germany); and UAM 529 (Spain), all from latitudes between 40° and 59° North were sequenced and compared with the sxt gene cluster of reference strain A. gracile NH-5 from the USA. All five sxt gene clusters are highly conserved with similarities exceeding 99.4%, but they differ slightly in the number and presence of single nucleotide polymorphisms (SNPs) and insertions/deletions (In/Dels). Altogether 178 variable sites (44 SNPs and 4 In/Dels, comprising 134 nucleotides) were found in the sxt gene clusters of the Norwegian, German and Spanish strains compared to the reference strain. Thirty-nine SNPs were located in 16 of the 27 coding regions. The sxt gene clusters of NIVA-CYA 851, NIVA-CYA 655, NIVA-CYA 676 and UAM 529, were characterized by 15, 16, 19 and 23 SNPs respectively. Only the Norwegian strain NIVA-CYA 851 possessed an insertion of 126 base pairs (bp) in the noncoding area between the sxtA and sxtE genes and a deletion of 6 nucleotides in the sxtN gene. The sxtI gene showed the highest variability and is recommended as the best genetic marker for further phylogenetic studies

  3. Insight into Shiga toxin genes encoded by Escherichia coli O157 from whole genome sequencing

    PubMed Central

    Ashton, Philip M.; Perry, Neil; Ellis, Richard; Petrovska, Liljana; Wain, John; Grant, Kathie A.; Jenkins, Claire

    2015-01-01

    The ability of Shiga toxin-producing Escherichia coli (STEC) to cause severe illness in humans is determined by multiple host factors and bacterial characteristics, including Shiga toxin (Stx) subtype. Given the link between Stx2a subtype and disease severity, we sought to identify the stx subtypes present in whole genome sequences (WGS) of 444 isolates of STEC O157. Difficulties in assembling the stx genes in some strains were overcome by using two complementary bioinformatics methods: mapping and de novo assembly. We compared the WGS analysis with the results obtained using a PCR approach and investigated the diversity within and between the subtypes. All strains of STEC O157 in this study had stx1a, stx2a or stx2c or a combination of these three genes. There was over 99% (442/444) concordance between PCR and WGS. When common source strains were excluded, 236/349 strains of STEC O157 had multiple copies of different Stx subtypes and 54 had multiple copies of the same Stx subtype. Of those strains harbouring multiple copies of the same Stx subtype, 33 had variants between the alleles while 21 had identical copies. Strains harbouring Stx2a only were most commonly found to have multiple alleles of the same subtype (42%). Both the PCR and WGS approach to stx subtyping provided a good level of sensitivity and specificity. In addition, the WGS data also showed there were a significant proportion of strains harbouring multiple alleles of the same Stx subtype associated with clinical disease in England. PMID:25737808

  4. Abundant toxin-related genes in the genomes of beneficial symbionts from deep-sea hydrothermal vent mussels

    PubMed Central

    Sayavedra, Lizbeth; Kleiner, Manuel; Ponnudurai, Ruby; Wetzel, Silke; Pelletier, Eric; Barbe, Valerie; Satoh, Nori; Shoguchi, Eiichi; Fink, Dennis; Breusing, Corinna; Reusch, Thorsten BH; Rosenstiel, Philip; Schilhabel, Markus B; Becher, Dörte; Schweder, Thomas; Markert, Stephanie; Dubilier, Nicole; Petersen, Jillian M

    2015-01-01

    Bathymodiolus mussels live in symbiosis with intracellular sulfur-oxidizing (SOX) bacteria that provide them with nutrition. We sequenced the SOX symbiont genomes from two Bathymodiolus species. Comparison of these symbiont genomes with those of their closest relatives revealed that the symbionts have undergone genome rearrangements, and up to 35% of their genes may have been acquired by horizontal gene transfer. Many of the genes specific to the symbionts were homologs of virulence genes. We discovered an abundant and diverse array of genes similar to insecticidal toxins of nematode and aphid symbionts, and toxins of pathogens such as Yersinia and Vibrio. Transcriptomics and proteomics revealed that the SOX symbionts express the toxin-related genes (TRGs) in their hosts. We hypothesize that the symbionts use these TRGs in beneficial interactions with their host, including protection against parasites. This would explain why a mutualistic symbiont would contain such a remarkable ‘arsenal’ of TRGs. DOI: http://dx.doi.org/10.7554/eLife.07966.001 PMID:26371554

  5. Staphylococcal food poisoning case and molecular analysis of toxin genes in Staphylococcus aureus strains isolated from food in Sicily, Italy.

    PubMed

    Vitale, Maria; Scatassa, Maria Luisa; Cardamone, Cinzia; Oliveri, Giuseppa; Piraino, Chiara; Alduina, Rosa; Napoli, Concetta

    2015-01-01

    A case of staphylococcal food poisoning was observed in two individuals of the same family after consumption of primosale, a semiripened sheep cheese produced in Sicily. Staphylococcus aureus isolated from the cheese produced enterotoxin C (SEC) and carried both the enterotoxin C (sec) and the toxic shock syndrome toxin (tsst-1) gene. Following this case, an extensive survey was conducted on 971 food samples (raw milk, cheese, meat, and food preparations). S. aureus was detected in 102 of 971 food samples, from all types of food with the exception of ricotta cheese. The tsst-1 gene was present in 42% of the strains, either alone or in combination with other toxin genes. The enterotoxin C gene was the most represented enterotoxin, but it was only found in dairy products. Six S. aureus isolates carried the sea gene alone, two isolates carried both sea and seb, and one isolate carried both sea and sec. A significant percentage (46%) of all isolates carried a toxin gene, creating significant concern that virulent S. aureus can be transmitted through food in Sicily.

  6. Food-animal related Staphylococcus aureus multidrug-resistant ST9 strains with toxin genes.

    PubMed

    He, Wenqiang; Liu, Yuqing; Qi, Jing; Chen, Hongbin; Zhao, Chunjiang; Zhang, Feifei; Li, Henan; Wang, Hui

    2013-09-01

    To determine whether methicillin-susceptible S. aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) are present in commercial pig farms and food products from supermarkets in China, we characterized S. aureus isolates from 250 samples associated with swine and animal-related food products in Shandong Province. The isolates were characterized by susceptibility testing, toxin gene detection, pulse-field gel electrophoresis (PFGE), multilocus sequence typing, and spa typing. MRSA were identified and typed by the staphylococcal cassette chromosome mec (SCCmec). The prevalence of S. aureus among all samples was 19.6% (49/250). MRSA and MSSA accounted for 16.7% (20/120) and 8.3% (10/120), respectively, of swine feces samples. Only MSSA was detected from swine carcass, pork, chicken, and raw milk, accounting for 15% (6/40), 10% (3/30), 20% (6/30), and 13.3% (4/30), respectively. The predominant MRSA clone was ST9-t899 SCCmecIVb/PFGE A (70.0%, 14/20). Among the MSSA isolates, ST9-t899/PFGE A was the most prevalent (27.6%), followed by ST15-t084 (17.2%), ST97-t2756 (10.3%), ST1-t127 (6.9%), and ST398-t899 (3.5%). Some lineages were found that are commonly detected in humans (e.g., ST1, ST5, ST7, ST59, ST88) or are human-specific (e.g., ST15). The toxin genes sec, seh, and enterotoxin gene cluster (egc) were significantly more prevalent among isolates of lineage ST9 (p<0.001) compared to other lineages, and the ST9 isolates were more resistant to erythromycin, clindamycin, ciprofloxacin, chloramphenicol, and gentamicin. The same lineage was identified from different sample types, indicating circulation of the related strains within the area of study. In conclusion, swine and food products of animal origin carried S. aureus, and the predominant ST9 clone possesses a multidrug-resistance profile and a high prevalence of sec, seh, and egc enterotoxin genes.

  7. General secretion pathway (eps) genes required for toxin secretion and outer membrane biogenesis in Vibrio cholerae.

    PubMed

    Sandkvist, M; Michel, L O; Hough, L P; Morales, V M; Bagdasarian, M; Koomey, M; DiRita, V J; Bagdasarian, M

    1997-11-01

    The general secretion pathway (GSP) of Vibrio cholerae is required for secretion of proteins including chitinase, enterotoxin, and protease through the outer membrane. In this study, we report the cloning and sequencing of a DNA fragment from V. cholerae, containing 12 open reading frames, epsC to -N, which are similar to GSP genes of Aeromonas, Erwinia, Klebsiella, Pseudomonas, and Xanthomonas spp. In addition to the two previously described genes, epsE and epsM (M. Sandkvist, V. Morales, and M. Bagdasarian, Gene 123: 81-86, 1993; L. J. Overbye, M. Sandkvist, and M. Bagdasarian, Gene 132:101-106, 1993), it is shown here that epsC, epsF, epsG, and epsL also encode proteins essential for GSP function. Mutations in the eps genes result in aberrant outer membrane protein profiles, which indicates that the GSP, or at least some of its components, is required not only for secretion of soluble proteins but also for proper outer membrane assembly. Several of the Eps proteins have been identified by use of the T7 polymerase-promoter system in Escherichia coli. One of them, a pilin-like protein, EpsG, was analyzed also in V. cholerae and found to migrate as two bands on polyacrylamide gels, suggesting that in this organism it might be processed or otherwise modified by a prepilin peptidase. We believe that TcpJ prepilin peptidase, which processes the subunit of the toxin-coregulated pilus, TcpA, is not involved in this event. This is supported by the observations that apparent processing of EpsG occurs in a tcpJ mutant of V. cholerae and that, when coexpressed in E. coli, TcpJ cannot process EpsG although the PilD peptidase from Neisseria gonorrhoeae can.

  8. Purification and characterization of neurotoxin complex from a dual toxin gene containing Clostridium Botulinum Strain PS-5.

    PubMed

    Singh, Ajay K; Sachdeva, Amita; Degrasse, Jeffrey A; Croley, Timothy R; Stanker, Larry H; Hodge, David; Sharma, Shashi K

    2013-04-01

    Botulinum neurotoxins are produced as a toxin complex (TC) which consists of neurotoxin (NT) and neurotoxin associated proteins. The characterization of NT in its native state is an essential step for developing diagnostics and therapeutic countermeasures against botulism. The presence of NT genes was validated by PCR amplification of toxin specific fragments from genomic DNA of Clostridium botulinum strain PS-5 which indicated the presence of both serotype A and B genes on PS-5 genome. Further, TC was purified and characterized by Western blotting, Digoxin-enzyme linked immunosorbent assay, endopeptidase activity assay, and Liquid chromatography-Mass spectrometry. The data showed the presence of serotype A specific neurotoxin. Based on the analysis of neurotoxin genes and characterization of TC, PS-5 strain appears as a serotype A (B) strain of C. botulinum which produces only serotype A specific TC in the cell culture medium.

  9. Transgenic cotton expressing synthesized scorpion insect toxin AaHIT gene confers enhanced resistance to cotton bollworm (Heliothis armigera) larvae.

    PubMed

    Wu, Jiahe; Luo, Xiaoli; Wang, Zhian; Tian, Yingchuan; Liang, Aihua; Sun, Yi

    2008-03-01

    A synthetic scorpion Hector Insect Toxin (AaHIT) gene, under the control of a CaMV35S promoter, was cloned into cotton via Agrobacterium tumefaciens-mediated transformation. Southern blot analyses indicated that integration of the transgene varied from one to more than three estimated copies per genome; seven homozygous transgenic lines with one copy of the T-DNA insert were then selected by PCR and Southern blot analysis. AaHIT expression was from 0.02 to 0.43% of total soluble protein determined by western blot. These homozygous transgenic lines killed larvae of cotton bollworm (Heliothis armigera) by 44-98%. The AaHIT gene could used therefore an alternative to Bt toxin and proteinase inhibitor genes for producing transgenic cotton crops with effective control of bollworm.

  10. Anti-cancer Parasporin Toxins are Associated with Different Environments: Discovery of Two Novel Parasporin 5-like Genes.

    PubMed

    Ammons, David R; Short, John D; Bailey, Jeffery; Hinojosa, Gabriela; Tavarez, Lourdes; Salazar, Martha; Rampersad, Joanne N

    2016-02-01

    Cry toxins are primarily a family of insecticidal toxins produced by the bacterium Bacillus thuringiensis (Bt). However, some Cry toxins, called parasporins (PSs), are non-insecticidal and have been shown to differentially kill human cancer cells. Based on amino acid homology, there are currently six different classes of parasporins (PS1-6). It is not known what role parasporins play in nature, nor if certain PSs are associated with Bt found in particular environments. Herein, we present ten parasporin-containing isolates of Bt from the Caribbean island of Trinidad. Genes coding for PS1 and PS6 were found in isolates associated mainly with artificial aquatic environments (e.g., barrels with rain water), while Bt possessing two novel PS5-like genes (ps5-1 and ps5-2), were isolated from manure collected directly from the rectum of cattle. The amino acid sequences inferred from the two PS5-like genes were 51 % homologous to each other, while being only 41 or 45 % similar to PS5Aa1/Cry64Aa, the only reported member of the parasporin five class. The low level of amino acid homology between the two PS5-like genes and PS5Aa1 indicate that the two PS5-like genes may represent a new class of parasporins, or greatly expand the level of diversity within the current parasporin 5 class.

  11. Cloning and characterization of a novel calcium channel toxin-like gene BmCa1 from Chinese scorpion Mesobuthus martensii Karsch.

    PubMed

    Zhijian, Cao; Yun, Xie; Chao, Dai; Shunyi, Zhu; Shijin, Yin; Yingliang, Wu; Wenxin, Li

    2006-06-01

    Many studies have been carried on peptides and genes encoding scorpion toxins from the venom of Mesobuthus martensii Karsch (synonym: Buthus martensii Karsch, BmK), such as Na+, K+ and Cl- channel modulators. In this study, a novel calcium channel toxin-like gene BmCa1 was isolated and characterized from the venom of Mesobuthus martensii Karsch. First, a partial cDNA sequence of the Ca2+ channel toxin-like gene was identified by random sequencing method from a venomous gland cDNA library of Mesobuthus martensii Karsch. The full-length sequence of BmCa1 was then obtained by 5'RACE technique. The peptide deduced from BmCa1 precursor nucleotide sequence contains a 27-residue signal peptide and a 37-residue mature peptide. Although BmCa1 and other scorpion toxins are different at the gene and protein primary structure levels, BmCa1 has the same precursor nucleotide organization and cysteine arrangement as that of the first subfamily members of calcium channel scorpion toxins. Genomic DNA sequence of BmCa1 was also cloned by PCR. Sequence analysis showed that BmCa1 gene consists of three exons separated by two introns of 72 bp and 1076 bp in length, respectively. BmCa1 is the first calcium channel toxin-like gene cloned from the venom of Mesobuthus martensii Karsch and potentially represents a novel class of calcium channel toxins in scorpion venoms.

  12. Molecular characterization of the PR-toxin gene cluster in Penicillium roqueforti and Penicillium chrysogenum: cross talk of secondary metabolite pathways.

    PubMed

    Hidalgo, Pedro I; Ullán, Ricardo V; Albillos, Silvia M; Montero, Olimpio; Fernández-Bodega, María Ángeles; García-Estrada, Carlos; Fernández-Aguado, Marta; Martín, Juan-Francisco

    2014-01-01

    The PR-toxin is a potent mycotoxin produced by Penicillium roqueforti in moulded grains and grass silages and may contaminate blue-veined cheese. The PR-toxin derives from the 15 carbon atoms sesquiterpene aristolochene formed by the aristolochene synthase (encoded by ari1). We have cloned and sequenced a four gene cluster that includes the ari1 gene from P. roqueforti. Gene silencing of each of the four genes (named prx1 to prx4) resulted in a reduction of 65-75% in the production of PR-toxin indicating that the four genes encode enzymes involved in PR-toxin biosynthesis. Interestingly the four silenced mutants overproduce large amounts of mycophenolic acid, an antitumor compound formed by an unrelated pathway suggesting a cross-talk of PR-toxin and mycophenolic acid production. An eleven gene cluster that includes the above mentioned four prx genes and a 14-TMS drug/H(+) antiporter was found in the genome of Penicillium chrysogenum. This eleven gene cluster has been reported to be very poorly expressed in a transcriptomic study of P. chrysogenum genes under conditions of penicillin production (strongly aerated cultures). We found that this apparently silent gene cluster is able to produce PR-toxin in P. chrysogenum under static culture conditions on hydrated rice medium. Noteworthily, the production of PR-toxin was 2.6-fold higher in P. chrysogenum npe10, a strain deleted in the 56.8kb amplifiable region containing the pen gene cluster, than in the parental strain Wisconsin 54-1255 providing another example of cross-talk between secondary metabolite pathways in this fungus. A detailed PR-toxin biosynthesis pathway is proposed based on all available evidence.

  13. Detection of beta2 and major toxin genes by PCR in Clostridium perfringens field isolates of domestic animals suffering from enteritis or enterotoxaemia.

    PubMed

    Sting, Reinhard

    2009-01-01

    The production of Clostridium (C.) perfringens toxins in the intestine is an important cause of enteritis and enterotoxaemia in livestock. In the present study, the alpha toxin and the genes encoding beta2 and epsilon toxin could be frequently detected by means of phenotypical and PCR examinations in these bacteria. The C. perfringens isolates originated from 1213 field samples taken from diseased or perished livestock located in the north-eastern administrative districts of Baden-Württemberg (Germany) from 2005 to 2008. The beta2 toxin gene of C perfringens was detected in all animal species examined, comprising pigs, the small ruminants sheep and goats, cattle, horses, rabbits, alpacas and lamas, and fallow deer. Among all the animal species included in this study, pigs attracted attention by a high quota of 74.2% (610 of 822) cpb2-positive C. perfringens isolates in comparison to the other animal species tested, revealing a quota of 20.8% (72 of 346). Beta2 toxigenic isolates could be predominantly cultivated from the faeces of young piglets. The beta toxin gene was detected in isolates from piglets and small ruminants only, amounting to 82.5% (33 of 40) in piglets in combination with the cpb2 gene. In this context, cpb2/cpb-positive C. perfringens isolates of piglets could be clearly detected more often in the intestine of perished animals (18 of 158) than in faeces (15 of 629). Furthermore, cpb2-bearing C. perfringens isolates were detected in cattle, horses, rabbits, alpacas and lamas, and fallow deer to a notable degree. The detection of C. perfringens isolates carrying the epsilon toxin gene was restricted to sheep and goats. Of a total of 242 small ruminants that succumbed to sudden death, 71 (29.3%) harboured epsilon toxin-positive C. perfringens isolates in their intestines. These cases clustered seasonally in the second quarter (April, May, and June) of the year. Neither the isolates bearing the beta2 nor beta toxin gene nor those carrying the epsilon

  14. Virulence genes in clinical and environmental Stenotrophomas maltophilia isolates: a genome sequencing and gene expression approach.

    PubMed

    Adamek, Martina; Linke, Burkhard; Schwartz, Thomas

    2014-01-01

    The rate of nosocomial infections with the opportunistic pathogen Stenotrophomonas maltophilia has remarkably increased in the last decade. To determine S. maltophilia virulence genes, the complete genome sequences of two S. maltophilia isolates were compared. The clinical strain SKK35 was proved virulent in an amoeba host-pathogen model, and wastewater strain RA8 was determined as non-virulent in the amoeba model. The genome sequences of three additional S. maltophilia strains, K279a (clinical, non-virulent against amoeba), R511-3 and SKA14 (both environmental, non-virulent against amoeba) were taken into account as reference strains. We were able to show that all clinical and environmental S. maltophilia strains presented comparable distribution of so far identified potential virulence genes, regardless to their virulence potential against amoebae. Aside from that, strain SKK35 was found harboring a putative, strain specific pathogenicity island, encoding two proteins from the RTX (repeats-in-toxin) family. The actual expression of the RTX genes was verified in growth experiments in different culture media containing blood or blood components and in co-cultures with amoeba.

  15. Quantitative effects of carbohydrates and aromatic amino acids on Clostridium botulinum toxin gene expression using a rapid competitive RT/PCR assay.

    PubMed

    Sharkey, Freddie H; Dooley, James S; Haylock, Richard W

    2005-01-01

    A rapid competitive RT/PCR assay was developed to determine the effects of nutrients on Clostridium botulinum type E toxin gene expression. The type E strain (EVH) was grown in a nutrient-rich broth containing 1% glucose (base medium). Toxin gene expression was quantified at both mid and late exponential phases of growth. It was found that toxin encoding mRNA levels were highly growth phase dependent with elevated levels found in late exponential phase compared to mid exponential phase. Changing the carbohydrate source had a smaller effect on toxin encoding mRNA levels but as earlier results have suggested, toxin encoding mRNA levels show a strong correlation with type E growth rate. The results have important implications for the food industry whereby risk of type E botulism could be correlated to the nutrient composition of the contaminated food or assessed from C. botulinum growth rates in challenged foodstuffs.

  16. The gene for type A streptococcal exotoxin (erythrogenic toxin) is located in bacteriophage T12.

    PubMed Central

    Weeks, C R; Ferretti, J J

    1984-01-01

    The infection of Streptococcus pyogenes T25(3) with the temperate bacteriophage T12 results in the conversion of the nontoxigenic strain to type A streptococcal exotoxin (erythrogenic toxin) production. Although previous research has established that integration of the bacteriophage genome into the host chromosome is not essential for exotoxin production, the location of the gene on the bacteriophage or bacterial chromosome had not been determined. In the present investigation, recombinant DNA techniques were used to determine whether the gene specifying type A streptococcal exotoxin (speA) production is located on the bacteriophage chromosome. Bacteriophage T12 was obtained from S. pyogenes T25(3)(T12) by induction with mitomycin C, and after isolation of bacteriophage DNA by phenol-chloroform extraction, the DNA was digested with restriction enzymes and ligated with Escherichia coli plasmid pHP34 or the Streptococcus-E. coli shuttle vector pSA3. Transformation of E. coli HB101 with the recombinant molecules allowed selection of E. coli clones containing bacteriophage T12 genes. Immunological assays with specific antibody revealed the presence of type A streptococcal exotoxin in sonicates of E. coli transformants. Subcloning experiments localized the speA gene to a 1.7-kilobase segment of the bacteriophage T12 genome flanked by SalI and HindIII sites. Introduction of the pSA3 vector containing the speA gene into Streptococcus sanguis (Challis) resulted in transformants that secreted the type A exotoxin. Immunological analysis showed that the type A streptococcal exotoxin produced by E. coli and S. sanguis transformants was identical to the type A exotoxin produced by S. pyogenes T25(3)(T12). Southern blot hybridizations with the cloned fragment confirmed its presence in the bacteriophage T12 genome and its absence in the T25(3) nonlysogen. Therefore, the gene for type A streptococcal exotoxin is located in the bacteriophage genome, and conversion of S. pyogenes T

  17. The High PMNs Phagocytosis Resistance of Enterococcal Isolates from RTx Patients

    PubMed Central

    Daca, Agnieszka; Witkowski, Jacek M.; Bryl, Ewa; Rutkowski, Bolesław

    2015-01-01

    Infections caused by opportunistic pathogens such as enterococci remain difficult to manage, especially in immunocompromised patients. Because of infections' limited symptoms in such patients the additional problems are to find proper diagnostic criteria and the management of infection. Here we aimed to compare the resistance of commensal enterococcal strains and RTx patients' isolates, to PMNs phagocytosis. Thirty-six enterococcal urine and faecal isolates from RTx patients and 17 faecal isolates from healthy volunteers were cultured in planktonic and biofilm forms in 37°C or 42°C. Another tested variable was the addition of immunosuppressant to the culture media. Bacterial cells were stained with fluorescent reporter (CFDA, PI) and incubated with PMNs. Results of phagocytosis were estimated as a mean fluorescence intensity (MFI) of PMNs using flow cytometry. Commensal enterococci cultured in all abovementioned (37°C and 42°C/the addition of immunosuppressant) conditions were less resistant to phagocytosis compared to RTx isolates. Observed significant difference in phagocytosis resistance suggests that patients in immunosuppression are colonized with high risk strains which may lead to the development of infection. PMID:25861625

  18. Regulation of toxin and bacteriocin gene expression in Clostridium by interchangeable RNA polymerase sigma factors.

    PubMed

    Dupuy, Bruno; Raffestin, Stéphanie; Matamouros, Susana; Mani, Nagraj; Popoff, Michel R; Sonenshein, Abraham L

    2006-05-01

    The production of major extracellular toxins by pathogenic strains of Clostridium botulinum, Clostridium tetani and Clostridium difficile, and a bacteriocin by Clostridium perfringens is dependent on a related group of RNA polymerase sigma-factors. These sigma-factors (BotR, TetR, TcdR and UviA) were shown to be sufficiently similar that they could substitute for one another in in vitro DNA binding and run-off transcription experiments. In cells, however, the sigma-factors fell into two subclasses. BotR and TetR were able to direct transcription of their target genes in a fully reciprocal manner. Similarly, UviA and TcdR were fully interchangeable. Neither BotR nor TetR could substitute for UviA or TcdR, however, and neither UviA nor TcdR could direct transcription of the natural targets of BotR or TetR. The extent of functional interchangeability of the sigma-factors was attributed to the strong conservation of their subregion 4.2 sequences and the conserved -35 sequences of their target promoters, while restrictions on interchangeability were attributed to variations in their subregion 2.4 sequences and the target site -10 sequences. The four sigma-factors have been assigned to group 5 of the sigma(70) family and seem to have arisen from a common ancestral protein that may have co-evolved with the genes whose transcription they direct. A fifth Clostridiumsigma-factor, sigma(Y) of Clostridium acetobutylicum, resembles the TcdR family, but was not functionally interchangeable with members of this family.

  19. A Double Selection Approach to Achieve Specific Expression of Toxin Genes for Ovarian Cancer Gene Therapy

    DTIC Science & Technology

    2005-11-01

    incorporating the CAV-A knob into the Ad5 fiber-shaft protein. The T-L-W-T peptide sequence joining the shaft and knob regions of both fibers is shown...base to new receptors through replacement of its RGD gene transfer assays of patient tissue slices, all viral motif with other receptor-specific peptide ...Institution of Bioregulation , Kyushu University, Fukuoka 812- 8582, Japan M.N. mnakayam@uab.edu G.W.B. gerry.both@csiro.au B.B. bbanizs@uab.edu Y.T

  20. Detection of Escherichia coli Shiga toxin (stx) and enterotoxin (estA and elt) genes in fecal samples from non-diarrheic and diarrheic greyhounds.

    PubMed

    Staats, Jacque J; Chengappa, M M; DeBey, Mary C; Fickbohm, Barry; Oberst, Richard D

    2003-07-30

    Virulence factors responsible for acute diarrhea in greyhounds have not been well established. The objective of this study was to determine if a correlation exists between disease and the presence of the Escherichia coli toxin genes in non-diarrheic and diarrheic greyhound feces. DNA extracted from broth cultures was evaluated for the presence of Shiga toxin and enterotoxin genes and broth samples were evaluated for Shiga toxin and heat-labile enterotoxin. Shiga toxin (stx1 and stx2) and enterotoxin (et and estA) genes were identified in both non-diarrheic and diarrheic samples after in vitro cultured of swabs at 37 degrees C for 16-24h. The stx1 gene was present in 3% of non-diarrheic and 15% diarrheic samples and the stx2 gene was identified in 36 and 23%, non-diarrheic and diarrheic samples, respectively. Shiga toxin was present in 48% diarrheic and 25% of the non-diarrheic in vitro cultured samples. The elt gene was detected in vitro cultured swabs in 12% of the non-diarrheic and 7% of the diarrheic samples. Labile toxin was present in the feces of small numbers of both groups of dogs. A significant correlation existed between the presence of both stx1 genes and Shiga toxin in feces, and lack of disease in non-diarrheic (P=0.01) and presence of disease in diarrheic (P=0.024) greyhounds. Correlation between production of Shiga toxin and detection of stx1 or stx2 was significant in both the diarrheic and non-diarrheic feces (P=0.03); however, only the presence of stx1 correlated with diarrhea in both groups of samples (P<0.008). The incidence of toxigenic E. coli in both non-diarrheic and diarrheic greyhounds indicates a zoonotic potential from dogs to humans and requires further study.

  1. Spatial, Temporal, and Matrix Variability of Clostridium botulinum Type E Toxin Gene Distribution at Great Lakes Beaches.

    PubMed

    Wijesinghe, Rasanthi U; Oster, Ryan J; Haack, Sheridan K; Fogarty, Lisa R; Tucker, Taaja R; Riley, Stephen C

    2015-07-01

    Clostridium botulinum type E toxin is responsible for extensive mortality of birds and fish in the Great Lakes. The C. botulinum bontE gene that produces the type E toxin was amplified with quantitative PCR from 150 sloughed algal samples (primarily Cladophora species) collected during summer 2012 from 10 Great Lakes beaches in five states; concurrently, 74 sediment and 37 water samples from four sites were also analyzed. The bontE gene concentration in algae was significantly higher than in water and sediment (P < 0.05), suggesting that algal mats provide a better microenvironment for C. botulinum. The bontE gene was detected most frequently in algae at Jeorse Park and Portage Lake Front beaches (Lake Michigan) and Bay City State Recreation Area beach on Saginaw Bay (Lake Huron), where 77, 100, and 83% of these algal samples contained the bontE gene, respectively. The highest concentration of bontE was detected at Bay City (1.98 × 10(5) gene copies/ml of algae or 5.21 × 10(6) g [dry weight]). This study revealed that the bontE gene is abundant in the Great Lakes but that it has spatial, temporal, and matrix variability. Further, embayed beaches, low wave height, low wind velocity, and greater average water temperature enhance the bontE occurrence.

  2. Spatial, Temporal, and Matrix Variability of Clostridium botulinum Type E Toxin Gene Distribution at Great Lakes Beaches

    PubMed Central

    Oster, Ryan J.; Haack, Sheridan K.; Fogarty, Lisa R.; Tucker, Taaja R.; Riley, Stephen C.

    2015-01-01

    Clostridium botulinum type E toxin is responsible for extensive mortality of birds and fish in the Great Lakes. The C. botulinum bontE gene that produces the type E toxin was amplified with quantitative PCR from 150 sloughed algal samples (primarily Cladophora species) collected during summer 2012 from 10 Great Lakes beaches in five states; concurrently, 74 sediment and 37 water samples from four sites were also analyzed. The bontE gene concentration in algae was significantly higher than in water and sediment (P < 0.05), suggesting that algal mats provide a better microenvironment for C. botulinum. The bontE gene was detected most frequently in algae at Jeorse Park and Portage Lake Front beaches (Lake Michigan) and Bay City State Recreation Area beach on Saginaw Bay (Lake Huron), where 77, 100, and 83% of these algal samples contained the bontE gene, respectively. The highest concentration of bontE was detected at Bay City (1.98 × 105 gene copies/ml of algae or 5.21 × 106 g [dry weight]). This study revealed that the bontE gene is abundant in the Great Lakes but that it has spatial, temporal, and matrix variability. Further, embayed beaches, low wave height, low wind velocity, and greater average water temperature enhance the bontE occurrence. PMID:25888178

  3. Prevalence of cytolethal distending toxin production in Campylobacter jejuni and relatedness of Campylobacter sp. cdtB gene.

    PubMed Central

    Pickett, C L; Pesci, E C; Cottle, D L; Russell, G; Erdem, A N; Zeytin, H

    1996-01-01

    Campylobacter jejuni produces a toxin called cytolethal distending toxin (CDT). The genes encoding this toxin in C. jejuni 81-176 were cloned and sequenced. The nucleotide sequence of the genes revealed that there are three genes, cdtA, cdtB, and cdtC, encoding proteins with predicted sizes of 30,11-6, 28,989, and 21,157 Da, respectively. All three proteins were found to be related to the Escherichia coli CDT proteins, yet the amino acid sequences have diverged significantly. All three genes were required for toxic activity in a HeLa cell assay. HeLa cell assays of a variety of C. jejuni and C. coli strains suggested that most C. jejuni strains produce significantly higher CDT titers than do C. coli strains. Southern hybridization experiments demonstrated that the cdtB gene is present on a 6.0-kb ClaI fragment in all but one of the C. jejuni strains tested; the cdtB gene was on a 6.9-kb ClaI fragment in one strain. The C. jejuni 81-176 cdtB probe hybridized weakly to DNAs from C. coli strains. The C. jejuni 81-176 cdtB probe did not hybridize to DNAs from representative C. fetus, C. lari, C. "upsaliensis," and C. hyointestinalis strains, although the HeLa cell assay indicated that these strains make CDT. PCR experiments indicated the probable presence of cdtB sequences in all of these Campylobacter species. PMID:8675309

  4. Identification of Actinobacillus actinomycetemcomitans by leukotoxin gene-specific hybridization and polymerase chain reaction assays.

    PubMed Central

    Tønjum, T; Haas, R

    1993-01-01

    Eleven strains of Actinobacillus actinomycetemcomitans isolated from cases of systemic infections, local abscesses, and periodontitis were identified by genetic assays using the leukotoxin gene as the target. We have developed a polymerase chain reaction (PCR) assay, based on the leukotoxin structural gene of this pathogen, which clearly identified all tested strains of A. actinomycetemcomitans and separated them from the closely related Haemophilus aphrophilus as well as other bacterial species. Furthermore, DNA-DNA hybridization was performed with the cloned partial leukotoxin structural gene (lktA) as a probe, which again clearly distinguished A. actinomycetemcomitans from H. aphrophilus, parts of the normal oral flora, and species harboring RTX (repeats in toxin) family-related cytotoxins. The PCR fragment amplified from the leukotoxin structural gene gave results similar to those given by the cloned leukotoxin gene when used as a probe in hybridization experiments. The hybridization and PCR assays described here are fundamental improvements for the identification of A. actinomycetemcomitans. Images PMID:8349764

  5. Distinct Roles of the Repeat-Containing Regions and Effector Domains of the Vibrio vulnificus Multifunctional-Autoprocessing Repeats-in-Toxin (MARTX) Toxin

    PubMed Central

    Kim, Byoung Sik; Gavin, Hannah E.

    2015-01-01

    ABSTRACT Vibrio vulnificus is a seafood-borne pathogen that destroys the intestinal epithelium, leading to rapid bacterial dissemination and death. The most important virulence factor is the multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin comprised of effector domains in the center region flanked by long repeat-containing regions which are well conserved among MARTX toxins and predicted to translocate effector domains. Here, we examined the role of the repeat-containing regions using a modified V. vulnificus MARTX (MARTXVv) toxin generated by replacing all the internal effector domains with β-lactamase (Bla). Bla activity was detected in secretions from the bacterium and also in the cytosol of intoxicated epithelial cells. The modified MARTXVv toxin without effector domains retained its necrotic activity but lost its cell-rounding activity. Further, deletion of the carboxyl-terminal repeat-containing region blocked toxin secretion from the bacterium. Deletion of the amino-terminal repeat-containing region had no effect on secretion but completely abolished translocation and necrosis. Neither secretion nor translocation was affected by enzymatically inactivating the cysteine protease domain of the toxin. These data demonstrate that the amino-terminal and carboxyl-terminal repeat-containing regions of the MARTXVv toxin are necessary and sufficient for the delivery of effector domains and epithelial cell lysis in vitro but that effector domains are required for other cytopathic functions. Furthermore, Ca2+-dependent secretion of the modified MARTXVv toxin suggests that nonclassical RTX-like repeats found in the carboxyl-terminal repeat-containing region are functionally similar to classical RTX repeats found in other RTX proteins. PMID:25827415

  6. Horizontal gene transfer of chromosomal Type II toxin-antitoxin systems of Escherichia coli.

    PubMed

    Ramisetty, Bhaskar Chandra Mohan; Santhosh, Ramachandran Sarojini

    2016-02-01

    Type II toxin-antitoxin systems (TAs) are small autoregulated bicistronic operons that encode a toxin protein with the potential to inhibit metabolic processes and an antitoxin protein to neutralize the toxin. Most of the bacterial genomes encode multiple TAs. However, the diversity and accumulation of TAs on bacterial genomes and its physiological implications are highly debated. Here we provide evidence that Escherichia coli chromosomal TAs (encoding RNase toxins) are 'acquired' DNA likely originated from heterologous DNA and are the smallest known autoregulated operons with the potential for horizontal propagation. Sequence analyses revealed that integration of TAs into the bacterial genome is unique and contributes to variations in the coding and/or regulatory regions of flanking host genome sequences. Plasmids and genomes encoding identical TAs of natural isolates are mutually exclusive. Chromosomal TAs might play significant roles in the evolution and ecology of bacteria by contributing to host genome variation and by moderation of plasmid maintenance.

  7. Quantitative Prevalence and Toxin Gene Profile of Bacillus cereus from Ready-to-Eat Vegetables in South Korea.

    PubMed

    Chon, Jung-Whan; Yim, Jin-Hyeok; Kim, Hong-Seok; Kim, Dong-Hyeon; Kim, Hyunsook; Oh, Deog-Hwan; Kim, Soo-Ki; Seo, Kun-Ho

    2015-09-01

    Ready-to-eat (RTE) foods such as prepared vegetables are becoming an increasingly popular food choice. Since RTE vegetables are not commonly sterilized by heat treatment, contamination with foodborne pathogens such as Bacillus cereus (B. cereus) is a major concern. The objective of this study was to assess the quantitative prevalence and toxin gene profiles of B. cereus strains isolated from RTE vegetables. We found that 70 of the 145 (48%) tested retail vegetable salad and sprout samples were positive for B. cereus. The B. cereus isolates harbored at least one enterotoxin gene. The detection rates of nheABC, hblCDA, cytK, and entFM enterotoxin genes among all isolates were 97.1%, 100%, 81.4%, and 98.6%, respectively. No strain carried the emetic toxin genes. Only 4 strains (5.7%) from the 70 isolates were psychrotrophic and were able to grow at 7°C. All of the psychrotrophic isolates possessed at least 1 enterotoxin gene.

  8. Construction of an alpha toxin gene knockout mutant of Clostridium perfringens type A by use of a mobile group II intron.

    PubMed

    Chen, Yue; McClane, Bruce A; Fisher, Derek J; Rood, Julian I; Gupta, Phalguni

    2005-11-01

    In developing Clostridium perfringens as a safe vaccine vector, the alpha toxin gene (plc) in the bacterial chromosome must be permanently inactivated. Disrupting genes in C. perfringens by traditional mutagenesis methods is very difficult. Therefore, we developed a new strategy using group II intron-based Target-Tron technology to inactivate the plc gene in C. perfringens ATCC 3624. Western blot analysis showed no production of alpha toxin protein in the culture supernatant of the plc mutant. Advantages of this technology, such as site specificity, relatively high frequency of insertion, and introduction of no antibiotic resistance genes into the chromosome, could facilitate construction of other C. perfringens mutants.

  9. Novel genes encoding six kinds of three-finger toxins in Ophiophagus hannah (king cobra) and function characterization of two recombinant long-chain neurotoxins.

    PubMed

    Li, Jing; Zhang, Huayuan; Liu, Jing; Xu, Kangsen

    2006-09-01

    Three-finger toxins are a family of low-molecular-mass toxins (<10 kDa) having very similar three-dimensional structures. In the present study, 19 novel cDNAs coding three-finger toxins were cloned from the venom gland of Ophiophagus hannah (king cobra). Alignment analysis showed that the putative peptides could be divided into six kinds of three-finger toxins: LNTXs (long-chain neurotoxins), short-chain neurotoxins, cardiotoxins (CTXs), weak neurotoxins, muscarinic toxins and a toxin with a free SH group. Furthermore, a phylogenetic tree was established on the basis of the toxin cDNAs and the previously reported similar nucleotide sequences from the same source venom. It indicated that three-finger-toxin genes in O. hannah diverged early in the course of evolution by long- and short-type pathways. Two LNTXs, namely rLNTX1 (recombinant LNTX1) and rLNTX3, were expressed and showed cytolytic activity in addition to their neurotoxic function. By comparing the functional residues, we offer some possible explanations for the differences in their neurotoxic function. Moreover, a plausible elucidation of the additonal cytolytic activity was achieved by hydropathy-profile analysis. This, to our knowledge, is the first observation that recombinant long chain alpha-neurotoxins have a CTX-like cytolytic activity.

  10. Two Polyketide Synthase-encoding Genes are Required for Biosynthesis of the Polyketide Virulence Factor, T-toxin, by Cochliobolus heterostrophus

    SciTech Connect

    Baker, Scott E.; Kroken, Scott; Inderbitzin, Patrik; Asvarak, Thipa; Li, Bi-Yu; Shi, Liang; Yoder, Olen C.; Turgeon, Barbara G.

    2006-03-01

    Cochliobolus heterostrophus race T, causal agent of Southern Corn Leaf Blight, requires T-toxin (a family of C35 – C49 polyketides) for high virulence on T-cytoplasm maize. Production of T-toxin is controlled by two unlinked loci, Tox1A and Tox1B, carried on 1.2 Mb of DNA not found in race O, a mildly virulent form of the fungus that does not produce T-toxin, or in any other Cochliobolus spp. or closely related fungus. PKS1, a polyketide synthase (PKS)-encoding gene at Tox1A and DEC1, a decarboxylase-encoding gene at Tox1B, are necessary for T-toxin production. Although there is evidence that additional genes are required for T-toxin production, efforts to clone them have been frustrated because the genes are located in highly repeated, A+T-rich DNA. To overcome this difficulty, Ligation specificity-based Expression Analysis Display (LEAD), a comparative AFLP/gel fractionation/capillary sequencing procedure was applied to cDNAs from a near isogenic pair of race T (Tox1+) and race O (Tox1-) strains. This led to discovery of PKS2, a second PKS-encoding gene that maps at Tox1A and is required for both T-toxin biosynthesis and high virulence to maize. Thus, the carbon chain of each T-toxin family member is likely assembled by action of two PKSs, which produce two polyketides, one of which may act as the starter unit for biosynthesis of the mature T-toxin molecule.

  11. Functional characterization of three genes encoding putative oxidoreductases required for cercosporin toxin biosynthesis in the fungus Cercospora nicotianae.

    PubMed

    Chen, Hui-Qin; Lee, Miin-Huey; Chung, Kuang-Ren

    2007-08-01

    Cercosporin is a non-host-selective, photoactivated polyketide toxin produced by many phytopathogenic Cercospora species, which plays a crucial role during pathogenesis on host plants. Upon illumination, cercosporin converts oxygen molecules to toxic superoxide and singlet oxygen that damage various cellular components and induce lipid peroxidation and electrolyte leakage. Three genes (CTB5, CTB6 and CTB7) encoding putative FAD/FMN- or NADPH-dependent oxidoreductases in the cercosporin toxin biosynthetic pathway of C. nicotianae were functionally analysed. Replacement of each gene via double recombination was utilized to create null mutant strains that were completely impaired in cercosporin production as a consequence of specific interruption at the CTB5, CTB6 or CTB7 locus. Expression of CTB1, CTB5, CTB6, CTB7 and CTB8 was drastically reduced or nearly abolished when CTB5, CTB6 or CTB7 was disrupted. Production of cercosporin was revived when a functional gene cassette was introduced into the respective mutants. All ctb5, ctb6 and ctb7 null mutants retained wild-type levels of resistance against toxicity of cercosporin or singlet-oxygen-generating compounds, indicating that none of the genes plays a role in self-protection.

  12. T-2 toxin inhibits gene expression and activity of key steroidogenesis enzymes in mouse Leydig cells.

    PubMed

    Yang, Jian Ying; Zhang, Yong Fa; Meng, Xiang Ping; Li, Yuan Xiao; Ma, Kai Wang; Bai, Xue Fei

    2015-08-01

    T-2 toxin is one of the mycotoxins, a group of type A trichothecenes produced by several fungal genera including Fusarium species, which may lead to the decrease of the testosterone secretion in the primary Leydig cells derived from the mouse testis. The previous study demonstrated the effects of T-2 toxin through direct decrease of the testosterone biosynthesis in the primary Leydig cells derived from the mouse testis. In this study, we further examined the direct biological effects of T-2 toxin on steroidogenesis production, primarily in Leydig cells of mice. Mature mouse Leydig cells were purified by Percoll gradient centrifugation and the cell purity was determined by 3β-hydroxysteroid dehydrogenase (3β-HSD) staining. To examine T-2 toxin-induced testosterone secretion decrease, we measured the transcription levels of 3 key steroidogenic enzymes and 5 enzyme activities including 3β-HSD-1, P450scc, StAR, CYP17A1, and 17β-HSD in T-2 toxin/human chorionicgonadotropin (hCG) co-treated cells. Our previous study showed that T-2 toxin (10(-7) M, 10(-8) M and 10(-9) M) significantly suppressed hCG (10 ng/ml)-induced testosterone secretion. The studies demonstrated that the suppressive effect is correlated with the decreases in the levels of transcription of 3β-HSD-1, P450scc, and StAR (P<0.05) and also in enzyme activities of 3β-HSD-1, P450scc, StAR, CYP17A1, and 17β-HSD (P<0.05).

  13. Genes essential for the morphogenesis of the Shiga toxin 2-transducing phage from Escherichia coli O157:H7.

    PubMed

    Mondal, Shakhinur Islam; Islam, Md Rakibul; Sawaguchi, Akira; Asadulghani, Md; Ooka, Tadasuke; Gotoh, Yasuhiro; Kasahara, Yasuhiro; Ogura, Yoshitoshi; Hayashi, Tetsuya

    2016-12-14

    Shiga toxin 2 (Stx2), one of the most important virulence factors of enterohaemorrhagic Escherichia coli (EHEC), is encoded by phages. These phages (Stx2 phages) are often called lambda-like. However, most Stx2 phages are short-tailed, thus belonging to the family Podoviridae, and the functions of many genes, especially those in the late region, are unknown. In this study, we performed a systematic genetic and morphological analysis of genes with unknown functions in Sp5, the Stx2 phage from EHEC O157:H7 strain Sakai. We identified nine essential genes, which, together with the terminase genes, determine Sp5 morphogenesis. Four of these genes most likely encoded portal, major capsid, scaffolding and tail fiber proteins. Although exact roles/functions of the other five genes are unknown, one was involved in head formation and four were required for tail formation. One of the four tail genes encoded an unusually large protein of 2,793 amino-acid residues. Two genes that are likely required to maintain the lysogenic state were also identified. Because the late regions of Stx2 phages from various origins are highly conserved, the present study provides an important basis for better understanding the biology of this unique and medically important group of bacteriophages.

  14. Genes essential for the morphogenesis of the Shiga toxin 2-transducing phage from Escherichia coli O157:H7

    PubMed Central

    Mondal, Shakhinur Islam; Islam, Md Rakibul; Sawaguchi, Akira; Asadulghani, Md; Ooka, Tadasuke; Gotoh, Yasuhiro; Kasahara, Yasuhiro; Ogura, Yoshitoshi; Hayashi, Tetsuya

    2016-01-01

    Shiga toxin 2 (Stx2), one of the most important virulence factors of enterohaemorrhagic Escherichia coli (EHEC), is encoded by phages. These phages (Stx2 phages) are often called lambda-like. However, most Stx2 phages are short-tailed, thus belonging to the family Podoviridae, and the functions of many genes, especially those in the late region, are unknown. In this study, we performed a systematic genetic and morphological analysis of genes with unknown functions in Sp5, the Stx2 phage from EHEC O157:H7 strain Sakai. We identified nine essential genes, which, together with the terminase genes, determine Sp5 morphogenesis. Four of these genes most likely encoded portal, major capsid, scaffolding and tail fiber proteins. Although exact roles/functions of the other five genes are unknown, one was involved in head formation and four were required for tail formation. One of the four tail genes encoded an unusually large protein of 2,793 amino-acid residues. Two genes that are likely required to maintain the lysogenic state were also identified. Because the late regions of Stx2 phages from various origins are highly conserved, the present study provides an important basis for better understanding the biology of this unique and medically important group of bacteriophages. PMID:27966628

  15. A novel positive regulatory element for exfoliative toxin A gene expression in Staphylococcus aureus.

    PubMed

    Sakurai, Susumu; Suzuki, Hitoshi; Hata, Toshiaki; Yoshizawa, Yukio; Nakayama, Ritsuko; Machida, Katsuhiko; Masuda, Shogo; Tsukiyama, Takashi

    2004-04-01

    A 1.4 kb positive regulatory element (ETA(exp)) that controls staphylococcal exfoliative toxin A (sETA) transcription was cloned from Staphylococcus aureus. ETA(exp) is located upstream of the cloned 5.8 kb eta gene (etaJ1) obtained from the chomosomal DNA of S. aureus ZM, the standard ETA-producing strain. The cETA prepared from an Escherichia coli transformant into which the recombinant plasmid petaJ1 (5.8 kb eta/pUC9) had been introduced was expressed at high levels in the culture supernatant and the ammonium-sulfate-precipitated culture supernatant fraction as shown by immunoblotting and the single radial immunodiffusion test. However, cETA produced by the recombinant plasmid petaJ3 containing the 1.7 kb eta sequence (etaJ3) with a 1.45 kb ETA(exp)-deficient eta fragment (1.7 kb eta/pUC9) obtained from the 5.8 kb eta sequence by subcloning was not detected in either the culture supernatant or the ammonium-sulfate-precipitated culture supernatant fraction (167-fold concentrate of the culture supernatant) by immunoblotting or the single radial immunodiffusion test. A large amount of cETA was produced by the 1.7 kb eta sequence when it was linked to ETA(exp) amplified by PCR (1.7 kb eta-ETA(exp)/pUC9), regardless of the orientation of ETA(exp) insertion. Northern blot hybridization showed lower levels of the transcripts of the 1.7 kb eta sequence than of the 5.8 kb eta sequence. The rsETA prepared from an S. aureus transformant into which the recombinant plasmid 3.4 kb eta-ETA(exp)/pYT3 (pYT3-etaJ6) had been introduced was expressed at high levels in the culture supernatant fraction as shown by the latex agglutination test. However, the agglutination titre in the culture supernatant fraction of rsETA produced by the recombinant plasmid (1.7 kb eta/pYT3) containing the 1.7 kb eta sequence carrying the 1.4 kb ETA(exp)-deficient eta fragment (pYT3-etaJ3) was 2500-4000 times lower than that of pYT3-etaJ6.

  16. The Genome Sequence of the Cyanobacterium Oscillatoria sp. PCC 6506 Reveals Several Gene Clusters Responsible for the Biosynthesis of Toxins and Secondary Metabolites▿

    PubMed Central

    Méjean, Annick; Mazmouz, Rabia; Mann, Stéphane; Calteau, Alexandra; Médigue, Claudine; Ploux, Olivier

    2010-01-01

    We report a draft sequence of the genome of Oscillatoria sp. PCC 6506, a cyanobacterium that produces anatoxin-a and homoanatoxin-a, two neurotoxins, and cylindrospermopsin, a cytotoxin. Beside the clusters of genes responsible for the biosynthesis of these toxins, we have found other clusters of genes likely involved in the biosynthesis of not-yet-identified secondary metabolites. PMID:20675499

  17. Neutralization of Bacterial YoeBSpn Toxicity and Enhanced Plant Growth in Arabidopsis thaliana via Co-Expression of the Toxin-Antitoxin Genes

    PubMed Central

    Abu Bakar, Fauziah; Yeo, Chew Chieng; Harikrishna, Jennifer Ann

    2016-01-01

    Bacterial toxin-antitoxin (TA) systems have various cellular functions, including as part of the general stress response. The genome of the Gram-positive human pathogen Streptococcus pneumoniae harbors several putative TA systems, including yefM-yoeBSpn, which is one of four systems that had been demonstrated to be biologically functional. Overexpression of the yoeBSpn toxin gene resulted in cell stasis and eventually cell death in its native host, as well as in Escherichia coli. Our previous work showed that induced expression of a yoeBSpn toxin-Green Fluorescent Protein (GFP) fusion gene apparently triggered apoptosis and was lethal in the model plant, Arabidopsis thaliana. In this study, we investigated the effects of co-expression of the yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic A. thaliana. When co-expressed in Arabidopsis, the YefMSpn antitoxin was found to neutralize the toxicity of YoeBSpn-GFP. Interestingly, the inducible expression of both yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic hybrid Arabidopsis resulted in larger rosette leaves and taller plants with a higher number of inflorescence stems and increased silique production. To our knowledge, this is the first demonstration of a prokaryotic antitoxin neutralizing its cognate toxin in plant cells. PMID:27104531

  18. Cloning and expression of Clostridium perfringens type D vaccine strain epsilon toxin gene in E. coli as a recombinant vaccine candidate

    PubMed Central

    Aziminia, Parastoo; Pilehchian-Langroudi, Reza; Esmaeilnia, Kasra

    2016-01-01

    Background and Objectives: Clostridium perfringens, a Gram-positive obligate anaerobic bacterium, is able to form resistant spores which are widely distributed in the environment. C. perfringens is subdivided into five types A to E based on its four major alpha, beta, epsilon and iota toxins. The aim of the present study was cloning and expression of C. perfringens type D vaccine strain epsilon toxin gene. Materials and Methods: Genomic DNA was extracted and the epsilon toxin gene was amplified using Pfu DNA polymerase. The PCR product was cloned into pJET1.2/blunt cloning vector. The recombinant vector (pJETε) was sequenced using universal primers. At the next step epsilon toxin gene was subcloned into pET22b(+) expression vector and transformed into E. coli Rosetta (DE3) host strain. Results: The recombinant protein has been expressed in E. coli Rosetta (DE3) cells after subcloning of C. perfringens etx gene (1008 bp) into the expression vector. Conclusion: We concluded that E. coli Rosetta strain was suitable for the expression of recombinant C. perfringens epsilon toxin protein from pET22ε expression vector. This recombinant cell can be used for further research on recombinant vaccine development. PMID:28210460

  19. Phospholipase C produced by Clostridium botulinum types C and D: comparison of gene, enzymatic, and biological activities with those of Clostridium perfringens alpha-toxin.

    PubMed

    Fatmawati, Ni Nengah Dwi; Sakaguchi, Yoshihiko; Suzuki, Tomonori; Oda, Masataka; Shimizu, Kenta; Yamamoto, Yumiko; Sakurai, Jun; Matsushita, Osamu; Oguma, Keiji

    2013-01-01

    Clostridium botulinum type C and D strains recently have been found to produce PLC on egg yolk agar plates. To characterize the gene, enzymatic and biological activities of C. botulinum PLCs (Cb-PLCs), the cb-plc genes from 8 strains were sequenced, and 1 representative gene was cloned and expressed as a recombinant protein. The enzymatic and hemolytic activities of the recombinant Cb-PLC were measured and compared with those of the Clostridium perfringens alpha-toxin. Each of the eight cb-plc genes encoded a 399 amino acid residue protein preceded by a 27 residue signal peptide. The protein consists of 2 domains, the N- and C-domains, and the overall amino acid sequence identity between Cb-PLC and alpha-toxin was greater than 50%, suggesting that Cb-PLC is homologous to the alpha-toxin. The key residues in the N-domain were conserved, whereas those in the C-domain which are important in membrane interaction were different than in the alpha-toxin. As expected, Cb-PLC could hydrolyze egg yolk phospholipid, p-nitrophenylphosphorylcholine, and sphingomyelin, and also exhibited hemolytic activity;however, its activities were about 4- to over 200-fold lower than those of alpha-toxin. Although Cb-PLC showed weak enzymatic and biological activities, it is speculated that Cb-PLC might play a role in the pathogenicity of botulism or for bacterial survival.

  20. Characterization of Toxin Complex Gene Clusters and Insect Toxicity of Bacteria Representing Four Subgroups of Pseudomonas fluorescens

    PubMed Central

    Rangel, Lorena I.; Henkels, Marcella D.; Shaffer, Brenda T.; Walker, Francesca L.; Davis, Edward W.; Stockwell, Virginia O.; Bruck, Denny; Taylor, Barbara J.; Loper, Joyce E.

    2016-01-01

    Ten strains representing four lineages of the Pseudomonas fluorescens group (P. chlororaphis, P. corrugata, P. koreensis, and P. fluorescens subgroups) were evaluated for toxicity to the tobacco hornworm Manduca sexta and the common fruit fly Drosophila melanogaster. The three strains within the P. chlororaphis subgroup exhibited both oral and injectable toxicity to the lepidopteran M. sexta. All three strains have the gene cluster encoding the FitD insect toxin and a ΔfitD mutant of P. protegens strain Pf-5 exhibited diminished oral toxicity compared to the wildtype strain. Only one of the three strains, P. protegens Pf-5, exhibited substantial levels of oral toxicity against the dipteran D. melanogaster. Three strains in the P. fluorescens subgroup, which lack fitD, consistently showed significant levels of injectable toxicity against M. sexta. In contrast, the oral toxicity of these strains against D. melanogaster was variable between experiments, with only one strain, Pseudomonas sp. BG33R, causing significant levels of mortality in repeated experiments. Toxin complex (Tc) gene clusters, which encode insecticidal properties in Photorhabdus luminescens, were identified in the genomes of seven of the ten strains evaluated in this study. Within those seven genomes, six types of Tc gene clusters were identified, distinguished by gene content, organization and genomic location, but no correlation was observed between the presence of Tc genes and insect toxicity of the evaluated strains. Our results demonstrate that members of the P. fluorescens group have the capacity to kill insects by both FitD-dependent and independent mechanisms. PMID:27580176

  1. A biomarker for the identification of cattle fecal pollution in water using the LTIIa toxin gene from enterotoxigenic Escherichia coli.

    PubMed

    Khatib, L A; Tsai, Y L; Olson, B H

    2002-06-01

    This research describes a method based on PCR to identify cattle fecal pollution in water using a portion of the heat labile toxin IIA (LTIIa) gene from enterotoxigenic Escherichia coli (ETEC). We describe the development of the primers and target. DNA extracts (221) from different animal fecal and human sewage samples were screened and showed no cross-reactivity. Minimum detection limits using centrifugation and filtration methods to concentrate E. coli seeded into stream, ocean, and secondary effluent waters were found to be at femtogram and attogram levels, respectively. Stability of the biomarker in stream, ocean, and secondary effluent waters was 2-4 weeks for all water types. Finally, 33 farm lagoon and waste samples were collected and 31 tested to validate the method; 93% were positive for the LTIIa trait when >1,000 E. coli were screened and 100% positive when >10(5) E. coli were screened. Prevalence of the toxin gene in the E. coli population affected the outcome of the analyses. The cow biomarker can be used in watershed studies to identify cattle waste with great accuracy if the appropriate numbers of E. coli are screened.

  2. Tradeoff between reproduction and resistance evolution to Bt-toxin in Helicoverpa armigera: regulated by vitellogenin gene expression.

    PubMed

    Zhang, W N; Xiao, H J; Liang, G M; Guo, Y Y; Wu, K M

    2014-08-01

    Evolution of resistance to insecticides usually has fitness tradeoffs associated with adaptation to the stress. The basic regulation mechanism of tradeoff between reproduction and resistance evolution to Bacillus thuringiensis (Bt) toxin in the cotton bollworm, Helicoverpa armigera (Ha), based on the vitellogenin (Vg) gene expression was analyzed here. The full-length cDNA of the Vg gene HaVg (JX504706) was cloned and identified. HaVg has 5704 base pairs (bp) with an open reading frame (ORF) of 5265 bp, which encoded 1756 amino acid protein with a predicted molecular mass of 197.28 kDa and a proposed isoelectric point of 8.74. Sequence alignment analysis indicated that the amino acid sequence of HaVg contained all of the conserved domains detected in the Vgs of the other insects and had a high similarity with the Vgs of the Lepidoptera insects, especially Noctuidae. The resistance level to Cry1Ac Bt toxin and relative HaVg mRNA expression levels among the following four groups: Cry1Ac-susceptible strain (96S), Cry1Ac-resistant strain fed on artificial diet with Bt toxin for 135 generations (BtR stands for the Cry1Ac Bt resistance), progeny of the Cry1Ac-resistant strain with a non-Bt-toxin artificial diet for 38 generations (CK1) and the direct descendants of the 135th-generation resistant larvae which were fed on an artificial diet without the Cry1Ac protein (CK2) were analyzed. Compared with the 96S strain, the resistance ratios of the BtR strain, the CK1 strain and the CK2 strain were 2917.15-, 2.15- and 2037.67-fold, respectively. The maximum relative HaVg mRNA expression levels of the BtR strain were approximately 50% less than that of the 96S strain, and the coming of maximum expression was delayed for approximately 4 days. The overall trend of the HaVg mRNA expression levels in the CK1 strain was similar to that in the 96S strain, and the overall trend of the HaVg mRNA expression levels in the CK2 strain was similar to that in the BtR strain. Our results

  3. Population-level variation of the preproricin gene contradicts expectation of neutral equilibrium for generalist plant defense toxins.

    PubMed

    Weeks, Andrea; Leshin, Jonathan A; Dretchen, Kenneth L; Skowronski, Evan W; O'Connell, Kevin P

    2010-07-01

    The preproricin gene encodes ricin, the highly toxic, type II ribosome-inactivating protein of castor bean (Ricinus communis L.). As a generalist plant defense gene, preproricin is expected to exhibit population-level variation consistent with the neutral equilibrium model and to comprise few functionally different alleles. We first test the hypothesis that the preproricin gene family should comprise six to eight members by searching the publicly available draft genome sequence of R. communis and analyzing its ricin-like loci. We then test the neutral equilibrium expectation for the preproricin gene by characterizing its allelic variation among 25 geographically diverse castor bean plants. We confirm the presence of six ricin-like loci that share with the preproricin gene 62.9-96.3% nucleotide identity and intact A-chains. DNA sequence variation among the preproricin haplotypes significantly rejects tests of the neutral equilibrium model. Replacement mutations preserve the 12 amino acids known to affect catalytic and electrostatic interactions of the native protein toxin, which suggests functional divergence among alleles has been minimal. Nucleotide polymorphism is maintained by purifying selection (omega < 0.3) yet includes an excess of rare silent mutations greater than predicted by the neutral equilibrium model. Development of robust detection methods for ricin contamination must account for the presence of these other ricin-like molecules and should leverage the specificity provided by the numerous single nucleotide polymorphisms in the preproricin gene.

  4. Cloning, Characterization, and Expression of Animal Toxin Genes for Vaccine Development

    DTIC Science & Technology

    1990-04-27

    viper, Echis carnatus) (5 3,5 6) VI. MISCELLANEOUS TOXINS A. 1ONOPHORES 1. Alpha-latrotoxin (black widow spider, Latrodectus mactans ) (57) 2. Diamphotoxin...143,144), the cloth-of-gold cone snail (Conus textile) (146), the honey bee (Apis mellifera)(147,148), and the black widow spi- der ( Latrodectus ... mactans (150). With mRNA isolated from the telsons of Androctonus australis Hector a cDNA library was constructed by using the Okayama/Berg cloning

  5. Detection of virulence, antibiotic resistance and toxin (VAT) genes in Campylobacter species using newly developed multiplex PCR assays.

    PubMed

    Laprade, Natacha; Cloutier, Michel; Lapen, David R; Topp, Edward; Wilkes, Graham; Villemur, Richard; Khan, Izhar U H

    2016-05-01

    Campylobacter species are one of the leading causes of bacterial gastroenteritis in humans worldwide. This twofold study was sought to: i) develop and optimize four single-tube multiplex PCR (mPCR) assays for the detection of six virulence (ciaB, dnaJ, flaA, flaB, pldA and racR), three toxin (cdtA, cdtB and cdtC) and one antibiotic resistance tet(O) genes in thermophilic Campylobacter spp. and ii) apply and evaluate the developed mPCR assays by testing 470 previously identified C. jejuni, C. coli and C. lari isolates from agricultural water. In each mPCR assay, a combination of two or three sets of primer pairs for virulence, antibiotic resistance and toxin (VAT) genes was used and optimized. Assay 1 was developed for the detection of dnaJ, racR and cdtC genes with expected amplification sizes of 720, 584 and 182bp. Assay 2 generated PCR amplicons for tet(O) and cdtA genes of 559 and 370bp. Assay 3 amplified cdtB ciaB, and pldA genes with PCR amplicon sizes of 620, 527 and 385bp. Assay 4 was optimized for flaA and flaB genes that generated PCR amplicons of 855 and 260bp. The primer pairs and optimized PCR protocols did not show interference and/or cross-amplification with each other and generated the expected size of amplification products for each target VAT gene for the C. jejuni ATCC 33291 reference strain. Overall, all ten target VAT genes were detected at a variable frequency in tested isolates of thermophilic Campylobacter spp. where cdtC, flaB, ciaB, cdtB, cdtA and pldA were commonly detected compared to the flaA, racR, dnaJ and tet(O) genes which were detected with less frequency. The developed mPCR assays are simple, rapid, reliable and sensitive tools for simultaneously assessing potential pathogenicity and antibiotic resistance profiling in thermophilic Campylobacter spp. The mPCR assays will be useful in diagnostic and analytical settings for routine screening of VAT characteristics of Campylobacter spp. as well as being applicable in epidemiological

  6. Characterization of Alpha-Toxin hla Gene Variants, Alpha-Toxin Expression Levels, and Levels of Antibody to Alpha-Toxin in Hemodialysis and Postsurgical Patients with Staphylococcus aureus Bacteremia

    PubMed Central

    Wu, Yuling; Tabor, David E.; Mok, Hoyin; Sellman, Bret R.; Jenkins, Amy; Yu, Li; Jafri, Hasan S.; Rude, Thomas H.; Ruffin, Felicia; Schell, Wiley A.; Park, Lawrence P.; Yan, Qin; Thaden, Joshua T.; Messina, Julia A.; Esser, Mark T.

    2014-01-01

    Alpha-toxin is a major Staphylococcus aureus virulence factor. This study evaluated potential relationships between in vitro alpha-toxin expression of S. aureus bloodstream isolates, anti-alpha-toxin antibody in serum of patients with S. aureus bacteremia (SAB), and clinical outcomes in 100 hemodialysis and 100 postsurgical SAB patients. Isolates underwent spa typing and hla sequencing. Serum anti-alpha-toxin IgG and neutralizing antibody levels were measured by using an enzyme-linked immunosorbent assay and a red blood cell (RBC)-based hemolysis neutralization assay. Neutralization of alpha-toxin by an anti-alpha-toxin monoclonal antibody (MAb MEDI4893) was tested in an RBC-based lysis assay. Most isolates encoded hla (197/200; 98.5%) and expressed alpha-toxin (173/200; 86.5%). In vitro alpha-toxin levels were inversely associated with survival (cure, 2.19 μg/ml, versus failure, 1.09 μg/ml; P < 0.01). Both neutralizing (hemodialysis, 1.26 IU/ml, versus postsurgical, 0.95; P < 0.05) and IgG (hemodialysis, 1.94 IU/ml, versus postsurgical, 1.27; P < 0.05) antibody levels were higher in the hemodialysis population. Antibody levels were also significantly higher in patients infected with alpha-toxin-expressing S. aureus isolates (P < 0.05). Levels of both neutralizing antibodies and IgG were similar among patients who were cured and those not cured (failures). Sequence analysis of hla revealed 12 distinct hla genotypes, and all genotypic variants were susceptible to a neutralizing monoclonal antibody in clinical development (MEDI4893). These data demonstrate that alpha-toxin is highly conserved in clinical S. aureus isolates. Higher in vitro alpha-toxin levels were associated with a positive clinical outcome. Although patients infected with alpha-toxin-producing S. aureus exhibited higher anti-alpha-toxin antibody levels, these levels were not associated with a better clinical outcome in this study. PMID:25392350

  7. Incidence, diversity and toxin gene characteristics of Bacillus cereus group strains isolated from food products marketed in Belgium.

    PubMed

    Samapundo, S; Heyndrickx, M; Xhaferi, R; Devlieghere, F

    2011-10-17

    The major objectives of this study were to determine the incidence, diversity and characteristics of Bacillus cereus group spp. isolated from food products marketed in Belgium. The food products investigated in this study included cooked pasta, lasagna, béchamel sauce, bolognaise sauce, fresh minced beef, fresh-cut vegetables and raw basmati rice. B. cereus group spp. were detected in 56.3% (324 of 575) of the samples giving rise to 380 strains. The highest incidence (100%) occurred in the raw basmati rice. Although only 10 (2.6%) of the 380 isolates were determined to be psychrotolerant (able to grow at ≤7°C), 25 (6.2%), 189 (49.7%) and 334 (87.9%) isolates were able to grow at mild temperature abuse conditions of 8°C, 9°C and 10°C, respectively. The large diversity of the isolates obtained (overall and between isolates obtained from the same product type) was highlighted by the results of the (GTG)(5) PCR fingerprinting of 80 selected isolates. Sixty-one of these 80 isolates belonged to 15 distinct clusters (≥85% Pearson correlation) whereas the remaining 19 were each clustered separately. Further diversity was also found in the distribution of toxin genes as 16 different profiles were observed in the 80 selected isolates. Whilst none of 80 selected strains harboured the ces gene required for the production of the emetic toxin cereulide, 42 strains (52.5%) carried all seven genes required for the production of the diarrhoeal enterotoxins: haemolytic BL, non-haemolytic enterotoxin and cytotoxin K. The results of this study highlight not only the omnipresence but also the highly diverse ecology of B. cereus spp. within and across several food product types available on the retail market in Belgium. They should also provide the impetus for more studies to enable detailed risk assessment studies to be performed.

  8. Development of a Tunable Wide-Range Gene Induction System Useful for the Study of Streptococcal Toxin-Antitoxin Systems

    PubMed Central

    Xie, Zhoujie; Qi, Fengxia

    2013-01-01

    Despite the plethora of genetic tools that have been developed for use in Streptococcus mutans, the S. mutans genetic system still lacks an effective gene induction system exhibiting low basal expression and strong inducibility. Consequently, we created two hybrid gene induction cassettes referred to as Xyl-S1 and Xyl-S2. Both Xyl-S cassettes are xylose inducible and controlled by the Bacillus megaterium xylose repressor. The Xyl-S cassettes each demonstrated >600-fold-increased reporter activity in the presence of 1.2% (wt/vol) xylose. However, the Xyl-S1 cassette yielded a much higher maximum level of gene expression, whereas the Xyl-S2 cassette exhibited much lower uninduced basal expression. The cassettes also performed similarly in Streptococcus sanguinis and Streptococcus gordonii, which suggests that they are likely to be useful in a variety of streptococci. We demonstrate how both Xyl-S cassettes are particularly useful for the study of toxin-antitoxin (TA) modules using both the previously characterized S. mutans mazEF TA module and a previously uncharacterized HicAB TA module in S. mutans. HicAB TA modules are widely distributed among bacteria and archaea, but little is known about their function. We show that HicA serves as the toxin component of the module, while HicB serves as the antitoxin. Our results suggest that, in contrast to that of typical TA modules, HicA toxicity in S. mutans is modest at best. The implications of these results for HicAB function are discussed. PMID:23934493

  9. A Simple and Rapid Procedure for the Detection of Genes Encoding Shiga Toxins and Other Specific DNA Sequences

    PubMed Central

    Nejman-Faleńczyk, Bożena; Bloch, Sylwia; Januszkiewicz, Aleksandra; Węgrzyn, Alicja; Węgrzyn, Grzegorz

    2015-01-01

    A novel procedure for the detection of specific DNA sequences has been developed. This procedure is based on the already known method employing PCR with appropriate primers and a sequence-specific DNA probe labeled with the fluorescent agent 6-carboxylfluorescein (FAM) at the 5′ end and the fluorescence quencher BHQ-1 (black hole quencher) at the 3′ end. However, instead of the detection of the fluorescence signal with the use of real-time PCR cyclers, fluorescence/luminescence spectrometers or fluorescence polarization readers, as in all previously-reported procedures, we propose visual observation of the fluorescence under UV light directly in the reaction tube. An example for the specific detection of the Shiga toxin-producing Escherichia coli (STEC) strains, by detecting Shiga toxin genes, is demonstrated. This method appears to be specific, simple, rapid and cost effective. It may be suitable for use in research laboratories, as well as in diagnostic units of medical institutions, even those equipped only with a thermocycler and a UV transilluminator, particularly if rapid identification of a pathogen is required. PMID:26580652

  10. A simple and rapid procedure for the detection of genes encoding Shiga toxins and other specific DNA sequences.

    PubMed

    Nejman-Faleńczyk, Bożena; Bloch, Sylwia; Januszkiewicz, Aleksandra; Węgrzyn, Alicja; Węgrzyn, Grzegorz

    2015-11-13

    A novel procedure for the detection of specific DNA sequences has been developed. This procedure is based on the already known method employing PCR with appropriate primers and a sequence-specific DNA probe labeled with the fluorescent agent 6-carboxylfluorescein (FAM) at the 5' end and the fluorescence quencher BHQ-1 (black hole quencher) at the 3' end. However, instead of the detection of the fluorescence signal with the use of real-time PCR cyclers, fluorescence/luminescence spectrometers or fluorescence polarization readers, as in all previously-reported procedures, we propose visual observation of the fluorescence under UV light directly in the reaction tube. An example for the specific detection of the Shiga toxin-producing Escherichia coli (STEC) strains, by detecting Shiga toxin genes, is demonstrated. This method appears to be specific, simple, rapid and cost effective. It may be suitable for use in research laboratories, as well as in diagnostic units of medical institutions, even those equipped only with a thermocycler and a UV transilluminator, particularly if rapid identification of a pathogen is required.

  11. Hemagglutinin gene shuffling among Clostridium botulinum serotypes C and D yields distinct sugar recognition of the botulinum toxin complex.

    PubMed

    Miyata, Keita; Suzuki, Tomonori; Hayashi, Shintaro; Miyashita, Shin-Ichiro; Ohyama, Tohru; Niwa, Koichi; Watanabe, Toshihiro; Sagane, Yoshimasa

    2015-10-01

    Clostridium botulinum strains produce a large-sized toxin complex (TC) that is composed of botulinum neurotoxin (BoNT), non-toxic non-hemagglutinin and three different hemagglutinins (HA-70, HA-33 and HA-17). HA components enhance toxin delivery across the intestinal cell wall in a sugar chain-dependent manner. Here we characterized the sugar recognition of serotype D strain 1873 (D-1873) botulinum L-TC. Most L-TCs produced by serotype C and D strains bind to cells via interactions between HA-33 and cell surface sialo-oligosaccharides. However, like the previously reported L-TC produced by serotype C strain Yoichi (C-Yoichi), D-1873 L-TC binds only to cells that have been treated with neuraminidase, indicating that they recognize asialo-oligosaccharides. The D-1873 HA-33 amino acid sequence is similar to that of C-Yoichi, but had lower similarity to the majority of serotype C and D HA-33s. A comparison of TC component primary structures for 12 serotype C and D strains suggested that at least three types of HA-33 genes exist, and these are shuffled among the serotype C and D strains independently of BoNT serotype. This shuffling produces the distinct sugar recognition of serotype C and D botulinum TCs.

  12. Precision Analysis of Trimble Rtx Surveying Technology with Xfill Function in the Context of Obtained Conversion Observations

    NASA Astrophysics Data System (ADS)

    Krzyżek, Robert

    2015-02-01

    As a result of traditional geodetic surveying we usually achieve observations which are then used for calculating rectangular coordinates onto a plane along with precision evaluation. In this article the surveying methods are presented in which the situation is different. Test measurements were carried out, consisting in the measurement of a fragment of detailed control network in RTK (Real Time Kinematic) and RTX (Real Time Extended) mode with xFill function. First, the rectangular coordinates onto a plane (through the transformation of data ellipsoidal) were obtained, on the basis of which the conversion observations were determined and they were compared with each other, as well as with reference parameters - conversion observations out of detailed control network adjustment with use of the method of least squares. The results of the study allow to verify the precision and application possibilities of conversion observations obtained thanks to Trimble RTX technology with xFill function. Application of this surveying method in typical geodetic tasks is fully justifiable. Nevertheless, it is recommendable to be aware of the correlations of absolute or relative values obtained in RTX procedure to reference parameters, which in turn will enable conclusive verification of the possibilities of Trimble RTX technology application in certain geodetic surveys.

  13. Staphylococcus aureus of phage type 187 isolated from people occurred to be a genes carrier of eneterotoxin C and toxic shock syndrome toxin-1 (TSST-1).

    PubMed

    Piechowicz, Lidia; Garbacz, Katarzyna; Galiński, Janusz

    2008-07-01

    The aim of this study was to examine the genotype properties of Staphylococcus aureus of phage type 187 strains that constitute a separate group among the strains of S. aureus. Sixteen strains were collected from the hospital patients (n=12) and the healthy carriers (n=4) in 13 medical centres in Poland during 1991 and 2005. Biotyping, antibiotic susceptibility, phage typing, detection the genes of enterotoxins and toxic shock syndrome toxin, genotyping of chromosomal DNA by pulsed-field gel electrophoresis (PFGE), also amplification and restriction analysis of the coagulase (coa) and the protein A genes (spa) (PCR/restriction fragment length polymorphism (RFLP)) was tested. The results of this study showed that all staphylococcus of phage type 187 belonged to the human biotype (A) and appeared to be sensitive to all of the tested antibiotics, including methicillin (MSSA). Finding out the toxin genes showed that almost all of them (93.8%) had the enterotoxin C gene (sec) and TSST-1 gene (tst). The PFGE typing proved that the phage type 187 strains (except for one) constitute one PFGE type. These results and the identical restriction patterns in the PCR/RFLP method, also the same biotype, sensitivity to antibiotics and the presence genes of the same type of toxins confirmed that the phage type 187 strains constitute one clone within our country. Additionally, the fact that almost all of them have the enterotoxin genes and tst gene allows to consider them the strains of potentially high virulence.

  14. Antigenic secreted proteins from Haemophilus paragallinarum. A 110-kDa putative RTX protein.

    PubMed

    Mena-Rojas, Erika; Vázquez Cruz, Candelario; Vaca Pacheco, Sergio; García González, Octavio; Pérez-Márquez, Víctor M; Pérez-Méndez, Alma; Ibarra-Caballero, Jorge; de la Garza, Mireya; Zenteno, Edgar; Negrete-Abascal, Erasmo

    2004-03-12

    Haemophilus paragallinarum is the causal agent of infectious coryza, an economically important disease for the poultry industry. This bacterium secreted proteins of 25-110 kDa during its growth in brain heart infusion, tryptic soy broth, or Luria-Bertani glucose phosphate media, all lacking serum. Some of these proteins were recognized by sera from chickens experimentally infected with H. paragallinarum. A 110-kDa protein was recognized by a serum pool from convalescent-phase pigs naturally infected with Actinobacillus pleuropneumoniae, and also by a rabbit polyclonal serum against Apx I as well as a rabbit serum against Mannheimia haemolytica leukotoxin, suggesting the presence of an RTX-like protein in H. paragallinarum. H. paragallinarum secreted proteins could be important immunogens in the control of infectious coryza.

  15. Identification of a novel Staphylococcus pseudintermedius exfoliative toxin gene and its prevalence in isolates from canines with pyoderma and healthy dogs.

    PubMed

    Iyori, Keita; Hisatsune, Junzo; Kawakami, Tetsuji; Shibata, Sanae; Murayama, Nobuo; Ide, Kaori; Nagata, Masahiko; Fukata, Tsuneo; Iwasaki, Toshiroh; Oshima, Kenshiro; Hattori, Masahira; Sugai, Motoyuki; Nishifuji, Koji

    2010-11-01

    Staphylococcal exfoliative toxins are involved in some cutaneous infections in mammals by targeting desmoglein 1 (Dsg1), a desmosomal cell-cell adhesion molecule. Recently, an exfoliative toxin gene (exi) was identified in Staphylococcus pseudintermedius isolated from canine pyoderma. The aim of this study was to identify novel exfoliative toxin genes in S. pseudintermedius. Here, we describe a novel orf in the genome of S. pseudintermedius isolated from canine impetigo, whose deduced amino acid sequence was homologous to that of the SHETB exfoliative toxin from Staphylococcus hyicus (70.4%). The ORF recombinant protein caused skin exfoliation and abolished cell surface staining of Dsg1 in canine skin. Moreover, the ORF protein degraded the recombinant extracellular domains of canine Dsg1, but not Dsg3, in vitro. PCR analysis revealed that the orf was present in 23.2% (23/99) of S. pseudintermedius isolates from dogs with superficial pyoderma exhibiting various clinical phenotypes, while the occurrence in S. pseudintermedius isolates from healthy dogs was 6.1% (3/49). In summary, this newly found orf in S. pseudintermedius encodes a novel exfoliative toxin, which targets a cell-cell adhesion molecule in canine epidermis and might be involved in a broad spectrum of canine pyoderma.

  16. Analysis of transcriptomes of three orb-web spider species reveals gene profiles involved in silk and toxin.

    PubMed

    Zhao, Ying-Jun; Zeng, Yan; Chen, Lei; Dong, Yang; Wang, Wen

    2014-12-01

    As an ancient arthropod with a history of 390 million years, spiders evolved numerous morphological forms resulting from adaptation to different environments. The venom and silk of spiders, which have promising commercial applications in agriculture, medicine and engineering fields, are of special interests to researchers. However, little is known about their genomic components, which hinders not only understanding spider biology but also utilizing their valuable genes. Here we report on deep sequenced and de novo assembled transcriptomes of three orb-web spider species, Gasteracantha arcuata, Nasoonaria sinensis and Gasteracantha hasselti which are distributed in tropical forests of south China. With Illumina paired-end RNA-seq technology, 54 871, 101 855 and 75 455 unigenes for the three spider species were obtained, respectively, among which 9 300, 10 001 and 10 494 unique genes are annotated, respectively. From these annotated unigenes, we comprehensively analyzed silk and toxin gene components and structures for the three spider species. Our study provides valuable transcriptome data for three spider species which previously lacked any genetic/genomic data. The results have laid the first fundamental genomic basis for exploiting gene resources from these spiders.

  17. Characterization of shiga toxin-producing Escherichia coli recovered from domestic animals to determine stx variants, virulence genes, and cytotoxicity in mammalian cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing Escherichia coli (STEC) can cause foodborne illnesses ranging from diarrhea to severe diseases such as hemorrhagic colitis (HC), and hemolytic uremic syndrome (HUS) in humans. In this study, we determined virulence genes, stx subtypes and we evaluated the cytotoxicity in mammal...

  18. Survival and expression of acid resistance genes in Shiga toxin-producing Escherichia coli acid adapted in pineapple juice and exposed to synthetic gastric fluid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aims: The aim of this research was to examine relative transcriptional expression of acid resistance (AR) genes, rpoS, gadA and adiA, in O157:H7 and non-O157 Shiga toxin-producing Escherichia coli (STEC) serotypes after adaptation to pineapple juice (PJ) and subsequently to determine survival with e...

  19. MvirDB: Microbial Database of Protein Toxins, Virulence Factors and Antibiotic Resistance Genes for Bio-Defense Applications

    DOE Data Explorer

    Zhou, C. E.; Smith, J.; Lam, M.; Zemla, M. D.; Slezak, T.

    MvirDB is a cenntralized resource (data warehouse) comprising all publicly accessible, organized sequence data for protein toxins, virulence factors, and antibiotic resistance genes. Protein entries in MvirDB are annotated using a high-throughput, fully automated computational annotation system; annotations are updated periodically to ensure that results are derived using current public database and open-source tool releases. Tools provided for using MvirDB include a web-based browser tool and BLAST interfaces. MvirDB serves researchers in the bio-defense and medical fields. (taken from page 3 of PI's paper of same title published in Nucleic Acids Research, 2007, Vol.35, Database Issue (Open Source)

  20. The Effector Domain Region of the Vibrio vulnificus MARTX Toxin Confers Biphasic Epithelial Barrier Disruption and Is Essential for Systemic Spread from the Intestine

    PubMed Central

    Gavin, Hannah E.; Beubier, Nike T.

    2017-01-01

    Vibrio vulnificus causes highly lethal bacterial infections in which the Multifunctional Autoprocessing Repeats-in-Toxins (MARTX) toxin product of the rtxA1 gene is a key virulence factor. MARTX toxins are secreted proteins up to 5208 amino acids in size. Conserved MARTX N- and C-terminal repeat regions work in concert to form pores in eukaryotic cell membranes, through which the toxin’s central region of modular effector domains is translocated. Upon inositol hexakisphosphate-induced activation of the of the MARTX cysteine protease domain (CPD) in the eukaryotic cytosol, effector domains are released from the holotoxin by autoproteolytic activity. We previously reported that the native MARTX toxin effector domain repertoire is dispensable for epithelial cellular necrosis in vitro, but essential for cell rounding and apoptosis prior to necrotic cell death. Here we use an intragastric mouse model to demonstrate that the effector domain region is required for bacterial virulence during intragastric infection. The MARTX effector domain region is essential for bacterial dissemination from the intestine, but dissemination occurs in the absence of overt intestinal tissue pathology. We employ an in vitro model of V. vulnificus interaction with polarized colonic epithelial cells to show that the MARTX effector domain region induces rapid intestinal barrier dysfunction and increased paracellular permeability prior to onset of cell lysis. Together, these results negate the inherent assumption that observations of necrosis in vitro directly predict bacterial virulence, and indicate a paradigm shift in our conceptual understanding of MARTX toxin function during intestinal infection. Results implicate the MARTX effector domain region in mediating early bacterial dissemination from the intestine to distal organs–a key step in V. vulnificus foodborne pathogenesis–even before onset of overt intestinal pathology. PMID:28060924

  1. [Protein toxins of Staphylococcus aureus].

    PubMed

    Shamsutdinov, A F; Tiurin, Iu A

    2014-01-01

    Main scientific-research studies regarding protein bacterial toxins of the most widespread bacteria that belong to Staphylococcus spp. genus and in particular the most pathogenic species for humans--Staphylococcus aureus, are analyzed. Structural and biological properties of protein toxins that have received the name of staphylococcus pyrogenic toxins (PTSAg) are presented. Data regarding genetic regulation of secretion and synthesis of these toxins and 3 main regulatory genetic systems (agr--accessory gene regulator, xpr--extracellular protein regulator, sar--staphylococcal accessory regulator) that coordinate synthesis of the most important protein toxins and enzymes for virulence of S. aureus, are presented.

  2. New secreted toxins and immunity proteins encoded within the Type VI secretion system gene cluster of Serratia marcescens

    PubMed Central

    English, Grant; Trunk, Katharina; Rao, Vincenzo A; Srikannathasan, Velupillai; Hunter, William N; Coulthurst, Sarah J

    2012-01-01

    Protein secretion systems are critical to bacterial virulence and interactions with other organisms. The Type VI secretion system (T6SS) is found in many bacterial species and is used to target either eukaryotic cells or competitor bacteria. However, T6SS-secreted proteins have proven surprisingly elusive. Here, we identified two secreted substrates of the antibacterial T6SS from the opportunistic human pathogen, Serratia marcescens. Ssp1 and Ssp2, both encoded within the T6SS gene cluster, were confirmed as antibacterial toxins delivered by the T6SS. Four related proteins encoded around the Ssp proteins (‘Rap’ proteins) included two specifically conferring self-resistance (‘immunity’) against T6SS-dependent Ssp1 or Ssp2 toxicity. Biochemical characterization revealed specific, tight binding between cognate Ssp–Rap pairs, forming complexes of 2:2 stoichiometry. The atomic structures of two Rap proteins were solved, revealing a novel helical fold, dependent on a structural disulphide bond, a structural feature consistent with their functional localization. Homologues of the Serratia Ssp and Rap proteins are found encoded together within other T6SS gene clusters, thus they represent founder members of new families of T6SS-secreted and cognate immunity proteins. We suggest that Ssp proteins are the original substrates of the S. marcescens T6SS, before horizontal acquisition of other T6SS-secreted toxins. Molecular insight has been provided into how pathogens utilize antibacterial T6SSs to overcome competitors and succeed in polymicrobial niches. PMID:22957938

  3. [Presence of enterotoxin C and toxic shock syndrome toxin--1 (TSST-1) genes in population of Staphylococcus aureus phage type 187].

    PubMed

    Garbacz, Katarzyna; Piechowicz, Lidia; Galiński, Janusz

    2006-01-01

    The aim of this study was to examine whether Staphylococcus aureus of phage type 187 possess the genes of enterotoxins and toxic shock syndrom toxin. Sixteen phage type 187 strains were isolated from the hospital patients (12) and the carriers (4) in twelve medical centres in Poland during 1991 and 2005. Biotyping, phage typing, antibiotic susceptibility, detection of the genes of enterotoxins (sea--sed) and toxic shock syndrome toxin (tst) was tested. The results of this study showed that all staphylococci of phage type 187 belonged to the human biotype (A) and appeared to be sensitive to all of the tested antibiotics, including methicillin (MSSA). Almost all of them (93.8%) had the enterotoxin C gene and TSST-1 gene. This fact allows to consider them the strains of potentially high virulence.

  4. Altered gene expression in Choristoneura fumiferana and Manduca sexta in response to sublethal intoxication by Bacillus thuringiensis Cry1Ab toxin.

    PubMed

    van Munster, M; Préfontaine, G; Meunier, L; Elias, M; Mazza, A; Brousseau, R; Masson, L

    2007-02-01

    In order to understand how lepidopteran insects react physiologically to Bacillus thuringiensis crystal toxin ingestion, transcriptional profiling of Choristoneura fumiferana larvae exposed to sublethal doses of Cry1Ab protoxin were monitored using a C. fumiferana-specific cDNA microarray derived from a protoxin-specific subtractive library. Differential gene expression occurred primarily between 2 and 5 h postingestion. Metabolic enzymes such as lipases and proteases were generally repressed, whereas genes involved in detoxification, immune system regulation or general stress response were upregulated. A similar protoxin-specific transcriptional pattern was also observed with Manduca sexta larvae, using three upregulated genes (serpin, cytochrome P450 and carboxyl/cholinesterase) and one downregulated gene (beta-glucosidase), suggesting that a susceptible larval response to Cry toxin exposure might be universal among lepidopterous insects.

  5. A PCR-RFLP assay to detect and type cytolethal distending toxin (cdt) genes in Campylobacter hyointestinalis

    PubMed Central

    HATANAKA, Noritoshi; KAMEI, Kazumasa; SOMROOP, Srinuan; AWASTHI, Sharda Prasad; ASAKURA, Masahiro; MISAWA, Naoaki; HINENOYA, Atsushi; YAMASAKI, Shinji

    2016-01-01

    Campylobacter hyointestinalis is considered as an emerging zoonotic pathogen. We have recently identified two types of cytolethal distending toxin (cdt) gene in C. hyointestinalis and designated them as Chcdt-I and Chcdt-II. In this study, we developed a PCR-restriction fragment length polymorphism (RFLP) assay that can differentiate Chcdt-I from Chcdt-II. When the PCR-RFLP assay was applied to 17 other Campylobacter strains and 25 non-Campylobacter strains, PCR products were not obtained irrespective of their cdt gene-possession, indicating that the specificity of the PCR-RFLP assay was 100%. In contrast, when the PCR-RFLP assay was applied to 35 C. hyointestinalis strains including 23 analyzed in the previous study and 12 newly isolated from pigs and bovines, all of them showed the presence of cdt genes. Furthermore, a restriction digest by EcoT14-I revealed that 29 strains contained both Chcdt-I and Chcdt-II and 6 strains contained only Chcdt-II, showing 100% sensitivity. Unexpectedly, however, PCR products obtained from 7 C. hyointestinalis strains were not completely digested by EcoT14-I. Nucleotide sequence analysis revealed that the undigested PCR product was homologous to cdtB but not to Chcdt-IB or Chcdt-IIB, indicating the presence of another cdt gene-variant. Then, we further digested the PCR products with DdeI in addition to EcoT14-I, showing that all three cdt genes, including a possible new Chcdt variant, could be clearly differentiated. Thus, the PCR-RFLP assay developed in this study is a valuable tool for evaluating the Chcdt gene-profile of bacteria. PMID:27916784

  6. Deletion of a MFS transporter-like gene in Cercospora nicotianae reduces cercosporin toxin accumulation and fungal virulence.

    PubMed

    Choquer, Mathias; Lee, Miin-Huey; Bau, Huey-Jiunn; Chung, Kuang-Ren

    2007-02-06

    Many phytopathogenic Cercospora species produce a host-nonselective polyketide toxin, called cercosporin, whose toxicity exclusively relies on the generation of reactive oxygen species. Here, we describe a Cercospora nicotianae CTB4 gene that encodes a putative membrane transporter and provide genetic evidence to support its role in cercosporin accumulation. The predicted CTB4 polypeptide has 12 transmembrane segments with four conserved motifs and has considerable similarity to a wide range of transporters belonging to the major facilitator superfamily (MFS). Disruption of the CTB4 gene resulted in a mutant that displayed a drastic reduction of cercosporin production and accumulation of an unknown brown pigment. Cercosporin was detected largely from fungal hyphae of ctb4 disruptants, but not from the surrounding medium, suggesting that the mutants were defective in both cercosporin biosynthesis and secretion. Cercosporin purified from the ctb4 disruptants exhibited toxicity to tobacco suspension cells, insignificantly different from wild-type, whereas the disruptants formed fewer lesions on tobacco leaves. The ctb4 null mutants retained normal resistance to cercosporin and other singlet oxygen-generating photosensitizers, indistinguishable from the parental strain. Transformation of a functional CTB4 clone into a ctb4 null mutant fully revived cercosporin production. Thus, we propose that the CTB4 gene encodes a putative MFS transporter responsible for secretion and accumulation of cercosporin.

  7. Expression pattern of three-finger toxin and phospholipase A2 genes in the venom glands of two sea snakes, Lapemis curtus and Acalyptophis peronii: comparison of evolution of these toxins in land snakes, sea kraits and sea snakes

    PubMed Central

    Pahari, Susanta; Bickford, David; Fry, Bryan G; Kini, R Manjunatha

    2007-01-01

    Background Snake venom composition varies widely both among closely related species and within the same species, based on ecological variables. In terrestrial snakes, such variation has been proposed to be due to snakes' diet. Land snakes target various prey species including insects (arthropods), lizards (reptiles), frogs and toads (amphibians), birds (aves), and rodents (mammals), whereas sea snakes target a single vertebrate class (fishes) and often specialize on specific types of fish. It is therefore interesting to examine the evolution of toxins in sea snake venoms compared to that of land snakes. Results Here we describe the expression of toxin genes in the venom glands of two sea snakes, Lapemis curtus (Spine-bellied Sea Snake) and Acalyptophis peronii (Horned Sea Snake), two members of a large adaptive radiation which occupy very different ecological niches. We constructed cDNA libraries from their venom glands and sequenced 214 and 192 clones, respectively. Our data show that despite their explosive evolutionary radiation, there is very little variability in the three-finger toxin (3FTx) as well as the phospholipase A2 (PLA2) enzymes, the two main constituents of Lapemis curtus and Acalyptophis peronii venom. To understand the evolutionary trends among land snakes, sea snakes and sea kraits, pairwise genetic distances (intraspecific and interspecific) of 3FTx and PLA2 sequences were calculated. Results show that these proteins appear to be highly conserved in sea snakes in contrast to land snakes or sea kraits, despite their extremely divergent and adaptive ecological radiation. Conclusion Based on these results, we suggest that streamlining in habitat and diet in sea snakes has possibly kept their toxin genes conserved, suggesting the idea that prey composition and diet breadth may contribute to the diversity and evolution of venom components. PMID:17900344

  8. Multiplex PCR for detection of superantigenic toxin genes in methicillin-sensitive and methicillin-resistant Staphylococcus aureus isolated from patients and carriers of a hospital in northeast Thailand.

    PubMed

    Wongboot, Warawan; Chomvarin, Chariya; Engchanil, Chulapan; Chaimanee, Prajuab

    2013-07-04

    The aims of this study were to develop multiplex PCR for simultaneous detection of five superantigenic toxin genes (sea, seb, sec, sed and tst-1) in Staphylococcus aureus isolated from 149 clinical samples and nasal swabs from 201 healthy subjects in Thailand, and to compare prevalence and expression of those genes between methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA). The sensitivity of multiplex PCR was 10(3) CFU/ml (60 CFU/PCR reaction) for DNA templates extracted by both boiling and extraction methods. S. aureus strains from patients (65%) harbored more superantigenic toxin genes than healthy subjects (54%). MRSA (80%) isolated from patients harbored more superantigenic toxin genes than MSSA (52%). Sea was the most frequently found gene in S. aureus strains from patients and carriers. MRSAisolates harbored sea and produced SEA more frequently than MSSA isolates (p <0.05) and MRSA isolates (59%) from blood samples consisted of a higher number of superantigenic toxin producers than MSSA (9%) (p < 0.05). More S. aureus strains isolated from patients with severe septicemia contained superantigenic toxin genes (94%) and produced toxins (82%) than those from non-severe patients (64% and 57%, respectively). The multiplex PCR method described here offers a reliable tool for simultaneous detection of various staphylococcal toxin genes.

  9. Oligonucleotide probes for detection and differentiation of Staphylococcus aureus strains containing genes for enterotoxins A, B, and C and toxic shock syndrome toxin 1.

    PubMed Central

    Neill, R J; Fanning, G R; Delahoz, F; Wolff, R; Gemski, P

    1990-01-01

    Different synthetic DNA nucleotide sequences were evaluated as gene probes for the specific detection and differentiation of Staphylococcus aureus strains encoding enterotoxins A (SEA), B (SEB), and C (SEC) and toxic shock syndrome toxin 1 (TSST-1). Identification of sequences unique to each toxin, based on knowledge of their nucleotide sequences, led to preparation of the specific 18-base oligonucleotide probes EA1 (encoding amino acids 177 to 182 of SEA), EB2 (encoding amino acids 105 to 110 of SEB), EC5 (encoding amino acids 125 to 131 of SEC1), and TS1 (encoding amino acids 160 to 166 of TSST-1). In colony blot hybridization analyses, these probes hybridized specifically with DNA from strains that produced the respective toxin serotypes. An excellent (greater than or equal to 93%) correlation between hybridization results (genotype) and toxin protein detection by an enzyme-linked immunosorbent assay (phenotype) was observed in the characterization of both reference and clinical strains of S. aureus for SEA, SEB, and TSST-1. A lower correlation (64%) for SEC reflected a lack of sensitivity in detecting toxin production. Our findings demonstrate that molecular DNA hybridization with synthetic oligonucleotide probes provides another approach for establishing the toxigenicity of S. aureus. Images PMID:2380378

  10. Molecular cloning, nucleotide sequence, and expression in Escherichia coli of a hemolytic toxin (aerolysin) gene from Aeromonas trota

    SciTech Connect

    Khan, A.A.; Kim, E.; Cerniglia, C.E.

    1998-07-01

    Aeromonas trota AK2, which was derived from ATCC 49659 and produces the extracellular pore-forming hemolytic toxin aerolysin, was mutagenized with the transposon mini-Tn5Km1 to generate a hemolysin-deficient mutant, designated strain AK253. Southern blotting data indicated that an 8.7-kb NotI fragment of the genomic DNA of strain AK253 contained the kanamycin resistance gene of mini-Tn5Km1. The 8.7-kb NotI DNA fragment was cloned into the vector pGEM5Zf({minus}) by selecting for kanamycin resistance, and the resultant clone, pAK71, showed aerolysin activity in Escherichia coli JM109. The nucleotide sequence of the aerA gene, located on the 1.8-kb ApaI-EcoRI fragment, was determined to consist of 1,479 bp and to have an ATG initiation codon and a TAA termination codon. An in vitro coupled transcription-translation analysis of the 1.8-kb region suggested that the aerA gene codes for a 54-kDa protein, in agreement with nucleotide sequence data. The deduced amino acid sequence of the aerA gene product of A. trota exhibited 99% homology with the amino acid sequence of the aerA product of Aeromonas sobria AB3 and 57% homology with the amino acid sequences of the products of the aerA genes of Aeromonas salmonicida 17-2 and A. sobria 33.

  11. Shiga toxin-producing Escherichia coli strains isolated from dairy products - Genetic diversity and virulence gene profiles.

    PubMed

    Douëllou, T; Delannoy, S; Ganet, S; Mariani-Kurkdjian, P; Fach, P; Loukiadis, E; Montel, Mc; Thevenot-Sergentet, D

    2016-09-02

    Shiga toxin-producing Escherichia coli (STEC) are widely recognized as pathogens causing food borne disease. Here we evaluate the genetic diversity of 197 strains, mainly STEC, from serotypes O157:H7, O26:H11, O103:H2, O111:H8 and O145:28 and compared strains recovered in dairy products against strains from human, meat and environment cases. For this purpose, we characterized a set of reference-collection STEC isolates from dairy products by PFGE DNA fingerprinting and a subset of these by virulence-gene profiling. PFGE profiles of restricted STEC total DNA showed high genomic variability (0.9976 on Simpson's discriminatory index), enabling all dairy isolates to be differentiated. High-throughput real-time PCR screening of STEC virulence genes were applied on the O157:H7 and O26:H11 STEC isolates from dairy products and human cases. The virulence gene profiles of dairy and human STEC strains were similar. Nevertheless, frequency-wise, stx1 was more prevalent among dairy O26:H11 isolates than in human cases ones (87% vs. 44%) while stx2 was more prevalent among O26:H11 human isolates (23% vs. 81%). For O157:H7 isolates, stx1 (0% vs. 39%), nleF (40% vs 94%) and Z6065 (40% vs 100%) were more prevalent among human than dairy strains. Our data point to differences between human and dairy strains but these differences were not sufficient to associate PFGE and virulence gene profiles to a putative lower pathogenicity of dairy strains based on their lower incidence in disease. Further comparison of whole-genome expression and virulence gene profiles should be investigated in cheese and intestinal tract samples.

  12. Incidence, Antimicrobial Susceptibility, and Toxin Genes Possession Screening of Staphylococcus aureus in Retail Chicken Livers and Gizzards

    PubMed Central

    Abdalrahman, Lubna S.; Fakhr, Mohamed K.

    2015-01-01

    Few recent outbreaks in Europe and the US involving Campylobacter and Salmonella were linked to the consumption of chicken livers. Studies investigating Staphylococcus aureus in chicken livers and gizzards are very limited. The objectives of this study were to determine the prevalence, antimicrobial resistance, and virulence of S. aureus and MRSA (Methicillin-Resistant Staphylococcus aureus) in retail chicken livers and gizzards in Tulsa, Oklahoma. In this study, 156 chicken livers and 39 chicken gizzards samples of two brands were collected. While one of the brands showed very low prevalence of 1% (1/100) for S. aureus in chicken livers and gizzards, the second brand showed prevalence of 37% (31/95). No MRSA was detected since none harbored the mecA or mecC gene. Eighty seven S. aureus isolates from livers and 28 from gizzards were screened for antimicrobial resistance to 16 antimicrobials and the possession of 18 toxin genes. Resistance to most of the antimicrobials screened including cefoxitin and oxacillin was higher in the chicken gizzards isolates. While the prevalence of enterotoxin genes seg and sei was higher in the gizzards isolates, the prevalence of hemolysin genes hla, hlb, and hld was higher in the livers ones. The lucocidin genes lukE-lukD was equally prevalent in chicken livers and gizzards isolates. Using spa typing, a subset of the recovered isolates showed that they are not known to be livestock associated and, hence, may be of a human origin. In conclusion, this study stresses the importance of thorough cooking of chicken livers and gizzards since it might contain multidrug resistant enterotoxigenic S. aureus. To our knowledge this is the first study to specifically investigate the prevalence of S. aureus in chicken livers and gizzards in the US. PMID:28231192

  13. Human tumor necrosis factor-alpha gene 3' untranslated region confers inducible toxin responsiveness to homologous promoter in monocytic THP-1 cells.

    PubMed

    Seiler-Tuyns, A; Dufour, N; Spertini, F

    1999-07-30

    To better define the role of 3' untranslated region (3'UTR) on transcriptional regulation of the human tumor necrosis factor (TNF)-alpha gene, monocytic human THP-1 cells were transfected with two TNF-alpha promoter constructs spanning base pairs -1897/-1 and -1214/-1, respectively, and linked to the rabbit beta-globin gene. Quantitative globin gene expression of chimerae was measured by reverse transcription-polymerase chain reaction. A construct linking the chicken beta-actin promoter and a deleted portion of the beta-globin gene was cotransfected and used as internal standard. Unexpectedly, when THP-1 cells were stimulated with lipopolysaccharide or toxic shock syndrome toxin-1, gene regulation was hardly detected. In contrast, endogenous TNF-alpha gene regulation measured by the same reverse transcription-polymerase chain reaction procedure was vigorous. Remarkably, ligation of 3'UTR to chimeric constructs led to a drastic drop in the basal level of chimeric gene expression, resulting in a 15- to 40-fold induction of the reporter gene. Consistently, when the TNF-alpha promoter was replaced by the cytomegalovirus early immediate promoter, gene expression was also uniformly reduced but was no longer up-regulated upon stimulation with lipopolysaccharide and toxic shock syndrome toxin-1. These data provide the first line of evidence that, in addition to its role in TNF-alpha transcript stability and translation, human TNF-alpha 3'UTR also participates in modulating gene expression at the transcriptional level.

  14. Molecular epidemiology of Escherichia coli O157:H7 by restriction fragment length polymorphism using Shiga-like toxin genes.

    PubMed Central

    Samadpour, M

    1995-01-01

    The purpose of this study was to assess a simplified method for interstrain differentiation of Escherichia coli O157:H7 and other Shiga-like toxin-producing E. coli (SLTEC) strains. A method based on the use of nucleic acid probes from Shiga-like toxin (SLT) I and II structural genes was used to generate restriction fragment length polymorphism (RFLP) patterns of SLTEC strains, (SLT-RFLP patterns) resulting from digestion of isolated genomic DNA with four different restriction enzymes (BamHI, EcoRI, HindIII, and PvuII) used separately. A total of 165 SLTEC strains from clinical, food, and environmental sources, including O157:H7 isolates from four food-borne outbreaks in Canada and the United States, were analyzed in the study. SLT-RFLP demonstrated that E. coli O157:H7 strains from each food-borne outbreak had the same unique SLT-RFLP pattern. Fifty-two SLT-RFLP types were found among 96 E. coli O157:H7 isolates from sporadic cases of hemorrhagic colitis and hemolytic uremic syndrome in Washington state. The use of the SLT probes proved to be a very powerful method for interstrain differentiation of SLTEC strains. Although the use of each of the enzymes alone did not give enough differentiative power to be used in epidemiological studies, the combination of patterns generated by two restriction enzymes (EcoRI and PvuII, used separately) provided the desired sensitivity for such studies. The results clearly demonstrate the usefulness of the method for studying the molecular epidemiology of E. coli O157:H7. The method is also suitable for establishing an epidemiological database, in terms of both sensitivity and ease of compilation and interpretation of results. PMID:7559966

  15. Quorum-sensing gene luxS regulates flagella expression and Shiga-like toxin production in F18ab Escherichia coli.

    PubMed

    Yang, Yang; Zhou, Mingxu; Hou, Huayan; Zhu, Jun; Yao, Fenghua; Zhang, Xinjun; Zhu, Xiaofang; Hardwidge, Philip R; Zhu, Guoqiang

    2014-06-01

    To investigate the effect of the luxS gene on the expression of virulence factors in Shiga-like toxin producing and verotoxin-producing Escherichia coli, the luxS gene from E. coli 107/86 (wild type, O139:H1:F18ab, Stx2e) was deleted. The successful deletion of luxS was confirmed by bioluminescence assays. The luxS deletion mutant exhibited changed flagella-related phenotypes, like impaired expression of flagella, decreased flagella motility, reduced biofilm formation, and reduced ability to induce pro-immunity response in host cells, which were restored after complementation with the intact luxS gene. The mutant strain also displayed attenuated production of Stx2e. This study provides new information to the crucial function of luxS in regulating Shiga-like toxin producing E. coli virulence.

  16. Identification of Functional Toxin/Immunity Genes Linked to Contact-Dependent Growth Inhibition (CDI) and Rearrangement Hotspot (Rhs) Systems

    PubMed Central

    Poole, Stephen J.; Diner, Elie J.; Aoki, Stephanie K.; Braaten, Bruce A.; t'Kint de Roodenbeke, Claire; Low, David A.; Hayes, Christopher S.

    2011-01-01

    Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiA/CdiB family of two-partner secretion proteins. Each CdiA protein exhibits a distinct growth inhibition activity, which resides in the polymorphic C-terminal region (CdiA-CT). CDI+ cells also express unique CdiI immunity proteins that specifically block the activity of cognate CdiA-CT, thereby protecting the cell from autoinhibition. Here we show that many CDI systems contain multiple cdiA gene fragments that encode CdiA-CT sequences. These “orphan” cdiA-CT genes are almost always associated with downstream cdiI genes to form cdiA-CT/cdiI modules. Comparative genome analyses suggest that cdiA-CT/cdiI modules are mobile and exchanged between the CDI systems of different bacteria. In many instances, orphan cdiA-CT/cdiI modules are fused to full-length cdiA genes in other bacterial species. Examination of cdiA-CT/cdiI modules from Escherichia coli EC93, E. coli EC869, and Dickeya dadantii 3937 confirmed that these genes encode functional toxin/immunity pairs. Moreover, the orphan module from EC93 was functional in cell-mediated CDI when fused to the N-terminal portion of the EC93 CdiA protein. Bioinformatic analyses revealed that the genetic organization of CDI systems shares features with rhs (rearrangement hotspot) loci. Rhs proteins also contain polymorphic C-terminal regions (Rhs-CTs), some of which share significant sequence identity with CdiA-CTs. All rhs genes are followed by small ORFs representing possible rhsI immunity genes, and several Rhs systems encode orphan rhs-CT/rhsI modules. Analysis of rhs-CT/rhsI modules from D. dadantii 3937 demonstrated that Rhs-CTs have growth inhibitory activity, which is specifically blocked by cognate RhsI immunity proteins. Together, these results suggest that Rhs plays a role in intercellular competition and that orphan gene modules expand the diversity of toxic activities deployed by both CDI and Rhs systems. PMID:21829394

  17. A biomarker for the identification of swine fecal pollution in water, using the STII toxin gene from enterotoxigenic Escherichia coli.

    PubMed

    Khatib, L A; Tsai, Y L; Olson, B H

    2003-12-01

    This research developed a PCR method to identify swine fecal pollution in water, using a portion of the STII toxin gene from enterotoxigenic Escherichia coli as the target sequence. This method showed the gene to have a wide-spread geographical distribution and temporal stability; and the primers demonstrated high specificity, sensitivity, and reliability. A total of 110 DNA extracts from different animal fecal and human sewage samples were screened using the primers and no positives resulted. Centrifugation and filtration methods for concentrating E. coli seeded into stream, ocean, secondary effluent, and dairy lagoon waters resulted in detection limits at the femtogram and attogram levels. E. coli with the biomarker seeded into stream, ocean, and secondary effluent waters remained stable for approximately 2 weeks for all water types. Of the farm lagoon and waste samples tested, 94% were positive for the STII trait, regardless of the number of E. coli screened and 100% were positive when > or =35 E. coli isolates were screened. As the PCR product of the target sequence yielded a single band, the method is applicable to dot blot detection methodology, yielding great accuracy in determining the presence of swine fecal sources.

  18. Prevalence of staphylococcal enterotoxins, toxin genes and genetic-relatedness of foodborne Staphylococcus aureus strains isolated in the Marmara Region of Turkey.

    PubMed

    Aydin, Ali; Sudagidan, Mert; Muratoglu, Karlo

    2011-08-02

    Staphylococcus aureus is a major foodborne pathogen and it has the ability to produce a number of extracellular toxins. We analyzed 1070 food samples obtained from retail markets and dairy farms in the Marmara Region of Turkey for the presence of S. aureus. Out of 147 isolates, 92 (62.6%) were enterotoxigenic. PCR was used to investigate the presence of staphylococcal enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, sej, sek, sel, sem, sen, seo, sep, seq and seu), exfoliative toxin genes (eta and etb) and the toxic-shock syndrome toxin gene (tst). The PCR results showed that 53.3% of the isolates contained staphylococcal enterotoxin-like (SEl) toxin genes (seg, seh, sei, sej, sek, sel, sem, sen, seo, sep, seq and seu) which were more frequent than classical enterotoxin genes (sea to see). Furthermore, seo, sei, sem, seg, seu and sec were found in 37.0, 32.7, 30.4, 29.3, 29.3 and 27.2% of the isolates, respectively. The tst gene was detected and confirmed by DNA sequencing in 9 isolates. The presence of eta and etb were not found in the isolates. Enterotoxigenic capabilities of isolates with SEA-SEE were investigated by ELISA. Enterotoxigenic S. aureus isolates produced one to three enterotoxins, with the most frequently produced types being enterotoxin A and C. There was a correlation of 72.1% between production of a specific toxin and the presence of the respective genes. PFGE analysis was used to identify genetic-relatedness of enterotoxigenic S. aureus isolates and the results revealed that 13 groups of isolates from different or the same origin that contained the same genes showed 100% homology with indistinguishable band patterns. The other enterotoxigenic isolates showed related band patterns with 72-86% homology in sea-, 61-90% homology in sec-, 80-96% homology in seh-, and 69-96% homology in sep-positive isolates. To our knowledge, this is the first study to examine enterotoxins and related gene contents of S. aureus food isolates in the Marmara

  19. Nucleotide sequence of the hemolysin I gene from Actinobacillus pleuropneumoniae.

    PubMed Central

    Frey, J; Meier, R; Gygi, D; Nicolet, J

    1991-01-01

    The DNA sequence of the gene encoding the structural protein of hemolysin I (HlyI) of Actinobacillus pleuropneumoniae serotype 1 strain 4074 was analyzed. The nucleotide sequence shows a 3,072-bp reading frame encoding a protein of 1,023 amino acids with a calculated molecular size of 110.1 kDa. This corresponds to the HlyI protein, which has an apparent molecular size on sodium dodecyl sulfate gels of 105 kDa. The structure of the protein derived from the DNA sequence shows three hydrophobic regions in the N-terminal part of the protein, 13 glycine-rich domains in the second half of the protein, and a hydrophilic C-terminal area, all of which are typical of the cytotoxins of the RTX (repeats in the structural toxin) toxin family. The derived amino acid sequence of HlyI shows 42% homology with the hemolysin of A. pleuropneumoniae serotype 5, 41% homology with the leukotoxin of Pasteurella haemolytica, and 56% homology with the Escherichia coli alpha-hemolysin. The 13 glycine-rich repeats and three hydrophobic areas of the HlyI sequence show more similarity to the E. coli alpha-hemolysin than to either the A. pleuropneumoniae serotype 5 hemolysin or the leukotoxin (while the last two are more similar to each other). Two types of RTX hemolysins therefore seem to be present in A. pleuropneumoniae, one (HlyI) resembling the alpha-hemolysin and a second more closely related to the leukotoxin. Ca(2+)-binding experiments using HlyI and recombinant A. pleuropneumoniae prohemolysin (HlyIA) that was produced in E. coli shows that HlyI binds 45Ca2+, probably because of the 13 glycine-rich repeated domains. Activation of the prohemolysin is not required for Ca2+ binding. Images PMID:1879928

  20. New variants of lepidoptericidal toxin genes encoding Bacillus thuringiensis Vip3Aa proteins.

    PubMed

    Sauka, Diego H; Rodriguez, Sonia E; Benintende, Graciela B

    2012-01-01

    Bacillus thuringiensis is an entomopathogenic bacterium characterized by producing parasporal proteinaceous insecticidal crystal inclusions during sporulation. Many strains are capable of also expressing other insecticidal proteins called Vip during the vegetative growing phase. Particularly, Vip3A proteins have activity against certain Lepidoptera species through a unique mechanism of action which emphasized their possible use in resistance management strategies against resistant pests. The aim of the work was to develop a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method that can distinguish between vip3A genes from B. thuringiensis strains. In addition, 4 novel vip3Aa genes were cloned and sequenced. The method was originally based on amplification of a single PCR amplicon and the use of 2 restriction enzymes with recognition sites that facilitate simultaneous detection. Subsequently, a third restriction enzyme was used to distinguish between vip3A variants. Thirteen vip3Aa genes were identified in strains belonging to 10 different B. thuringiensis serovars. Three intra-subclass variants of vip3Aa genes could be differentiated. The presented method can serve as an invaluable tool for the investigation of known and novel vip3A genes in B. thuringiensis strains. To the best of our knowledge, this is the first report where variants of a same subclass of insecticidal genes could be distinguished following PCR-RFLP.

  1. Analysis of the enterohemorrhagic Escherichia coli O157 DNA region containing lambdoid phage gene p and Shiga-like toxin structural genes.

    PubMed Central

    Datz, M; Janetzki-Mittmann, C; Franke, S; Gunzer, F; Schmidt, H; Karch, H

    1996-01-01

    In this study, we determined the nucleotide sequence of the p gene contained within a 5-kb EcoRI restriction fragment cloned from Shiga-like toxin II (SLT-II)-converting phage 933W of Escherichia coli O157:H7 strain EDL933. The p gene was 702 bp long and had 95.3% sequence similarity to the p gene of phage lambda. Multiple hybridization patterns were obtained when genomic DNA fragments were hybridized with both p and slt-I, slt-II, or slt-IIc sequences. All O157 isolates also possessed an analog of lambda gene p which was not linked with either slt-I or slt-II. Restriction fragment length polymorphism comparisons of clinical O157 isolates and derivates undergoing genotype turnover during infection were made, and loss of large DNA fragments that hybridized with slt-II and p sequences was observed. To further analyze the DNA region containing the p and slt genes, we amplified fragments by using a PCR with one primer complementary to p and the other complementary to either the slt-I or the slt-II gene. PCR analysis with enterohemorrhagic E. coli O157 and non-O157 strains yielded PCR products that varied in size between 5.1 and 7.8 kb. These results suggest that even within O157 isolates, the genomes of SLT-converting phages differ. The methods described here may assist in further investigation of SLT-encoding phages and their role in the epidemiology of infection with enterohemorrhagic E. coli. PMID:8975608

  2. RNA-Seq Analysis Identifies New Genes Regulated by the Histone-Like Nucleoid Structuring Protein (H-NS) Affecting Vibrio cholerae Virulence, Stress Response and Chemotaxis

    PubMed Central

    Wang, Hongxia; Ayala, Julio C.; Benitez, Jorge A.; Silva, Anisia J.

    2015-01-01

    The histone-like nucleoid structuring protein (H-NS) functions as a transcriptional silencer by binding to AT-rich sequences at bacterial promoters. However, H-NS repression can be counteracted by other transcription factors in response to environmental changes. The identification of potential toxic factors, the expression of which is prevented by H-NS could facilitate the discovery of new regulatory proteins that may contribute to the emergence of new pathogenic variants by anti-silencing. Vibrio cholerae hns mutants of the El Tor biotype exhibit altered virulence, motility and environmental stress response phenotypes compared to wild type. We used an RNA-seq analysis approach to determine the basis of the above hns phenotypes and identify new targets of H-NS transcriptional silencing. H-NS affected the expression of 18% of all predicted genes in a growth phase-dependent manner. Loss of H-NS resulted in diminished expression of numerous genes encoding methyl-accepting chemotaxis proteins as well as chemotaxis toward the attractants glycine and serine. Deletion of hns also induced an endogenous envelope stress response resulting in elevated expression of rpoE encoding the extracytoplamic sigma factor E (σE). The RNA-seq analysis identified new genes directly repressed by H-NS that can affect virulence and biofilm development in the El Tor biotype cholera bacterium. We show that H-NS and the quorum sensing regulator HapR silence the transcription of the vieSAB three-component regulatory system in El Tor biotype V. cholerae. We also demonstrate that H-NS directly represses the transcription of hlyA (hemolysin), rtxCA (the repeat in toxin or RTX), rtxBDE (RTX transport) and the biosynthesis of indole. Of these genes, H-NS occupancy at the hlyA promoter was diminished by overexpression of the transcription activator HlyU. We discuss the role of H-NS transcriptional silencing in phenotypic differences exhibited by V. cholerae biotypes. PMID:25679988

  3. Discovery of toxin-encoding genes from the false viper Macropisthodon rudis, a rear-fanged snake, by transcriptome analysis of venom gland.

    PubMed

    Zhang, Zhixiao; Zhang, Xi; Hu, Tingsong; Zhou, Weiguo; Cui, Qinghua; Tian, Jing; Zheng, Ying; Fan, Quanshui

    2015-11-01

    Although rear-fanged snakes are often considered as non-threatening to humans, some species are lethal or medically hazardous. The toxin components and bioactivities of front-fanged snakes have been extensively studied; however, only limited research has explored the venoms of rear-fanged snakes. The false viper, Macropisthodon rudis, is widespread in southern China, but little is known about the toxins that this snake produces. Here, we analyzed the transcriptome of the venom gland of M. rudis using high-throughput sequencing with an illumina HiSeq 2000. The raw data were assembled and annotated using public databases. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and gene ontology (GO) were analyzed. Using sequence comparisons, snake venom metalloproteinases (SVMPs) and a phosphodiesterase (PDE) were discovered in the venom gland of M. rudis.

  4. Toxin Plasmids of Clostridium perfringens

    PubMed Central

    Li, Jihong; Adams, Vicki; Bannam, Trudi L.; Miyamoto, Kazuaki; Garcia, Jorge P.; Uzal, Francisco A.; Rood, Julian I.

    2013-01-01

    SUMMARY In both humans and animals, Clostridium perfringens is an important cause of histotoxic infections and diseases originating in the intestines, such as enteritis and enterotoxemia. The virulence of this Gram-positive, anaerobic bacterium is heavily dependent upon its prolific toxin-producing ability. Many of the ∼16 toxins produced by C. perfringens are encoded by large plasmids that range in size from ∼45 kb to ∼140 kb. These plasmid-encoded toxins are often closely associated with mobile elements. A C. perfringens strain can carry up to three different toxin plasmids, with a single plasmid carrying up to three distinct toxin genes. Molecular Koch's postulate analyses have established the importance of several plasmid-encoded toxins when C. perfringens disease strains cause enteritis or enterotoxemias. Many toxin plasmids are closely related, suggesting a common evolutionary origin. In particular, most toxin plasmids and some antibiotic resistance plasmids of C. perfringens share an ∼35-kb region containing a Tn916-related conjugation locus named tcp (transfer of clostridial plasmids). This tcp locus can mediate highly efficient conjugative transfer of these toxin or resistance plasmids. For example, conjugative transfer of a toxin plasmid from an infecting strain to C. perfringens normal intestinal flora strains may help to amplify and prolong an infection. Therefore, the presence of toxin genes on conjugative plasmids, particularly in association with insertion sequences that may mobilize these toxin genes, likely provides C. perfringens with considerable virulence plasticity and adaptability when it causes diseases originating in the gastrointestinal tract. PMID:23699255

  5. Genetic profiles of Shiga toxin and intimin genes found in stool broth cultures: a 2-year reference laboratory study.

    PubMed

    Michel, Pierre A; Kase, Julie A

    2009-10-01

    Shiga toxin (Stx)-producing Escherichia coli (STEC) are associated with potentially serious illness in humans. STEC detection is often based on the presence of Stxs, Stx(1) and/or Stx(2), and intimin, encoded by the eae gene. A 2-year collection of stool broth cultures was tested for variants of stx(1), stx(2), and eae. Approximately 80% (138 of 174) were positive for stx(1) and/or stx(2), with stx(1) as the most prevalent (66%). Of the stx(1) variants, stx(1) was the most common (76%) followed by stx(1c) (22%). Analysis of stx(2)-positive isolates found 20 (53%) stx(2), 13 (34%) stx(2)/stx(2v-ha), 3 (8%) stx(2v-ha), 1 (3%) stx(2v-hb), and 1 (3%) stx(2d-activatable). Findings of stx(2)/stx(2v-ha) and stx(2d-activatable) are noteworthy given associations with hemolytic uremic syndrome and increased cytotoxicity, respectively. Of the Stx positive, 94 (68%) were eae positive with 31 (33%) eae(varepsilon1), 19 (20%) eae(gamma1), and 18 (19%) eae(beta1). A predominance of eae(varepsilon1) may suggest a new pathogenic significance because, reportedly, eae(beta1) is one of the most widespread variants.

  6. Heat-induced transcription of diphtheria toxin A or its variants, CRM176 and CRM197: implications for pancreatic cancer gene therapy.

    PubMed

    Fogar, P; Navaglia, F; Basso, D; Zambon, C-F; Moserle, L; Indraccolo, S; Stranges, A; Greco, E; Fadi, E; Padoan, A; Pantano, G; Sanzari, M C; Pedrazzoli, S; Montecucco, C; Plebani, M

    2010-01-01

    Vectors combining the heat shock proteins (HSPs) promoter with the catalytic subunit A of the diphtheria toxin (DTA) or its variants, cross-reacting material (CRM) 176 and 197, were engineered to investigate the effect of bacterial toxins on pancreatic cancer (PC) cells. Three heat-inducible enhanced green fluorescent protein (eGFP)-expression vectors were obtained: V1 (91% homology to HSPA6), V2 (five heat shock elements upstream the minimal HSPA6 promoter) and V3 (V1 and V2 combined). The highest eGFP transcription and translation levels were found in V3 transfected PC cells. The V3 promoter was used to control DTA, CRM176 and CRM197 expression, treatment response being investigated in four PC cell lines. DTAwt or CRM176 transfected cell growth was completely arrested after heat shock. CRM197 toxin presumed to be inactive, caused mild distress at 37 degrees C and induced a 25-50% reduction in cell growth after heat shock. Preliminary in vivo findings showed that heat treatment arrests tumor growth in DTA197 stably transfected PSN1 cells. In conclusion, the efficient HSP promoter identified in this study may be extremely useful in controlling the transcription of toxins such as CRM197, which have lethal dose-related effects, and may thus be a promising tool in PC gene therapy in vivo.

  7. Tribolium castaneum immune defense genes are differentially expressed in response to Bacillus thuringiensis toxins sharing common receptor molecules and exhibiting disparate toxicity.

    PubMed

    Contreras, Estefanía; Benito-Jardón, María; López-Galiano, M José; Real, M Dolores; Rausell, Carolina

    2015-06-01

    In Tribolium castaneum larvae we have demonstrated by RNA interference knockdown that the Bacillus thuringiensis Cry3Ba toxin receptors Cadherin-like and Sodium solute symporter proteins are also functional receptors of the less active Cry3Aa toxin. Differences in susceptibility to B. thuringiensis infection might not only rely on toxin-receptor interaction but also on host defense mechanisms. We compared the expression of the immune related genes encoding Apolipophorin-III and two antimicrobial peptides, Defensin3 and Defensin2 after B. thuringiensis challenge. All three genes were up-regulated following Cry3Ba spore-crystal intoxication whereas only Defensins gene expression was induced upon Cry3Aa spore-crystal treatment, evidencing a possible association between host immune response and larval susceptibility to B. thuringiensis. We assessed the antimicrobial activity spectra of T. castaneum defensins peptide fragments and found that a peptide fragment of Defensin3 was effective against the human microbial pathogens, Escherichia coli, Staphylococcus aureus and Candida albicans, being S. aureus the most susceptible one.

  8. Comprehensive Analysis of Gene Expression Profiles of the Beet Armyworm Spodoptera exigua Larvae Challenged with Bacillus thuringiensis Vip3Aa Toxin

    PubMed Central

    Bel, Yolanda; Jakubowska, Agata K.; Costa, Juliana; Herrero, Salvador; Escriche, Baltasar

    2013-01-01

    Host-pathogen interactions result in complex relationship, many aspects of which are not completely understood. Vip proteins, which are Bacillus thuringensis (Bt) insecticidal toxins produced during the vegetative stage, are selectively effective against specific insect pests. This new group of Bt proteins represents an interesting alternative to the classical Bt Cry toxins because current data suggests that they do not share the same mode of action. We have designed and developed a genome-wide microarray for the beet armyworm Spodoptera exigua, a serious lepidopteran pest of many agricultural crops, and used it to better understand how lepidopteran larvae respond to the treatment with the insecticidal protein Vip3Aa. With this approach, the goal of our study was to evaluate the changes in gene expression levels caused by treatment with sublethal doses of Vip3Aa (causing 99% growth inhibition) at 8 and 24 h after feeding. Results indicated that the toxin provoked a wide transcriptional response, with 19% of the microarray unigenes responding significantly to treatment. The number of up- and down-regulated unigenes was very similar. The number of genes whose expression was regulated at 8 h was similar to the number of genes whose expression was regulated after 24 h of treatment. The up-regulated sequences were enriched for genes involved in innate immune response and in pathogen response such as antimicrobial peptides (AMPs) and repat genes. The down-regulated sequences were mainly unigenes with homology to genes involved in metabolism. Genes related to the mode of action of Bt Cry proteins were found, in general, to be slightly overexpressed. The present study is the first genome-wide analysis of the response of lepidopteran insects to Vip3Aa intoxication. An insight into the molecular mechanisms and components related to Vip intoxication will allow designing of more effective management strategies for pest control. PMID:24312604

  9. Comprehensive analysis of gene expression profiles of the beet armyworm Spodoptera exigua larvae challenged with Bacillus thuringiensis Vip3Aa toxin.

    PubMed

    Bel, Yolanda; Jakubowska, Agata K; Costa, Juliana; Herrero, Salvador; Escriche, Baltasar

    2013-01-01

    Host-pathogen interactions result in complex relationship, many aspects of which are not completely understood. Vip proteins, which are Bacillus thuringensis (Bt) insecticidal toxins produced during the vegetative stage, are selectively effective against specific insect pests. This new group of Bt proteins represents an interesting alternative to the classical Bt Cry toxins because current data suggests that they do not share the same mode of action. We have designed and developed a genome-wide microarray for the beet armyworm Spodoptera exigua, a serious lepidopteran pest of many agricultural crops, and used it to better understand how lepidopteran larvae respond to the treatment with the insecticidal protein Vip3Aa. With this approach, the goal of our study was to evaluate the changes in gene expression levels caused by treatment with sublethal doses of Vip3Aa (causing 99% growth inhibition) at 8 and 24 h after feeding. Results indicated that the toxin provoked a wide transcriptional response, with 19% of the microarray unigenes responding significantly to treatment. The number of up- and down-regulated unigenes was very similar. The number of genes whose expression was regulated at 8 h was similar to the number of genes whose expression was regulated after 24 h of treatment. The up-regulated sequences were enriched for genes involved in innate immune response and in pathogen response such as antimicrobial peptides (AMPs) and repat genes. The down-regulated sequences were mainly unigenes with homology to genes involved in metabolism. Genes related to the mode of action of Bt Cry proteins were found, in general, to be slightly overexpressed. The present study is the first genome-wide analysis of the response of lepidopteran insects to Vip3Aa intoxication. An insight into the molecular mechanisms and components related to Vip intoxication will allow designing of more effective management strategies for pest control.

  10. Clostridium perfringens type A-E toxin plasmids.

    PubMed

    Freedman, John C; Theoret, James R; Wisniewski, Jessica A; Uzal, Francisco A; Rood, Julian I; McClane, Bruce A

    2015-05-01

    Clostridium perfringens relies upon plasmid-encoded toxin genes to cause intestinal infections. These toxin genes are associated with insertion sequences that may facilitate their mobilization and transfer, giving rise to new toxin plasmids with common backbones. Most toxin plasmids carry a transfer of clostridial plasmids locus mediating conjugation, which likely explains the presence of similar toxin plasmids in otherwise unrelated C. perfringens strains. The association of many toxin genes with insertion sequences and conjugative plasmids provides virulence flexibility when causing intestinal infections. However, incompatibility issues apparently limit the number of toxin plasmids maintained by a single cell.

  11. Clostridium perfringens type A–E toxin plasmids

    PubMed Central

    Freedman, John C.; Theoret, James R.; Wisniewski, Jessica A.; Uzal, Francisco A.; Rood, Julian I.; McClane, Bruce A.

    2014-01-01

    Clostridium perfringens relies upon plasmid-encoded toxin genes to cause intestinal infections. These toxin genes are associated with insertion sequences that may facilitate their mobilization and transfer, giving rise to new toxin plasmids with common backbones. Most toxin plasmids carry a transfer of clostridial plasmids locus mediating conjugation, which likely explains the presence of similar toxin plasmids in otherwise unrelated C. perfringens strains. The association of many toxin genes with insertion sequences and conjugative plasmids provides virulence flexibility when causing intestinal infections. However, incompatibility issues apparently limit the number of toxin plasmids maintained by a single cell. PMID:25283728

  12. Early direct and transneuronal effects in mice with targeted expression of a toxin gene to D1 dopamine receptor neurons.

    PubMed

    Padungchaichot, P; Wong, J Y; Natoli, A L; Massalas, J S; Finkelstein, D I; Lawrence, A L; Drago, J

    2000-01-01

    The neurochemical profile was examined at postnatal day 3-4 in mutant mice generated by in vivo Cre mediated activation of an attenuated diphtheria toxin gene inserted into the D1 dopamine receptor gene locus. An earlier study of this model had shown that D1 dopamine receptor, substance P and dynorphin were not expressed in the striatum. Quantitative in situ hybridization analysis showed an increase in D2 dopamine receptor and enkephalin messenger RNA expression. The nigrostriatal pathway in the mutant pups was intact with a normal number of dopaminergic neurons in the substantia nigra and the ventral tegmental area in addition to a normal pattern of striatal dopamine transporter and tyrosine hydroxylase immunoreactivity. Quantitative analysis of striatal dopamine transporter density using [3H]mazindol showed a reduction of 26% suggesting a degree of transneuronal down-regulation. There was also a 49% reduction of striatal GABA receptor binding and a 36% reduction of striatal muscarinic receptor binding in mutant pups. The number of healthy striatal neuropeptide Y-containing interneurons was also substantially down-regulated in the mutant striatum. In contrast, there was an increase in the number of striatal cholinergic interneurons. Down-regulated cortical GABA receptor and muscarinic receptor binding was also observed in addition to subtle morphological changes in the neuropeptide Y-expressing population of cortical neurons. The changes reflect the early cascade of events which follows the ablation of D1 dopamine receptor-positive cells. Although extensive changes in a number of striatal and cortical neurons were demonstrated, only subtle transneuronal effects were seen in the nigrostriatal pathway.

  13. Stool C difficile toxin

    MedlinePlus

    Antibiotic associated colitis - toxin; Colitis - toxin; Pseudomembranous - toxin; Necrotizing colitis - toxin; C difficile - toxin ... provider thinks that diarrhea is caused by the antibiotic medicines you have taken recently. Antibiotics change the ...

  14. Pertussis toxin

    SciTech Connect

    Sekura, R.D.; Moss, J.; Vaughan, M.

    1985-01-01

    This book contains 13 selections. Some of the titles are: Genetic and Functional Studies of Pertussis Toxin Substrates; Effect of Pertussis Toxin on the Hormonal Responsiveness of Different Tissues; Extracellular Adenylate Cyclase of Bordetella pertussis; and GTP-Regulatory Proteins are Introcellular Messagers: A Model for Hormone Action.

  15. Screening, detection, and serotyping methods for toxin genes and enterotoxins in Staphylococcus strains.

    PubMed

    Hait, Jennifer M; Tallent, Sandra M; Bennett, Reginald W

    2014-01-01

    Staphylococcus aureus continues to play a significant role in foodborne outbreak investigations, with numerous individuals sickened each year after ingesting assorted foods contaminated with staphylococcal enterotoxins. The purpose of this study was to evaluate the use of several methods for the screening, detection, and enterotoxin serotyping of staphylococcal bacterial strains for classical staphylococcal enterotoxins (SEs; SEA, SEB, SEC, SED, and SEE) and the newly described SE and SE-like enterotoxin genes (seg, seh, sei, sej, sek, sel, sem, sen, seo, sep, seq, ser, ses, set, and seu). Inclusivity and exclusivity panels of staphylococcal strains were tested using a multiplex PCR method in addition to three polyvalent commercially prepared ELISA systems for the detection of SEA-SEE and one monovalent assay for the identification of classical SE serotypes. The results indicate an overall agreement between serological detection methods with a few exceptions, and molecular characterization identified an abundance of SE and SE-like enterotoxin genes including several potentially enterotoxigenic isolates that would have otherwise been missed by ELISA-based methods. These findings demonstrate the significance of PCR for future screening purposes and the use of ELISA systems for the detection and enterotoxin serotyping of staphylococcal bacterial strains.

  16. PCR amplfication on a microarray of gel-immobilized oligonucleotides : detection of bacterial toxin- and drug-resistent genes and their mutations.

    SciTech Connect

    Strizhkov, B. N.; Drobyshev, A. L.; Mikhailovich, V. M.; Mirzabekov, A. D.; Biochip Technology Center; Engelhardt Inst. of Molecular Biology

    2000-10-01

    PCR amplification on a microarray of gel-immobilized primers (microchip) has been developed. One of a pair of PCR primers was immobilized inside a separate microchip polyacrylamide porous gel pad of 0.1 x 0.1 x 0.02 (or 0.04) micron in size and 0.2 (or 0.4) nL in volume. The amplification was carried out simultaneously both in solution covering the microchip array and inside gel pads. Each gel pad contained the immobilized forward primers, while the fluorescently labeled reverse primers, as well as all components of the amplification reaction, diffused into the gel pads from the solution. To increase the amplification efficiency, the forward primers were also added into the solution. The kinetics of amplification was measured in real time in parallel for all gel pads with a fluorescent microscope equipped with a charge-coupled device (CCD) camera. The accuracy of the amplification was assessed by using the melting curves obtained for the duplexes formed by the labeled amplification product and the gel-immobilized primers during the amplification process; alternatively, the duplexes were produced by hybridization of the extended immobilized primers with labeled oligonucleotide probes. The on-chip amplification was applied to detect the anthrax toxin genes and the plasmid-borne beta-lactamase gene responsible for bacterial ampicillin resistance. The allele-specific type of PCR amplification was used to identify the Shiga toxin gene and discriminate it from the Shiga-like one. The genomic mutations responsible for rifampicin resistance of the Mycobacterium tuberculosis strains were detected by the same type of PCR amplification of the rpoB gene fragment isolated from sputum of tuberculosis patients. The on-chip PCR amplification has been shown to be a rapid, inexpensive and powerful tool to test genes responsible for bacterial toxin production and drug resistance, as well as to reveal point nucleotide mutations.

  17. A large cholera outbreak due to a new cholera toxin variant of the Vibrio cholerae O1 El Tor biotype in Orissa, Eastern India.

    PubMed

    Kumar, P; Jain, M; Goel, A K; Bhadauria, S; Sharma, S K; Kamboj, D V; Singh, L; Ramamurthy, T; Nair, G B

    2009-02-01

    A total of 32 Vibrio cholerae isolates were collected during a recent large cholera outbreak in Eastern India. Biochemical and serological studies revealed that all of the isolates belonged to serogroup O1, biotype El Tor, serotype Ogawa. Two multiplex PCR assays confirmed the presence of various toxigenic and pathogenic genes - ace, ctxAB, hlyA, ompU, ompW, rfbO1, rtx, tcp, toxR and zot - in all of the isolates. Sequencing of the ctxB gene from the isolates revealed a novel mutation in the gene. Sequencing also confirmed the presence of altered cholera toxin B of the classical biotype in all of the El Tor isolates, suggesting infection of isolates by classical CTXPhi. The molecular diversity of V. cholerae isolates studied by enterobacterial repetitive intergenic consensus sequence PCR, BOX-PCR and randomly amplified polymorphic DNA analysis uniformly showed the clonal relationship among the outbreak V. cholerae O1 isolates. The results of this study suggest that cholera-causing V. cholerae strains are constantly evolving in epidemic areas, highlighting the potential of the emergence of more virulent strains.

  18. Occurrence, genotyping, shiga toxin genes and associated risk factors of E. coli isolated from dairy farms, handlers and milk consumers.

    PubMed

    Awadallah, M A; Ahmed, H A; Merwad, A M; Selim, M A

    2016-11-01

    The objectives of the current study were to determine the occurrence and genotypes of E. coli in dairy farms, workers and milk consumers and to evaluate risk factors associated with contamination of milk in dairy farms. Molecular characterization of shiga toxin associated genes and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) finger printing of E. coli from different sources were also studied. Paired milk samples and rectal swabs from 125 dairy cows, rectal swabs from 82 calves and hand swabs from 45 dairy workers from five dairy farms were collected. In addition, 100 stool samples from 70 diarrheic and 30 healthy humans were collected and examined for the presence of E. coli. E. coli was isolated from milk (22.4%), dairy cattle feces (33.6%), calf feces (35.4%), dairy worker hand swabs (11.1%) and stools of milk consumers (2%, from diarrheic patients only). Only stx1 was identified in seven of 12 E. coli O125 isolated from different sources. High genetic diversity was determined (Simpson's index of diversity, D = 1) and E. coli O125 isolates were classified into 12 distinct profiles, E1-E12. The dendrogram analysis showed that two main clusters were generated. Mastitis in dairy cows was considered a risk factor associated with contamination of the produced milk with E. coli. The isolation of E. coli from rectal swabs of dairy cows and calves poses a zoonotic risk through consumption of unpasteurized contaminated dairy milk. Educational awareness should be developed to address risks related to consumption of raw milk.

  19. A new pyrosequencing assay for rapid detection and genotyping of Shiga toxin, intimin and O157-specific rfbE genes of Escherichia coli.

    PubMed

    Goji, Noriko; Mathews, Amit; Huszczynski, George; Laing, Chad R; Gannon, Victor P J; Graham, Morag R; Amoako, Kingsley K

    2015-02-01

    Shiga toxin (stx)-producing Escherichia coli (STEC) contamination in food and water is one of the most recognized concerns and a major financial burden in human hygiene control worldwide. Rapid and highly reliable methods of detecting and identifying STEC causing gastroenteric illnesses are crucial to prevent foodborne outbreaks. A number of tests have been developed and commercialized to detect STEC using molecular microbiology techniques. Most of these are designed to identify virulence factors such as Shiga toxin and intimin as well as E. coli O and H antigen serotype specific genes. In order to screen pathogenic STEC without relying on O:H serotyping, we developed a rapid detection and genotyping assay for STEC virulence genes using a PCR-pyrosequencing application. We adapted the PyroMark Q24 Pyrosequencing platform for subtyping 4 major virulence genes, Shiga toxin 1 and 2 (stx1 and stx2), intimin (eae) and O157-antigen gene cluster target rfbE, using Single Nucleotide Polymorphism (SNP) analysis. A total of 224 E. coli strains including isolates from Canadian environment, food and clinical cases were examined. Based on the multiple alignment analysis of 30-80 base nucleotide pyrogram reads, three alleles of the Shiga toxin 1a gene (stx1a) (stx1a-I, stx1a-II, stx1a-III) were identified. Results of the stx1, stx2, eae and rfbE genotyping revealed that each group of O:H serotype shares distinctive characteristics that could be associated with the virulence of each genotype. O157:H7/NM carries stx1a-II (94%), stx2a (82%), λ/γ1-eae (100%) and rfbE type-H7/NM (100%). Whereas isolates of the "Top-6" serotypes (O26, O45, O103, O111, O121, O145) had a high incidence of stx1a-I (90%) and stx2a (100%). stx1a-III (60%) was only observed in non Top-7 (Top-6 plus O157) STEC and Shigella spp. The entire assay, from extracting DNA from colonies on a plate to the generation of sequence information, can be completed in 5h. The method of profiling these 4 STEC pathogenic

  20. Distribution of virulence-associated genes and genetic relationships in non-O1/O139 Vibrio cholerae aquatic isolates from China.

    PubMed

    Li, Fengjuan; Du, Pengcheng; Li, Baisheng; Ke, Changwen; Chen, Aiping; Chen, Jie; Zhou, Haijian; Li, Jie; Morris, J Glenn; Kan, Biao; Wang, Duochun

    2014-08-01

    Non-O1/O139 Vibrio cholerae is naturally present in aquatic ecosystems and has been linked with cholera-like diarrhea and local outbreaks. The distribution of virulence-associated genes and genetic relationships among aquatic isolates from China are largely unknown. In this study, 295 aquatic isolates of V. cholerae non-O1/O139 serogroups from different regions in China were investigated. Only one isolate was positive for ctxB and harbored a rare genotype; 10 (3.4%) isolates carried several types of rstR sequences, eight of which carried rare types of toxin-coregulated pili (tcpA). Furthermore, 16 (5.4%) isolates carried incomplete (with partial open reading frames [ORFs]) vibrio seventh pandemic island I (VSP-I) or VSP-II clusters, which were further classified as 11 novel types. PCR-based analyses revealed remarkable variations in the distribution of putative virulence genes, including mshA (95.6%), hlyA (95.3%), rtxC (89.8%), rtxA (82.7%), IS1004 (52.9%), chxA (30.2%), SXT (15.3%), type III secretion system (18.0%), and NAG-ST (3.7%) genes. There was no correlation between the prevalence of putative virulence genes and that of CTX prophage or TCP genes, whereas there were correlations among the putative virulence genes. Further multilocus sequence typing (MLST) placed selected isolates (n = 70) into 69 unique sequence types (STs), which were different from those of the toxigenic O1 and O139 counterparts, and each isolate occupied a different position in the MLST tree. The V. cholerae non-O1/O139 aquatic isolates predominant in China have high genotypic diversity; these strains constitute a reservoir of potential virulence genes, which may contribute to evolution of pathogenic isolates.

  1. Spatial soil heterogeneity has a greater effect on symbiotic arbuscular mycorrhizal fungal communities and plant growth than genetic modification with Bacillus thuringiensis toxin genes.

    PubMed

    Cheeke, Tanya E; Schütte, Ursel M; Hemmerich, Chris M; Cruzan, Mitchell B; Rosenstiel, Todd N; Bever, James D

    2015-05-01

    Maize, genetically modified with the insect toxin genes of Bacillus thuringiensis (Bt), is widely cultivated, yet its impacts on soil organisms are poorly understood. Arbuscular mycorrhizal fungi (AMF) form symbiotic associations with plant roots and may be uniquely sensitive to genetic changes within a plant host. In this field study, the effects of nine different lines of Bt maize and their corresponding non-Bt parental isolines were evaluated on AMF colonization and community diversity in plant roots. Plants were harvested 60 days after sowing, and data were collected on plant growth and per cent AMF colonization of roots. AMF community composition in roots was assessed using 454 pyrosequencing of the 28S rRNA genes, and spatial variation in mycorrhizal communities within replicated experimental field plots was examined. Growth responses, per cent AMF colonization of roots and AMF community diversity in roots did not differ between Bt and non-Bt maize, but root and shoot biomass and per cent colonization by arbuscules varied by maize cultivar. Plot identity had the most significant effect on plant growth, AMF colonization and AMF community composition in roots, indicating spatial heterogeneity in the field. Mycorrhizal fungal communities in maize roots were autocorrelated within approximately 1 m, but at greater distances, AMF community composition of roots differed between plants. Our findings indicate that spatial variation and heterogeneity in the field has a greater effect on the structure of AMF communities than host plant cultivar or modification by Bt toxin genes.

  2. MAPK signaling pathway alters expression of midgut ALP and ABCC genes and causes resistance to Bacillus thuringiensis Cry1Ac toxin in diamondback moth.

    PubMed

    Guo, Zhaojiang; Kang, Shi; Chen, Defeng; Wu, Qingjun; Wang, Shaoli; Xie, Wen; Zhu, Xun; Baxter, Simon W; Zhou, Xuguo; Jurat-Fuentes, Juan Luis; Zhang, Youjun

    2015-04-01

    Insecticidal crystal toxins derived from the soil bacterium Bacillus thuringiensis (Bt) are widely used as biopesticide sprays or expressed in transgenic crops to control insect pests. However, large-scale use of Bt has led to field-evolved resistance in several lepidopteran pests. Resistance to Bt Cry1Ac toxin in the diamondback moth, Plutella xylostella (L.), was previously mapped to a multigenic resistance locus (BtR-1). Here, we assembled the 3.15 Mb BtR-1 locus and found high-level resistance to Cry1Ac and Bt biopesticide in four independent P. xylostella strains were all associated with differential expression of a midgut membrane-bound alkaline phosphatase (ALP) outside this locus and a suite of ATP-binding cassette transporter subfamily C (ABCC) genes inside this locus. The interplay between these resistance genes is controlled by a previously uncharacterized trans-regulatory mechanism via the mitogen-activated protein kinase (MAPK) signaling pathway. Molecular, biochemical, and functional analyses have established ALP as a functional Cry1Ac receptor. Phenotypic association experiments revealed that the recessive Cry1Ac resistance was tightly linked to down-regulation of ALP, ABCC2 and ABCC3, whereas it was not linked to up-regulation of ABCC1. Silencing of ABCC2 and ABCC3 in susceptible larvae reduced their susceptibility to Cry1Ac but did not affect the expression of ALP, whereas suppression of MAP4K4, a constitutively transcriptionally-activated MAPK upstream gene within the BtR-1 locus, led to a transient recovery of gene expression thereby restoring the susceptibility in resistant larvae. These results highlight a crucial role for ALP and ABCC genes in field-evolved resistance to Cry1Ac and reveal a novel trans-regulatory signaling mechanism responsible for modulating the expression of these pivotal genes in P. xylostella.

  3. Resiniferatoxin and botulinum toxin type A for treatment of lower urinary tract symptoms.

    PubMed

    Cruz, Francisco; Dinis, Paulo

    2007-10-01

    Resiniferatoxin (RTX) and botulinum toxin subtype A (BTX-A) are increasingly viewed as potential treatments for lower urinary tract symptoms (LUTS) refractory to conventional therapy. RTX, a capsaicin analogue devoid of severe pungent properties, acts by desensitizing the transient receptor potential vanilloid type 1 (TRPV1) receptor and inactivating C-fibers. BTX-A cleaves soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in afferent and efferent nerve endings, therefore impeding the fusion of synaptic vesicles with the neuronal membrane necessary for the release of neurotransmitters. In patients with neurogenic and idiopathic detrusor overactivity, RTX and BTX-A have been shown to increase the volume to first detrusor contraction, increase bladder capacity, and improve urinary incontinence and quality of life. Recent data also suggest a role for these neurotoxins in treating urgency, the primary symptom in overactive bladder (OAB) syndrome. Furthermore, experimental data strongly support the use of both neurotoxins in the treatment of pain and frequency in patients with interstitial cystitis/painful bladder syndrome (IC/PBS), although the results from available clinical trials for this use are still inconclusive. In spite of promising results overall, it should be made clear that the administration of these neurotoxins is still considered an experimental procedure and that more clinical studies are necessary before a license for their use will be issued by health authorities.

  4. Virulence gene profiles of shiga toxin-producing Escherichia coli isolated from fecal samples of finishing swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing Escherichia coli (STEC) are important pathogens responsible for food-borne outbreaks and serious illness including hemorrhagic colitis and hemolytic uremic syndrome. Certain STEC serogroups may cause edema disease in swine; and similar to cattle, swine have been shown to be a ...

  5. Purification and characterization of neurotoxin complex from a dual toxin gene containing Clostridium botulinum strain PS-5

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Botulinum neurotoxins (BoNTs) are produced as a toxin complex (TC) which consists of neurotoxin (NT) and neurotoxin associated proteins (NAPs). The characterization of NT in its native state is an essential step for developing diagnostics and therapeutic countermeasures against botulism. The presenc...

  6. Comparative possession of Shiga toxin, intimin, enterohaemolysin and major extended spectrum beta lactamase (ESBL) genes in Escherichia coli isolated from backyard and farmed poultry

    PubMed Central

    Samanta, I.; Joardar, S. N.; Das, P. K.; Sar, T. K.

    2015-01-01

    The present work was conducted to compare the occurrence of Escherichia coli possessing virulence and ESBL genes in backyard and farmed poultry. Three hundred and sixty samples from the poultry kept in backyard system and 120 samples from the farmed birds were collected from West Bengal, India. Among the E. coli isolates of backyard poultry (O2, O10, O25, O55, O60, O106, UT), none of them possessed any of the Shiga toxin genes and eight E. coli isolates (8/272; 2.9%) harboured eaeA gene alone. Whereas among the E. coli isolated from the farmed poultry (O17, O20, O22, O102, O114, O119, rough, UT), four isolates (4/78, 5.1%) harboured stx1/stx2 gene and 11 isolates (11/78, 14.1%) possessed eaeA gene. None of the E. coli isolates from the backyard poultry harboured any studied ESBL gene. Whereas 29.4% of E. coli isolates from the farmed poultry were found to possess the ESBL genes. PMID:27175158

  7. TetR is a positive regulator of the tetanus toxin gene in Clostridium tetani and is homologous to botR.

    PubMed

    Marvaud, J C; Eisel, U; Binz, T; Niemann, H; Popoff, M R

    1998-12-01

    The TetR gene immediately upstream from the tetanus toxin (TeTx) gene was characterized. It encodes a 21,562-Da protein which is related (50 to 65% identity) to the equivalent genes (botR) in Clostridium botulinum. TetR has the feature of a DNA binding protein with a basic pI (9.53). It contains a helix-turn-helix motif and shows 29% identity with other putative regulatory genes in Clostridium, i.e., uviA from C. perfringens and txeR from C. difficile. We report for the first time the transformation of C. tetani by electroporation, which permitted us to investigate the function of tetR. Overexpression of tetR in C. tetani induced an increase in TeTx production and in the level of the corresponding mRNA. This indicates that TetR is a transcriptional activator of the TeTx gene. Overexpression of botR/A (60% identity with TetR at the amino acid level) in C. tetani induced an increase in TeTx production comparable to that for overexpression of tetR. However, botR/C (50% identity with TetR at the amino acid level) was less efficient. This supports that TetR positively regulates the TeTx gene in C. tetani and that a conserved mechanism of regulation of the neurotoxin genes is involved in C. tetani and C. botulinum.

  8. Stability, structural and functional properties of a monomeric, calcium–loaded adenylate cyclase toxin, CyaA, from Bordetella pertussis

    PubMed Central

    Cannella, Sara E.; Ntsogo Enguéné, Véronique Yvette; Davi, Marilyne; Malosse, Christian; Sotomayor Pérez, Ana Cristina; Chamot-Rooke, Julia; Vachette, Patrice; Durand, Dominique; Ladant, Daniel; Chenal, Alexandre

    2017-01-01

    Bordetella pertussis, the causative agent of whooping cough, secretes an adenylate cyclase toxin, CyaA, which invades eukaryotic cells and alters their physiology by cAMP overproduction. Calcium is an essential cofactor of CyaA, as it is the case for most members of the Repeat-in-ToXins (RTX) family. We show that the calcium-bound, monomeric form of CyaA, hCyaAm, conserves its permeabilization and haemolytic activities, even in a fully calcium-free environment. In contrast, hCyaAm requires sub-millimolar calcium in solution for cell invasion, indicating that free calcium in solution is involved in the CyaA toxin translocation process. We further report the first in solution structural characterization of hCyaAm, as deduced from SAXS, mass spectrometry and hydrodynamic studies. We show that hCyaAm adopts a compact and stable state that can transiently conserve its conformation even in a fully calcium-free environment. Our results therefore suggest that in hCyaAm, the C-terminal RTX-domain is stabilized in a high-affinity calcium-binding state by the N-terminal domains while, conversely, calcium binding to the C-terminal RTX-domain strongly stabilizes the N-terminal regions. Hence, the different regions of hCyaAm appear tightly connected, leading to stabilization effects between domains. The hysteretic behaviour of CyaA in response to calcium is likely shared by other RTX cytolysins. PMID:28186111

  9. Safety assessment of the Clostridium butyricum MIYAIRI 588® probiotic strain including evaluation of antimicrobial sensitivity and presence of Clostridium toxin genes in vitro and teratogenicity in vivo.

    PubMed

    Isa, K; Oka, K; Beauchamp, N; Sato, M; Wada, K; Ohtani, K; Nakanishi, S; McCartney, E; Tanaka, M; Shimizu, T; Kamiya, S; Kruger, C; Takahashi, M

    2016-08-01

    Probiotics are live microorganisms ingested for the purpose of conferring a health benefit on the host. Development of new probiotics includes the need for safety evaluations that should consider factors such as pathogenicity, infectivity, virulence factors, toxicity, and metabolic activity. Clostridium butyricum MIYAIRI 588(®) (CBM 588(®)), an anaerobic spore-forming bacterium, has been developed as a probiotic for use by humans and food animals. Safety studies of this probiotic strain have been conducted and include assessment of antimicrobial sensitivity, documentation of the lack of Clostridium toxin genes, and evaluation of CBM 588(®) on reproductive and developmental toxicity in a rodent model. With the exception of aminoglycosides, to which anaerobes are intrinsically resistant, CBM 588(®) showed sensitivity to all antibiotic classes important in human and animal therapeutics. In addition, analysis of the CBM 588(®) genome established the absence of genes for encoding for α, β, or ε toxins and botulin neurotoxins types A, B, E, or F. There were no deleterious reproductive and developmental effects observed in mice associated with the administration of CBM 588(®) These data provide further support for the safety of CBM 588(®) for use as a probiotic in animals and humans.

  10. Key role of LaeA and velvet complex proteins on expression of β-lactam and PR-toxin genes in Penicillium chrysogenum: cross-talk regulation of secondary metabolite pathways.

    PubMed

    Martín, Juan F

    2016-08-26

    Penicillium chrysogenum is an excellent model fungus to study the molecular mechanisms of control of expression of secondary metabolite genes. A key global regulator of the biosynthesis of secondary metabolites is the LaeA protein that interacts with other components of the velvet complex (VelA, VelB, VelC, VosA). These components interact with LaeA and regulate expression of penicillin and PR-toxin biosynthetic genes in P. chrysogenum. Both LaeA and VelA are positive regulators of the penicillin and PR-toxin biosynthesis, whereas VelB acts as antagonist of the effect of LaeA and VelA. Silencing or deletion of the laeA gene has a strong negative effect on penicillin biosynthesis and overexpression of laeA increases penicillin production. Expression of the laeA gene is enhanced by the P. chrysogenum autoinducers 1,3 diaminopropane and spermidine. The PR-toxin gene cluster is very poorly expressed in P. chrysogenum under penicillin-production conditions (i.e. it is a near-silent gene cluster). Interestingly, the downregulation of expression of the PR-toxin gene cluster in the high producing strain P. chrysogenum DS17690 was associated with mutations in both the laeA and velA genes. Analysis of the laeA and velA encoding genes in this high penicillin producing strain revealed that both laeA and velA acquired important mutations during the strain improvement programs thus altering the ratio of different secondary metabolites (e.g. pigments, PR-toxin) synthesized in the high penicillin producing mutants when compared to the parental wild type strain. Cross-talk of different secondary metabolite pathways has also been found in various Penicillium spp.: P. chrysogenum mutants lacking the penicillin gene cluster produce increasing amounts of PR-toxin, and mutants of P. roqueforti silenced in the PR-toxin genes produce large amounts of mycophenolic acid. The LaeA-velvet complex mediated regulation and the pathway cross-talk phenomenon has great relevance for improving the

  11. The role of Cercospora zeae-maydis homologs of Rhodobacter sphaeroides 1O2-resistance genes in resistance to the photoactivated toxin cercosporin.

    PubMed

    Beseli, Aydin; Goulart da Silva, Marilia; Daub, Margaret E

    2015-01-01

    The photosynthetic bacterium Rhodobacter sphaeroides and plant pathogenic fungus Cercospora nicotianae have been used as models for understanding resistance to singlet oxygen ((1)O(2)), a highly toxic reactive oxygen species. In Rhodobacter and Cercospora, (1)O(2) is derived, respectively, from photosynthesis and from the (1)O(2)-generating toxin cercosporin which the fungus produces to parasitize plants. We identified common genes recovered in transcriptome studies of putative (1)O(2)-resistance genes in these two systems, suggesting common (1)O(2)-resistance mechanisms. To determine if the Cercospora homologs of R. sphaeroides (1)O(2)-resistance genes are involved in resistance to cercosporin, we expressed the genes in the cercosporin-sensitive fungus Neurospora crassa and assayed for increases in cercosporin resistance. Neurospora crassa transformants expressing genes encoding aldo/keto reductase, succinyl-CoA ligase, O-acetylhomoserine (thiol) lyase, peptide methionine sulphoxide reductase and glutathione S-transferase did not have elevated levels of cercosporin resistance. Several transformants expressing aldehyde dehydrogenase were significantly more resistant to cercosporin. Expression of the transgene and enzyme activity did not correlate with resistance, however. We conclude that although the genes tested in this study are important in (1)O(2) resistance in R. sphaeroides, their Cercospora homologs are not involved in resistance to (1)O(2) generated from cercosporin.

  12. Functional validation of putative toxin-antitoxin genes from the Gram-positive pathogen Streptococcus pneumoniae: phd-doc is the fourth bona-fide operon.

    PubMed

    Chan, Wai Ting; Yeo, Chew Chieng; Sadowy, Ewa; Espinosa, Manuel

    2014-01-01

    Bacterial toxin-antitoxin (TAs) loci usually consist of two genes organized as an operon, where their products are bound together and inert under normal conditions. However, under stressful circumstances the antitoxin, which is more labile, will be degraded more rapidly, thereby unleashing its cognate toxin to act on the cell. This, in turn, causes cell stasis or cell death, depending on the type of TAs and/or time of toxin exposure. Previously based on in silico analyses, we proposed that Streptococcus pneumoniae, a pathogenic Gram-positive bacterium, may harbor between 4 and 10 putative TA loci depending on the strains. Here we have chosen the pneumococcal strain Hungary(19A)-6 which contains all possible 10 TA loci. In addition to the three well-characterized operons, namely relBE2, yefM-yoeB, and pezAT, we show here the functionality of a fourth operon that encodes the pneumococcal equivalent of the phd-doc TA. Transcriptional fusions with gene encoding Green Fluorescent Protein showed that the promoter was slightly repressed by the Phd antitoxin, and exhibited almost background values when both Phd-Doc were expressed together. These findings demonstrate that phd-doc shows the negative self-regulatory features typical for an authentic TA. Further, we also show that the previously proposed TAs XreA-Ant and Bro-XreB, although they exhibit a genetic organization resembling those of typical TAs, did not appear to confer a functional behavior corresponding to bona fide TAs. In addition, we have also discovered new interesting bioinformatics results for the known pneumococcal TAs RelBE2 and PezAT. A global analysis of the four identified toxins-antitoxins in the pneumococcal genomes (PezAT, RelBE2, YefM-YoeB, and Phd-Doc) showed that RelBE2 and Phd-Doc are the most conserved ones. Further, there was good correlation among TA types, clonal complexes and sequence types in the 48 pneumococcal strains analyzed.

  13. Characterization of the genetic environment of blaESBL genes, integrons and toxin-antitoxin systems identified on large transferrable plasmids in multi-drug resistant Escherichia coli

    PubMed Central

    Wang, Juan; Stephan, Roger; Zurfluh, Katrin; Hächler, Herbert; Fanning, Séamus

    2015-01-01

    Objectives: Previously 14 conjugative plasmids from multi-drug resistant (MDR) Escherichia coli from healthy humans and food-producing animals in Switzerland were sequenced. The aim of this study was to extend the genetic characterization of these plasmids with a focus on blaESBL genes including blaCTX-M-1 and blaTEM, class 1 integrons and toxin-antitoxin (TA) systems contained therein. Methods: The nucleotide sequences and subsequent annotation therein of 14 conjugative plasmids were previously determined from their corresponding transconjugants. The TA loci were confirmed by RASTA-Bacteria. Results: Eight of the conjugative plasmids identified were found to encode genes expressing ESBLs. Structural heterogeneity was noted in the regions flanking both the blaCTX-M-1 and blaTEM genes. The blaCTX-M-1 genes were associated with the common insertion sequences ISEcp1 and IS26, and uniquely with an IS5 element in one case; while blaTEM genes were found to be associated with IS26 and Tn2. A new blaTEM-210 gene was identified. Seven class 1 integrons were also identified and assigned into 3 groups, denoted as In54, In369 and In501. Sixteen TA loci belonging to 4 of the TA gene families (relBE, vapBC, ccd and mazEF) were identified on 11 of these plasmids. Conclusions: Comparative sequence analysis of these plasmids provided data on the structures likely to contribute to sequence diversity associated with these accessory genes, including IS26, ISEcp1 and Tn2. All of them contribute to the dissemination of the corresponding resistance genes located on the different plasmids. There appears to be no association between β-lactam encoding genes and TA systems. PMID:25610429

  14. Adeno-associated virus transfer of a gene encoding SNAP-25 resistant to botulinum toxin A attenuates neuromuscular paralysis associated with botulism.

    PubMed

    Raghunath, Arvind; Perez-Branguli, Francesc; Smith, Leonard; Dolly, J Oliver

    2008-04-02

    Advances in viral gene therapy have opened new possibilities for treating a range of motor neuron diseases, but these have not yet been translated into clinically applicable therapies because of difficulties in delivery to susceptible/damaged neurons, ambiguities in the identity of gene(s) implicated, and a paucity of means to quantify any physiological improvement. Most of these hurdles can be overcome by using the neuromuscular paralysis induced by botulinum neurotoxin type A (BoNT/A) as a prototype disease. Furthermore, because human botulism, occasionally fatal, causes prolonged muscle disablement as a result of the intraneuronal persistence of the toxin's SNAP-25 (S25)-cleaving protease, development of a genetic approach could lead to a potential treatment for this debilitating disease. Adeno-associated viral delivery of a cleavage-resistant S25 gene (S25-R198T) to chromaffin cells in vitro yielded exocytotically active S25-R198T that diminished subsequent blockade by BoNT/A of evoked catecholamine release. Evaluation in vivo, by administering this virus into rat spinal cord before injecting BoNT/A, showed a decreased inhibition of acetylcholine release as reflected in elevated retention of neuromuscular transmission. A similar, although smaller, protection of synaptic transmission from the toxin was seen after peripherally injecting the therapeutic virus. Such therapy also curtailed nerve sprouting normally induced by BoNT/A. This first demonstration of the utility of a DNA-based therapy for botulism paves the way for further advances in its treatment and for application to genetic disorders of motor neurons.

  15. An Integrative Approach to Computational Modelling of the Gene Regulatory Network Controlling Clostridium botulinum Type A1 Toxin Production

    PubMed Central

    Walshaw, John; Peck, Michael W.; Barker, Gary C.

    2016-01-01

    Clostridium botulinum produces botulinum neurotoxins (BoNTs), highly potent substances responsible for botulism. Currently, mathematical models of C. botulinum growth and toxigenesis are largely aimed at risk assessment and do not include explicit genetic information beyond group level but integrate many component processes, such as signalling, membrane permeability and metabolic activity. In this paper we present a scheme for modelling neurotoxin production in C. botulinum Group I type A1, based on the integration of diverse information coming from experimental results available in the literature. Experiments show that production of BoNTs depends on the growth-phase and is under the control of positive and negative regulatory elements at the intracellular level. Toxins are released as large protein complexes and are associated with non-toxic components. Here, we systematically review and integrate those regulatory elements previously described in the literature for C. botulinum Group I type A1 into a population dynamics model, to build the very first computational model of toxin production at the molecular level. We conduct a validation of our model against several items of published experimental data for different wild type and mutant strains of C. botulinum Group I type A1. The result of this process underscores the potential of mathematical modelling at the cellular level, as a means of creating opportunities in developing new strategies that could be used to prevent botulism; and potentially contribute to improved methods for the production of toxin that is used for therapeutics. PMID:27855161

  16. An Integrative Approach to Computational Modelling of the Gene Regulatory Network Controlling Clostridium botulinum Type A1 Toxin Production.

    PubMed

    Ihekwaba, Adaoha E C; Mura, Ivan; Walshaw, John; Peck, Michael W; Barker, Gary C

    2016-11-01

    Clostridium botulinum produces botulinum neurotoxins (BoNTs), highly potent substances responsible for botulism. Currently, mathematical models of C. botulinum growth and toxigenesis are largely aimed at risk assessment and do not include explicit genetic information beyond group level but integrate many component processes, such as signalling, membrane permeability and metabolic activity. In this paper we present a scheme for modelling neurotoxin production in C. botulinum Group I type A1, based on the integration of diverse information coming from experimental results available in the literature. Experiments show that production of BoNTs depends on the growth-phase and is under the control of positive and negative regulatory elements at the intracellular level. Toxins are released as large protein complexes and are associated with non-toxic components. Here, we systematically review and integrate those regulatory elements previously described in the literature for C. botulinum Group I type A1 into a population dynamics model, to build the very first computational model of toxin production at the molecular level. We conduct a validation of our model against several items of published experimental data for different wild type and mutant strains of C. botulinum Group I type A1. The result of this process underscores the potential of mathematical modelling at the cellular level, as a means of creating opportunities in developing new strategies that could be used to prevent botulism; and potentially contribute to improved methods for the production of toxin that is used for therapeutics.

  17. Botulinum Toxin

    DTIC Science & Technology

    2009-01-01

    resulting from antibiotic treatment (Cherington, 1998). Foodbome and inhalational have noninfectious etiologies and are the result of ingesting or...infants Self- Antibiotic -treated All exposed All exposed individuals Patients treated with local toxin injections (2 to 4 months of age) prior to...typically take place except under circumstances where the normal flora has been altered by antibiotic treatment (Cherington, 1998). Botulism results

  18. The influence of intravesical administration of resiniferatoxin (RTX) on the chemical coding of sympathetic chain ganglia (SChG) neurons supplying the porcine urinary bladder.

    PubMed

    Lepiarczyk, Ewa; Majewski, Mariusz; Bossowska, Agnieszka

    2015-11-01

    Resiniferatoxin (RTX) is used as an experimental drug in therapy of neurogenic urinary bladder disorders. The present study investigated the chemical coding of sympathetic chain ganglia (SChG) neurons supplying porcine urinary bladder after intravesical RTX instillation. The SChG neurons were visualized with retrograde tracing method and their chemical profile was disclosed with double-labeling immunohistochemistry using antibodies against dopamine β-hydroxylase (DβH; marker of noradrenergic neurons), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), galanin, Leu(5)-enkephalin and neuronal nitric oxide synthase (nNOS). It was found that in both the control (n = 5) and RTX-treated pigs (n = 5), the vast majority (90.4 ± 2.8 and 89.7 ± 2.3%, respectively) of FB-positive (FB+) nerve cells were DβH+. RTX instillation caused a decrease in the number of FB+/DβH+ neurons immunopositive to NPY (71.1 ± 12.1 vs 43.2 ± 6.7%), VIP (21.3 ± 10.7 vs 5.3 ± 4.3%) or SOM (16.5 ± 4.6 vs 2.3 ± 2.6%) and a distinct increase in the number of FB+/DβH+ neurons immunoreactive to nNOS (0.8 ± 1 vs 5.3 ± 1.9 %). The present study for the first time has provided some information that therapeutic effects of RTX on the mammalian urinary bladder can be partly mediated by SChG neurons.

  19. Toxins-antitoxins: diversity, evolution and function.

    PubMed

    Hayes, Finbarr; Van Melderen, Laurence

    2011-10-01

    Genes for toxin-antitoxin (TA) complexes are widespread in prokaryote genomes, and species frequently possess tens of plasmid and chromosomal TA loci. The complexes are categorized into three types based on genetic organization and mode of action. The toxins universally are proteins directed against specific intracellular targets, whereas the antitoxins are either proteins or small RNAs that neutralize the toxin or inhibit toxin synthesis. Within the three types of complex, there has been extensive evolutionary shuffling of toxin and antitoxin genes leading to considerable diversity in TA combinations. The intracellular targets of the protein toxins similarly are varied. Numerous toxins, many of which are sequence-specific endoribonucleases, dampen protein synthesis levels in response to a range of stress and nutritional stimuli. Key resources are conserved as a result ensuring the survival of individual cells and therefore the bacterial population. The toxin effects generally are transient and reversible permitting a set of dynamic, tunable responses that reflect environmental conditions. Moreover, by harboring multiple toxins that intercede in protein synthesis in response to different physiological cues, bacteria potentially sense an assortment of metabolic perturbations that are channeled through different TA complexes. Other toxins interfere with the action of topoisomersases, cell wall assembly, or cytoskeletal structures. TAs also play important roles in bacterial persistence, biofilm formation and multidrug tolerance, and have considerable potential both as new components of the genetic toolbox and as targets for novel antibacterial drugs.

  20. Real-time microfluidic recombinase polymerase amplification for the toxin B gene of Clostridium difficile on a SlipChip platform.

    PubMed

    Tsaloglou, M-N; Watson, R J; Rushworth, C M; Zhao, Y; Niu, X; Sutton, J M; Morgan, H

    2015-01-07

    Clostridium difficile is one of the key bacterial pathogens that cause infectious diarrhoea both in the developed and developing world. Isothermal nucleic acid amplification methods are increasingly used for identification of toxinogenic infection by clinical labs. For this purpose, we developed a low-cost microfluidic platform based on the SlipChip concept and implemented real-time isothermal recombinase polymerase amplification (RPA). The on-chip RPA assay targets the Clostridium difficile toxin B gene (tcdB) coding for toxin B, one of the proteins responsible for bacterial toxicity. The device was fabricated in clear acrylic using rapid prototyping methods. It has six replicate 500 nL reaction wells as well as two sets of 500 nL control wells. The reaction can be monitored in real-time using exonuclease fluorescent probes with an initial sample volume of as little as 6.4 μL. We demonstrated a limit of detection of 1000 DNA copies, corresponding to 1 fg, at a time-to-result of <20 minutes. This miniaturised platform for pathogen detection has potential for use in resource-limited environments or at the point-of-care because of its ease of use and low cost, particularly if combined with preserved reagents.

  1. Legionella Toxin.

    DTIC Science & Technology

    1981-04-29

    of a cytotoxin produced by Legionella pneumophila. Infect. Immun. 29:271-274. Fumarola, D. (1978) Legionnaires ’ disease agent and Limulus endotoxin... Legionnaires ’ disease bacterium in the AKR/J mouse. Ann. intern. Med. 90:676-679. Hedlund, K. W. and Larson, R. (1981) Legionella pneumophila toxin, isolation... Legionella Species New Name Old Name Lpneumophila Legionnaires ’ disease organism, OLDA L. bozemanil WIGA, MI 15 L. dumoffii NY 23, TEX-KL *L. micdadei

  2. The CRG1 gene required for resistance to the singlet oxygen-generating cercosporin toxin in Cercospora nicotianae encodes a putative fungal transcription factor.

    PubMed

    Chung, Kuang-Ren; Daub, Margaret E; Kuchler, Karl; Schüller, Christoph

    2003-03-07

    The Cercospora nicotianae CRG1 gene is involved in cellular resistance to the perylenequinone toxin, cercosporin, that generates highly toxic singlet oxygen upon exposure to light. The entire open reading frame (ORF) of CRG1 was isolated and sequenced. The gene contains an ORF of 1950bp including a 65-bp intron. The predicted 650 amino acid CRG1 protein contains a Cys(6)Zn(2) binuclear cluster DNA-binding motif with homology to various fungal regulatory proteins, indicating that CRG1 may act functionally as a transcription activator. Targeted gene disruption of CRG1 resulted in mutants that are partially sensitive to cercosporin and reduced in cercosporin production. Genetic complementation revealed that CRG1 fully restored cercosporin resistance, but only slightly restored cercosporin production in a UV-derived mutant (CS10) containing a single nucleotide substitution in crg1. Complementation of a crg1-null mutant, however, yielded strains that are similar to the wild-type in both phenotypes. These results indicate that the transcription regulator CRG1 is involved in the activation of genes associated with cercosporin resistance and production in the fungus Cercospora nicotianae.

  3. Distribution of Shiga toxin genes subtypes in B1 phylotypes of Escherichia coli isolated from calves suffering from diarrhea in Tehran suburb using DNA oligonucleotide arrays

    PubMed Central

    Staji, Hamid; Tonelli, Alfreda; Javaheri-Vayeghan, Abbas; Changizi, Emad; Salimi-Bejestani, Mohammad Reza

    2015-01-01

    Background and Objectives: Shiga toxin-producing Escherichia coli (STEC) have emerged as human pathogens and contamination via animal origin has been a major public health concern. We compared the distribution of phylogenetic groups and prevalence of stx gene variants among the pathogenic strains of Escherichia coli isolated from feces of diarrheatic calves in Tehran suburb farms. Materials and Methods: In this study we screened 140 diarrheatic calves (1–15 days old) for E. coli strains during a 3 months period of time. The isolated strains were grouped into different phylotypes according to the presence of chuA, yjaA and TSPE4.C2 genes. Then, the prevalence of stx gene subtypes was evaluated in the B1 phylotypes. Results: From diarrheatic calves, 51 bacterial isolates were biochemically identified as E. coli and 31 isolates out of 51 were considered B1 phylotype using DNA Microarray technology. Of these isolates, 20 contained stx1a and stx1b and one harbored all mentioned variants of stx genes except stx2b2. Conclusion: This study showed that in Tehran suburb, the B1 phylotype of E. coli is prevalent as a causative agent of diarrhea in calves and the prevalence of stx1 gene subtypes is dominant in comparison with other subtypes. Considering the possibility that these stx genes can be spread to other strains, bovine E. coli strains are an important source of stx genes for other strains and further study and surveillance seems to be required for the exact identification of virulence profile of E. coli phylotypes in different hosts. PMID:26697157

  4. Coexpression of the silent cry2Ab27 together with cry1 genes in Bacillus thuringiensis subsp. aizawai SP41 leads to formation of amorphous crystal toxin and enhanced toxicity against Helicoverpa armigera.

    PubMed

    Somwatcharajit, Rasapirose; Tiantad, Itsares; Panbangred, Watanalai

    2014-02-01

    The unexpressed cry2Ab27 gene of Bacillus thuringiensis subsp. aizawai SP41 (SP41) consists of a single open reading frame (ORF) of 1902bp encoding for 634 amino acid residues. The cry2Ab27 gene appears to be silent due to the lack of promoter and terminator sequences. In this study we fused the cry2Ab27 ORF with the cry1Ab promoter (500bp) and the terminator (300bp) in vector pHT304-18Z in order to drive the expression of cry2Ab27 in both SP41 and an acrystaliferous, B. thuringiensis subsp. thuringiensis 407 (407). A protein with a molecular mass of 65kDa, consistent with the Cry2Ab protein, was detected in both transformants using SDS-PAGE and Western blot analysis. Bipyramidal crystals were observed in SP41 and its transformant containing the pHT304-18Z vector (SPHT) in contrast, cells expressing cry2Ab27 (SPC2) exhibited crystal proteins with irregular shapes. No inclusion protein was detected in the 407 transformant expressing the cry2Ab27 gene. Cry2Ab27 was found in the purified crystal toxin from strain SPC2. The solubilized crystal toxin proteins from SPC2 were 6.9-fold more toxic toward the larvae of Helicoverpa armigera compared to toxin proteins from SPHT. However SPC2 crystal toxin displayed only slightly higher toxicity against the larvae of Spodoptera litura and S. exigua compared to SPHT produced toxin. Our data support the use of Cry2Ab in combination with the Cry1 toxin for enhanced control of heliothine insect pests.

  5. Detection of a group II intron without an open reading frame in the alpha-toxin gene of Clostridium perfringens isolated from a broiler chicken.

    PubMed

    Ma, Menglin; Ohtani, Kaori; Shimizu, Tohru; Misawa, Naoaki

    2007-03-01

    A DNA insertion of 834 bp, designated CPF-G2Im, was identified within the alpha toxin gene (cpa) of Clostridium perfringens strain CPBC16ML, isolated from a broiler chicken. Sequence analysis of CPF-G2Im indicated that it was integrated 340 nucleotides downstream of the start codon of cpa. However, the insertion did not abolish the phospholipase C and hemolytic activities of CPBC16ML. To investigate the expression of its alpha toxin, the intact copy of cpa was cloned into an expression vector and transformed into Escherichia coli M15 cells. Immunoblotting analysis showed that the protein expressed from the transformant as well as in the culture supernatant of C. perfringens strain CPBC16ML had the expected molecular weight detected in reference strains of C. perfringens. Northern hybridization and reverse transcriptase PCR (RT-PCR) analysis revealed that the entire CPF-G2Im insertion was completely spliced from the cpa precursor mRNA transcripts. The sequence of the insertion fragment has 95% and 97% identity to two noncoding regions corresponding to sequences that flank a predicted group II RT gene present in the pCPF4969 plasmid of C. perfringens. However, an RT was not encoded by the CPF-G2Im fragment. Based on the secondary structure prediction analysis, CPF-G2Im revealed typical features of group II introns. The present study shows that CPF-G2Im is capable of splicing in both C. perfringens and E. coli. To our knowledge, this is the first report that a group II intron without an open reading frame (ORF) is located in the cpa ORF of C. perfringens.

  6. A Novel Pathogenicity Island Integrated Adjacent to the thrW tRNA Gene of Avian Pathogenic Escherichia coli Encodes a Vacuolating Autotransporter Toxin

    PubMed Central

    Parreira, V. R.; Gyles, C. L.

    2003-01-01

    We report the complete nucleotide sequence and genetic organization of the Vat-encoding pathogenicity island (PAI) of avian pathogenic Escherichia coli strain Ec222. The 22,139-bp PAI is situated adjacent to the 3′ terminus of the thrW tRNA gene, has a G+C content of 41.2%, and includes a bacteriophage SfII integrase gene, mobile genetic elements, two open reading frames with products exhibiting sequence similarity to known proteins, and several other open reading frames of unknown function. The PAI encodes an autotransporter protein, Vat (vacuolating autotransporter toxin), which induces the formation of intracellular vacuoles resulting in cytotoxic effects similar to those caused by the VacA toxin from Helicobacter pylori. The predicted 148.3-kDa protein product possesses the three domains that are typical of serine protease autotransporters of Enterobacteriaceae: an N-terminal signal sequence of 55 amino acids, a 111.8-kDa passenger domain containing a modified serine protease site (ATSGSG), and a C-terminal outer membrane translocator of 30.5 kDa. Vat has 75% protein homology with the hemagglutinin Tsh, an autotransporter of avian pathogenic E. coli. A vat deletion mutant of Ec222 showed no virulence in respiratory and cellulitis infection models of disease in broiler chickens. We conclude that the newly described PAI and Vat may be involved in the pathogenicity of avian septicemic E. coli strain Ec222 and other avian pathogenic E. coli strains. PMID:12933851

  7. Genomics Study of the Exposure Effect of Gymnodinium catenatum, a Paralyzing Toxin Producer, on Crassostrea gigas' Defense System and Detoxification Genes

    PubMed Central

    García-Lagunas, Norma; Romero-Geraldo, Reyna; Hernández-Saavedra, Norma Y.

    2013-01-01

    Background Crassostrea gigas accumulates paralytic shellfish toxins (PST) associated with red tide species as Gymnodinium catenatum. Previous studies demonstrated bivalves show variable feeding responses to toxic algae at physiological level; recently, only one study has reported biochemical changes in the transcript level of the genes involved in C. gigas stress response. Principal Findings We found that 24 h feeding on toxic dinoflagellate cells (acute exposure) induced a significant decrease in clearance rate and expression level changes of the genes involved in antioxidant defense (copper/zinc superoxide dismutase, Cu/Zn-SOD), cell detoxification (glutathione S-transferase, GST and cytochrome P450, CPY450), intermediate immune response activation (lipopolysaccharide and beta glucan binding protein, LGBP), and stress responses (glutamine synthetase, GS) in Pacific oysters compared to the effects with the non-toxic microalga Isochrysis galbana. A sub-chronic exposure feeding on toxic dinoflagellate cells for seven and fourteen days (30×103 cells mL−1) showed higher gene expression levels. A significant increase was observed in Cu/Zn-SOD, GST, and LGBP at day 7 and a major increase in GS and CPY450 at day 14. We also observed that oysters fed only with G. catenatum (3×103 cells mL−1) produced a significant increase on the transcription level than in a mixed diet (3×103 cells mL−1 of G. catenatum+0.75×106 cells mL−1 I. galbana) in all the analyzed genes. Conclusions Our results provide gene expression data of PST producer dinoflagellate G. catenatum toxic effects on C. gigas, a commercially important bivalve. Over expressed genes indicate the activation of a potent protective mechanism, whose response depends on both cell concentration and exposure time against these toxic microalgae. Given the importance of dinoflagellate blooms in coastal environments, these results provide a more comprehensive overview of how oysters respond to stress generated by

  8. Two different Bacillus thuringiensis toxin genes confer resistance to beet armyworm (Spodoptera exigua Hübner) in transgenic Bt-shallots (Allium cepa L.).

    PubMed

    Zheng, Si-Jun; Henken, Betty; de Maagd, Ruud A; Purwito, Agus; Krens, Frans A; Kik, Chris

    2005-06-01

    Agrobacterium-mediated genetic transformation was applied to produce beet armyworm (Spodoptera exigua Hübner) resistant tropical shallots (Allium cepa L. group Aggregatum). A cry1Ca or a H04 hybrid gene from Bacillus thuringiensis, driven by the chrysanthemum ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (Rubisco SSU) promoter, along with the hygromycin phosphotransferase gene (hpt) driven by the CaMV 35S promoter, was employed for genetic transformation. An average transformation frequency of 3.68% was obtained from two shallot cultivars, Tropix and Kuning. After transfer of the in vitro plants to the greenhouse 69% of the cry1Ca and 39% of the H04 transgenic shallots survived the first half year. After one year of cultivation in the greenhouse the remaining cry1Ca and H04 transgenic plants grew vigorously and had a normal bulb formation, although the cry1Ca transgenic plants (and controls) had darker green leaves compared to their H04 counterparts. Standard PCR, adaptor ligation PCR and Southern analyses confirmed the integration of T-DNA into the shallot genome. Northern blot and ELISA analyses revealed expression of the cry1Ca or H04 gene in the transgenic plants. The amount of Cry1Ca expressed in transgenic plants was higher than the expression levels of H04 (0.39 vs. 0.16% of the total soluble leaf proteins, respectively). There was a good correlation between protein expression and beet armyworm resistance. Cry1Ca or H04 gene expression of at least 0.22 or 0.08% of the total soluble protein in shallot leaves was sufficient to give a complete resistance against beet armyworm. This confirms earlier observations that the H04 toxin is more toxic to S. exigua than the Cry1Ca toxin. The results from this study suggest that the cry1Ca and H04 transgenic shallots developed could be used for introducing resistance to beet armyworm in (sub) tropical shallot.

  9. Evidence for Contemporary Switching of the O-Antigen Gene Cluster between Shiga Toxin-Producing Escherichia coli Strains Colonizing Cattle

    PubMed Central

    Geue, Lutz; Menge, Christian; Eichhorn, Inga; Semmler, Torsten; Wieler, Lothar H.; Pickard, Derek; Berens, Christian; Barth, Stefanie A.

    2017-01-01

    Shiga toxin-producing Escherichia coli (STEC) comprise a group of zoonotic enteric pathogens with ruminants, especially cattle, as the main reservoir. O-antigens are instrumental for host colonization and bacterial niche adaptation. They are highly immunogenic and, therefore, targeted by the adaptive immune system. The O-antigen is one of the most diverse bacterial cell constituents and variation not only exists between different bacterial species, but also between individual isolates/strains within a single species. We recently identified STEC persistently infecting cattle and belonging to the different serotypes O156:H25 (n = 21) and O182:H25 (n = 15) that were of the MLST sequence types ST300 or ST688. These STs differ by a single nucleotide in purA only. Fitness-, virulence-associated genome regions, and CRISPR/CAS (clustered regularly interspaced short palindromic repeats/CRISPR associated sequence) arrays of these STEC O156:H25 and O182:H25 isolates were highly similar, and identical genomic integration sites for the stx converting bacteriophages and the core LEE, identical Shiga toxin converting bacteriophage genes for stx1a, identical complete LEE loci, and identical sets of chemotaxis and flagellar genes were identified. In contrast to this genomic similarity, the nucleotide sequences of the O-antigen gene cluster (O-AGC) regions between galF and gnd and very few flanking genes differed fundamentally and were specific for the respective serotype. Sporadic aEPEC O156:H8 isolates (n = 5) were isolated in temporal and spatial proximity. While the O-AGC and the corresponding 5′ and 3′ flanking regions of these aEPEC isolates were identical to the respective region in the STEC O156:H25 isolates, the core genome, the virulence associated genome regions and the CRISPR/CAS elements differed profoundly. Our cumulative epidemiological and molecular data suggests a recent switch of the O-AGC between isolates with O156:H8 strains having served as DNA donors. Such

  10. Subtilase cytotoxin encoding genes are present in human, sheep and deer intimin-negative, Shiga toxin-producing Escherichia coli O128:H2.

    PubMed

    Sánchez, Sergio; Beristain, Xabier; Martínez, Remigio; García, Alfredo; Martín, Carmen; Vidal, Dolors; Díaz-Sánchez, Sandra; Rey, Joaquín; Alonso, Juan M; Herrera-León, Silvia

    2012-10-12

    Shiga toxin-producing Escherichia coli (STEC) O128:H2 is recognised worldwide to be an important non-O157 STEC associated with human illness and in particular with causing haemolytic uraemic syndrome. This serotype is commonly isolated from sheep and is being increasingly isolated from deer. We determined the virulence profile and genetic relationships of one human, six sheep and five deer intimin-negative STEC O128:H2 strains isolated in Spain over a 7-year period. Our goals were to establish the presence of other virulence-associated factors, such as SubAB, in intimin-negative STEC O128:H2 strains involved in human disease and in that case, to determine if sheep and/or deer represent a reservoir of SubAB-positive STEC O128:H2. All the strains lacked the eae gene and carried subtilase cytotoxin (SubAB) encoding genes (subAB) and tia genes, but not saa gene, suggesting the presence of the recently identified new variant of SubAB, encoded on a putative pathogenicity island together with tia. We report for the first time the presence of subtilase cytotoxin encoding genes in intimin-negative STEC O128:H2 strains pathogenic for humans and how this finding might explain their clinical relevance despite neither carrying eae nor stx subtypes associated with severe clinical outcomes, but only stx1c and stx2b. Multilocus sequence typing analysis revealed that STEC O128:H2 strains from sheep and deer belong to the clonal lineage of STEC O128:H2 strains involved in diarrhoeal and haemorrhagic diseases in humans. Our results indicate that sheep and deer represent a reservoir of SubAB-positive STEC O128:H2 strains and thus a potential source of human infection.

  11. Transcriptional Profiling of Type II Toxin-Antitoxin Genes of Helicobacter pylori under Different Environmental Conditions: Identification of HP0967-HP0968 System.

    PubMed

    Cárdenas-Mondragón, María G; Ares, Miguel A; Panunzi, Leonardo G; Pacheco, Sabino; Camorlinga-Ponce, Margarita; Girón, Jorge A; Torres, Javier; De la Cruz, Miguel A

    2016-01-01

    Helicobacter pylori is a Gram-negative bacterium that colonizes the human gastric mucosa and is responsible for causing peptic ulcers and gastric carcinoma. The expression of virulence factors allows the persistence of H. pylori in the stomach, which results in a chronic, sometimes uncontrolled inflammatory response. Type II toxin-antitoxin (TA) systems have emerged as important virulence factors in many pathogenic bacteria. Three type II TA systems have previously been identified in the genome of H. pylori 26695: HP0315-HP0316, HP0892-HP0893, and HP0894-HP0895. Here we characterized a heretofore undescribed type II TA system in H. pylori, HP0967-HP0968, which is encoded by the bicistronic operon hp0968-hp0967 and belongs to the Vap family. The predicted HP0967 protein is a toxin with ribonuclease activity whereas HP0968 is an antitoxin that binds to its own regulatory region. We found that all type II TA systems were expressed in H. pylori during early stationary growth phase, and differentially expressed in the presence of urea, nickel, and iron, although, the hp0968-hp0967 pair was the most affected under these environmental conditions. Transcription of hp0968-hp0967 was strongly induced in a mature H. pylori biofilm and when the bacteria interacted with AGS epithelial cells. Kanamycin and chloramphenicol considerably boosted transcription levels of all the four type II TA systems. The hp0968-hp0967 TA system was the most frequent among 317 H. pylori strains isolated from all over the world. This study is the first report on the transcription of type II TA genes in H. pylori under different environmental conditions. Our data show that the HP0967 and HP0968 proteins constitute a bona fide type II TA system in H. pylori, whose expression is regulated by environmental cues, which are relevant in the context of infection of the human gastric mucosa.

  12. Design and implementation of laser target simulator in hardware-in-the-loop simulation system based on LabWindows/CVI and RTX

    NASA Astrophysics Data System (ADS)

    Tong, Qiujie; Wang, Qianqian; Li, Xiaoyang; Shan, Bin; Cui, Xuntai; Li, Chenyu; Peng, Zhong

    2016-11-01

    In order to satisfy the requirements of the real-time and generality, a laser target simulator in semi-physical simulation system based on RTX+LabWindows/CVI platform is proposed in this paper. Compared with the upper-lower computers simulation platform architecture used in the most of the real-time system now, this system has better maintainability and portability. This system runs on the Windows platform, using Windows RTX real-time extension subsystem to ensure the real-time performance of the system combining with the reflective memory network to complete some real-time tasks such as calculating the simulation model, transmitting the simulation data, and keeping real-time communication. The real-time tasks of simulation system run under the RTSS process. At the same time, we use the LabWindows/CVI to compile a graphical interface, and complete some non-real-time tasks in the process of simulation such as man-machine interaction, display and storage of the simulation data, which run under the Win32 process. Through the design of RTX shared memory and task scheduling algorithm, the data interaction between the real-time tasks process of RTSS and non-real-time tasks process of Win32 is completed. The experimental results show that this system has the strongly real-time performance, highly stability, and highly simulation accuracy. At the same time, it also has the good performance of human-computer interaction.

  13. Botulinum toxin, Quo Vadis?

    PubMed

    Lim, Erle C H; Seet, Raymond C S

    2007-01-01

    Botulinum toxin (BTX), derived from the exotoxin of Clostridium botulinum, cleaves Soluble N-ethylmaleimide-sensitive factor-Attachment protein REceptor (SNARE) proteins, causing chemodenervation of cholinergic neurons. BTX also inhibits exocytosis of vesicles containing norepinephrine, glutamate, substance P and calcitonin gene-related peptide (CGRP) and inhibits expression of the vanilloid receptor. Clinical applications of BTX, which include the treatment of overactive skeletal and smooth muscles, hypersecretory and painful disorders, have increased exponentially since it was first used clinically to treat strabismus more than two decades ago. In this editorial, we discuss reports of new therapeutic indications of BTX, and propose new areas for research.

  14. PolySearch2: a significantly improved text-mining system for discovering associations between human diseases, genes, drugs, metabolites, toxins and more.

    PubMed

    Liu, Yifeng; Liang, Yongjie; Wishart, David

    2015-07-01

    PolySearch2 (http://polysearch.ca) is an online text-mining system for identifying relationships between biomedical entities such as human diseases, genes, SNPs, proteins, drugs, metabolites, toxins, metabolic pathways, organs, tissues, subcellular organelles, positive health effects, negative health effects, drug actions, Gene Ontology terms, MeSH terms, ICD-10 medical codes, biological taxonomies and chemical taxonomies. PolySearch2 supports a generalized 'Given X, find all associated Ys' query, where X and Y can be selected from the aforementioned biomedical entities. An example query might be: 'Find all diseases associated with Bisphenol A'. To find its answers, PolySearch2 searches for associations against comprehensive collections of free-text collections, including local versions of MEDLINE abstracts, PubMed Central full-text articles, Wikipedia full-text articles and US Patent application abstracts. PolySearch2 also searches 14 widely used, text-rich biological databases such as UniProt, DrugBank and Human Metabolome Database to improve its accuracy and coverage. PolySearch2 maintains an extensive thesaurus of biological terms and exploits the latest search engine technology to rapidly retrieve relevant articles and databases records. PolySearch2 also generates, ranks and annotates associative candidates and present results with relevancy statistics and highlighted key sentences to facilitate user interpretation.

  15. Immunogenicity and virulence of attenuated vaccinia virus Tian Tan encoding HIV-1 muti-epitope genes, p24 and cholera toxin B subunit in mice.

    PubMed

    Du, Shouwen; Wang, Yuhang; Liu, Cunxia; Wang, Maopeng; Zhu, Yilong; Tan, Peng; Ren, Dayong; Li, Xiao; Tian, Mingyao; Yin, Ronglan; Li, Chang; Jin, Ningyi

    2015-07-01

    No effective prophylactic or therapeutic vaccine against HIV-1 in humans is currently available. This study analyzes the immunogenicity and safety of a recombinant attenuated vaccinia virus. A chimeric gene of HIV-1 multi-epitope genes containing CpG ODN and cholera toxin B subunit (CTB) was inserted into Chinese vaccinia virus Tian Tan strain (VTT) mutant strain. The recombinant virus rddVTT(-CCMp24) was assessed for immunogenicity and safety in mice. Results showed that the protein CCMp24 was expressed stably in BHK-21 infected with rddVTT(-CCMp24). And the recombinant virus induced the production of HIV-1 p24 specific immunoglobulin G (IgG), IL-2 and IL-4. The recombinant vaccine induced γ-interferon secretion against HIV peptides, and elicited a certain levels of immunological memory. Results indicated that the recombinant virus had certain immunogenicity to HIV-1. Additionally, the virulence of the recombinant virus was been attenuated in vivo of mice compared with wild type VTT (wtVTT), and the introduction of CTB and HIV Mp24 did not alter the infectivity and virulence of defective vaccinia virus.

  16. PolySearch2: a significantly improved text-mining system for discovering associations between human diseases, genes, drugs, metabolites, toxins and more

    PubMed Central

    Liu, Yifeng; Liang, Yongjie; Wishart, David

    2015-01-01

    PolySearch2 (http://polysearch.ca) is an online text-mining system for identifying relationships between biomedical entities such as human diseases, genes, SNPs, proteins, drugs, metabolites, toxins, metabolic pathways, organs, tissues, subcellular organelles, positive health effects, negative health effects, drug actions, Gene Ontology terms, MeSH terms, ICD-10 medical codes, biological taxonomies and chemical taxonomies. PolySearch2 supports a generalized ‘Given X, find all associated Ys’ query, where X and Y can be selected from the aforementioned biomedical entities. An example query might be: ‘Find all diseases associated with Bisphenol A’. To find its answers, PolySearch2 searches for associations against comprehensive collections of free-text collections, including local versions of MEDLINE abstracts, PubMed Central full-text articles, Wikipedia full-text articles and US Patent application abstracts. PolySearch2 also searches 14 widely used, text-rich biological databases such as UniProt, DrugBank and Human Metabolome Database to improve its accuracy and coverage. PolySearch2 maintains an extensive thesaurus of biological terms and exploits the latest search engine technology to rapidly retrieve relevant articles and databases records. PolySearch2 also generates, ranks and annotates associative candidates and present results with relevancy statistics and highlighted key sentences to facilitate user interpretation. PMID:25925572

  17. Expression of the cercosporin toxin resistance gene ( CRG1) as a dicistronic mRNA in the filamentous fungus Cercospora nicotianae.

    PubMed

    Chung, Kuang-Ren; Daub, Margaret E; Ehrenshaft, Marilyn

    2003-09-01

    The CRG1 gene in Cercospora nicotianae encodes a transcription factor and is required for cercosporin toxin resistance and production. Cloning and sequencing of the downstream region of the CRG1 gene led to the discovery of an adjacent gene ( PUT1) encoding a putative uracil transporter. Expression of CRG1 and PUT1 as assessed by Northern analysis indicated that, in addition to the expected monocistronic mRNAs (2.6 kb and 2.0 kb, respectively), a common 4.5-kb mRNA could be identified, using either a CRG1 or a PUT1 gene probe. The 2.6-kb transcript identified only by the CRG1 probe was expressed constitutively, whereas the 2.0-kb transcript identified only by the PUT1 probe was differentially expressed in various media. Four cDNA clones containing CRG1, PUT1, and the CRG1- PUT1 intergenic region were identified as part of the products from the 4.5-kb transcript. Both the 4.5-kb and 2.6-kb transcripts were not detectable in three crg1-disrupted mutants, using the CRG1 probe. The 2.0-kb transcript, but not the 4.5-kb one was detected using the PUT1 probe in the three crg1-disrupted mutants. Taken together, we conclude that the 4.5-kb transcript is a dicistronic mRNA of both CRG1 and PUT1 in the fungus C. nicotianae. This is the first example of a dicistronic mRNA identified in filamentous fungi.

  18. Gene and antigen markers of Shiga-toxin producing E. coli from Michigan and Indiana river water: Occurrence and relation to recreational water quality criteria

    USGS Publications Warehouse

    Duris, J.W.; Haack, S.K.; Fogarty, L.R.

    2009-01-01

    The relation of bacterial pathogen occurrence to fecal indicator bacteria (FIB) concentrations used for recreational water quality criteria (RWQC) is poorly understood. This study determined the occurrence of Shiga-toxin producing Escherichia coli (STEC) markers and their relation to FIB concentrations in Michigan and Indiana river water. Using 67 fecal coliform (FC) bacteria cultures from 41 river sites in multiple watersheds, we evaluated the occurrence of five STEC markers: the Escherichia coli (EC) O157 antigen and gene, and the STEC virulence genes eaeA, stx1, and stx2. Simple isolations from selected FC cultures yielded viable EC O157. By both antigen and gene assays, EC O157 was detected in a greater proportion of samples exceeding rather than meeting FC RWQC (P < 0.05), but was unrelated to EC and enterococci RWQC. The occurrence of all other STEC markers was unrelated to any FIB RWQC. The eaeA, stx2, and stx1 genes were found in 93.3, 13.3, and in 46.7% of samples meeting FC RWQC and in 91.7, 0.0, and 37.5% of samples meeting the EC RWQC. Although not statistically significant, the percentage of samples positive for each STEC marker except stx1 was lower in samples that met, as opposed to exceeded, FIB RWQC. Viable STEC were common members of the FC communities in river water throughout southern Michigan and northern Indiana, regardless of FIB RWQC. Our study indicates that further information on the occurrence of pathogens in recreational waters, and research on alternative indicators of their occurrence, may help inform water-resource management and public health decision-making. Copyright ?? 2009 by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America. All rights reserved.

  19. Activation of syndecan-1 ectodomain shedding by Staphylococcus aureus alpha-toxin and beta-toxin.

    PubMed

    Park, Pyong Woo; Foster, Timothy J; Nishi, Eiichiro; Duncan, Sheila J; Klagsbrun, Michael; Chen, Ye

    2004-01-02

    Exploitation of host components by microbes to promote their survival in the hostile host environment has been a recurring theme in recent years. Available data indicate that bacterial pathogens activate ectodomain shedding of host cell surface molecules to enhance their virulence. We reported previously that several major bacterial pathogens activate ectodomain shedding of syndecan-1, the major heparan sulfate proteoglycan of epithelial cells. Here we define the molecular basis of how Staphylococcus aureus activates syndecan-1 shedding. We screened mutant S. aureus strains devoid of various toxin and protease genes and found that only strains lacking both alpha-toxin and beta-toxin genes do not stimulate shedding. Mutations in the agr global regulatory locus, which positively regulates expression of alpha- and beta-toxins and other exoproteins, also abrogated the capacity to stimulate syndecan-1 shedding. Furthermore, purified S. aureus alpha- and beta-toxins, but not enterotoxin A and toxic shock syndrome toxin-1, rapidly potentiated shedding in a concentration-dependent manner. These results establish that S. aureus activates syndecan-1 ectodomain shedding via its two virulence factors, alpha- and beta-toxins. Toxin-activated shedding was also selectively inhibited by antagonists of the host cell shedding mechanism, indicating that alpha- and beta-toxins shed syndecan-1 ectodomains through stimulation of the host cell's shedding machinery. Interestingly, beta-toxin, but not alpha-toxin, also enhanced ectodomain shedding of syndecan-4 and heparin-binding epidermal growth factor. Because shedding of these ectodomains has been implicated in promoting bacterial pathogenesis, activation of ectodomain shedding by alpha-toxin and beta-toxin may be a previously unknown virulence mechanism of S. aureus.

  20. Target-Driven Evolution of Scorpion Toxins.

    PubMed

    Zhang, Shangfei; Gao, Bin; Zhu, Shunyi

    2015-10-07

    It is long known that peptide neurotoxins derived from a diversity of venomous animals evolve by positive selection following gene duplication, yet a force that drives their adaptive evolution remains a mystery. By using maximum-likelihood models of codon substitution, we analyzed molecular adaptation in scorpion sodium channel toxins from a specific species and found ten positively selected sites, six of which are located at the core-domain of scorpion α-toxins, a region known to interact with two adjacent loops in the voltage-sensor domain (DIV) of sodium channels, as validated by our newly constructed computational model of toxin-channel complex. Despite the lack of positive selection signals in these two loops, they accumulated extensive sequence variations by relaxed purifying selection in prey and predators of scorpions. The evolutionary variability in the toxin-bound regions of sodium channels indicates that accelerated substitutions in the multigene family of scorpion toxins is a consequence of dealing with the target diversity. This work presents an example of atypical co-evolution between animal toxins and their molecular targets, in which toxins suffered from more prominent selective pressure from the channels of their competitors. Our discovery helps explain the evolutionary rationality of gene duplication of toxins in a specific venomous species.

  1. Characterization of clinical Clostridium difficile isolates by PCR ribotyping and detection of toxin genes in Austria, 2006-2007.

    PubMed

    Indra, A; Schmid, D; Huhulescu, S; Hell, M; Gattringer, R; Hasenberger, P; Fiedler, A; Wewalka, G; Allerberger, F

    2008-06-01

    In order to assess the lethality of Clostridium difficile-associated disease (CDAD) and the PCR ribotypes prevalent in Austria, the Austrian Agency for Health and Food Safety requested isolates of C. difficile from patients in a structured but arbitrary sampling scheme. In the allocated period from February 2006 to January 2007, local hospital laboratories within each of the nine provinces were asked to submit C. difficile isolates from at least ten cases of CDAD. Confirmation of species identification, toxin detection, susceptibility testing against four antimicrobial agents and typing using a PCR ribotyping method were performed at the reference laboratory. In total, 149 isolates of putative C. difficile were submitted, from which 142 were included for study. Antimicrobial susceptibility patterns revealed resistance to clindamycin in 57% and high-level resistance to moxifloxacin in 38% of isolates tested. CDAD manifested as diarrhoea (including eight cases of bloody diarrhoea) in 126 cases (88.7%), as pseudomembranous colitis in 15 cases (10.6%) and as toxic megacolon in one case. Twelve of the 142 patients died within 30 days of specimen collection (8.45% lethality). A lethal outcome occurred in 2/15 cases (13.3%) when pseudomembranous colitis was present and in 10/126 cases (7.9%) in the absence of pseudomembranous colitis or toxic megacolon. Among the 142 isolates from 25 health-care facilities, 41 PCR ribotype patterns were found. The most frequent ribotypes were AI-5 (including six lethal cases out of 26 patients), 014 (two out of 24) and 053 (one out of 24). The typing patterns demonstrated the occurrence of clusters in hospitals.

  2. Role of the RS1 sequence of the cholera vibrio in amplification of the segment of plasmid DNA carrying the gene of resistance to tetracycline and the genes of cholera toxin

    SciTech Connect

    Fil'kova, S.L.; Il'ina, T.S.; Gintsburg, A.L.; Yanishevskii, N.V.; Smirnov, G.B.

    1988-11-01

    The hybrid plasmid pCO107, representing cointegrate 14(2)-5(2) of two plasmids, an F-derivative (pOX38) and a PBR322-derivative (pCT105) with an RS1 sequence of the cholera vibrio cloned in its makeup, contains two copes of RS1 at the sites of union of the two plasmids. Using a tetracycline resistance marker (Tc/sup R/) of the plasmid pCT105, clones were isolated which have an elevated level of resistance to tetracycline (an increase of from 4- to 30-fold). Using restriction analysis and the Southern blot method of hybridization it was shown that the increase in the level of resistance of tetracycline is associated with the amplification of pCT105 portion of the cointegrate, and that the process of amplification is governed by the presence of direct repeats of the RS1 sequence at its ends. The increase in the number of copies of the pCT105 segment, which contains in its composition the genes of cholera toxin (vct), is accompanied by an increase in toxin production.

  3. Effects of In Vitro Exposure to Diarrheic Toxin Producer Prorocentrum lima on Gene Expressions Related to Cell Cycle Regulation and Immune Response in Crassostrea gigas

    PubMed Central

    de Jesús Romero-Geraldo, Reyna; García-Lagunas, Norma; Hernández-Saavedra, Norma Yolanda

    2014-01-01

    Background Crassostrea gigas accumulates diarrheic shellfish toxins (DSP) associated to Prorocentrum lima of which Okadaic acid (OA) causes specific inhibitions of serine and threonine phosphatases 1 and 2A. Its toxic effects have been extensively reported in bivalve mollusks at cellular and physiological levels, but genomic approaches have been scarcely studied. Methodology/Principal Findings Acute and sub-chronic exposure effects of P. lima were investigated on farmed juvenile C. gigas (3–5 mm). The Pacific oysters were fed with three dinoflagellate concentrations: 0.3, 3, and 30×103 cells mL−1 along with a nontoxic control diet of Isochrysis galbana. The effects of P. lima on C. gigas were followed by analyzing expression levels of a total of four genes, three involved in cell cycle regulation and one in immune response by polymerase chain reaction and real time quantitative PCR, where changes in time and cell concentration were found. The highest expression levels were found in oysters fed 3×103 cells mL−1 at 168 h for the cycle regulator p21 protein (9 fold), chromatin assembly factor 1 p55 subunit (8 fold), elongation factor 2 (2 fold), and lipopolysaccharide/β-1, 3 glucan binding protein (13 fold above base line). Additionally, the transcript level of all the genes decreased in oysters fed wich the mixed diet 30×103 cells mL−1 of dinoflagellate after 72 h and was lowest in the chromatin assembly factor 1 p55 subunit (0.9 fold below baseline). Conclusions On C. gigas the whole cell ingestion of P lima caused a clear mRNA modulation expression of the genes involved in cell cycle regulation and immune system. Over-expression could be related to DNA damage, disturbances in cell cycle continuity, probably a genotoxic effect, as well as an activation of its innate immune system as first line of defense. PMID:24825133

  4. High Specificity of a Quantitative PCR Assay Targeting a Saxitoxin Gene for Monitoring Toxic Algae Associated with Paralytic Shellfish Toxins in the Yellow Sea

    PubMed Central

    Gao, Yan; Murray, Shauna A.; Chen, Jian-Hua; Kang, Zhen-Jun; Zhang, Qing-Chun; Kong, Fan-Zhou; Zhou, Ming-Jiang

    2015-01-01

    The identification of core genes involved in the biosynthesis of saxitoxin (STX) offers a great opportunity to detect toxic algae associated with paralytic shellfish toxins (PST). In the Yellow Sea (YS) in China, both toxic and nontoxic Alexandrium species are present, which makes it a difficult issue to specifically monitor PST-producing toxic algae. In this study, a quantitative PCR (qPCR) assay targeting sxtA4, a domain in the sxt gene cluster that encodes a unique enzyme involved in STX biosynthesis, was applied to analyze samples collected from the YS in spring of 2012. The abundance of two toxic species within the Alexandrium tamarense species complex, i.e., A. fundyense and A. pacificum, was also determined with TaqMan-based qPCR assays, and PSTs in net-concentrated phytoplankton samples were analyzed with high-performance liquid chromatography coupled with a fluorescence detector. It was found that the distribution of the sxtA4 gene in the YS was consistent with the toxic algae and PSTs, and the quantitation results of sxtA4 correlated well with the abundance of the two toxic species (r = 0.857). These results suggested that the two toxic species were major PST producers during the sampling season and that sxtA-based qPCR is a promising method to detect toxic algae associated with PSTs in the YS. The correlation between PST levels and sxtA-based qPCR results, however, was less significant (r = 0.552), implying that sxtA-based qPCR is not accurate enough to reflect the toxicity of PST-producing toxic algae. The combination of an sxtA-based qPCR assay and chemical means might be a promising method for monitoring toxic algal blooms. PMID:26231652

  5. Neuroprotective Effect of Non-viral Gene Therapy Treatment Based on Tetanus Toxin C-fragment in a Severe Mouse Model of Spinal Muscular Atrophy.

    PubMed

    Oliván, Sara; Calvo, Ana C; Rando, Amaya; Herrando-Grabulosa, Mireia; Manzano, Raquel; Zaragoza, Pilar; Tizzano, Eduardo F; Aquilera, Jose; Osta, Rosario

    2016-01-01

    Spinal muscular atrophy (SMA) is a hereditary childhood disease that causes paralysis and progressive degeneration of skeletal muscles and spinal motor neurons. SMA is associated with reduced levels of full-length Survival of Motor Neuron (SMN) protein, due to mutations in the Survival of Motor Neuron 1 gene. Nowadays there are no effective therapies available to treat patients with SMA, so our aim was to test whether the non-toxic carboxy-terminal fragment of tetanus toxin heavy chain (TTC), which exhibits neurotrophic properties, might have a therapeutic role or benefit in SMA. In this manuscript, we have demonstrated that TTC enhance the SMN expression in motor neurons "in vitro" and evaluated the effect of intramuscular injection of TTC-encoding plasmid in the spinal cord and the skeletal muscle of SMNdelta7 mice. For this purpose, we studied the weight and the survival time, as well as, the survival and cell death pathways and muscular atrophy. Our results showed that TTC treatment reduced the expression of autophagy markers (Becn1, Atg5, Lc3, and p62) and pro-apoptotic genes such as Bax and Casp3 in spinal cord. In skeletal muscle, TTC was able to downregulate the expression of the main marker of autophagy, Lc3, to wild-type levels and the expression of the apoptosis effector protein, Casp3. Regarding the genes related to muscular atrophy (Ankrd1, Calm1, Col19a1, Fbox32, Mt2, Myod1, NogoA, Pax7, Rrad, and Sln), TTC suggest a compensatory effect for muscle damage response, diminished oxidative stress and modulated calcium homeostasis. These preliminary findings suggest the need for further experiments to depth study the effect of TTC in SMA disease.

  6. Neuroprotective Effect of Non-viral Gene Therapy Treatment Based on Tetanus Toxin C-fragment in a Severe Mouse Model of Spinal Muscular Atrophy

    PubMed Central

    Oliván, Sara; Calvo, Ana C.; Rando, Amaya; Herrando-Grabulosa, Mireia; Manzano, Raquel; Zaragoza, Pilar; Tizzano, Eduardo F.; Aquilera, Jose; Osta, Rosario

    2016-01-01

    Spinal muscular atrophy (SMA) is a hereditary childhood disease that causes paralysis and progressive degeneration of skeletal muscles and spinal motor neurons. SMA is associated with reduced levels of full-length Survival of Motor Neuron (SMN) protein, due to mutations in the Survival of Motor Neuron 1 gene. Nowadays there are no effective therapies available to treat patients with SMA, so our aim was to test whether the non-toxic carboxy-terminal fragment of tetanus toxin heavy chain (TTC), which exhibits neurotrophic properties, might have a therapeutic role or benefit in SMA. In this manuscript, we have demonstrated that TTC enhance the SMN expression in motor neurons “in vitro” and evaluated the effect of intramuscular injection of TTC-encoding plasmid in the spinal cord and the skeletal muscle of SMNdelta7 mice. For this purpose, we studied the weight and the survival time, as well as, the survival and cell death pathways and muscular atrophy. Our results showed that TTC treatment reduced the expression of autophagy markers (Becn1, Atg5, Lc3, and p62) and pro-apoptotic genes such as Bax and Casp3 in spinal cord. In skeletal muscle, TTC was able to downregulate the expression of the main marker of autophagy, Lc3, to wild-type levels and the expression of the apoptosis effector protein, Casp3. Regarding the genes related to muscular atrophy (Ankrd1, Calm1, Col19a1, Fbox32, Mt2, Myod1, NogoA, Pax7, Rrad, and Sln), TTC suggest a compensatory effect for muscle damage response, diminished oxidative stress and modulated calcium homeostasis. These preliminary findings suggest the need for further experiments to depth study the effect of TTC in SMA disease. PMID:27605908

  7. Inactivation of allergens and toxins.

    PubMed

    Morandini, Piero

    2010-11-30

    Plants are replete with thousands of proteins and small molecules, many of which are species-specific, poisonous or dangerous. Over time humans have learned to avoid dangerous plants or inactivate many toxic components in food plants, but there is still room for ameliorating food crops (and plants in general) in terms of their allergens and toxins content, especially in their edible parts. Inactivation at the genetic rather than physical or chemical level has many advantages and classical genetic approaches have resulted in significant reduction of toxin content. The capacity, offered by genetic engineering, of turning off (inactivating) specific genes has opened up the possibility of altering the plant content in a far more precise manner than previously available. Different levels of intervention (genes coding for toxins/allergens or for enzymes, transporters or regulators involved in their metabolism) are possible and there are several tools for inactivating genes, both direct (using chemical and physical mutagens, insertion of transposons and other genetic elements) and indirect (antisense RNA, RNA interference, microRNA, eventually leading to gene silencing). Each level/strategy has specific advantages and disadvantages (speed, costs, selectivity, stability, reversibility, frequency of desired genotype and regulatory regime). Paradigmatic examples from classical and transgenic approaches are discussed to emphasize the need to revise the present regulatory process. Reducing the content of natural toxins is a trade-off process: the lesser the content of natural toxins, the higher the susceptibility of a plant to pests and therefore the stronger the need to protect plants. As a consequence, more specific pesticides like Bt are needed to substitute for general pesticides.

  8. Bullous impetigo in children infected with methicillin-resistant Staphylococcus aureus alone or in combination with methicillin-susceptible S. aureus: analysis of genetic characteristics, including assessment of exfoliative toxin gene carriage.

    PubMed

    Shi, Da; Higuchi, Wataru; Takano, Tomomi; Saito, Kohei; Ozaki, Kyoko; Takano, Misao; Nitahara, Yoshiyuki; Yamamoto, Tatsuo

    2011-05-01

    Among bullous impetigo isolates, exfoliative toxin (ET) gene carriage was found in 61.5% of methicillin-resistant Staphylococcus aureus (MRSA) isolates versus 90.6% of methicillin-susceptible S. aureus (MSSA) isolates. MRSA-only cases were ETB or ETA positive, while MRSA/MSSA coinfection cases were ET negative for MRSA but ETA positive for MSSA. Collagen adhesin may facilitate some MRSA infections.

  9. Expression of tetanus toxin fragment C in yeast: gene synthesis is required to eliminate fortuitous polyadenylation sites in AT-rich DNA.

    PubMed Central

    Romanos, M A; Makoff, A J; Fairweather, N F; Beesley, K M; Slater, D E; Rayment, F B; Payne, M M; Clare, J J

    1991-01-01

    Fragment C is a non-toxic 50 kDa fragment of tetanus toxin which is a candidate subunit vaccine against tetanus. The AT-rich Clostridium tetani DNA encoding fragment C could not be expressed in Saccharomyces cerevisiae due to the presence of several fortuitous polyadenylation sites which gave rise to truncated mRNAs. The polyadenylation sites were eliminated by chemically synthesising the DNA with increased GC-content (from 29% to 47%). Synthesis of the entire gene (1400 base pairs) was necessary to generate full-length transcripts and for protein production in yeast. Using a GAL1 promoter vector, fragment C was expressed to 2-3% of soluble cell protein. Fragment C could also be secreted using the alpha-factor leader peptide as a secretion signal. The protein was present at 5-10 mg/l in the culture medium in two forms: a high molecular mass hyper-glycosylated protein (75-200 kDa) and a core-glycosylated protein (65 kDa). Intracellular fragment C was as effective in vaccinating mice against tetanus authentic fragment C. The glycosylated material was inactive, though it was rendered fully active by de-glycosylation. Images PMID:2027754

  10. Expression profile of eight glutathione S-transferase genes in Crassostrea ariakensis after exposure to DSP toxins producing dinoflagellate Prorocentrum lima.

    PubMed

    Zou, Ying; Wei, Xiao-Meng; Weng, Hui-Wen; Li, Hong-Ye; Liu, Jie-Sheng; Yang, Wei-Dong

    2015-10-01

    In this study, changes in eight GSTs mRNA level including GST-α, GST-σ, GST-ω, GST-π, GST-μ, GST-ρ, GST-θ and microsomal GST (mGST) in the oyster Crassostrea ariakensis after exposure to Prorocentrum lima have been evaluated by quantitative real-time PCR. Additionally, the contents of five GST isoforms were detected by ELISA. After exposure to P. lima at density of 2 × 10(5) cells/L, mGST mRNA significantly increased in gill, while GST-σ was induced in digestive gland. After exposure to P. lima at density of 2 × 10(6) cells/L, GST-ω and mGST expressions increased in gill, whereas GST-α and GST-σ were induced in digestive gland. The GST content and activity in oysters exposed to P. lima also showed a different pattern when the different isoforms and organs were compared. After exposure to P. lima (2 × 10(6) cell/L), GST-π increased in gill but decreased in digestive gland. The total GST enzyme activity increased in gill, while remained unchanged in digestive gland. These various regulation of GST gene expressions indicated that the GSTs isoenzymes might play divergent physiological roles in the detoxification of DSP toxins in C. ariakensis.

  11. Plasmids from Shiga Toxin-Producing Escherichia coli Strains with Rare Enterohemolysin Gene (ehxA) Subtypes Reveal Pathogenicity Potential and Display a Novel Evolutionary Path

    PubMed Central

    Lorenz, Sandra C.; Monday, Steven R.; Hoffmann, Maria; Fischer, Markus

    2016-01-01

    ABSTRACT Most Shiga toxin-producing Escherichia coli (STEC) strains associated with severe disease, such as hemolytic-uremic syndrome (HUS), carry large enterohemolysin-encoding (ehxA) plasmids, e.g., pO157 and pO103, that contribute to STEC clinical manifestations. Six ehxA subtypes (A through F) exist that phylogenetically cluster into eae-positive (B, C, F), a mix of eae-positive (E) and eae-negative (A), and a third, more distantly related, cluster of eae-negative (D) STEC strains. While subtype B, C, and F plasmids share a number of virulence traits that are distinct from those of subtype A, sequence data have not been available for subtype D and E plasmids. Here, we determined and compared the genetic composition of four subtype D and two subtype E plasmids to establish their evolutionary relatedness among ehxA subtypes and define their potential role in pathogenicity. We found that subtype D strains carry one exceptionally large plasmid (>200 kbp) that carries a variety of virulence genes that are associated with enterotoxigenic and enterohemorrhagic E. coli, which, quite possibly, enables these strains to cause disease despite being food isolates. Our data offer further support for the hypothesis that this subtype D plasmid represents a novel virulence plasmid, sharing very few genetic features with other plasmids; we conclude that these plasmids have evolved from a different evolutionary lineage than the plasmids carrying the other ehxA subtypes. In contrast, the 50-kbp plasmids of subtype E (pO145), although isolated from HUS outbreak strains, carried only few virulence-associated determinants, suggesting that the clinical presentation of subtype E strains is largely a result of chromosomally encoded virulence factors. IMPORTANCE Bacterial plasmids are known to be key agents of change in microbial populations, promoting the dissemination of various traits, such as drug resistance and virulence. This study determined the genetic makeup of virulence

  12. Species diversity and peptide toxins blocking selectivity of ether-a-go-go-related gene subfamily K+ channels in the central nervous system.

    PubMed

    Restano-Cassulini, Rita; Korolkova, Yuliya V; Diochot, Sylvie; Gurrola, Georgina; Guasti, Leonardo; Possani, Lourival D; Lazdunski, Michel; Grishin, Eugene V; Arcangeli, Annarosa; Wanke, Enzo

    2006-05-01

    The ether-à-go-go-related gene (erg) K+ channels are known to be crucial for life in Caenorhabditis elegans (mating), Drosophila melanogaster (seizure), and humans (LQT syndrome). The erg genes known to date (erg1, erg2, and erg3) are highly expressed in various areas of the rat and mouse central nervous system (CNS), and ERG channel blockers alter firing accommodation. To assign physiological roles to each isoform, it is necessary to design pharmacological strategies to distinguish individual currents. To this purpose, we have investigated the blocking properties of specific peptide inhibitors of hERG1 channels on the human and rat isoforms. In particular, we have tested ErgTx1 (from the scorpion Centruroides noxious), BeKm-1 (from the scorpion Buthus eupeus), and APETx1 (from the sea anemone Anthopleura elegantissima). Because these peptides had different species-specific effects on the six different channels, we have also carried out a biophysical characterization of hERG2 and hERG3 channels that turned out to be different from the rat homologs. It emerged that APETx1 is exquisitely selective for ERG1 and does not compete with the other two toxins. BeKm-1 discriminates well among the three rat members. ErgTx1 is unable to block hERG2, but blocks rERG2 and has the lowest KD for hERG3. BeKm-1 and ErgTx1 compete for hERG3 but not for rERG2 blockade. Our findings should be helpful for structure-function studies and for novel CNS ERG-specific drug design.

  13. Gene Discovery and Expression Profiling in the Toxin-Producing Marine Diatom, Pseudo-nitzschia Multiseries (Hasle) Hasle

    DTIC Science & Technology

    2004-09-01

    measuring mRNAs. Comparing the amount of specific mRNAs between two samples provides a mechanism to screen for genes that are turned on or off under defined ...were based on a comparison of different criteria and software packages that revealed that Seqman (DNAStar) yielded the most consistent results using...branches of the eukaryotic tree and to further define the root of the tree. Multiple copies of actin, beta-tubulin, and EF- 1 alpha were identified in P

  14. Multiplex Real-Time PCR Assays for Screening of Shiga Toxin 1 and 2 Genes, Including All Known Subtypes, and Escherichia coli O26-, O111-, and O157-Specific Genes in Beef and Sprout Enrichment Cultures.

    PubMed

    Harada, Tetsuya; Iguchi, Atsushi; Iyoda, Sunao; Seto, Kazuko; Taguchi, Masumi; Kumeda, Yuko

    2015-10-01

    Shiga toxin family members have recently been classified using a new nomenclature into three Stx1 subtypes (Stx1a, Stx1c, and Stx1d) and seven Stx2 subtypes (Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f, and Stx2g). To develop screening methods for Stx genes, including all of these subtype genes, and Escherichia coli O26-, O111-, and O157-specific genes in laboratory investigations of Shiga toxin-producing E. coli (STEC) foodborne cases, we developed multiplex real-time PCR assays and evaluated their specificity and quantitative accuracy using STEC and non-STEC isolates, recombinant plasmids, and food enrichment cultures and by performing STEC spiking experiments with beef and sprout enrichment cultures. In addition, we evaluated the relationship between the recovery rates of the target strains by direct plating and immunomagnetic separation and the cycle threshold (CT) values of the real-time PCR assays for the Stx subtypes and STEC O26, O111, and O157 serogroups. All three stx1- and seven stx2-subtype genes were detected by real-time PCR with high sensitivity and specificity, and the quantitative accuracy of this assay was confirmed using control plasmids and STEC spiking experiments. The results of the STEC spiking experiments suggest that it is not routinely possible to isolate STEC from enrichment cultures with real-time PCR CT values greater than 30 by direct plating on MacConkey agar, although highly selective media and immunomagnetic beads were able to isolate the inoculated strains from the enrichment cultures. These data suggest that CT values obtained from the highly quantitative real-time PCR assays developed in this study provide useful information to develop effective isolation strategies for STEC from food samples. The real-time PCR assays developed here are expected to aid in investigations of infections or outbreaks caused by STEC harboring any of the stx-subtype genes in the new Stx nomenclature, as well as STEC O26, O111, and O157.

  15. Hfr-2, a wheat cytolytic toxin-like gene, is up-regulated by virulent Hessian fly larval feedingdouble dagger.

    PubMed

    Puthoff, David P; Sardesai, Nagesh; Subramanyam, Subhashree; Nemacheck, Jill A; Williams, Christie E

    2005-07-01

    SUMMARY Both yield and grain-quality are dramatically decreased when susceptible wheat (Triticum aestivum) plants are infested by Hessian fly (Mayetiola destructor) larvae. Examination of the changes in wheat gene expression during infestation by virulent Hessian fly larvae has identified the up-regulation of a gene, Hessian fly responsive-2 (Hfr-2), which contains regions similar to genes encoding seed-specific agglutinin proteins from Amaranthus. Hfr-2, however, did not accumulate in developing seeds, as do other wheat seed storage proteins. Additionally, a separate region of the HFR-2 predicted amino acid sequence is similar to haemolytic proteins, from both mushroom and bacteria, that are able to form pores in cell membranes of mammalian red blood cells. The involvement of Hfr-2 in interactions with insects was supported by experiments demonstrating its up-regulation by both fall armyworm (Spodoptera frugiperda) and bird cherry-oat aphid (Rhopalosiphum padi) infestations but not by virus infection. Examination of wheat defence response pathways showed Hfr-2 up-regulation following methyl jasmonate treatment and only slight up-regulation in response to salicylic acid, abscisic acid and wounding treatments. Like related proteins, HFR-2 may normally function in defence against certain insects or pathogens. However, we propose that as virulent Hessian fly larvae manipulate the physiology of the susceptible host, the HFR-2 protein inserts in plant cell membranes at the feeding sites and by forming pores provides water, ions and other small nutritive molecules to the developing larvae.

  16. Verification of the Usefulness of the Trimble Rtx Extended Satellite Technology with the Xfill Function in the Local Network Implementing Rtk Measurements

    NASA Astrophysics Data System (ADS)

    Siejka, Zbigniew

    2014-12-01

    The paper presents the method of satellite measurements, which gives users the ability of GNSS continuous precise positioning in real time, even in the case of short interruptions in receiving the correction of the local ground system of measurements support. The proposed method is a combination of two satellite positioning technologies RTN GNSS and RTX Extended. In technology RTX Extended the xFill function was used for precise positioning in real time and in the local reference system. This function provides the ability to perform measurement without the need for constant communication with the ground support satellite system. Test measurements were performed on a test basis located in Krakow, and RTN GNSS positioning was done based on the national network of reference stations of the ASGEUPOS. The solution allows for short (up to 5 minutes) interruptions in radio or internet communication. When the primary stream of RTN correction is not available, then the global corrections Trimble xFill broadcasted by satellite are used. The new technology uses in the real-time data from the global network of tracking stations and contributes significantly to improving the quality and efficiency of surveying works. At present according to the authors, technology Trimble CenterPoint RTX can guarantee repeatability of measurements not worse than 3.8 cm (Trimble Survey Division, 2012). In the paper the comparative analysis of measurement results between the two technologies was performed: RTN carried out in the classic way, which was based on the corrections of the terrestrial local network of the Polish system of active geodetic network (ASG-EUPOS) and RTK xFill technology. The results were related to the data of test network, established as error free. The research gave satisfactory results and confirmed the great potential of the use of the new technology in the geodetic work realization. By combining these two technologies of GNSS surveying the user can greatly improve the

  17. *CYANOBACTERIA AND THEIR TOXINS

    EPA Science Inventory

    Cyanobacteria, or blue-green algae, are naturally-occurring contaminants of surface waters worldwide. These photosynthesizing prokaryotes thrive in warm, shallow, nutrient-rich waters. Many produce potent toxins as secondary metabolites. Cyanobacteria toxins have been document...

  18. Molecular characterization of high-level-cholera-toxin-producing El Tor variant Vibrio cholerae strains in the Zanzibar Archipelago of Tanzania.

    PubMed

    Naha, A; Chowdhury, G; Ghosh-Banerjee, J; Senoh, M; Takahashi, T; Ley, B; Thriemer, K; Deen, J; Seidlein, L V; Ali, S M; Khatib, A; Ramamurthy, T; Nandy, R K; Nair, G B; Takeda, Y; Mukhopadhyay, A K

    2013-03-01

    Analysis of 1,180 diarrheal stool samples in Zanzibar detected 247 Vibrio cholerae O1, Ogawa strains in 2009. Phenotypic traits and PCR-based detection of rstR, rtxC, and tcpA alleles showed that they belonged to the El Tor biotype. Genetic analysis of ctxB of these strains revealed that they were classical type, and production of classical cholera toxin B (CTB) was confirmed by Western blotting. These strains produced more CT than the prototype El Tor and formed a separate cluster by pulsed-field gel electrophoresis (PFGE) analysis.

  19. Molecular Characterization of High-Level-Cholera-Toxin-Producing El Tor Variant Vibrio cholerae Strains in the Zanzibar Archipelago of Tanzania

    PubMed Central

    Naha, A.; Chowdhury, G.; Ghosh-Banerjee, J.; Senoh, M.; Takahashi, T.; Ley, B.; Thriemer, K.; Deen, J.; Seidlein, L. V.; Ali, S. M.; Khatib, A.; Ramamurthy, T.; Nandy, R. K.; Nair, G. B.; Takeda, Y.

    2013-01-01

    Analysis of 1,180 diarrheal stool samples in Zanzibar detected 247 Vibrio cholerae O1, Ogawa strains in 2009. Phenotypic traits and PCR-based detection of rstR, rtxC, and tcpA alleles showed that they belonged to the El Tor biotype. Genetic analysis of ctxB of these strains revealed that they were classical type, and production of classical cholera toxin B (CTB) was confirmed by Western blotting. These strains produced more CT than the prototype El Tor and formed a separate cluster by pulsed-field gel electrophoresis (PFGE) analysis. PMID:23325815

  20. Functional characterization of SOR1, a gene required for resistance to photosensitizing toxins in the fungus Cercospora nicotianae.

    PubMed

    Ehrenshaft, M; Chung, K R; Jenns, A E; Daub, M E

    1999-01-01

    The Cercospora nicotianae SOR1 gene is required for resistance to singlet oxygen-generating photosensitizers. SOR1 was characterized in the wild-type and in five photosensitizer-sensitive mutant strains which are complemented to photosensitizer resistance by transformation with SOR1. Sequence analysis determined that three of the mutants contain SOR1 copies with mutations encoding substitutions in the protein-coding sequence; however, two other mutants had wild-type SOR1 protein and promoter sequences. All five mutants accumulate SOR1 mRNA at levels comparable to that of the wild-type strain. In the wild-type strain, SOR1 accumulation is enhanced two-fold by light, but is unaffected by the presence of cercosporin, the photosensitizer synthesized by C. nicotianae. Southern analysis indicates that SOR1 is present in other fungi that synthesize structurally related perylenequinone photosensitizers.

  1. Vibrio cholerae cytolysin is essential for high enterotoxicity and apoptosis induction produced by a cholera toxin gene-negative V. cholerae non-O1, non-O139 strain.

    PubMed

    Saka, Hector Alex; Bidinost, Carla; Sola, Claudia; Carranza, Pablo; Collino, Cesar; Ortiz, Susana; Echenique, Jose Ricardo; Bocco, José Luis

    2008-02-01

    Cholera toxin (CT) gene-negative Vibrio cholerae non-O1, non-O139 strains may cause severe diarrhea though their pathogenic mechanism remains unclear. V. cholerae cytolysin (VCC) is a pore-forming exotoxin encoded in the hlyA gene of V. cholerae whose contribution to the pathogenesis is not fully understood. In this work, the virulence properties of a CT gene-negative V. cholerae non-O1, non-O139 strain causing a cholera-like syndrome were analyzed. Inoculation of rabbit ileal loops with the wild type strain induced extensive fluid accumulation, accompanied by severe histopathological damage characterized by villus shortening, lymphangiectasia and focal areas of necrosis. These pathogenic effects were abrogated by mutation of the hlyA gene thus pointing out the main role of VCC in the virulence of the strain. Interestingly, this toxin was capable of triggering apoptosis in human intestinal cell lines due to its anion channel activity. Moreover, the wild type strain also induced increased apoptosis of the intestinal epithelium cells which was not observed upon inoculation of the VCC null mutant strain, indicating that VCC may trigger apoptotic cell death during infection in vivo. Altogether, these results support a main role of VCC in the pathogenesis of the CT gene-negative V. cholerae non-O1, non-O139 strain and identify apoptosis as a previously unrecognized cell death pathway triggered by VCC.

  2. Detection of Protein Toxins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have focused on ricin, shiga-like toxin, botulinum neurotoxin (BoNT), and staphylococcal enterotoxin A (SEA), developing sensitive test methods for toxins and marker compounds in food matrices. Although animal models provide the best means for risk assessment, especially for crude toxins in compl...

  3. Plasmids from Shiga Toxin-Producing Escherichia coli Strains with Rare Enterohemolysin Gene (ehxA) Subtypes Reveal Pathogenicity Potential and Display a Novel Evolutionary Path.

    PubMed

    Lorenz, Sandra C; Monday, Steven R; Hoffmann, Maria; Fischer, Markus; Kase, Julie A

    2016-11-01

    Most Shiga toxin-producing Escherichia coli (STEC) strains associated with severe disease, such as hemolytic-uremic syndrome (HUS), carry large enterohemolysin-encoding (ehxA) plasmids, e.g., pO157 and pO103, that contribute to STEC clinical manifestations. Six ehxA subtypes (A through F) exist that phylogenetically cluster into eae-positive (B, C, F), a mix of eae-positive (E) and eae-negative (A), and a third, more distantly related, cluster of eae-negative (D) STEC strains. While subtype B, C, and F plasmids share a number of virulence traits that are distinct from those of subtype A, sequence data have not been available for subtype D and E plasmids. Here, we determined and compared the genetic composition of four subtype D and two subtype E plasmids to establish their evolutionary relatedness among ehxA subtypes and define their potential role in pathogenicity. We found that subtype D strains carry one exceptionally large plasmid (>200 kbp) that carries a variety of virulence genes that are associated with enterotoxigenic and enterohemorrhagic E. coli, which, quite possibly, enables these strains to cause disease despite being food isolates. Our data offer further support for the hypothesis that this subtype D plasmid represents a novel virulence plasmid, sharing very few genetic features with other plasmids; we conclude that these plasmids have evolved from a different evolutionary lineage than the plasmids carrying the other ehxA subtypes. In contrast, the 50-kbp plasmids of subtype E (pO145), although isolated from HUS outbreak strains, carried only few virulence-associated determinants, suggesting that the clinical presentation of subtype E strains is largely a result of chromosomally encoded virulence factors.

  4. Bacteriophage ΦM1 of Pectobacterium evolves to escape two bifunctional Type III toxin-antitoxin and abortive infection systems through mutations in a single viral gene.

    PubMed

    Blower, Tim R; Chai, Ray; Przybilski, Rita; Chindhy, Shahzad; Fang, Xinzhe; Kidman, Samuel E; Tan, Hui; Luisi, Ben F; Fineran, Peter C; Salmond, George P C

    2017-02-03

    Some bacteria, when infected by their viral parasites (bacteriophages), undergo a suicidal response that also terminates productive viral replication (abortive infection; Abi). This response can be viewed as an altruistic act protecting the uninfected bacterial clonal population. Abortive infection can occur through the action of Type III protein-RNA toxin-antitoxin (TA) systems, such as ToxINPa from the phytopathogen, Pectobacterium atrosepticum Rare spontaneous mutants evolved in the generalized transducing phage, ΦM1, which escaped ToxINPa-mediated abortive infection in P. atrosepticum ΦM1 is a member of the Podoviridae and member of the "KMV-like viruses", a subset of the T7 supergroup. Genomic sequencing of ΦM1 escape mutants revealed single-base changes which clustered in a single open reading frame. The "escape" gene product, M1-23, was highly toxic to the host bacterium when over-expressed, but mutations in M1-23 that enabled an escape phenotype caused M1-23 to be less toxic. M1-23 is encoded within the DNA metabolism modular section of the phage genome, and when it was over-expressed, it co-purified with the host nucleotide excision repair protein, UvrA. While the M1-23 protein interacted with UvrA in co-immunoprecipitation assays, a UvrA mutant strain still aborted ΦM1, suggesting that the interaction is not critical for the Type III TA Abi activity. Additionally, ΦM1 escaped a heterologous Type III TA system (TenpINPl) from Photorhabdus luminescens (reconstituted in P. atrosepticum) through mutations in the same protein, M1-23. The mechanistic action of M1-23 is currently unknown but further analysis of this protein could provide insights into the mode of activation of both systems.

  5. The Cercospora nicotianae gene encoding dual O-methyltransferase and FAD-dependent monooxygenase domains mediates cercosporin toxin biosynthesis.

    PubMed

    Dekkers, Katherine L; You, Bang-Jau; Gowda, Vivek S; Liao, Hui-Ling; Lee, Miin-Huey; Bau, Huey-Jiunn; Ueng, Peter P; Chung, Kuang-Ren

    2007-05-01

    Cercosporin, a photo-activated, non-host-selective phytotoxin produced by many species of the plant pathogenic fungus Cercospora, causes peroxidation of plant cell membranes by generating reactive oxygen species and is an important virulence determinant. Here we report a new gene, CTB3 that is involved in cercosporin biosynthesis in Cercospora nicotianae. CTB3 is adjacent to a previously identified CTB1 encoding a polyketide synthase which is also required for cercosporin production. CTB3 contains a putative O-methyltransferase domain in the N-terminus and a putative flavin adenine dinucleotide (FAD)-dependent monooxygenase domain in the C-terminus. The N-terminal amino acid sequence also is similar to that of the transcription enhancer AFLS (formerly AFLJ) involved in aflatoxin biosynthesis. Expression of CTB3 was differentially regulated by light, medium, nitrogen and carbon sources and pH. Disruption of the N- or C-terminus of CTB3 yielded mutants that failed to accumulate the CTB3 transcript and cercosporin. The Deltactb3 disruptants produced a yellow pigment that is not toxic to tobacco suspension cells. Production of cercosporin in a Deltactb3 null mutant was fully restored when transformed with a functional CTB3 clone or when paired with a Deltactb1-null mutant (defective in polyketide synthase) by cross feeding of the biosynthetic intermediates. Pathogenicity assays using detached tobacco leaves revealed that the Deltactb3 disruptants drastically reduced lesion formation.

  6. Prevalence of Shiga toxin-producing Escherichia coli stx1, stx2, eaeA, and rfbE genes and survival of E. coli O157:H7 in manure from organic and low-input conventional dairy farms.

    PubMed

    Franz, Eelco; Klerks, Michel M; De Vos, Oscar J; Termorshuizen, Aad J; van Bruggen, Ariena H C

    2007-04-01

    Manure samples were collected from 16 organic (ORG) and 9 low-input conventional (LIC) Dutch dairy farms during August and September 2004 to determine the prevalence of the STEC virulence genes stx(1) (encoding Shiga toxin 1), stx(2) (encoding Shiga toxin 2), and eaeA (encoding intimin), as well as the rfbE gene, which is specific for Escherichia coli O157. The rfbE gene was present at 52% of the farms. The prevalence of rfbE was higher at ORG farms (61%) than at LIC farms (36%), but this was not significant. Relatively more LIC farms were positive for all Shiga toxin-producing E. coli (STEC) virulence genes eaeA, stx(1), and stx(2), which form a potentially highly virulent combination. Species richness of Enterobacteriaceae, as determined by DGGE, was significantly lower in manure positive for rfbE. Survival of a green fluorescent protein-expressing E. coli O157:H7 strain was studied in the manure from all farms from which samples were obtained and was modeled by a biphasic decline model. The time needed to reach the detection limit was predominantly determined by the level of native coliforms and the pH (both negative relationships). Initial decline was faster for ORG manure but leveled off earlier, resulting in longer survival than in LIC manure. Although the nonlinear decline curve could theoretically be explained as the cumulative distribution of an underlying distribution of decline kinetics, it is proposed that the observed nonlinear biphasic pattern of the survival curve is the result of changing nutrient status of the manure over time (and thereby changing competition pressure), instead of the presence of subpopulations differing in the level of resistance.

  7. Identification and molecular characterization of three new K+-channel specific toxins from the Chinese scorpion Mesobuthus martensii Karsch revealing intronic number polymorphism and alternative splicing in duplicated genes.

    PubMed

    Zeng, Xian-Chun; Zhang, Lei; Nie, Yao; Luo, Xuesong

    2012-04-01

    K(+)-channel specific toxins from scorpions are powerful probes used in the structural and functional characterization of different subfamilies of K(+)-channels which are thought to be the most diverse ion channels. However, only a limited number of K(+)-channel toxins have been identified from scorpions so far; moreover, little is known about the mechanisms for the generation of a combinatorial peptide library in a venom gland of a scorpion. Here, we identified and characterized three new K(+)-channel toxin-like peptides from the scorpion Mesobuthus martensii Karsch, which were referred to as BmKcug1, BmKcug2 and BmKcugx, respectively. BmKcug1 and BmKcug2 are two new members of α-KTx1 subfamily, and have been classified as α-KTx1.14 and α-KTx1.15, respectively. BmKcugx represents a new subfamily of K(+)-channel specific toxins which was classified into α-KTx22. BmKcugx was thus classified as α-KTx22.1. Genomic analysis demonstrated that BmKcugx gene has two exons interrupted by an intron inserted in the signal peptide encoding region, whereas BmKcug1a (a close homologue of BmKcug1)/BmKcug2 gene was interrupted by two introns, located within the 5'UTR of the gene and in the signal peptide encoding region, respectively. Transcriptomic analysis for the venom glands of M. martensii Karsch indicated that the abundances of the transcripts of BmKcug1a and BmKcug2 are much higher than that of BmKcugx; it suggests that the intron in 5'UTR could markedly increase the expression level of the K(+)-channel toxins. Alignment of the genomic sequences of BmKcug1a and BmKcug2 revealed that an alternative splicing event occurred at the intron 1-exon 2 junction in the 5'UTR of BmKcug2 transcript.

  8. Clonal spread of catalase-negative ST5/SCCmec II Staphylococcus aureus carrying the staphylococcal enterotoxin A (sea), staphylococcal enterotoxin b (seb), and toxic shock toxin (tst) virulence genes.

    PubMed

    Lee, Hae Kyung; Kim, Jung-Beom; Kim, Hyunjung; Jekarl, Dong Wook; Kim, Yang Ree; Yu, Jin Kyung; Park, Yeon-Joon

    2014-01-01

    17 catalase-negative methicillin-resistant Staphylococcus aureus (MRSA) isolates were recovered from respiratory specimens of patients at a 700-bed hospital in Korea. The goal of this study was to determine the molecular characteristics of catalase-negative MRSA strains in Korea for the first time. Characteristics that we explored included kat A gene mutation sequence, sequence type, staphylococcal cassette chromosome (SCC) mec subtype classification, and toxin gene profiles. All 17 isolates showed similar pulsed field gel electrophoresis (PFGE) pattern. Four mutations were identified in the kat A gene of a representative catalase-negative MRSA strain: A602G causing a histidine 201 to arginine change, A695T causing a glutamic acid 232 to valine change, T778A causing a tryptophan 260 to arginine change, and G1438A causing a glycine 480 to serine change. Previous studies suggest that the A695T and T778A mutations may have strong effects on the catalase activity of catalase-negative MRSA. The sequence type (ST) and SCCmec type of this isolate were ST 5 and SCCmec type II, respectively. All 17 isolates harbored toxic shock toxin (tst), staphylococcal enterotoxin A (sea), and staphylococcal enterotoxin B (seb) virulence genes. The mortality rate of the present study was 11.8%, suggesting that the clinical relevance of catalase-negative MRSA requires further study in the future.

  9. Sea anemone (Cnidaria, Anthozoa, Actiniaria) toxins: an overview.

    PubMed

    Frazão, Bárbara; Vasconcelos, Vitor; Antunes, Agostinho

    2012-08-01

    The Cnidaria phylum includes organisms that are among the most venomous animals. The Anthozoa class includes sea anemones, hard corals, soft corals and sea pens. The composition of cnidarian venoms is not known in detail, but they appear to contain a variety of compounds. Currently around 250 of those compounds have been identified (peptides, proteins, enzymes and proteinase inhibitors) and non-proteinaceous substances (purines, quaternary ammonium compounds, biogenic amines and betaines), but very few genes encoding toxins were described and only a few related protein three-dimensional structures are available. Toxins are used for prey acquisition, but also to deter potential predators (with neurotoxicity and cardiotoxicity effects) and even to fight territorial disputes. Cnidaria toxins have been identified on the nematocysts located on the tentacles, acrorhagi and acontia, and in the mucous coat that covers the animal body. Sea anemone toxins comprise mainly proteins and peptides that are cytolytic or neurotoxic with its potency varying with the structure and site of action and are efficient in targeting different animals, such as insects, crustaceans and vertebrates. Sea anemones toxins include voltage-gated Na⁺ and K⁺ channels toxins, acid-sensing ion channel toxins, Cytolysins, toxins with Kunitz-type protease inhibitors activity and toxins with Phospholipase A2 activity. In this review we assessed the phylogentic relationships of sea anemone toxins, characterized such toxins, the genes encoding them and the toxins three-dimensional structures, further providing a state-of-the-art description of the procedures involved in the isolation and purification of bioactive toxins.

  10. Quality and safety evaluation of genetically modified potatoes spunta with Cry V gene: compositional analysis, determination of some toxins, antinutrients compounds and feeding study in rats.

    PubMed

    El Sanhoty, Rafaat; El-Rahman, Ahamed Ali Abd; Bögl, Klaus Werner

    2004-02-01

    The aim of this study was to evaluate the composition, nutritional and toxicology safety of GM potato Spunta lines compared to that of conventional potato Spunta. Compositional analyses were conducted to measure the proximate chemical composition with references to 14 components, total solid, protein, lipid, crude fibre, ash, carbohydrate, starch, reducing sugar, nonreducing sugar, sodium, calcium, potassium, phosphorus, and ascorbic acid. Some toxins and anti-nutrients compounds were determined. Feeding study of GM potatoes line (G2 and G3) in rats were done for 30 days. Four groups of albino rats were used for studying the effect and the safety assessment of GM potatoes Spunta G2 and G3. Group (I) was fed on control basal diet, group (II) was fed on control diet plus 30% freeze-dried nongenetically modified potato Spunta, group (III) was fed on control diet plus 30% freeze-dried genetically modified potato Spunta, and group (IV) was fed on control diet plus 30% freeze-dried genetically modified potato Spunta GMO G3. There were no significant differences between GM potatoes G2, G3, and Spunta control potato line in the proximate chemical composition. The levels of glycoalkaloids in transgenic potato tubers and nontransgenic were determined and there were also no significant differences between the GM potatoes and conventional potato line, the levels were in agreement with a safety level recommended by FAO/WHO (200 mg/ kg) for acute toxicity. Protease inhibitor activity and total phenol were estimated and no significant differences between the GM potatoes line and conventional potato Spunta line were found. During the period tested, rats in each group (I, II, III, IV) grew well without marked differences in appearance. No statistical difference were found in food intake, daily body weight gain and feed efficiency. But there is a slightly significant difference in finally body weight between the control group and experimental groups. No significant difference were

  11. [Intoxication of botulinum toxin].

    PubMed

    Chudzicka, Aleksandra

    2015-09-01

    Botulinum toxin is an egzotoxin produced by Gram positive bacteria Clostridium botulinum. It is among the most potent toxins known. The 3 main clinical presentations of botulism are as follows: foodborne botulism, infant botulism and wound botulism. The main symptom of intoxication is flat muscles paralysis. The treatment is supportive care and administration of antitoxin. In prevention the correct preparing of canned food is most important. Botulinum toxin is accepted as a biological weapon.

  12. Regulation of Toxin Production in Clostridium perfringens

    PubMed Central

    Ohtani, Kaori; Shimizu, Tohru

    2016-01-01

    The Gram-positive anaerobic bacterium Clostridium perfringens is widely distributed in nature, especially in soil and the gastrointestinal tracts of humans and animals. C. perfringens causes gas gangrene and food poisoning, and it produces extracellular enzymes and toxins that are thought to act synergistically and contribute to its pathogenesis. A complicated regulatory network of toxin genes has been reported that includes a two-component system for regulatory RNA and cell-cell communication. It is necessary to clarify the global regulatory system of these genes in order to understand and treat the virulence of C. perfringens. We summarize the existing knowledge about the regulatory mechanisms here. PMID:27399773

  13. Chorea caused by toxins.

    PubMed

    Miyasaki, Janis M

    2011-01-01

    Chorea is uncommonly caused by toxins. Anecdotal evidence from cases of toxin-induced chorea assists in our understanding of neurodegenerative diseases associated with chorea. Beginning in medieval Europe with ergotism and the "fire that twisted people," spanning to crack dancing in contemporary times and the coexistence of alcohol abuse with chorea, toxins may exert direct effects to enhance mesolimbic dopamine transmission or indirect effects through gamma-aminobutyric acid modulation. The following chapter will discuss toxins associated with chorea and the presumed pathophysiology underlying the movement disorders in these case series.

  14. Botulinum toxin injection - larynx

    MedlinePlus

    Injection laryngoplasty; Botox-larynx: spasmodic dysphonia-BTX; Essential voice tremor (EVT)-btx; Glottic insufficiency; Percutaneous electromyography-guided botulinum toxin treatment; Percutaneous indirect laryngoscopy- ...

  15. 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

  16. Overview of scorpion species from China and their toxins.

    PubMed

    Cao, Zhijian; Di, Zhiyong; Wu, Yingliang; Li, Wenxin

    2014-02-26

    Scorpions are one of the most ancient groups of terrestrial animals. They have maintained a steady morphology over more than 400 million years of evolution. Their venom arsenals for capturing prey and defending against predators may play a critical role in their ancient and conservative appearance. In the current review, we present the scorpion fauna of China: 53 species covering five families and 12 genera. We also systematically list toxins or genes from Chinese scorpion species, involving eight species covering four families. Furthermore, we review the diverse functions of typical toxins from Chinese scorpion species, involving Na+ channel modulators, K+ channel blockers, antimicrobial peptides and protease inhibitors. Using scorpion species and their toxins from China as an example, we build the bridge between scorpion species and their toxins, which helps us to understand the molecular and functional diversity of scorpion venom arsenal, the dynamic and functional evolution of scorpion toxins, and the potential relationships of scorpion species and their toxins.

  17. Overview of Scorpion Species from China and Their Toxins

    PubMed Central

    Cao, Zhijian; Di, Zhiyong; Wu, Yingliang; Li, Wenxin

    2014-01-01

    Scorpions are one of the most ancient groups of terrestrial animals. They have maintained a steady morphology over more than 400 million years of evolution. Their venom arsenals for capturing prey and defending against predators may play a critical role in their ancient and conservative appearance. In the current review, we present the scorpion fauna of China: 53 species covering five families and 12 genera. We also systematically list toxins or genes from Chinese scorpion species, involving eight species covering four families. Furthermore, we review the diverse functions of typical toxins from Chinese scorpion species, involving Na+ channel modulators, K+ channel blockers, antimicrobial peptides and protease inhibitors. Using scorpion species and their toxins from China as an example, we build the bridge between scorpion species and their toxins, which helps us to understand the molecular and functional diversity of scorpion venom arsenal, the dynamic and functional evolution of scorpion toxins, and the potential relationships of scorpion species and their toxins. PMID:24577583

  18. Comparative validation study to demonstrate the equivalence of a minor modification to AOAC Official Method 2005.05 Assurance GDS shiga Toxin Genes (O157) method to the reference culture method: 375 gram sample size.

    PubMed

    Feldsine, Philip T; Montgomery-Fullerton, Megan; Roa, Nerie; Kaur, Mandeep; Kerr, David E; Lienau, Andrew H; Jucker, Markus

    2013-01-01

    The Assurance GDS Shiga Toxin Genes (0157), AOAC Official MethodsM 2005.05, has been modified to include a larger sample size of 375 g. A methods comparison study was conducted to demonstrate the equivalence of this modification to the reference culture method. Ninety samples and controls, representing three foods, were analyzed. Results show no statistically detectable difference between the Assurance GDS Escherichia coli O157:H7 assay and the reference culture methods for the detection of E. coli O157:H7, other than the low level of inoculation for leaf lettuce for which the GDS gave noticeably higher recovery [difference in Probability of Detection between candidate methods (dPODc = +0.45)]. There were also suggestions of moderate differences (dPODc = +0.15 to +0.20) for ground beef and the high level of leaf lettuce, but the study size was too small to detect differences of this size. Results showed that the Assurance GDS Shiga Toxin Genes (0157) method is equivalent to the reference culture methods for the detection of Shiga toxigenic E. coli O157:H7.

  19. Sequence Analysis of Toxin Gene–Bearing Corynebacterium diphtheriae Strains, Australia

    PubMed Central

    Mazins, Adam; Graham, Rikki M.A.; Fang, Ning-Xia; Smith, Helen V.; Jennison, Amy V.

    2017-01-01

    By conducting a molecular characterization of Corynebacterium diphtheriae strains in Australia, we identified novel sequences, nonfunctional toxin genes, and 5 recent cases of toxigenic cutaneous diphtheria. These findings highlight the importance of extrapharyngeal infections for toxin gene–bearing (functional or not) and non–toxin gene–bearing C. diphtheriae strains. Continued surveillance is recommended. PMID:27983494

  20. Toxin-Antitoxin Systems of Staphylococcus aureus

    PubMed Central

    Schuster, Christopher F.; Bertram, Ralph

    2016-01-01

    Toxin-antitoxin (TA) systems are small genetic elements found in the majority of prokaryotes. They encode toxin proteins that interfere with vital cellular functions and are counteracted by antitoxins. Dependent on the chemical nature of the antitoxins (protein or RNA) and how they control the activity of the toxin, TA systems are currently divided into six different types. Genes comprising the TA types I, II and III have been identified in Staphylococcus aureus. MazF, the toxin of the mazEF locus is a sequence-specific RNase that cleaves a number of transcripts, including those encoding pathogenicity factors. Two yefM-yoeB paralogs represent two independent, but auto-regulated TA systems that give rise to ribosome-dependent RNases. In addition, omega/epsilon/zeta constitutes a tripartite TA system that supposedly plays a role in the stabilization of resistance factors. The SprA1/SprA1AS and SprF1/SprG1 systems are post-transcriptionally regulated by RNA antitoxins and encode small membrane damaging proteins. TA systems controlled by interaction between toxin protein and antitoxin RNA have been identified in S. aureus in silico, but not yet experimentally proven. A closer inspection of possible links between TA systems and S. aureus pathophysiology will reveal, if these genetic loci may represent druggable targets. The modification of a staphylococcal TA toxin to a cyclopeptide antibiotic highlights the potential of TA systems as rather untapped sources of drug discovery. PMID:27164142

  1. Toxin-Antitoxin Systems of Staphylococcus aureus.

    PubMed

    Schuster, Christopher F; Bertram, Ralph

    2016-05-05

    Toxin-antitoxin (TA) systems are small genetic elements found in the majority of prokaryotes. They encode toxin proteins that interfere with vital cellular functions and are counteracted by antitoxins. Dependent on the chemical nature of the antitoxins (protein or RNA) and how they control the activity of the toxin, TA systems are currently divided into six different types. Genes comprising the TA types I, II and III have been identified in Staphylococcus aureus. MazF, the toxin of the mazEF locus is a sequence-specific RNase that cleaves a number of transcripts, including those encoding pathogenicity factors. Two yefM-yoeB paralogs represent two independent, but auto-regulated TA systems that give rise to ribosome-dependent RNases. In addition, omega/epsilon/zeta constitutes a tripartite TA system that supposedly plays a role in the stabilization of resistance factors. The SprA1/SprA1AS and SprF1/SprG1 systems are post-transcriptionally regulated by RNA antitoxins and encode small membrane damaging proteins. TA systems controlled by interaction between toxin protein and antitoxin RNA have been identified in S. aureus in silico, but not yet experimentally proven. A closer inspection of possible links between TA systems and S. aureus pathophysiology will reveal, if these genetic loci may represent druggable targets. The modification of a staphylococcal TA toxin to a cyclopeptide antibiotic highlights the potential of TA systems as rather untapped sources of drug discovery.

  2. Defense against toxin weapons

    SciTech Connect

    Franz, D.R.

    1994-01-01

    The purpose of this manual is to provide basic information on biological toxins to military leaders and health-care providers at all levels to help them make informed decisions on protecting their troops from toxins. Much of the information contained herein will also be of interest to individuals charged with countering domestic and international terrorism. We typically fear what we do not understand.

  3. Protection against Shiga Toxins

    PubMed Central

    Kavaliauskiene, Simona; Dyve Lingelem, Anne Berit; Skotland, Tore; Sandvig, Kirsten

    2017-01-01

    Shiga toxins consist of an A-moiety and five B-moieties able to bind the neutral glycosphingolipid globotriaosylceramide (Gb3) on the cell surface. To intoxicate cells efficiently, the toxin A-moiety has to be cleaved by furin and transported retrogradely to the Golgi apparatus and to the endoplasmic reticulum. The enzymatically active part of the A-moiety is then translocated to the cytosol, where it inhibits protein synthesis and in some cell types induces apoptosis. Protection of cells can be provided either by inhibiting binding of the toxin to cells or by interfering with any of the subsequent steps required for its toxic effect. In this article we provide a brief overview of the interaction of Shiga toxins with cells, describe some compounds and conditions found to protect cells against Shiga toxins, and discuss whether they might also provide protection in animals and humans. PMID:28165371

  4. Association of Clostridium difficile ribotype 078 with detectable toxin in human stool specimens.

    PubMed

    Fairley, Derek J; McKenna, James P; Stevenson, Mike; Weaver, Jeremy; Gilliland, Carol; Watt, Alison; Coyle, Peter V

    2015-11-01

    Using a Clostridium difficile glutamate dehydrogenase (GDH) immunoassay and a sensitive C. difficile toxin A/B immunoassay, human stool specimens from patients with diarrhoea (n = 1085) were classified as either GDH positive/toxin negative, or GDH positive/toxin positive. Overall, 528/725 (73%) of the GDH-positive/toxin-negative specimens contained viable C. difficile, and 433/528 (82%) of these C. difficile isolates were PCR positive for the toxin gene pathogenicity locus. Overall, 867/1078 (80%) of the GDH-positive specimens contained viable C. difficile, and 433/725 (60%) of the GDH-positive/toxin-negative specimens contained a toxigenic C. difficile strain. The diversity of toxigenic C. difficile ribotypes isolated from toxin-negative specimens (n = 433) and toxin-positive specimens (n = 339) was significantly different (P < 0.0001). Specifically, the presence of ribotype 078 strains was very strongly associated (P < 0.0001) with detection of toxin in clinical specimens using a sensitive toxin immunoassay. Specimens positive for ribotype 078 were almost twice as likely to be toxin positive as opposed to toxin negative (risk ratio = 1.90, 95% confidence interval 1.64-2.19). In contrast, other circulating ribotypes were seen with similar frequency in specimens with and without detectable toxin. This supports the view that ribotype 078 strains may be more virulent than other common ribotypes in terms of toxin production.

  5. Role of Receptors in Bacillus thuringiensis Crystal Toxin Activity

    PubMed Central

    Pigott, Craig R.; Ellar, David J.

    2007-01-01

    Bacillus thuringiensis produces crystalline protein inclusions with insecticidal or nematocidal properties. These crystal (Cry) proteins determine a particular strain's toxicity profile. Transgenic crops expressing one or more recombinant Cry toxins have become agriculturally important. Individual Cry toxins are usually toxic to only a few species within an order, and receptors on midgut epithelial cells have been shown to be critical determinants of Cry specificity. The best characterized of these receptors have been identified for lepidopterans, and two major receptor classes have emerged: the aminopeptidase N (APN) receptors and the cadherin-like receptors. Currently, 38 different APNs have been reported for 12 different lepidopterans. Each APN belongs to one of five groups that have unique structural features and Cry-binding properties. While 17 different APNs have been reported to bind to Cry toxins, only 2 have been shown to mediate toxin susceptibly in vivo. In contrast, several cadherin-like proteins bind to Cry toxins and confer toxin susceptibility in vitro, and disruption of the cadherin gene has been associated with toxin resistance. Nonetheless, only a small subset of the lepidopteran-specific Cry toxins has been shown to interact with cadherin-like proteins. This review analyzes the interactions between Cry toxins and their receptors, focusing on the identification and validation of receptors, the molecular basis for receptor recognition, the role of the receptor in resistant insects, and proposed models to explain the sequence of events at the cell surface by which receptor binding leads to cell death. PMID:17554045

  6. Indicators: Algal Toxins (microcystin)

    EPA Pesticide Factsheets

    Algal toxins are toxic substances released by some types of algae (phytoplankton) when they are present in large quantities (blooms) and decay or degrade. High nutrient levels and warm temperatures often result in favorable conditions for algae blooms.

  7. Characterisation of multidrug-resistant Shiga toxin-producing Escherichia coli cultured from pigs in China: co-occurrence of extended-spectrum β-lactamase- and mcr-1-encoding genes on plasmids.

    PubMed

    Bai, Li; Hurley, Daniel; Li, Juan; Meng, Qiong; Wang, Juan; Fanning, Séamus; Xiong, Yanwen

    2016-10-01

    Identification of Enterobacteriaceae harbouring the plasmid-mediated transferable colistin resistance gene mcr-1 presents a new challenge to public health. The aim of this study was to characterise multidrug-resistant Shiga toxin-producing Escherichia coli (STEC) harbouring the mcr-1 gene on plasmids cultured from pigs in China. Using CHROMagar™ ECC plates combined with stx gene detection by PCR, 93 STEC were recovered from 326 faecal, 351 small intestine content and 326 colon content samples taken from healthy pigs in 2011 and 2012 in China. This study, in which ten colistin-resistant isolates with minimum inhibitory concentrations (MICs) of 8-12 mg/L were identified and found to be positive by PCR for the mcr-1 gene, is a follow-up to an earlier investigation. Plasmid profiling by S1-nuclease digestion followed by pulsed-field gel electrophoresis (PFGE) identified several high-molecular-weight plasmids and these were typed by PCR-based replicon typing (PBRT). Two of the ten isolates, namely STEC-CQ09 (O116:H11/CC23/ST88) and CQ10 (O2:H32/ST3628), were selected for further study as described in this report.

  8. A regulatory role for Staphylococcus aureus toxin-antitoxin system PemIKSa.

    PubMed

    Bukowski, Michal; Lyzen, Robert; Helbin, Weronika M; Bonar, Emilia; Szalewska-Palasz, Agnieszka; Wegrzyn, Grzegorz; Dubin, Grzegorz; Dubin, Adam; Wladyka, Benedykt

    2013-01-01

    Toxin-antitoxin systems were shown to be involved in plasmid maintenance when they were initially discovered, but other roles have been demonstrated since. Here we identify and characterize a novel toxin-antitoxin system (pemIKSa) located on Staphylococcus aureus plasmid pCH91. The toxin (PemKSa) is a sequence-specific endoribonuclease recognizing the tetrad sequence U↓AUU, and the antitoxin (PemISa) inhibits toxin activity by physical interaction. Although the toxin-antitoxin system is responsible for stable plasmid maintenance our data suggest the participation of pemIKSa in global regulation of staphylococcal virulence by alteration of the translation of large pools of genes. We propose a common mechanism of reversible activation of toxin-antitoxin systems based on antitoxin transcript resistance to toxin cleavage. Elucidation of this mechanism is particularly interesting because reversible activation is a prerequisite for the proposed general regulatory role of toxin-antitoxin systems.

  9. 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

  10. Targeted silencing of anthrax toxin receptors protects against anthrax toxins.

    PubMed

    Arévalo, Maria T; Navarro, Ashley; Arico, Chenoa D; Li, Junwei; Alkhatib, Omar; Chen, Shan; Diaz-Arévalo, Diana; Zeng, Mingtao

    2014-05-30

    Anthrax spores can be aerosolized and dispersed as a bioweapon. Current postexposure treatments are inadequate at later stages of infection, when high levels of anthrax toxins are present. Anthrax toxins enter cells via two identified anthrax toxin receptors: tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2). We hypothesized that host cells would be protected from anthrax toxins if anthrax toxin receptor expression was effectively silenced using RNA interference (RNAi) technology. Thus, anthrax toxin receptors in mouse and human macrophages were silenced using targeted siRNAs or blocked with specific antibody prior to challenge with anthrax lethal toxin. Viability assays were used to assess protection in macrophages treated with specific siRNA or antibody as compared with untreated cells. Silencing CMG2 using targeted siRNAs provided almost complete protection against anthrax lethal toxin-induced cytotoxicity and death in murine and human macrophages. The same results were obtained by prebinding cells with specific antibody prior to treatment with anthrax lethal toxin. In addition, TEM8-targeted siRNAs also offered significant protection against lethal toxin in human macrophage-like cells. Furthermore, silencing CMG2, TEM8, or both receptors in combination was also protective against MEK2 cleavage by lethal toxin or adenylyl cyclase activity by edema toxin in human kidney cells. Thus, anthrax toxin receptor-targeted RNAi has the potential to be developed as a life-saving, postexposure therapy against anthrax.

  11. Selective alteration of sodium channel gating by Australian funnel-web spider toxins.

    PubMed

    Nicholson, G M; Little, M J; Tyler, M; Narahashi, T

    1996-01-01

    The actions of potent mammalian neurotoxins isolated from the venom of two Australian funnel-web spiders were investigated using both electrophysiological and neurochemical techniques. Whole-cell patch clamp recording of sodium currents in rat dorsal root ganglion neurons revealed that versutoxin (VTX), isolated from the venom of Hadronyche versuta, produced a concentration-dependent slowing or removal of tetrodotoxin-sensitive (TTX-S) sodium current inactivation and a reduction in peak TTX-S sodium current. In contrast, VTX had no effect on tetrodotoxin-resistant (TTX-R) sodium currents or potassium currents. VTX also shifted the voltage dependence of sodium channel activation in the hyperpolarizing direction and increased the rate of recovery from inactivation. Ion flux studies performed in rat brain synaptosomes also revealed that robustoxin (RTX), from the venom of Atrax robustus, and VTX both produced a partial activation of 22Na+ flux and an inhibition of batrachotoxin-activated 22Na+ flux. This inhibition of flux through batrachotoxin-activated channels was not due to an interaction with neurotoxin receptor site 1 since [3H]saxitoxin binding was unaffected. In addition, the partial activation of 22Na+ flux was not enhanced in the presence of alpha-scorpion toxin and further experiments suggest that VTX also enhances [3H]batrachotoxin binding. These selective actions of funnel-web spider toxins on sodium channel function are comparable to those of alpha-scorpion and sea anemone toxins which bind to neurotoxin receptor site 3 on the channel to slow channel inactivation profoundly. Also, these modifications of sodium channel gating and kinetics are consistent with actions of the spider toxins to produce repetitive firing of action potentials.

  12. [Botulism: structure and function of botulinum toxin and its clinical application].

    PubMed

    Oguma, Keiji; Yamamoto, Yumiko; Suzuki, Tomonori; Fatmawati, Ni Nengah Dwi; Fujita, Kumiko

    2012-08-01

    Clostridium botulinum produces seven immunological distinct poisonous neurotoxins, A to G, with molecular masses of approximately 150kDa. In acidic foods and culture fluid, the neurotoxins associate with non-toxic components, and form large complexes designated progenitor toxins. The progenitor toxins are found in three forms named LL, L, and M. These neurotoxins and progenitor toxins were purified, and whole nucleotide sequences of their structure genes were determined. In this manuscript, the structure and function of these toxins, and the application of these toxins to clinical usage have been described.

  13. Expression of the p20 Gene from Bacillus thuringiensis H-14 Increases Cry11A Toxin Production and Enhances Mosquito-Larvicidal Activity in Recombinant Gram-Negative Bacteria

    PubMed Central

    Xu, Y.; Nagai, M.; Bagdasarian, M.; Smith, T. W.; Walker, E. D.

    2001-01-01

    Experimental analyses with recombinant Escherichia coli and Pseudomonas putida transformed with plasmids bearing genes coding for the Cry11A toxin and P20 protein from Bacillus thuringiensis H-14 showed that cells producing both proteins were more toxic when fed to third-instar Aedes aegypti larvae than were cells expressing cry11A alone; the 50% lethal concentrations were in the range of 104 to 105 cells/ml. Western blots revealed a higher production of Cry11A when the p20 gene was coexpressed. Cry11A was detected primarily in insoluble form in recombinant cells. Cry11A was not detected in P. putida when P20 was not coproduced, and these recombinants were not toxic to larvae, whereas P. putida recombinants producing both proteins were toxic at concentrations similar to those for E. coli. A coelution experiment was conducted, in which a p20 gene construct producing the P20 protein with an extension of six histidines on the C terminus was mixed with the Cry11A protein. The results showed that Cry11A bound to the P20(His6) on a nickel chelating column, whereas Cry11A produced without the P20(His6) protein was washed through the column, thus indicating that Cry11A and P20 physically interact. Thus, P20 protein either stabilizes Cry11A or helps it attain the folding important for its toxic activity. PMID:11425714

  14. [UV-induction of the LT-toxin operon depending on genes lexA, recA, and umuD].

    PubMed

    Tiganova, I G; Rusina, O Iu; Andreeva, I V; Brukhanskiĭ, G V; Skavronskaia, A G

    1994-06-01

    UV induction of the elt operon (the LT-toxin operon in Escherichia coli) was demonstrated in experiments using fusion of elt::lac operons with the help of Mud1(Ap lac) phage. UV induction of the elt operon is lexA-dependent; thus, the possibility of SOS regulation of this process may be assumed. However, UV induction of the elt operon turned out to be recA-independent, which makes it impossible to consider this induction as a typical SOS response. UV induction of the elt operon is also observed in Salmonella typhimurium, which differs from E. coli in the product of umuD, which suggests that the UV induction of the elt operon is umuD independent.

  15. Naturally Occurring Food Toxins

    PubMed Central

    Dolan, Laurie C.; Matulka, Ray A.; Burdock, George A.

    2010-01-01

    Although many foods contain toxins as a naturally-occurring constituent or, are formed as the result of handling or processing, the incidence of adverse reactions to food is relatively low. The low incidence of adverse effects is the result of some pragmatic solutions by the US Food and Drug Administration (FDA) and other regulatory agencies through the creative use of specifications, action levels, tolerances, warning labels and prohibitions. Manufacturers have also played a role by setting limits on certain substances and developing mitigation procedures for process-induced toxins. Regardless of measures taken by regulators and food producers to protect consumers from natural food toxins, consumption of small levels of these materials is unavoidable. Although the risk for toxicity due to consumption of food toxins is fairly low, there is always the possibility of toxicity due to contamination, overconsumption, allergy or an unpredictable idiosyncratic response. The purpose of this review is to provide a toxicological and regulatory overview of some of the toxins present in some commonly consumed foods, and where possible, discuss the steps that have been taken to reduce consumer exposure, many of which are possible because of the unique process of food regulation in the United States. PMID:22069686

  16. A New Family of Secreted Toxins in Pathogenic Neisseria Species

    PubMed Central

    Jamet, Anne; Jousset, Agnès B.; Euphrasie, Daniel; Mukorako, Paulette; Boucharlat, Alix; Ducousso, Alexia; Charbit, Alain; Nassif, Xavier

    2015-01-01

    The genus Neisseria includes both commensal and pathogenic species which are genetically closely related. However, only meningococcus and gonococcus are important human pathogens. Very few toxins are known to be secreted by pathogenic Neisseria species. Recently, toxins secreted via type V secretion system and belonging to the widespread family of contact-dependent inhibition (CDI) toxins have been described in numerous species including meningococcus. In this study, we analyzed loci containing the maf genes in N. meningitidis and N. gonorrhoeae and proposed a novel uniform nomenclature for maf genomic islands (MGIs). We demonstrated that mafB genes encode secreted polymorphic toxins and that genes immediately downstream of mafB encode a specific immunity protein (MafI). We focused on a MafB toxin found in meningococcal strain NEM8013 and characterized its EndoU ribonuclease activity. maf genes represent 2% of the genome of pathogenic Neisseria, and are virtually absent from non-pathogenic species, thus arguing for an important biological role. Indeed, we showed that overexpression of one of the four MafB toxins of strain NEM8013 provides an advantage in competition assays, suggesting a role of maf loci in niche adaptation. PMID:25569427

  17. Genetic analysis of host-toxin interactions in the wheat-Stagonospora nodorum pathosystem

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Stagonospora nodorum, causal agent of Stagonospora nodorum blotch (SNB) in wheat, produces multiple necrosis-inducing host-selective toxins (HSTs) that interact with dominant host sensitivity genes to cause disease. Absence of either the toxin or the dominant host gene precludes recognition and resu...

  18. Toxin Diversity Revealed by a Transcriptomic Study of Ornithoctonus huwena

    PubMed Central

    He, Quanze; Liu, Jinyan; Luo, Ji; Zhu, Li; Lu, Shanshan; Huang, Pengfei; Chen, Xinyi; Zeng, Xiongzhi; Liang, Songping

    2014-01-01

    Spider venom comprises a mixture of compounds with diverse biological activities, which are used to capture prey and defend against predators. The peptide components bind a broad range of cellular targets with high affinity and selectivity, and appear to have remarkable structural diversity. Although spider venoms have been intensively investigated over the past few decades, venomic strategies to date have generally focused on high-abundance peptides. In addition, the lack of complete spider genomes or representative cDNA libraries has presented significant limitations for researchers interested in molecular diversity and understanding the genetic mechanisms of toxin evolution. In the present study, second-generation sequencing technologies, combined with proteomic analysis, were applied to determine the diverse peptide toxins in venom of the Chinese bird spider Ornithoctonus huwena. In total, 626 toxin precursor sequences were retrieved from transcriptomic data. All toxin precursors clustered into 16 gene superfamilies, which included six novel superfamilies and six novel cysteine patterns. A surprisingly high number of hypermutations and fragment insertions/deletions were detected, which accounted for the majority of toxin gene sequences with low-level expression. These mutations contribute to the formation of diverse cysteine patterns and highly variable isoforms. Furthermore, intraspecific venom variability, in combination with variable transcripts and peptide processing, contributes to the hypervariability of toxins in venoms, and associated rapid and adaptive evolution of toxins for prey capture and defense. PMID:24949878

  19. Marine Toxins: An Overview

    NASA Astrophysics Data System (ADS)

    Fusetani, Nobuhiro

    Oceans provide enormous and diverse space for marine life. Invertebrates are conspicuous inhabitants in certain zones such as the intertidal; many are soft-bodied, relatively immobile and lack obvious physical defenses. These animals frequently have evolved chemical defenses against predators and overgrowth by fouling organisms. Marine animals may accumulate and use a variety of toxins from prey organisms and from symbiotic microorganisms for their own purposes. Thus, toxic animals are particularly abundant in the oceans. The toxins vary from small molecules to high molecular weight proteins and display unique chemical and biological features of scientific interest. Many of these substances can serve as useful research tools or molecular models for the design of new drugs and pesticides. This chapter provides an initial survey of these toxins and their salient properties.

  20. Secondary metabolite toxins and nutrition of plant pathogenic fungi.

    PubMed

    Howlett, Barbara J

    2006-08-01

    Fungal pathogens derive nutrition from the plants they invade. Some fungi can subvert plant defence responses such as programmed cell death to provide nutrition for their growth and colonisation. Secondary metabolite toxins produced by fungi often play a role in triggering these responses. Knowledge of the biosynthesis of these toxins, and the availability of fungal genome sequences and gene disruption techniques, allows the development of tools for experiments aimed at discovering the role of such toxins in triggering plant cell death and plant disease.

  1. 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.

  2. Polymyositis after ciguatera toxin exposure.

    PubMed

    Stommel, E W; Parsonnet, J; Jenkyn, L R

    1991-08-01

    Biopsy-proved polymyositis subsequently developed in two patients who were severely poisoned by ciguatera fish toxin. Ciguatera toxin may have several mechanisms of action and may represent more than one toxin. The patients' clinical courses and the unlikelihood of coincidence of contracting both diseases suggested to us a causal relationship. Although we cannot prove this relationship, we suggest a mechanism by which the toxin predisposed the muscle to inflammation.

  3. Integrated optical toxin sensor

    NASA Astrophysics Data System (ADS)

    Kelly, Dan; Song, Xuedong; Frayer, Daniel K.; Mendes, Sergio B.; Peyghambarian, Nasser; Swanson, Basil I.; Grace, Karen M.

    1999-12-01

    We have developed a method for simple and highly sensitive detection of multivalent proteins using an optical waveguide sensor. The optical biosensor is based on optically tagged glycolipid receptors imbedded within a fluid phospholipid bilayer membrane formed on the surface of a planar optical waveguide. The binding of multivalent toxin initiates a fluorescence resonance energy transfer resulting in a distinctive spectral signature that is monitored by measuring emitted luminescence above the waveguide surface. The sensor methodology is highly sensitive and specific, and requires no additional reagents or washing steps. Demonstration of the utility of protein-receptor recognition using planar optical waveguides is shown here by the detection of cholera toxin.

  4. A DNA vaccine targeting the receptor-binding domain of Clostridium difficile toxin A.

    PubMed

    Gardiner, David F; Rosenberg, Talia; Zaharatos, Jerry; Franco, David; Ho, David D

    2009-06-02

    Clostridium difficile is a pathogen with increasing severity for which host antibody responses provide protection from disease. DNA vaccination has several advantages compared to traditional vaccine methods, however no study has examined this platform against C. difficile toxins. A synthetic gene was created encoding the receptor-binding domain (RBD) of C. difficile toxin A, optimized for expression in human cells. Gene expression was examined in vitro. Mice were inoculated and then challenged with parenteral toxin A. Vaccination provided high titer antibodies and protected mice from death. This represents the first report of DNA vaccine inducing neutralizing antibodies to C. difficile toxin A.

  5. Increased pathogenicity against coffee berry borer, Hypothenemus hampei (Coleoptera: Curculionidae) by Metarhizium anisopliae expressing the scorpion toxin (AaIT) gene.

    PubMed

    Pava-Ripoll, Monica; Posada, Francisco J; Momen, Bahram; Wang, Chengshu; St Leger, Raymond

    2008-10-01

    Coffee berry borer (CBB) is the Worlds most devastating coffee pest causing an estimated US$500 million worth of losses annually through damage and control costs. Beauveria bassiana and Metarhizium anisopliae have been employed to control this pest but their low virulence (slow kill and large inoculums) is an important factor constraining their use. M. anisopliae (AaIT-Ma549) has been modified to express the scorpion toxin (AaIT) in insect hemolymph and this greatly increased pathogenicity against Manduca sexta and Aedes aegypti. Here, we demonstrate that AaIT-Ma549 was also dramatically more virulent against CBB, and we provide a much more comprehensive analysis of infection processes and post-mortality development than in the previous research. We evaluated several spore concentrations (10(1) through 10(7)spores/ml) of both the wild type and recombinant strain. At concentrations of 10(1), 10(2) and 10(3)spores/ml, the recombinant strain significantly increased mortality of CBB by 32.2%, 56.6% and 24.6%, respectively. The medial lethal concentration (LC(50)) was reduced 15.7-fold and the average survival time (AST) was reduced by 20.1% to 2.98+/-0.1 days with 10(7)spores/ml. This is the first occasion that an entomopathogenic fungus has been found to kill CBB in less than 3 days. However, AaIT-Ma549 produces significantly fewer spores on cadavers than the parental strain.

  6. Diffusion of Botulinum Toxins

    PubMed Central

    Brodsky, Matthew A.; Swope, David M.; Grimes, David

    2012-01-01

    Background It is generally agreed that diffusion of botulinum toxin occurs, but the extent of the spread and its clinical importance are disputed. Many factors have been suggested to play a role but which have the most clinical relevance is a subject of much discussion. Methods This review discusses the variables affecting diffusion, including protein composition and molecular size as well as injection factors (e.g., volume, dose, injection method). It also discusses data on diffusion from comparative studies in animal models and human clinical trials that illustrate differences between the available botulinum toxin products (onabotulinumtoxinA, abobotulinumtoxinA, incobotulinumtoxinA, and rimabotulinumtoxinB). Results Neither molecular weight nor the presence of complexing proteins appears to affect diffusion; however, injection volume, concentration, and dose all play roles and are modifiable. Both animal and human studies show that botulinum toxin products are not interchangeable, and that some products are associated with greater diffusion and higher rates of diffusion-related adverse events than others. Discussion Each of the botulinum toxins is a unique pharmacologic entity. A working knowledge of the different serotypes is essential to avoid unwanted diffusion-related adverse events. In addition, clinicians should be aware that the factors influencing diffusion may range from properties intrinsic to the drug to accurate muscle selection as well as dilution, volume, and dose injected. PMID:23440162

  7. CYANOBACTERIA AND THEIR TOXINS.

    EPA Science Inventory

    Science Questions

    Harmful algal blooms (HAB) of cyanobacteria, also known as blue-green algae, have recently become more spatially and temporally prevalent in the US and worldwide. Cyanobacteria and their highly potent toxins are a significant hazard for human health and ...

  8. CYANOBACTERIA AND THEIR TOXINS

    EPA Science Inventory

    Science Questions

    Harmful algal blooms (HAB) of cyanobacteria, also known as blue-green algae, have recently become more spatially and temporally prevalent in the US and worldwide. Cyanobacteria and their highly potent toxins are a significant hazard for human health and ...

  9. Plasmapheresis as preconditioning protocol in an extremely high titer ABO incompatible renal transplant (ABOiRTx) case: A new prospect for chronic kidney disease patients in India.

    PubMed

    Pandey, Prashant; Tiwari, Aseem Kumar; Sharma, Jyoti; Dixit, Surbhi; Raina, Vimarsh

    2013-08-01

    The biggest hurdle in renal transplantation is the ABO blood group system. But recently ABO incompatible renal transplants have been performed using plasmapheresis (PP) as a part of the preconditioning protocol. In the present study, the objective of PP along with immunosuppression was to bring down the antibody titer of the patient to ≤ 16 during the transplant and keep it low, around 32, until post-operative 4-14 weeks. The patient (O Negative) had his mother (B Positive) as the ABO non-identical donor. The PP was performed with an apheresis equipment Com.Tec (Fresenius Kabi, Germany) to lower the anti-B antibody titer in the recipient. An Antihuman globulin (AHG) titer was performed for anti-B antibody following the departmental standard operating procedure. A total of 11 plasmapheresis procedures was performed preoperatively and four procedures were performed post-operatively to maintain the titer of the anti-B antibody at or below the desired level. The baseline anti-B antibody titer in the recipient was 512. The baseline titer came down to 8 after the end of the 11th procedure. Post-operatively we performed four plasmapheresis procedures to keep the titer at 32. During the post-operative follow up the titer has been maintained at 32 and the serum creatinine level has been maintained at approximately 1.0mg/dl and other parameters relevant to graft function were within normal limits. Our case could be the first reported case from India in which we used a plasmapheresis procedure as a part of preconditioning protocol instead of using an immunoadsorption column. Furthermore, it could be one of the few ABOiRTx cases, which has been performed at an isoagglutinin titer of 512 using plasma exchange as part of a preconditioning regime.

  10. Expression in sugar beet of the introduced cercosporin toxin export (CFP) gene from Cercospora kikuchii, the causative organism of purple seed stain in soybean.

    PubMed

    Kuykendall, L David; Upchurch, Robert G

    2004-05-01

    The Cercospora kikuchii cercosporin export gene, CFP, introduced into Beta vulgaris L. by conjugation with Rhizobium radiobacter, was stably maintained during vegetative propagation as verified by PCR using primers specific for the CFP gene. Transcriptional expression of the CFP gene in leaves was determined by RT-PCR using CFP-specific primers. CFP protein was detected using Western analysis with an affinity-purified polypeptide-specifc antibody. Analysis of the relative susceptibility of CFP-transgenic and non-transgenic sugar beet plants is planned but will probably take several years to complete.

  11. An SOS-regulated type 2 toxin-antitoxin system.

    PubMed

    Singletary, Larissa A; Gibson, Janet L; Tanner, Elizabeth J; McKenzie, Gregory J; Lee, Peter L; Gonzalez, Caleb; Rosenberg, Susan M

    2009-12-01

    The Escherichia coli chromosome encodes seven demonstrated type 2 toxin-antitoxin (TA) systems: cassettes of two or three cotranscribed genes, one encoding a stable toxin protein that can cause cell stasis or death, another encoding a labile antitoxin protein, and sometimes a third regulatory protein. We demonstrate that the yafNO genes constitute an additional chromosomal type 2 TA system that is upregulated during the SOS DNA damage response. The yafNOP genes are part of the dinB operon, of which dinB underlies stress-induced mutagenesis mechanisms. yafN was identified as a putative antitoxin by homology to known antitoxins, implicating yafO (and/or yafP) as a putative toxin. Using phage-mediated cotransduction assays for linkage disruption, we show first that yafN is an essential gene and second that it is essential only when yafO is present. Third, yafP is not a necessary part of either the toxin or the antitoxin. Fourth, although DinB is required, the yafNOP genes are not required for stress-induced mutagenesis in the Escherichia coli Lac assay. These results imply that yafN encodes an antitoxin that protects cells against a yafO-encoded toxin and show a protein-based TA system upregulated by the SOS response.

  12. TcdC Does Not Significantly Repress Toxin Expression in Clostridium difficile 630ΔErm

    PubMed Central

    Bakker, Dennis; Smits, Wiep Klaas; Kuijper, Ed J.; Corver, Jeroen

    2012-01-01

    In the past decade, Clostridium difficile has emerged as an important gut pathogen. Symptoms of C. difficile infection range from mild diarrhea to pseudomembranous colitis, sometimes resulting in colectomy or death. The main virulence factors of C. difficile are toxin A and toxin B. Besides the genes encoding these toxins (tcdA and tcdB), the pathogenicity locus (PaLoc) also contains genes encoding a sigma factor (tcdR) and a putative anti-sigma factor (tcdC). The important role of TcdR as a sigma factor for toxin expression is undisputed, whereas the role of TcdC as an anti-sigma factor, inhibiting toxin expression, is currently the subject of debate. To clarify the role of TcdC in toxin expression, we generated an isogenic ClosTron-based mutant of tcdC in Clostridium difficile strain 630Δ Erm (CT::tcdC) and determined the transcription levels of the PaLoc genes and the expression levels of the toxins in the wild type strain and the tcdC mutant strain. We found only minor differences in transcription levels of the PaLoc genes between the wild type and CT::tcdC strains and total toxin levels did not significantly differ either. These results suggest that in C. difficile 630Δerm TcdC is not a major regulator of toxin expression under the conditions tested. PMID:22912837

  13. Toxin-associated and other genes in Clostridium perfringens type A isolates from bovine clostridial abomasitis (BCA) and jejunal hemorrhage syndrome (JHS).

    PubMed

    Schlegel, Benjamin J; Nowell, Victoria J; Parreira, Valeria R; Soltes, Glenn; Prescott, John F

    2012-10-01

    This study examined known or possible virulence-associated genes in type A Clostridium perfringens from cases of both bovine clostridial abomasitis (BCA) and jejunal hemorrhage syndrome (JHS) and compared these to isolates from calves that were healthy or had undifferentiated diarrheal illness. A real-time polymerase chain reaction (PCR) assay was used to genotype the 218 C. perfringens isolates. Isolates were sourced from healthy and diarrheic young and mature cattle (n = 191), from calves with confirmed or suspected BCA (n = 22), and from mature cattle with JHS (n = 5). Of 216 isolates (96%), 208 were positive for the cpa gene and 13% (29/218) were positive for atypical cpb2. Three of 8 (37.5%) confirmed BCA isolates, 2 of 13 (15.4%) suspected BCA isolates, and no JHS isolates tested positive for atypical cpb2. As all isolates were negative for cpb, cpb2, cpe, etx, netB, and tpeL, the results of the present study do not support a role for these genes in BCA or JHS. A subset of unique genes identified in 1 bovine clostridial abomasitis isolate (F262), for which a genome sequence is available, was searched for in 8 BCA isolates by PCR. None of the 10 genes was consistently present in all or even in a majority of BCA isolates. Many of these genes were also variably and inconsistently present in type A isolates from calves that did not have BCA. Although a virulence signature to aid in the diagnosis of BCA caused by C. perfringens type A was not identified, further work may discover a gene or group of genes that would constitute such a signature.

  14. A Four-Plex Real-Time PCR Assay, Based on rfbE, stx1, stx2, and eae Genes, for the Detection and Quantification of Shiga Toxin-Producing Escherichia coli O157 in Cattle Feces.

    PubMed

    Noll, Lance W; Shridhar, Pragathi B; Shi, Xiaorong; An, Baoyan; Cernicchiaro, Natalia; Renter, David G; Nagaraja, Tiruvoor G; Bai, Jianfa

    2015-09-01

    Several real-time polymerase chain reaction (PCR) assays have been developed to detect and quantify Shiga toxin-producing Escherichia coli (STEC) O157:H7, but none have targeted the O-antigen specific gene (rfbEO157) in combination with the three major virulence genes, stx1, stx2, and eae. Our objectives were to develop and validate a four-plex, quantitative PCR (mqPCR) assay targeting rfbE(O157), stx1, stx2, and eae for the detection and quantification of STEC O157 in cattle feces, and compare the applicability of the assay to detect STEC O157 to a culture method and conventional PCR (cPCR) targeting the same four genes. Specificity of the mqPCR assay to differentially detect the four genes was confirmed with strains of O157 and non-O157 STEC with different profiles of target genes. In cattle feces spiked with pure cultures, detection limits were 2.8×10(4) and 2.8×10(0) colony-forming units/g before and after enrichment, respectively. Detection of STEC O157 in feedlot cattle fecal samples (n=278) was compared between mqPCR, cPCR, and a culture method. The mqPCR detected 48.9% (136/278) of samples as positive for E. coli O157. Of the 100 samples that were randomly picked from 136 mqPCR-positive samples, 35 and 48 tested positive by cPCR and culture method, respectively. Of the 100 samples randomly chosen from 142 mqPCR-negative samples, all were negative by cPCR, but 21 samples tested positive by the culture method. McNemar's chi-square tests indicated significant disagreement between the proportions of positive samples detected by the three methods. In conclusion, the mqPCR assay that targets four genes is a novel and more sensitive method than the cPCR or culture method to detect STEC O157 in cattle feces. However, the use of real-time PCR as a screening method to identify positive samples and then subjecting only positive samples to a culture method may underestimate the presence of STEC O157 in fecal samples.

  15. Bacillus thuringiensis (Bt) toxin susceptibility and isolation of resistance mutants in the nematode Caenorhabditis elegans.

    PubMed Central

    Marroquin, L D; Elyassnia, D; Griffitts, J S; Feitelson, J S; Aroian, R V

    2000-01-01

    The protein toxins produced by Bacillus thuringiensis (Bt) are the most widely used natural insecticides in agriculture. Despite successful and extensive use of these toxins in transgenic crops, little is known about toxicity and resistance pathways in target insects since these organisms are not ideal for molecular genetic studies. To address this limitation and to investigate the potential use of these toxins to control parasitic nematodes, we are studying Bt toxin action and resistance in Caenorhabditis elegans. We demonstrate for the first time that a single Bt toxin can target a nematode. When fed Bt toxin, C. elegans hermaphrodites undergo extensive damage to the gut, a decrease in fertility, and death, consistent with toxin effects in insects. We have screened for and isolated 10 recessive mutants that resist the toxin's effects on the intestine, on fertility, and on viability. These mutants define five genes, indicating that more components are required for Bt toxicity than previously known. We find that a second, unrelated nematicidal Bt toxin may utilize a different toxicity pathway. Our data indicate that C. elegans can be used to undertake detailed molecular genetic analysis of Bt toxin pathways and that Bt toxins hold promise as nematicides. PMID:10924467

  16. Toxins and drug discovery.

    PubMed

    Harvey, Alan L

    2014-12-15

    Components from venoms have stimulated many drug discovery projects, with some notable successes. These are briefly reviewed, from captopril to ziconotide. However, there have been many more disappointments on the road from toxin discovery to approval of a new medicine. Drug discovery and development is an inherently risky business, and the main causes of failure during development programmes are outlined in order to highlight steps that might be taken to increase the chances of success with toxin-based drug discovery. These include having a clear focus on unmet therapeutic needs, concentrating on targets that are well-validated in terms of their relevance to the disease in question, making use of phenotypic screening rather than molecular-based assays, and working with development partners with the resources required for the long and expensive development process.

  17. The Influence of Low Doses of Zearalenone and T-2 Toxin on Calcitonin Gene Related Peptide-Like Immunoreactive (CGRP-LI) Neurons in the ENS of the Porcine Descending Colon

    PubMed Central

    Makowska, Krystyna; Obremski, Kazimierz; Zielonka, Lukasz; Gonkowski, Slawomir

    2017-01-01

    The enteric nervous system (ENS) can undergo adaptive and reparative changes in response to physiological and pathological stimuli. These manifest primarily as alterations in the levels of active substances expressed by the enteric neuron. While it is known that mycotoxins can affect the function of the central and peripheral nervous systems, knowledge about their influence on the ENS is limited. Therefore, the aim of the present study was to investigate the influence of low doses of zearalenone (ZEN) and T-2 toxin on calcitonin gene related peptide-like immunoreactive (CGRP-LI) neurons in the ENS of the porcine descending colon using a double immunofluorescence technique. Both mycotoxins led to an increase in the percentage of CGRP-LI neurons in all types of enteric plexuses and changed the degree of co-localization of CGRP with other neuronal active substances, such as substance P, galanin, nitric oxide synthase, and cocaine- and amphetamine-regulated transcript peptide. The obtained results demonstrate that even low doses of ZEN and T-2 can affect living organisms and cause changes in the neurochemical profile of enteric neurons. PMID:28287437

  18. The long polar fimbriae genes identified in Shiga toxin-producing Escherichia coli are present in other diarrheagenic E. coli and in the standard E. coli collection of reference (ECOR) strains.

    PubMed

    Toma, Claudia; Higa, Naomi; Iyoda, Sunao; Rivas, Marta; Iwanaga, Masaaki

    2006-03-01

    Long polar fimbriae (LPF) are related to type I fimbriae in genetic organization and were first identified in Salmonella enterica serovar Typhimurium. Four lpfA genetic variants designated lpfA(O157/OI-141), lpfA(O157/OI-154), lpfA(O26) and lpfA(O113) have been identified in Shiga toxin-producing Escherichia coli (STEC). In this study, PCR was employed to determine the distribution of STEC-lpfAs in enteropathogenic, enteroaggregative, enterotoxigenic and enteroinvasive E. coli (EPEC, EAEC, ETEC and EIEC) and in the standard E. coli collection of reference (ECOR). Among the 97 diarrheagenic strains from our collection, only 2 EPEC strains of serotypes O55:H7 and O119:NM were positive for both lpfA(O157/OI-141) and lpfA(O157/OI-154). lpfA(O157/OI-141) was also positive in 1 of 25 ETEC strains. lpfA(O113) was present in 51 of 97 strains and lpfA(O26) in 13 of 97 strains of diverse diarrheagenic categories. STEC-lpfAs were also present in non-pathogenic ECOR strains of all phylogenetic groups. This study showed that the lpfA genes identified in the genome of STEC strains are not specific to this category. Our results suggest that there is a relationship between the lpfA variant and the phylogenetic group.

  19. Influence of primer sequences and DNA extraction method on detection of non-O157 Shiga toxin-producing Escherichia coli in ground beef by real-time PCR targeting the eae, stx, and serogroup-specific genes.

    PubMed

    Wasilenko, Jamie L; Fratamico, Pina M; Narang, Neelam; Tillman, Glenn E; Ladely, Scott; Simmons, Mustafa; Cray, William C

    2012-11-01

    Non-O157 Shiga toxin-producing Escherichia coli (STEC) infections, particularly those caused by the "big six" or "top six" non-O157 serogroups (O26, O45, O103, O111, O121, and O145) can result in severe illness and complications. Because of their significant public health impact and the notable prevalence of STEC in cattle, methods for detection of the big six non-O157 STEC in ground beef have been established. Currently, the U.S. Department of Agriculture, Food Safety and Inspection Service detection methods for screening beef samples for non-O157 STEC target the stx(1), stx(2), and eae virulence genes, with the 16S rRNA gene as an internal control, in a real-time PCR multiplex assay. Further, the serogroup is determined by PCR targeting genes in the E. coli O-antigen gene clusters of the big six non-O157 serogroups. The method that we previously reported was improved so that additional stx variants, stx(1d), stx(2e), and stx(2g), are detected. Additionally, alignments of the primers targeting the eae gene were used to improve the detection assay so that eae subtypes that could potentially be of clinical significance would also be detected. Therefore, evaluation of alternative real-time PCR assay primers and probes for the stx and eae reactions was carried out in order to increase the stx and eae subtypes detected. Furthermore, a Tris-EDTA DNA extraction method was compared with a previously used procedure that was based on a commercially available reagent. The Tris-EDTA DNA extraction method significantly decreased the cycle threshold values for the stx assay (P < 0.0001) and eae assay (P < 0.0001), thereby increasing the ability to detect the targets. The use of different stx primers and probes increased the subtypes detected to include stx(1d), stx(2e), and stx(2g), and sequence data showed that modification of the eae primer should allow the known eae subtypes to be detected.

  20. [Cytolethal distending toxins].

    PubMed

    Curová, K; Kmeťová, M; Siegfried, L

    2014-06-01

    Cytolethal distending toxins (CDT) are intracellularly acting proteins which interfere with the eukaryotic cell cycle. They are produced by Gram-negative bacteria with affinity to mucocutaneous surfaces and could play a role in the pathogenesis of various mammalian diseases. The functional toxin is composed of three proteins: CdtB entering the nucleus and by its nuclease activity inducing nuclear fragmentation and chromatin disintegration, CdtA, and CdtC, the two latter being responsible for toxin attachment to the surface of the target cell. Cytotoxic effect of CDT leads to the cell cycle arrest before the cell enters mitosis and to further changes (cell distension and death, apoptosis) depending on the cell type. Thus, CDT may function as a virulence factor in pathogenic bacteria that produce it and thus may contribute to the initiation of certain diseases. Most important are inflammatory bowel diseases caused by intestinal bacteria, periodontitis with Aggregatibacter actinomycetemcomitans as the aetiologic agent and ulcus molle where Haemophilus ducreyi is the causative agent.

  1. Ribosomally encoded cyclic peptide toxins from mushrooms.

    PubMed

    Walton, Jonathan D; Luo, Hong; Hallen-Adams, Heather

    2012-01-01

    The cyclic peptide toxins of poisonous Amanita mushrooms are chemically unique among known natural products. Furthermore, they differ from other fungal cyclic peptides in being synthesized on ribosomes instead of by nonribosomal peptide synthetases. Because of their novel structures and biogenic origins, elucidation of the biosynthetic pathway of the Amanita cyclic peptides presents both challenges and opportunities. In particular, a full understanding of the pathway should lead to the ability to direct synthesis of a large number of novel cyclic peptides based on the Amanita toxin scaffold by genetic engineering of the encoding genes. Here, we highlight some of the principal methods for working with the Amanita cyclic peptides and the known steps in their biosynthesis.

  2. Method for detecting biological toxins

    SciTech Connect

    Ligler, F.S.; Campbell, J.R.

    1992-01-01

    Biological toxins are indirectly detected by using polymerase chain reaction to amplify unique nucleic acid sequences coding for the toxins or enzymes unique to toxin synthesis. Buffer, primers coding for the unique nucleic acid sequences and an amplifying enzyme are added to a sample suspected of containing the toxin. The mixture is then cycled thermally to exponentially amplify any of these unique nucleic acid sequences present in the sample. The amplified sequences can be detected by various means, including fluorescence. Detection of the amplified sequences is indicative of the presence of toxin in the original sample. By using more than one set of labeled primers, the method can be used to simultaneously detect several toxins in a sample.

  3. [Today's threat of ricin toxin].

    PubMed

    From, Sławomir; Płusa, Tadeusz

    2015-09-01

    Since the late 70s of the last century there were more than 700 incidents related to the use of the ricin toxin. For this reason, CDC (Center of Disease Control and Prevention) recognized toxin as a biological weapon category B. The lethal dose of ricin toxin after parenteral administration is 0.0001 mg/kg and after oral administration 0.2 mg. The first symptoms of poisoning occur within a few hours after application of toxin as a nausea, vomiting and abdominal pain. In the final stage there are observed: cardiac arrhythmia, collapse and symptoms suggestive of involvement of the central nervous system. Stage immediately preceding death is a state of coma. The ricin toxin is still the substance against which action has no optimal antidote. Developed a vaccine called RiVax is waiting for its registration. It should be pointed out that the availability of a ricin toxin makes it possible to use it for real bioterrorists.

  4. Response of the Gypsy Moth, Lymantria dispar to Transgenic Poplar, Populus simonii x P. nigra, Expressing Fusion Protein Gene of the Spider Insecticidal Peptide and Bt-toxin C-peptide

    PubMed Central

    Cao, Chuan-Wang; Liu, Gui-Feng; Wang, Zhi-Ying; Yan, Shan-Chun; Ma, Ling; Yang, Chuan-Ping

    2010-01-01

    The response of the Asian gypsy moth Lymantria dispar (L.) (Lepidoptera: Lymantriidae) to a fusion gene consisting of the spider, Atrax robustus Simon (Araneae: Hexanthelidae) ω?-ACTX-Ar1 sequence coding for an ω?-atracotoxin and a sequence coding for the Bt-toxin C-peptide, expressed in transgenic poplar Populus simonii x P. nigra L. (Malphigiales: Salicaceae) was investigated. Individual performance, feeding selection, midgut proteinase activity and nutrition utilization were monitored. The growth and development of L. dispar were significantly affected by continually feeding on the transgenic poplar, with the larval instars displaying significantly shorter developmental times than those fed on nontransgenic poplar, but pupation was delayed. Mortality was higher in populations fed transgenic poplar leaves, than for larvae fed nontransgenic poplar leaves. The cumulative mortality during all stages of larvae fed transgenic leaves was 92% compared to 16.7% of larvae on nontransgenic leaves. The highest mortality observed was 71.7% in the last larval instar stage. A two-choice test showed that fifth-instar larvae preferred to feed on nontransgenic leaves at a ratio of 1:1.4. Feeding on transgenic leaves had highly significant negative effects on relative growth of larvae, and the efficiency of conversion of ingested and digested food. Activity of major midgut proteinases was measured using substrates TAME and BTEE showed significant increases in tryptase and chymotrypsinlike activity (9.2- and 9.0-fold, respectively) in fifth-instar larvae fed on transgenic leaves over control. These results suggest transgenic poplar is resistant to L. dispar, and the mature L. dispar may be weakened by the transgenic plants due to Bt protoxins activated by elevated major midgut proteinase activity. The new transgenic poplar expressing fusion protein genes of Bt and a new spider insecticidal peptide are good candidates for managing gypsy moth. PMID:21268699

  5. Response of the gypsy moth, Lymantria dispar to transgenic poplar, Populus simonii x P. nigra, expressing fusion protein gene of the spider insecticidal peptide and Bt-toxin C-peptide.

    PubMed

    Cao, Chuan-Wang; Liu, Gui-Feng; Wang, Zhi-Ying; Yan, Shan-Chun; Ma, Ling; Yang, Chuan-Ping

    2010-01-01

    The response of the Asian gypsy moth Lymantria dispar (L.) (Lepidoptera: Lymantriidae) to a fusion gene consisting of the spider, Atrax robustus Simon (Araneae: Hexanthelidae) ω-ACTX-Ar1 sequence coding for an ω-atracotoxin and a sequence coding for the Bt-toxin C-peptide, expressed in transgenic poplar Populus simonii x P. nigra L. (Malphigiales: Salicaceae) was investigated. Individual performance, feeding selection, midgut proteinase activity and nutrition utilization were monitored. The growth and development of L. dispar were significantly affected by continually feeding on the transgenic poplar, with the larval instars displaying significantly shorter developmental times than those fed on nontransgenic poplar, but pupation was delayed. Mortality was higher in populations fed transgenic poplar leaves, than for larvae fed nontransgenic poplar leaves. The cumulative mortality during all stages of larvae fed transgenic leaves was 92% compared to 16.7% of larvae on nontransgenic leaves. The highest mortality observed was 71.7% in the last larval instar stage. A two-choice test showed that fifth-instar larvae preferred to feed on nontransgenic leaves at a ratio of 1:1.4. Feeding on transgenic leaves had highly significant negative effects on relative growth of larvae, and the efficiency of conversion of ingested and digested food. Activity of major midgut proteinases was measured using substrates TAME and BTEE showed significant increases in tryptase and chymotrypsinlike activity (9.2- and 9.0-fold, respectively) in fifth-instar larvae fed on transgenic leaves over control. These results suggest transgenic poplar is resistant to L. dispar, and the mature L. dispar may be weakened by the transgenic plants due to Bt protoxins activated by elevated major midgut proteinase activity. The new transgenic poplar expressing fusion protein genes of Bt and a new spider insecticidal peptide are good candidates for managing gypsy moth.

  6. Inhibitory effect of eugenol on aflatoxin B1 production in Aspergillus parasiticus by downregulating the expression of major genes in the toxin biosynthetic pathway.

    PubMed

    Jahanshiri, Zahra; Shams-Ghahfarokhi, Masoomeh; Allameh, Abdolamir; Razzaghi-Abyaneh, Mehdi

    2015-07-01

    Aflatoxin contamination of grains and agro-products is a serious food safety issue and a significant economic concern worldwide. In the present study, the effects of eugenol on Aspergillus parasiticus growth and aflatoxin production were studied in relation to the expression of some essential genes involved in aflatoxin biosynthetic pathway. The fungus was cultured in presence of serial two-fold concentrations of eugenol (15.62-500 μg mL(-1)) for 3 days at 28 °C. Mycelia dry weight was determined as an index of fungal growth, while aflatoxin production was assessed by high performance liquid chromatography. The expression of aflatoxin biosynthetic genes including ver-1, nor-1, pksA, omtA and aflR were evaluated by real-time PCR. Eugenol strongly inhibited A. parasiticus growth in the range of 19.16-95.83 % in a dose-dependent manner. Aflatoxin B1 production was also inhibited by the compound in the range of 15.07-98.0 %. The expressions of ver-1, nor-1, pksA, omtA and aflR genes were significantly suppressed by eugenol at concentrations of 62.5 and 125 μg mL(-1). These results indicate that eugenol may be considered as a good candidate to control toxigenic fungal growth and the subsequent contamination of food, feed and agricultural commodities by carcinogenic aflatoxins.

  7. Expression of pertussis toxin in Bordetella bronchiseptica and Bordetella parapertussis carrying recombinant plasmids.

    PubMed Central

    Lee, C K; Roberts, A; Perrin, S

    1989-01-01

    Pertussis toxin is produced only by strains of Bordetella pertussis. Cloned genes encoding pertussis toxin from B. pertussis were transferred into Bordetella bronchiseptica and Bordetella parapertussis by conjugation. These transconjugants expressed pertussis toxin at levels comparable to those expressed by B. pertussis. The toxin made by these strains was biologically active in the Chinese hamster cell clumping assay, contained all five subunits, and was mostly periplasmic. Toxin expression appeared to be modulated in the same way as are the vir-regulated genes of B. pertussis. Introduction of these plasmids into B. pertussis failed to produce hypertoxigenic strains. Instead, these transconjugants underwent plasmid loss, gene deletions, or conversion to the avirulent phase. Images PMID:2707851

  8. Pore formation by Cry toxins.

    PubMed

    Soberón, Mario; Pardo, Liliana; Muñóz-Garay, Carlos; Sánchez, Jorge; Gómez, Isabel; Porta, Helena; Bravo, Alejandra

    2010-01-01

    Bacillus thuringiensis (Bt) bacteria produce insecticidal Cry and Cyt proteins used in the biological control of different insect pests. In this review, we will focus on the 3d-Cry toxins that represent the biggest group of Cry proteins and also on Cyt toxins. The 3d-Cry toxins are pore-forming toxins that induce cell death by forming ionic pores into the membrane of the midgut epithelial cells in their target insect. The initial steps in the mode of action include ingestion of the protoxin, activation by midgut proteases to produce the toxin fragment and the interaction with the primary cadherin receptor. The interaction of the monomeric CrylA toxin with the cadherin receptor promotes an extra proteolytic cleavage, where helix alpha-1 of domain I is eliminated and the toxin oligomerization is induced, forming a structure of 250 kDa. The oligomeric structure binds to a secondary receptor, aminopeptidase N or alkaline phosphatase. The secondary receptor drives the toxin into detergent resistant membrane microdomains formingpores that cause osmotic shock, burst of the midgut cells and insect death. Regarding to Cyt toxins, these proteins have a synergistic effect on the toxicity of some Cry toxins. Cyt proteins are also proteolytic activated in the midgut lumen of their target, they bind to some phospholipids present in the mosquito midgut cells. The proposed mechanism of synergism between Cry and Cyt toxins is that Cyt1Aa function as a receptor for Cry toxins. The Cyt1A inserts into midgut epithelium membrane and exposes protein regions that are recognized by Cry11Aa. It was demonstrated that this interaction facilitates the oligomerization of Cry11Aa and also its pore formation activity.

  9. Ricin detection: tracking active toxin.

    PubMed

    Bozza, William P; Tolleson, William H; Rosado, Leslie A Rivera; Zhang, Baolin

    2015-01-01

    Ricin is a plant toxin with high bioterrorism potential due to its natural abundance and potency in inducing cell death. Early detection of the active toxin is essential for developing appropriate countermeasures. Here we review concepts for designing ricin detection methods, including mechanism of action of the toxin, advantages and disadvantages of current detection assays, and perspectives on the future development of rapid and reliable methods for detecting ricin in environmental samples.

  10. The chromosomal nature of LT-II enterotoxins solved: a lambdoid prophage encodes both LT-II and one of two novel pertussis-toxin-like toxin family members in type II enterotoxigenic Escherichia coli

    PubMed Central

    Jobling, Michael G.

    2016-01-01

    Heat-labile enterotoxins (LT) of enterotoxigenic Escherichia coli (ETEC) are structurally and functionally related to cholera toxin (CT). LT-I toxins are plasmid-encoded and flanked by IS elements, while LT-II toxins of type II ETEC are chromosomally encoded with flanking genes that appear phage related. Here, I determined the complete genomic sequence of the locus for the LT-IIa type strain SA53, and show that the LT-IIa genes are encoded by a 51 239 bp lambdoid prophage integrated at the rac locus, the site of a defective prophage in E. coli K12 strains. Of 50 LT-IIa and LT-IIc, 46 prophages also encode one member of two novel two-gene ADP-ribosyltransferase toxin families that are both related to pertussis toxin, which I named eplBA or ealAB, respectively. The eplBA and ealAB genes are syntenic with the Shiga toxin loci in their lambdoid prophages of the enteric pathogen enterohemorrhagic E. coli. These novel AB5 toxins show pertussis-toxin-like activity on tissue culture cells, and like pertussis toxin bind to sialic acid containing glycoprotein ligands. Type II ETEC are the first mucosal pathogens known to simultaneously produce two ADP-ribosylating toxins predicted to act on and modulate activity of both stimulatory and inhibitory alpha subunits of host cell heterotrimeric G-proteins. PMID:26755534

  11. The chromosomal nature of LT-II enterotoxins solved: a lambdoid prophage encodes both LT-II and one of two novel pertussis-toxin-like toxin family members in type II enterotoxigenic Escherichia coli.

    PubMed

    Jobling, Michael G

    2016-04-01

    Heat-labile enterotoxins (LT) of enterotoxigenic Escherichia coli (ETEC) are structurally and functionally related to cholera toxin (CT). LT-I toxins are plasmid-encoded and flanked by IS elements, while LT-II toxins of type II ETEC are chromosomally encoded with flanking genes that appear phage related. Here, I determined the complete genomic sequence of the locus for the LT-IIa type strain SA53, and show that the LT-IIa genes are encoded by a 51 239 bp lambdoid prophage integrated at the rac locus, the site of a defective prophage in E. coli K12 strains. Of 50 LT-IIa and LT-IIc, 46 prophages also encode one member of two novel two-gene ADP-ribosyltransferase toxin families that are both related to pertussis toxin, which I named eplBA or ealAB, respectively. The eplBA and ealAB genes are syntenic with the Shiga toxin loci in their lambdoid prophages of the enteric pathogen enterohemorrhagic E. coli. These novel AB(5) toxins show pertussis-toxin-like activity on tissue culture cells, and like pertussis toxin bind to sialic acid containing glycoprotein ligands. Type II ETEC are the first mucosal pathogens known to simultaneously produce two ADP-ribosylating toxins predicted to act on and modulate activity of both stimulatory and inhibitory alpha subunits of host cell heterotrimeric G-proteins.

  12. THE SYNERGY OF BACTERIAL TOXINS,

    DTIC Science & Technology

    TOXINS AND ANTITOXINS, STRENGTH(PHYSIOLOGY), BACTERIA , CLOSTRIDIUM PERFRINGENS, CLOSTRIDIUM TETANI, CLOSTRIDIUM, STAPHYLOCOCCUS, ESCHERICHIA COLI, PROTEUS, ETIOLOGY, ANTIGENS, ANTIBODIES, AMINO ACIDS.

  13. The DinJ/RelE toxin-antitoxin system suppresses virulence in Xylella fastidiosa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Xylella fastidiosa, the causal agent of a number agriculturally important plant diseases, encodes multiple toxin-antitoxin (TA) systems. TA modules consist of a toxin protein co-expressed with a specific antitoxin, and are often acquired through horizontal gene transfer. Antitoxin molecules (RNA or ...

  14. Comparison of non-O157 Shiga toxin-producing E. coli detection systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Category: methodology improvements Objective: To identify strengths and weaknesses of commercial Shiga toxin-producing E. coli detection systems and kits in a side by side fashion. Experimental Design: Three commercial Shiga toxin-producing E. coli detection tests (BAX, GDS, and GeneDisc) and two t...

  15. Effect of botulinum toxin A-induced paralysis and exercise training on mechanosensing and signalling gene expression in juvenile rat gastrocnemius muscle.

    PubMed

    Velders, Martina; Legerlotz, Kirsten; Falconer, Shelley J; Stott, N Susan; McMahon, Christopher D; Smith, Heather K

    2008-12-01

    Intramuscular injections of the paralytic botulinum neurotoxin A (Btx) and physical exercise are used in the treatment of chronic spasticity in children with cerebral palsy. We tested whether Btx-induced paralysis and/or exercise training would have differential effects on the expression of mechanosensing and signalling genes implicated in the adaptive remodelling of skeletal muscle. Juvenile (29-day-old) male rats were injected with Btx or saline (NoBtx) into the right gastrocnemius and housed in standard cages (NoEx) or with running wheels (Ex), for 3 weeks (n = 6 per group). The mRNA expression of nine sarcomere-associated genes in the medial gastrocnemius was then determined by quantitative reverse transcriptase-polymerase chain reaction. The Btx-injected muscles weighed 50% less than NoBtx muscles, but Ex had no effect on the wet mass of Btx or NoBtx muscles. Atrogenic MuRF1, sarcomeric Titin and myogenic MyoD were upregulated (2-fold) with the elimination of contractile activity in Btx muscle. Expression of CARP, Ankrd2 and MLP was increased with mechanical stimuli associated with Btx (5- to 10-fold) or Ex (2- to 4-fold). Expression of CARP and Ankrd2 increased synergistically in Btx-Ex muscle (> or = 20-fold), indicating that these genes may be sensitive to passive stretch of the sarcomeric I-band region of titin to which their proteins bind. Tcap, Myopalladin and Atrogin1 were not, or were no longer responsive to the altered mechanical stimuli after 3 weeks of Btx or Ex. The expression of Ankrd2, CARP and MLP may thus be enhanced by passive stretch within the Btx-paralysed and/or exercising gastrocnemius and contribute to adaptations, other than muscle mass, in juvenile rats.

  16. Geraniol synthase whose mRNA is induced by host-selective ACT-toxin in the ACT-toxin-insensitive rough lemon (Citrus jambhiri).

    PubMed

    Shishido, Hodaka; Miyamoto, Yoko; Ozawa, Rika; Taniguchi, Shiduku; Takabayashi, Junji; Akimitsu, Kazuya; Gomi, Kenji

    2012-09-15

    Host-selective toxins (HSTs) produced by some strains of Alternaria alternata are selectively toxic to certain cultivars of plants. However, the role of HSTs in toxin-insensitive plants is currently unknown. Here, we studied the role of ACT-toxin using an ACT-toxin producing A. alternata strain SH20 and the ACT-toxin-insensitive plant rough lemon. Induction of some defense related genes in response to SH20 were faster or stronger than in response to the ACT-toxin deficient SH20 mutant. By sequencing subtractive PCR clones obtained from mRNA of rough lemon leaves inoculated with SH20 after subtraction with that of the ACT-toxin deficient SH20 mutant, we isolated the SH20-responsive genes in rough lemon. Among the SH20-responsive genes analyzed in this study, we isolated a terpene synthase (TPS) gene, RlemTPS3. We also determined that RlemTPS3 localizes to the chloroplast and produces the monoterpene geraniol.

  17. Antibacterial resistance, genes encoding toxins and genetic background among Staphylococcus aureus isolated from community-acquired skin and soft tissue infections in France: a national prospective survey.

    PubMed

    Lamy, B; Laurent, F; Gallon, O; Doucet-Populaire, F; Etienne, J; Decousser, J-W

    2012-06-01

    The epidemiology of staphylococcal community-acquired skin and soft tissues infections (CA-SSTIs) has changed dramatically. We described prospectively the characteristics of the Staphylococcus aureus isolated from 71 non-teaching French hospitals and implicated in CA-SSTIs: antimicrobial susceptibility (mecA polymerase chain reaction [PCR], disk diffusion method), virulence factor gene (sea, tst, pvl) prevalence and genetic background (agr allele). During November 2006, 235 strains were collected (wound infection: 51%, abscess: 21%, whitlow: 8%, diabetic foot: 7%, furunculosis: 3%). sea, tst and pvl were identified in 22.1, 13.2 and 8.9% strains, respectively. agr allele 1 was the most frequently encountered genetic background, whatever the methicillin susceptibility. Among the 34 methicillin-resistant S. aureus (MRSA, 14.5% of all S. aureus), only one strain (2.9%) harboured pvl (belonging to the European ST80 clone), four (11.8%) tst (belonging to two endemic French clones) and 18 (52.9%) sea gene (mainly the Lyon clone). According to their in vitro activity, pristinamycin or trimethoprim/sulfamethoxazole could be considered as first-choice antibiotics. To date, the international pvl-positive MRSA clones have not spread in France. MRSA strains isolated from putative CA-SSTIs exhibited a genetic and phenotypic background of hospital-acquired (HA) clones. National survey should be continued, in order to monitor the emergence of virulent clones.

  18. Transcription of the toxin genes present within the Staphylococcal phage phiSa3ms is intimately linked with the phage's life cycle.

    PubMed

    Sumby, Paul; Waldor, Matthew K

    2003-12-01

    phiSa3ms, a lysogenic bacteriophage encoding the staphylococcal enterotoxins SEA, SEG, and SEK and the fibrinolytic enzyme staphylokinase (Sak), was identified in the unannotated genome sequence of the hypervirulent community-acquired Staphylococcus aureus strain 476. We found that mitomycin C induction of phiSa3ms led to increased transcription of all four virulence factors. The increase in sea and sak transcription was a result of read-through transcription from upstream latent phage promoters and an increase in phage copy number. The majority of the seg2 and sek2 transcripts were shown to initiate from the upstream phage cI promoter and hence were regulated by factors influencing cI transcription. The lysogeny module of phiSa3ms was shown to have some lambda-like features with divergent cI and cro genes. Band shift assays were used to identify binding sites for both CI and Cro within the region between these genes, suggesting a mechanism of control for the phiSa3ms lytic-lysogenic switch. Our findings suggest that the production of phage-encoded virulence factors in S. aureus may be regulated by processes that govern lysogeny.

  19. Functional significance of the highly conserved Glu(570) in the putative pore-forming helix 3 of the Bordetella pertussis haemolysin toxin.

    PubMed

    Kurehong, Chattip; Powthongchin, Busaba; Thamwiriyasati, Niramon; Angsuthanasombat, Chanan

    2011-05-01

    Adenylate cyclase-haemolysin toxin (CyaA) is a virulence factor secreted from the etiologic agent of whooping cough, Bordetella pertussis. Previously, the haemolysin or pore-forming domain (CyaA-PF) has been shown to cause cell lysis of sheep erythrocytes independently, and the predicted helix 3((570-593)) within the PF-hydrophobic stretch could be a pore-lining constituent. Here, a plausible involvement in haemolytic activity of polar or charged residues (Glu(570), Gln(574), Glu(581), Ser(584) and Ser(585)) lining the hydrophilic side of CyaA-PF helix 3 was investigated via single-alanine substitutions. All the 126-kDa mutant proteins over-expressed in Escherichia coli were verified for toxin acylation as the results are corresponding to the wild-type toxin. When haemolytic activity of E. coli lysates containing soluble mutant proteins was tested against sheep erythrocytes, the importance of Glu(570), which is highly conserved among the pore-forming RTX cytotoxin family, was revealed for pore formation, conceivably for a general pore-lining residue involved in ion conduction.

  20. Sensitivity to Alternaria alternata toxin in citrus because of altered mitochondrial RNA processing

    PubMed Central

    Ohtani, Kouhei; Yamamoto, Hiroyuki; Akimitsu, Kazuya

    2002-01-01

    Specificity in the interaction between rough lemon (Citrus jambhiri Lush.) and the fungal pathogen Alternaria alternata rough lemon pathotype is determined by a host-selective toxin, ACR-toxin. Mitochondria from rough lemon are sensitive to ACR-toxin whereas mitochondria from resistant plants, including other citrus species, are resistant. We have identified a C. jambhiri mitochondrial DNA sequence, designated ACRS (ACR-toxin sensitivity gene), that confers toxin sensitivity to Escherichia coli. ACRS is located in the group II intron of the mitochondrial tRNA-Ala and is translated into a SDS-resistant oligomeric protein in C. jambhiri mitochondria but is not translated in the toxin-insensitive mitochondria. ACRS is present in the mitochondrial genome of both toxin-sensitive and -insensitive citrus. However, in mitochondria of toxin-insensitive plants, the transcripts from ACRS are shorter than those in mitochondria of sensitive plants. These results demonstrate that sensitivity to ACR-toxin and hence specificity of the interaction between A. alternata rough lemon pathotype and C. jambhiri is due to differential posttranscriptional processing of a mitochondrial gene. PMID:11842194

  1. Bacillus thuringiensis insecticidal three-domain Cry toxins: mode of action, insect resistance and consequences for crop protection.

    PubMed

    Pardo-López, Liliana; Soberón, Mario; Bravo, Alejandra

    2013-01-01

    Bacillus thuringiensis bacteria are insect pathogens that produce different Cry and Cyt toxins to kill their hosts. Here we review the group of three-domain Cry (3d-Cry) toxins. Expression of these 3d-Cry toxins in transgenic crops has contributed to efficient control of insect pests and a reduction in the use of chemical insecticides. The mode of action of 3d-Cry toxins involves sequential interactions with several insect midgut proteins that facilitate the formation of an oligomeric structure and induce its insertion into the membrane, forming a pore that kills midgut cells. We review recent progress in our understanding of the mechanism of action of these Cry toxins and focus our attention on the different mechanisms of resistance that insects have evolved to counter their action, such as mutations in cadherin, APN and ABC transporter genes. Activity of Cry1AMod toxins, which are able to form toxin oligomers in the absence of receptors, against different resistant populations, including those affected in the ABC transporter and the role of dominant negative mutants as antitoxins, supports the hypothesis that toxin oligomerization is a limiting step in the Cry insecticidal activity. Knowledge of the action of 3d-Cry toxin and the resistance mechanisms to these toxins will set the basis for a rational design of novel toxins to overcome insect resistance, extending the useful lifespan of Cry toxins in insect control programs.

  2. Botulinum toxin: bioweapon & magic drug.

    PubMed

    Dhaked, Ram Kumar; Singh, Manglesh Kumar; Singh, Padma; Gupta, Pallavi

    2010-11-01

    Botulinum neurotoxins, causative agents of botulism in humans, are produced by Clostridium botulinum, an anaerobic spore-former Gram positive bacillus. Botulinum neurotoxin poses a major bioweapon threat because of its extreme potency and lethality; its ease of production, transport, and misuse; and the need for prolonged intensive care among affected persons. A single gram of crystalline toxin, evenly dispersed and inhaled, can kill more than one million people. The basis of the phenomenal potency of botulinum toxin is enzymatic; the toxin is a zinc proteinase that cleaves neuronal vesicle associated proteins responsible for acetylcholine release into the neuromuscular junction. As a military or terrorist weapon, botulinum toxin could be disseminated via aerosol or by contamination of water or food supplies, causing widespread casualties. A fascinating aspect of botulinum toxin research in recent years has been development of the most potent toxin into a molecule of significant therapeutic utility . It is the first biological toxin which is licensed for treatment of human diseases. In the late 1980s, Canada approved use of the toxin to treat strabismus, in 2001 in the removal of facial wrinkles and in 2002, the FDA in the United States followed suit. The present review focuses on both warfare potential and medical uses of botulinum neurotoxin.

  3. Lymphocyte receptors for pertussis toxin

    SciTech Connect

    Clark, C.G.; Armstrong, G.D. )

    1990-12-01

    We have investigated human T-lymphocyte receptors for pertussis toxin by affinity isolation and photoaffinity labeling procedures. T lymphocytes were obtained from peripheral human blood, surface iodinated, and solubilized in Triton X-100. The iodinated mixture was then passed through pertussis toxin-agarose, and the fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography of the fixed, dried gels revealed several bands in the pertussis toxin-bound fraction that were not observed in fractions obtained from histone or fetuin-agarose. Further investigations employed a photoaffinity labeling reagent, sulfosuccinimidyl 2-(p-azido-salicylamido)-1,3'-dithiopropionate, to identify pertussis toxin receptors in freshly isolated peripheral blood monocytic cells, T lymphocytes, and Jurkat cells. In all three cell systems, the pertussis toxin affinity probe specifically labeled a single protein species with an apparent molecular weight of 70,000 that was not observed when the procedure was performed in the presence of excess unmodified pertussis toxin. A protein comparable in molecular weight to the one detected by the photoaffinity labeling technique was also observed among the species that bound to pertussis toxin-agarose. The results suggest that pertussis toxin may bind to a 70,000-Da receptor in human T lymphocytes.

  4. Botulinum toxin: Bioweapon & magic drug

    PubMed Central

    Dhaked, Ram Kumar; Singh, Manglesh Kumar; Singh, Padma; Gupta, Pallavi

    2010-01-01

    Botulinum neurotoxins, causative agents of botulism in humans, are produced by Clostridium botulinum, an anaerobic spore-former Gram positive bacillus. Botulinum neurotoxin poses a major bioweapon threat because of its extreme potency and lethality; its ease of production, transport, and misuse; and the need for prolonged intensive care among affected persons. A single gram of crystalline toxin, evenly dispersed and inhaled, can kill more than one million people. The basis of the phenomenal potency of botulinum toxin is enzymatic; the toxin is a zinc proteinase that cleaves neuronal vesicle associated proteins responsible for acetylcholine release into the neuromuscular junction. As a military or terrorist weapon, botulinum toxin could be disseminated via aerosol or by contamination of water or food supplies, causing widespread casualties. A fascinating aspect of botulinum toxin research in recent years has been development of the most potent toxin into a molecule of significant therapeutic utility. It is the first biological toxin which is licensed for treatment of human diseases. In the late 1980s, Canada approved use of the toxin to treat strabismus, in 2001 in the removal of facial wrinkles and in 2002, the FDA in the United States followed suit. The present review focuses on both warfare potential and medical uses of botulinum neurotoxin. PMID:21149997

  5. Evidence for antinociceptive activity of botulinum toxin type A in pain management.

    PubMed

    Aoki, K Roger

    2003-01-01

    The neurotoxin, botulinum toxin type A, has been used successfully, in some patients, as an analgesic for myofascial pain syndromes, migraine, and other headache types. The toxin inhibits the release of the neurotransmitter, acetylcholine, at the neuromuscular junction thereby inhibiting striated muscle contractions. In the majority of pain syndromes where botulinum toxin type A is effective, inhibiting muscle spasms is an important component of its activity. Even so, the reduction of pain often occurs before the decrease in muscle contractions suggesting that botulinum toxin type A has a more complex mechanism of action than initially hypothesized. Current data points to an antinociceptive effect of botulinum toxin type A that is separate from its neuromuscular activity. The common biochemical mechanism, however, remains the same between botulinum toxin type A's effect on the motor nerve or the sensory nerve: enzymatic blockade of neurotransmitter release. The antinociceptive effect of the toxin was reported to block substance P release using in vitro culture systems. The current investigation evaluated the in vivo mechanism of action for the antinociceptive action of botulinum toxin type A. In these studies, botulinum toxin type A was found to block the release of glutamate. Furthermore, Fos, a product of the immediate early gene, c-fos, expressed with neuronal stimuli was prevented upon peripheral exposure to the toxin. These findings suggest that botulinum toxin type A blocks peripheral sensitization and, indirectly, reduces central sensitization. The recent hypothesis that migraine involves both peripheral and central sensitization may help explain how botulinum toxin type A inhibits migraine pain by acting on these two pathways. Further research is needed to determine whether the antinociceptive mechanism mediated by botulinum toxin type A affects the neuronal signaling pathways that are activated during migraine.

  6. Preponderance of toxigenic Escherichia coli in stool pathogens correlates with toxin detection in accessible drinking-water sources.

    PubMed

    Igbokwe, H; Bhattacharyya, S; Gradus, S; Khubbar, M; Griswold, D; Navidad, J; Igwilo, C; Masson-Meyers, D; Azenabor, A A

    2015-02-01

    Since early detection of pathogens and their virulence factors contribute to intervention and control strategies, we assessed the enteropathogens in diarrhoea disease and investigated the link between toxigenic strains of Escherichia coli from stool and drinking-water sources; and determined the expression of toxin genes by antibiotic-resistant E. coli in Lagos, Nigeria. This was compared with isolates from diarrhoeal stool and water from Wisconsin, USA. The new Luminex xTAG GPP (Gastroplex) technique and conventional real-time PCR were used to profile enteric pathogens and E. coli toxin gene isolates, respectively. Results showed the pathogen profile of stool and indicated a relationship between E. coli toxin genes in water and stool from Lagos which was absent in Wisconsin isolates. The Gastroplex technique was efficient for multiple enteric pathogens and toxin gene detection. The co-existence of antibiotic resistance with enteroinvasive E. coli toxin genes suggests an additional prognostic burden on patients.

  7. Toxin-induced hepatic injury.

    PubMed

    Lopez, Annette M; Hendrickson, Robert G

    2014-02-01

    Toxins such as pharmaceuticals, herbals, foods, and supplements may lead to hepatic damage. This damage may range from nonspecific symptoms in the setting of liver test abnormalities to acute hepatic failure. The majority of severe cases of toxin-induced hepatic injury are caused by acetaminophen and ethanol. The most important step in the patient evaluation is to gather an extensive history that includes toxin exposure and exclude common causes of liver dysfunction. Patients whose hepatic dysfunction progresses to acute liver failure may benefit from transfer to a transplant service for further management. Currently, the mainstay in management for most exposures is discontinuing the offending agent. This manuscript will review the incidence, pathophysiology, diagnosis and management of the different forms of toxin-induced hepatic injury and exam in-depth the most common hepatic toxins.

  8. Toxin production by Campylobacter spp.

    PubMed Central

    Wassenaar, T M

    1997-01-01

    Of all the virulence factors that were proposed for Campylobacter jejuni and related species to cause disease in humans, the discovery of toxin production was the most promising but led to a rather confusing and even disappointing stream of data. The discussion of whether proteinaceous exotoxins are relevant in disease remains open. One important reason for this lack of consensus is the anecdotal nature of the literature reports. To provide a basis for an unbiased opinion, this review compiles all described exotoxins, compares their reported properties, and provides a summary of animal model studies and clinical data. The toxins are divided into enterotoxins and cytotoxins and are sorted according to their biochemical properties. Since many Campylobacter toxins have been compared with toxins of other species, some key examples of the latter are also discussed. Future directions of toxin research that appear promising are defined. PMID:9227862

  9. Uremic toxins and oral adsorbents.

    PubMed

    Goto, Shunsuke; Yoshiya, Kunihiko; Kita, Tomoyuki; Fujii, Hideki; Fukagawa, Masafumi

    2011-04-01

    Uremic toxins are associated with various disorders in patients with end-stage renal disease and it is difficult to remove some of these toxins by dialysis. Since some uremic toxins are generated by bacterial metabolites in the colon, oral adsorbents that interfere with the absorption of uremic toxins or their precursors are believed to prevent their accumulation in the body. AST-120 adsorbs various uremic retention solutes in the gastrointestinal system and has potential for providing clinical benefit. Sevelamer hydrochloride binds some harmful compounds in addition to phosphate and seems to have pleiotropic effects that include lowering serum LDL cholesterol levels and reduction of inflammation. The effect of sevelamer hydrochloride on indoxyl sulfate and p-cresol has been shown in an in vitro study; however, in vivo studies in mice or humans did not demonstrate this effect on protein-binding uremic toxins. Oral adsorbents are thus one of the important modalities in the treatment of uremic syndrome.

  10. Immunogenicity of Staphylococcus aureus delta-toxin.

    PubMed Central

    Nolte, F S; Kapral, F A

    1981-01-01

    Studies were conducted to determine the immunogenicity of purified Staphylococcus aureus delta-toxin. Rabbits and guinea pigs immunized with delta-toxin incorporated into a multiple antibody, whereas animals given toxin in saline or toxin in saline with Tween 80 did not produce antibody. The immunoglobulin G (IgG) fraction isolated by chromatography on protein A-Sepharose was examined for the presence of anti-delta-toxin antibody by immunoelectrophoresis, immunodiffusion, quantitative precipitation tests, affinity chromatography, and toxin neutralization tests. Although delta-toxin-specific IgG precipitated the toxin in agar gels, the antibody did not neutralize the toxin's hemolytic activity. Delta-toxin binding to human erythrocyte membranes was demonstrated by indirect immunofluorescent staining of toxin-treated erythrocytes. Images PMID:7014461

  11. Photoactivated perylenequinone toxins in fungal pathogenesis of plants.

    PubMed

    Daub, Margaret E; Herrero, Sonia; Chung, Kuang-Ren

    2005-11-15

    Several genera of plant pathogenic fungi produce photoactivated perylenequinone toxins involved in pathogenesis of their hosts. These toxins are photosensitizers, absorbing light energy and generating reactive oxygen species that damage the membranes of the host cells. Studies with toxin-deficient mutants and on the involvement of light in symptom development have documented the importance of these toxins in successful pathogenesis of plants. This review focuses on the well studied perylenequinone toxin, cercosporin, produced by species in the genus Cercospora. Significant progress has been made recently on the biosynthetic pathway of cercosporin, with the characterization of genes encoding a polyketide synthase and a major facilitator superfamily transporter, representing the first and last steps of the biosynthetic pathway, as well as important regulatory genes. In addition, the resistance of Cercospora fungi to cercosporin and to the singlet oxygen that it generates has led to the use of these fungi as models for understanding cellular resistance to photosensitizers and singlet oxygen. These studies have shown that resistance is complex, and have documented a role for transporters, transient reductive detoxification, and quenchers in cercosporin resistance.

  12. Binding of ATP by pertussis toxin and isolated toxin subunits

    SciTech Connect

    Hausman, S.Z.; Manclark, C.R.; Burns, D.L. )

    1990-07-03

    The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of ({sup 3}H)ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP > GTP > CTP > TTP for pertussis toxin and ATP > GTP > TTP > CTP for the B oligomer. Phosphate ions inhibited the binding of ({sup 3}H)ATP to pertussis toxin in a competitive manner; however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of ({sup 3}H)ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site.

  13. Botulinum toxin: mechanisms of action.

    PubMed

    Dressler, Dirk; Adib Saberi, Fereshte

    2005-01-01

    Botulinum toxin (BT) has been perceived as a lethal threat for many centuries. In the early 1980s, this perception completely changed when BT's therapeutic potential suddenly became apparent. We wish to give an overview over BT's mechanisms of action relevant for understanding its therapeutic use. BT's molecular mode of action includes extracellular binding to glycoprotein structures on cholinergic nerve terminals and intracellular blockade of the acetylcholine secretion. BT affects the spinal stretch reflex by blockade of intrafusal muscle fibres with consecutive reduction of Ia/II afferent signals and muscle tone without affecting muscle strength (reflex inhibition). This mechanism allows for antidystonic effects not only caused by target muscle paresis. BT also blocks efferent autonomic fibres to smooth muscles and to exocrine glands. Direct central nervous system effects are not observed, since BT does not cross the blood-brain barrier and since it is inactivated during its retrograde axonal transport. Indirect central nervous system effects include reflex inhibition, normalisation of reciprocal inhibition, intracortical inhibition and somatosensory evoked potentials. Reduction of formalin-induced pain suggests direct analgesic BT effects possibly mediated by blockade of substance P, glutamate and calcitonin gene-related peptide.

  14. Phage-mediated Shiga toxin (Stx) horizontal gene transfer and expression in non-Shiga toxigenic Enterobacter and Escherichia coli strains.

    PubMed

    Khalil, Rowaida K S; Skinner, Craig; Patfield, Stephanie; He, Xiaohua

    2016-07-01

    Enterobacter cloacae M12X01451 strain recently identified from a clinical specimen produces a new Stx1 subtype (Stx1e) that was not neutralized by existing anti-Stx1 monoclonal antibodies. Acquisition of stx by Ent. cloacae is rare and origin/stability of stx1e in M12X01451 is not known. In this study, we confirmed the ability of Stx1a- and Stx1e-converting phages from an Escherichia coli O157:H7 strain RM8530 and M12X01451 respectively to infect several E. coli and Ent. cloacae strains. stx1e was detected in 97.5% and 72.5% of progenies of strains lysogenized by stx1e phage after 10 (T10) and 20 (T20) subcultures, versus 65% and 17.5% for stx1a gene. Infection of M12X01451 and RM8530 with each other's phages generated double lysogens containing both phages. stx1a was lost after T10, whereas the stx1e was maintained even after T20 in M12X01451 lysogens. In RM8530 lysogens, the acquired stx1e was retained with no mutations, but 20% of stx1a was lost after T20 ELISA and western blot analyses demonstrated that Stx1e was produced in all strains lysogenized by stx1e phage; however, Stx1a was not detected in any lysogenized strain. The study results highlight the potential risks of emerging Stx-producing strains via bacteriophages either in the human gastrointestinal tract or in food production environments, which are matters of great concern and may have serious impacts on human health.

  15. Suppression subtractive hybridization and comparative expression of a pore-forming toxin and glycosyl hydrolase genes in Rhizoctonia solani during potato sprout infection.

    PubMed

    Chamoun, Rony; Samsatly, Jamil; Pakala, Suman B; Cubeta, Marc A; Jabaji, Suha

    2015-06-01

    Rhizoctonia solani is a plant pathogenic fungus that causes black scurf on tubers and stem and stolon canker on underground parts of potato plant. Early in the season, the fungus attacks germinating sprouts underground before they emerge from the soil. Damage at this stage results in delayed emergence of weakened plants with poor and uneven stands. The mechanism underlying this phenomenon has been investigated in this study by coupling a cDNA-suppression subtractive hybridization (SSH) library to differential screening to identify transcripts of R. solani that are down-regulated during infection of potato sprouts. We report on the identification of 33 unique genes with functions related to carbohydrate binding, vitamin synthesis, pathogenicity, translation, ATP and nucleic acid binding and other categories. RACE-PCR was used to clone and characterize the first full-length cDNA clones, RSENDO1 and RSGLYC1 that encode for an eukaryotic delta-endotoxin CytB protein and an intracellular glycosyl hydrolase, respectively. Quantitative real-time PCR revealed the down-regulation of RSENDO1 during infection of potato sprouts and the up-regulation of RSGLYC1 when the fungus was grown on a cellulose-based nutrient medium. In contrast, additional experiments have highlighted the down-regulation of RSENDO1 when R. solani was co-cultured with the mycoparasite Stachybotrys elegans and the bacterial antagonist Bacillus subtilis B26. These results advance our understanding of R. solani-potato interaction in subterranean parts of the plant. Such approaches could be considered in building an efficient integrated potato disease management program.

  16. Stoichiometric regulation of phytoplankton toxins.

    PubMed

    Van de Waal, Dedmer B; Smith, Val H; Declerck, Steven A J; Stam, Eva C M; Elser, James J

    2014-06-01

    Ecological Stoichiometry theory predicts that the production, elemental structure and cellular content of biomolecules should depend on the relative availability of resources and the elemental composition of their producer organism. We review the extent to which carbon- and nitrogen-rich phytoplankton toxins are regulated by nutrient limitation and cellular stoichiometry. Consistent with theory, we show that nitrogen limitation causes a reduction in the cellular quota of nitrogen-rich toxins, while phosphorus limitation causes an increase in the most nitrogen-rich paralytic shellfish poisoning toxin. In addition, we show that the cellular content of nitrogen-rich toxins increases with increasing cellular N : P ratios. Also consistent with theory, limitation by either nitrogen or phosphorus promotes the C-rich toxin cell quota or toxicity of phytoplankton cells. These observed relationships may assist in predicting and managing toxin-producing phytoplankton blooms. Such a stoichiometric regulation of toxins is likely not restricted to phytoplankton, and may well apply to carbon- and nitrogen-rich secondary metabolites produced by bacteria, fungi and plants.

  17. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications.

    PubMed

    Yeo, Chew Chieng; Abu Bakar, Fauziah; Chan, Wai Ting; Espinosa, Manuel; Harikrishna, Jennifer Ann

    2016-02-19

    Toxin-antitoxin (TA) systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies.

  18. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications

    PubMed Central

    Yeo, Chew Chieng; Abu Bakar, Fauziah; Chan, Wai Ting; Espinosa, Manuel; Harikrishna, Jennifer Ann

    2016-01-01

    Toxin-antitoxin (TA) systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies. PMID:26907343

  19. Food toxin detection with atomic force microscope

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Externally introduced toxins or internal spoilage correlated pathogens and their metabolites are all potential sources of food toxins. To prevent and protect unsafe food, many food toxin detection techniques have been developed to detect various toxins for quality control. Although several routine m...

  20. Detecting and discriminating among Shiga toxins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The virulence associated with Shiga toxin producing Escherichia coli (STEC) infections is from the Shiga toxins produced by the E. coli strain. Although Shiga toxins are associated with E. coli, the expression of the toxins is actually controlled by a temperate lambdoid phage that infects the host. ...

  1. Novel Structure and Function of Typhoid Toxin

    MedlinePlus

    ... toxin, it caused symptoms similar to those in humans with typhoid fever, implicating typhoid toxin in the illness caused by S. typhi. Typhoid toxin can bind to a wide variety of cells. Experiments revealed that the toxin attaches to certain types ...

  2. Relevance of Bt toxin interaction studies for environmental risk assessment of genetically modified crops.

    PubMed

    De Schrijver, Adinda; De Clercq, Patrick; de Maagd, Ruud A; van Frankenhuyzen, Kees

    2015-12-01

    In recent years, different Bacillus thuringiensis (Bt) toxin-encoding genes have been combined or 'stacked' in genetically modified (GM) crops. Synergism between Bt proteins may occur and thereby increase the impact of the stacked GM event on nontarget invertebrates compared to plants expressing a single Bt gene. On the basis of bioassay data available for Bt toxins alone or in combination, we argue that the current knowledge of Bt protein interactions is of limited relevance in environmental risk assessment (ERA).

  3. Clostridium difficile Toxins A and B: Insights into Pathogenic Properties and Extraintestinal Effects

    PubMed Central

    Di Bella, Stefano; Ascenzi, Paolo; Siarakas, Steven; Petrosillo, Nicola; di Masi, Alessandra

    2016-01-01

    Clostridium difficile infection (CDI) has significant clinical impact especially on the elderly and/or immunocompromised patients. The pathogenicity of Clostridium difficile is mainly mediated by two exotoxins: toxin A (TcdA) and toxin B (TcdB). These toxins primarily disrupt the cytoskeletal structure and the tight junctions of target cells causing cell rounding and ultimately cell death. Detectable C. difficile toxemia is strongly associated with fulminant disease. However, besides the well-known intestinal damage, recent animal and in vitro studies have suggested a more far-reaching role for these toxins activity including cardiac, renal, and neurologic impairment. The creation of C. difficile strains with mutations in the genes encoding toxin A and B indicate that toxin B plays a major role in overall CDI pathogenesis. Novel insights, such as the role of a regulator protein (TcdE) on toxin production and binding interactions between albumin and C. difficile toxins, have recently been discovered and will be described. Our review focuses on the toxin-mediated pathogenic processes of CDI with an emphasis on recent studies. PMID:27153087

  4. Strategies to improve the insecticidal activity of Cry toxins from Bacillus thuringiensis.

    PubMed

    Pardo-López, L; Muñoz-Garay, C; Porta, H; Rodríguez-Almazán, C; Soberón, M; Bravo, A

    2009-03-01

    Bacillus thuringiensis Cry toxins have been widely used in the control of insect pests either as spray products or expressed in transgenic crops. These proteins are pore-forming toxins with a complex mechanism of action that involves the sequential interaction with several toxin-receptors. Cry toxins are specific against susceptible larvae and although they are often highly effective, some insect pests are not affected by them or show low susceptibility. In addition, the development of resistance threatens their effectiveness, so strategies to cope with all these problems are necessary. In this review we will discuss and compare the different strategies that have been used to improve insecticidal activity of Cry toxins. The activity of Cry toxins can be enhanced by using additional proteins in the bioassay like serine protease inhibitors, chitinases, Cyt toxins, or a fragment of cadherin receptor containing a toxin-binding site. On the other hand, different modifications performed in the toxin gene such as site-directed mutagenesis, introduction of cleavage sites in specific regions of the protein, and deletion of small fragments from the amino-terminal region lead to improved toxicity or overcome resistance, representing interesting alternatives for insect pest control.

  5. Daphnia magna negatively affected by chronic exposure to purified Cry-toxins.

    PubMed

    Bøhn, Thomas; Rover, Carina Macagnan; Semenchuk, Philipp Robert

    2016-05-01

    Cry-toxin genes originating from Bacillus thuringiensis are inserted into genetically modified (GM) plants, often called Bt-plants, to provide insect resistance to pests. Significant amounts of Bt-plant residues, and thus Cry-toxins, will be shed to soil and aquatic environments. We exposed Daphnia magna to purified Cry1Ab and Cry2Aa toxins for the full life-span of the animals. We used single toxins in different doses and combinations of toxins and Roundup(®), another potential stressor on the rise in agricultural ecosystems. Animals exposed to 4.5 mg/L (ppm) of Cry1Ab, Cry2Aa and the combination of both showed markedly higher mortality, smaller body size and very low juvenile production compared to controls. Animals exposed to 0.75 mg/L also showed a tendency towards increased mortality but with increased early fecundity compared to the controls. Roundup(®) stimulated animals to strong early reproductive output at the cost of later rapid mortality. We conclude that i) purified Cry-toxins in high concentrations are toxic to D. magna, indicating alternative modes-of-action for these Cry-toxins; ii) Cry-toxins act in combination, indicating that 'stacked events' may have stronger effects on non-target organisms; iii) further studies need to be done on combinatorial effects of multiple Cry-toxins and herbicides that co-occur in the environment.

  6. Regulation of toxin and bacteriocin synthesis in Clostridium species by a new subgroup of RNA polymerase sigma-factors.

    PubMed

    Dupuy, Bruno; Matamouros, Susana

    2006-04-01

    Many Clostridium species are pathogenic for humans and animals, and most of the resulting diseases, such as tetanus, botulism, gas gangrene and pseudomembranous colitis, are due to the production of potent extracellular toxins. The biochemical mechanisms of action of Clostridium toxins have been extensively studied in the past ten years. However, detailed information about the regulation of toxin gene expression has only recently emerged. TcdR, BotR, TetR and UviA are now known to be related alternative RNA polymerase sigma factors that drive transcription of toxin A and toxin B genes in C. difficile, the neurotoxin genes in C. botulinum and C. tetani, and a bacteriocin gene in C. perfringens. Although the Clostridium sigma factors have some similarity to members of the ECF sigma factor group, they differ sufficiently in structure and function so that they have been assigned to a new group within the sigma(70)-family.

  7. Carvacrol and trans-Cinnamaldehyde Reduce Clostridium difficile Toxin Production and Cytotoxicity in Vitro

    PubMed Central

    Mooyottu, Shankumar; Kollanoor-Johny, Anup; Flock, Genevieve; Bouillaut, Laurent; Upadhyay, Abhinav; Sonenshein, Abraham L.; Venkitanarayanan, Kumar

    2014-01-01

    Clostridium difficile is a nosocomial pathogen that causes a serious toxin-mediated enteric disease in humans. Reducing C. difficile toxin production could significantly minimize its pathogenicity and improve disease outcomes in humans. This study investigated the efficacy of two, food-grade, plant-derived compounds, namely trans-cinnamaldehyde (TC) and carvacrol (CR) in reducing C. difficile toxin production and cytotoxicity in vitro. Three hypervirulent C. difficile isolates were grown with or without the sub-inhibitory concentrations of TC or CR, and the culture supernatant and the bacterial pellet were collected for total toxin quantitation, Vero cell cytotoxicity assay and RT-qPCR analysis of toxin-encoding genes. The effect of CR and TC on a codY mutant and wild type C. difficile was also investigated. Carvacrol and TC substantially reduced C. difficile toxin production and cytotoxicity on Vero cells. The plant compounds also significantly down-regulated toxin production genes. Carvacrol and TC did not inhibit toxin production in the codY mutant of C. difficile, suggesting a potential codY-mediated anti-toxigenic mechanism of the plant compounds. The antitoxigenic concentrations of CR and TC did not inhibit the growth of beneficial gut bacteria. Our results suggest that CR and TC could potentially be used to control C. difficile, and warrant future studies in vivo. PMID:24625665

  8. Carvacrol and trans-cinnamaldehyde reduce Clostridium difficile toxin production and cytotoxicity in vitro.

    PubMed

    Mooyottu, Shankumar; Kollanoor-Johny, Anup; Flock, Genevieve; Bouillaut, Laurent; Upadhyay, Abhinav; Sonenshein, Abraham L; Venkitanarayanan, Kumar

    2014-03-12

    Clostridium difficile is a nosocomial pathogen that causes a serious toxin-mediated enteric disease in humans. Reducing C. difficile toxin production could significantly minimize its pathogenicity and improve disease outcomes in humans. This study investigated the efficacy of two, food-grade, plant-derived compounds, namely trans-cinnamaldehyde (TC) and carvacrol (CR) in reducing C. difficile toxin production and cytotoxicity in vitro. Three hypervirulent C. difficile isolates were grown with or without the sub-inhibitory concentrations of TC or CR, and the culture supernatant and the bacterial pellet were collected for total toxin quantitation, Vero cell cytotoxicity assay and RT-qPCR analysis of toxin-encoding genes. The effect of CR and TC on a codY mutant and wild type C. difficile was also investigated. Carvacrol and TC substantially reduced C. difficile toxin production and cytotoxicity on Vero cells. The plant compounds also significantly down-regulated toxin production genes. Carvacrol and TC did not inhibit toxin production in the codY mutant of C. difficile, suggesting a potential codY-mediated anti-toxigenic mechanism of the plant compounds. The antitoxigenic concentrations of CR and TC did not inhibit the growth of beneficial gut bacteria. Our results suggest that CR and TC could potentially be used to control C. difficile, and warrant future studies in vivo.

  9. Epsilon toxin: a fascinating pore-forming toxin.

    PubMed

    Popoff, Michel R

    2011-12-01

    Epsilon toxin (ETX) is produced by strains of Clostridium perfringens classified as type B or type D. ETX belongs to the heptameric β-pore-forming toxins including aerolysin and Clostridium septicum alpha toxin, which are characterized by the formation of a pore through the plasma membrane of eukaryotic cells consisting in a β-barrel of 14 amphipatic β strands. By contrast to aerolysin and C. septicum alpha toxin, ETX is a much more potent toxin and is responsible for enterotoxemia in animals, mainly sheep. ETX induces perivascular edema in various tissues and accumulates in particular in the kidneys and brain, where it causes edema and necrotic lesions. ETX is able to pass through the blood-brain barrier and stimulate the release of glutamate, which accounts for the symptoms of nervous excitation observed in animal enterotoxemia. At the cellular level, ETX causes rapid swelling followed by cell death involving necrosis. The precise mode of action of ETX remains to be determined. ETX is a powerful toxin, however, it also represents a unique tool with which to vehicle drugs into the central nervous system or target glutamatergic neurons.

  10. Botulinum toxin in clinical practice

    PubMed Central

    Jankovic, J

    2004-01-01

    Botulinum toxin, the most potent biological toxin, has become a powerful therapeutic tool for a growing number of clinical applications. This review draws attention to new findings about the mechanism of action of botulinum toxin and briefly reviews some of its most frequent uses, focusing on evidence based data. Double blind, placebo controlled studies, as well as open label clinical trials, provide evidence that, when appropriate targets and doses are selected, botulinum toxin temporarily ameliorates disorders associated with excessive muscle contraction or autonomic dysfunction. When injected not more often than every three months, the risk of blocking antibodies is slight. Long term experience with this agent suggests that it is an effective and safe treatment not only for approved indications but also for an increasing number of off-label indications. PMID:15201348

  11. Gut microbes of mammalian herbivores facilitate intake of plant toxins.

    PubMed

    Kohl, Kevin D; Weiss, Robert B; Cox, James; Dale, Colin; Dearing, M Denise

    2014-10-01

    The foraging ecology of mammalian herbivores is strongly shaped by plant secondary compounds (PSCs) that defend plants against herbivory. Conventional wisdom holds that gut microbes facilitate the ingestion of toxic plants; however, this notion lacks empirical evidence. We investigated the gut microbiota of desert woodrats (Neotoma lepida), some populations of which specialise on highly toxic creosote bush (Larrea tridentata). Here, we demonstrate that gut microbes are crucial in allowing herbivores to consume toxic plants. Creosote toxins altered the population structure of the gut microbiome to facilitate an increase in abundance of genes that metabolise toxic compounds. In addition, woodrats were unable to consume creosote toxins after the microbiota was disrupted with antibiotics. Last, ingestion of toxins by naïve hosts was increased through microbial transplants from experienced donors. These results demonstrate that microbes can enhance the ability of hosts to consume PSCs and therefore expand the dietary niche breadth of mammalian herbivores.

  12. The Future of Bladder Control—Intravesical Drug Delivery, a Pinch of Pepper, and Gene Therapy

    PubMed Central

    Fraser, Matthew O; Lavelle, John P; Sacks, Michael S; Chancellor, Michael B

    2002-01-01

    The incidence of urinary incontinence and overactive bladder problems will continue to grow as the population ages. Future treatments are likely to include an implantable drug delivery system, gene therapy, and the intravesical use of the vallinoids capsaicin and resiniferatoxin (RTX). An understanding of the urothelium is essential for effective design of these therapies. Intravesical anticholinergic drug treatment is currently not widely used, but intravesical pumps are under development to provide less cumbersome treatment methods and will provide nonsurgical options for patients who cannot tolerate oral anticholinergic agents. Research on the use of capsaicin as an intravesicular drug has had limited success, but trials have confirmed the efficacy of intravesical capsaicin for detrusor hyperreflexia. RTX is as effective as capsaicin but without side effects, such as pain and inflammatory neuropeptide release. RTX treatment may eliminate the need for surgical and other drug treatments of lower urinary tract dysfunction in patients with spinal cord injuries. Gene therapy will change the practice of urology by addressing the deficiencies that cause symptoms rather than attacking the symptoms themselves. PMID:16985646

  13. [Biological and toxin terrorism weapons].

    PubMed

    Bokan, Slavko

    2003-03-01

    The use of biological agents and toxins in warfare and terrorism has a long history. Human, animal and plant pathogens and toxins can cause disease and can be used as a threat to humans, animals and staple crops. The same is true for biological agents. Although the use of biological agents and toxins in military conflicts has been a concern of military communities for many years, several recent events have increased the awareness of terrorist use of these weapons against civilian population. A Mass Casualty Biological (Toxin) Weapon (MCBTW) is any biological and toxin weapon capable of causing death or disease on a large scale, such that the military or civilian infrastructure of the state or organization being attacked is overwhelmed. A militarily significant (or terrorist) weapon is any weapon capable of affecting, directly or indirectly, that is physically or psychologically, the outcome of a military operation. Although many biological agents such as toxins and bioregulators can be used to cause diseases, there are only a few that can truly threaten civilian populations on a large scale. Bioregulators or modulators are biochemical compounds, such as peptides, that occur naturally in organisms. They are new class of weapons that can damage nervous system, alter moods, trigger psychological changes and kill. The potential military or terrorist use of bioregulators is similar to that of toxins. Some of these compounds are several hundred times more potent than traditional chemical warfare agents. Important features and military advantages of new bioregulators are novel sites of toxic action; rapid and specific effects; penetration of protective filters and equipment, and militarily effective physical incapacitation. This overview of biological agents and toxins is largely intended to help healthcare providers on all levels to make decisions in protecting general population from these agents.

  14. The Regulatory Networks That Control Clostridium difficile Toxin Synthesis

    PubMed Central

    Martin-Verstraete, Isabelle; Peltier, Johann; Dupuy, Bruno

    2016-01-01

    The pathogenic clostridia cause many human and animal diseases, which typically arise as a consequence of the production of potent exotoxins. Among the enterotoxic clostridia, Clostridium difficile is the main causative agent of nosocomial intestinal infections in adults with a compromised gut microbiota caused by antibiotic treatment. The symptoms of C. difficile infection are essentially caused by the production of two exotoxins: TcdA and TcdB. Moreover, for severe forms of disease, the spectrum of diseases caused by C. difficile has also been correlated to the levels of toxins that are produced during host infection. This observation strengthened the idea that the regulation of toxin synthesis is an important part of C. difficile pathogenesis. This review summarizes our current knowledge about the regulators and sigma factors that have been reported to control toxin gene expression in response to several environmental signals and stresses, including the availability of certain carbon sources and amino acids, or to signaling molecules, such as the autoinducing peptides of quorum sensing systems. The overlapping regulation of key metabolic pathways and toxin synthesis strongly suggests that toxin production is a complex response that is triggered by bacteria in response to particular states of nutrient availability during infection. PMID:27187475

  15. Influence of primer sequences and DNA extraction method on detection of non-O157 Shiga toxin-producing Escherichia coli in ground beef by real-time PCR targeting the eae, stx, and serogroup-specific genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Non-O157 Shiga toxin-producing E. coli (STEC) infections, particularly those caused by the “big six”/”top six” non-O157 serogroups (O26, O45, O103, O111, O121, and O145) can result in severe illness and complications. Due to their significant public health impact and the notable prevalence of STEC ...

  16. Fusarial toxins: secondary metabolites of Fusarium fungi.

    PubMed

    Nesic, Ksenija; Ivanovic, Snezana; Nesic, Vladimir

    2014-01-01

    Exposure to mycotoxins occurs worldwide, even though there are geographic and climatic differences in the amounts produced and occurrence of these substances.Mycotoxins are secondary chemical metabolites of different fungi. They are natural contaminants of cereals, so their presence is often inevitable. Among many genera that produce mycotoxins, Fusarium fungi are the most widespread in cereal-growing areas of the planet. Fusarium fungi produce a diversity of mycotoxin types, whose distributions are also diverse. What is produced and where it is produced is influenced primarily by environmental conditions, and crop production and storage methods. The amount of toxin produced depends on physical (viz., moisture, relative humidity, temperature, and mechanical damage), chemical (viz., carbon dioxide,oxygen, composition of substrate, insecticides and fungicides), and biological factors (viz., plant variety, stress, insects, spore load, etc.). Moisture and temperature have a major influence on mold growth rate and mycotoxin production.Among the most toxic and prevalent fusaria) toxins are the following: zearalenone,fumonisins, moniliformin and trichothecenes (T-2/HT-2 toxin, deoxynivalenol,diacetoxyscirpenol, nivalenol). Zearalenone (ZEA; ZON, F-2 toxin) isaphy to estrogenic compound, primarily a field contaminant, which exhibits estrogenic activity and has been implicated in numerous mycotoxicoses of farm animals,especially pigs. Recently, evidence suggests that ZEA has potential to stimulate the growth of human breast cancer cells. Fumonisins are also cancer-promoting metabolites,of which Fumonisin 8 I (FBI) is the most important. Moniliformin (MON) isalso highly toxic to both animals and humans. Trichothecenes are classified as gastrointestinal toxins, dermatotoxins, immunotoxins, hematotoxins, and gene toxins.T-2 and HT-2 toxin, and diacetoxyscirpenol (DAS, anguidine) are the most toxic mycotoxins among the trichothecene group. Deoxynivalenol (DON, vomitoxin) and

  17. A genetic switch controls the production of flagella and toxins in Clostridium difficile.

    PubMed

    Anjuwon-Foster, Brandon R; Tamayo, Rita

    2017-03-27

    In the human intestinal pathogen Clostridium difficile, flagella promote adherence to intestinal epithelial cells. Flagellar gene expression also indirectly impacts production of the glucosylating toxins, which are essential to diarrheal disease development. Thus, factors that regulate the expression of the flgB operon will likely impact toxin production in addition to flagellar motility. Here, we report the identification a "flagellar switch" that controls the phase variable production of flagella and glucosylating toxins. The flagellar switch, located upstream of the flgB operon containing the early stage flagellar genes, is a 154 bp invertible sequence flanked by 21 bp inverted repeats. Bacteria with the sequence in one orientation expressed flagellum and toxin genes, produced flagella, and secreted the toxins ("flg phase ON"). Bacteria with the sequence in the inverse orientation were attenuated for flagellar and toxin gene expression, were aflagellate, and showed decreased toxin secretion ("flg phase OFF"). The orientation of the flagellar switch is reversible during growth in vitro. We provide evidence that gene regulation via the flagellar switch occurs post-transcription initiation and requires a C. difficile-specific regulatory factor to destabilize or degrade the early flagellar gene mRNA when the flagellar switch is in the OFF orientation. Lastly, through mutagenesis and characterization of flagellar phase locked isolates, we determined that the tyrosine recombinase RecV, which catalyzes inversion at the cwpV switch, is also responsible for inversion at the flagellar switch in both directions. Phase variable flagellar motility and toxin production suggests that these important virulence factors have both advantageous and detrimental effects during the course of infection.

  18. Antibody-based biological toxin detection

    SciTech Connect

    Menking, D.E.; Goode, M.T.

    1995-12-01

    Fiber optic evanescent fluorosensors are under investigation in our laboratory for the study of drug-receptor interactions for detection of threat agents and antibody-antigen interactions for detection of biological toxins. In a direct competition assay, antibodies against Cholera toxin, Staphylococcus Enterotoxin B or ricin were noncovalently immobilized on quartz fibers and probed with fluorescein isothiocyanate (FITC) - labeled toxins. In the indirect competition assay, Cholera toxin or Botulinum toxoid A was immobilized onto the fiber, followed by incubation in an antiserum or partially purified anti-toxin IgG. These were then probed with FITC-anti-IgG antibodies. Unlabeled toxins competed with labeled toxins or anti-toxin IgG in a dose dependent manner and the detection of the toxins was in the nanomolar range.

  19. Ribosomal biosynthesis of the cyclic peptide toxins of Amanita mushrooms.

    PubMed

    Walton, Jonathan D; Hallen-Adams, Heather E; Luo, Hong

    2010-01-01

    Some species of mushrooms in the genus Amanita are extremely poisonous and frequently fatal to mammals including humans and dogs. Their extreme toxicity is due to amatoxins such as alpha- and beta-amanitin. Amanita mushrooms also biosynthesize a chemically related group of toxins, the phallotoxins, such as phalloidin. The amatoxins and phallotoxins (collectively known as the Amanita toxins) are bicyclic octa- and heptapeptides, respectively. Both contain an unusual Trp-Cys crossbridge known as tryptathionine. We have shown that, in Amanita bisporigera, the amatoxins and phallotoxins are synthesized as proproteins on ribosomes and not by nonribosomal peptide synthetases. The proproteins are 34-35 amino acids in length and have no predicted signal peptides. The genes for alpha-amanitin (AMA1) and phallacidin (PHA1) are members of a large family of related genes, characterized by highly conserved amino acid sequences flanking a hypervariable "toxin" region. The toxin regions are flanked by invariant proline (Pro) residues. An enzyme that could cleave the proprotein of phalloidin was purified from the phalloidin-producing lawn mushroom Conocybe apala. The enzyme is a serine protease in the prolyl oligopeptidase (POP) subfamily. The same enzyme cuts at both Pro residues to release the linear hepta- or octapeptide.

  20. Tightly Bound Binary Toxin in the Cell Wall of Bacillus sphaericus

    PubMed Central

    Klein, Daniela; Uspensky, Igor; Braun, Sergei

    2002-01-01

    We have shown that urea-extracted cell wall of entomopathogenic Bacillus sphaericus 2297 and some other strains is a potent larvicide against Culex pipiens mosquitoes, with 50% lethal concentrations comparable to that of the well-known B. sphaericus binary toxin, with which it acts synergistically. The wall toxicity develops in B. sphaericus 2297 cultures during the late logarithmic stage, earlier than the appearance of the binary toxin crystal. It disappears with sporulation when the binary toxin activity reaches its peak. Disruption of the gene for the 42-kDa protein (P42) of the binary toxin abolishes both cell wall toxicity and crystal formation. However, the cell wall of B. sphaericus 2297, lacking P42, kills C. pipiens larvae when mixed with Escherichia coli cells expressing P42. Thus, the cell wall toxicity in strongly toxic B. sphaericus strains must be attributed to the presence in the cell wall of tightly bound 51-kDa (P51) and P42 binary toxin proteins. The synergism between binary toxin crystals and urea-treated cell wall preparations reflects suboptimal distribution of binary toxin subunits in both compartments. Binary toxin crystal is slightly deficient in P51, while cell wall is lacking in P42. PMID:12089007

  1. Tightly bound binary toxin in the cell wall of Bacillus sphaericus.

    PubMed

    Klein, Daniela; Uspensky, Igor; Braun, Sergei

    2002-07-01

    We have shown that urea-extracted cell wall of entomopathogenic Bacillus sphaericus 2297 and some other strains is a potent larvicide against Culex pipiens mosquitoes, with 50% lethal concentrations comparable to that of the well-known B. sphaericus binary toxin, with which it acts synergistically. The wall toxicity develops in B. sphaericus 2297 cultures during the late logarithmic stage, earlier than the appearance of the binary toxin crystal. It disappears with sporulation when the binary toxin activity reaches its peak. Disruption of the gene for the 42-kDa protein (P42) of the binary toxin abolishes both cell wall toxicity and crystal formation. However, the cell wall of B. sphaericus 2297, lacking P42, kills C. pipiens larvae when mixed with Escherichia coli cells expressing P42. Thus, the cell wall toxicity in strongly toxic B. sphaericus strains must be attributed to the presence in the cell wall of tightly bound 51-kDa (P51) and P42 binary toxin proteins. The synergism between binary toxin crystals and urea-treated cell wall preparations reflects suboptimal distribution of binary toxin subunits in both compartments. Binary toxin crystal is slightly deficient in P51, while cell wall is lacking in P42.

  2. Uremic toxins and peritoneal dialysis.

    PubMed

    Lameire, N; Vanholder, R; De Smet, R

    2001-02-01

    Uremic toxicity is related in part to the accumulation of toxic substances, the nature of which has only partly been characterized. Because of the use of a highly permeable membrane and better preservation of the residual renal function, it could be anticipated that some of these uremic toxins are more efficiently cleared across the peritoneal membrane, and that the plasma and tissue levels of these compounds are lower than in hemodialysis patients. This article analyzes the generation and removal of several uremic toxins in peritoneal dialysis patients. The following uremic toxins are discussed: beta2-microglobulin, advanced glycation end products, advanced oxidation protein products, granulocyte inhibitory proteins, p-Cresol, and hyperhomocysteinemia. Some recent studies are reviewed suggesting that uremic toxins are involved in the progression of renal failure and are at least partially removed by peritoneal dialysis. We conclude that, although the plasma levels of some of these compounds are lower in peritoneal dialysis versus hemodialysis patients, it does not mean that the peritoneal dialysis patient is "better" protected against the numerous disturbances caused by these toxins.

  3. Protective antibody responses against Clostridium difficile elicited by a DNA vaccine expressing the enzymatic domain of toxin B.

    PubMed

    Jin, Ke; Wang, Shixia; Zhang, Chunhua; Xiao, Yanling; Lu, Shan; Huang, Zuhu

    2013-01-01

    A DNA vaccination approach was used in the current study to screen for the immunogenicity of different fragments of toxin A and toxin B from Clostridium difficile. With this approach, protein antigens do not need to be produced in vitro and the immunogenicity of candidate C. difficile antigens can be identified directly in animals. Codon optimized toxin gene fragments were individually cloned into the DNA vaccine vector and tested in mice and rabbits for their ability to elicit C. difficile toxin-specific antibody responses. Only a subset of the C. difficile toxin fragments, including the C-terminal receptor binding domain of toxin A and a novel N-terminal enzymatic domain of toxin B, were able to elicit protective antibody responses as determined by protection of target cells in a cytotoxicity assay or by preventing death of mice in a passive antibody protection study. Significantly, antibodies elicited by the novel N-terminus of the toxin B DNA vaccine were able to increase the level of protection when used in combination with anti-toxin A antibodies in a toxin challenge model in mice.

  4. Discovery of Functional Toxin/Antitoxin Systems in Bacteria by Shotgun Cloning

    SciTech Connect

    Sberro, Hila; Leavitt, Azita; Kiro, Ruth; Koh, Eugene; Peleg, Yoav; Qimron, Udi; Sorek, Rotem

    2013-04-01

    Toxin-antitoxin (TA) modules, composed of a toxic protein and a counteracting antitoxin, play important roles in bacterial physiology. We examined the experimental insertion of 1.5 million genes from 388 microbial genomes into an Escherichia coli host using over 8.5 million random clones. This revealed hundreds of genes (toxins) that could only be cloned when the neighboring gene (antitoxin) was present on the same clone. Clustering of these genes revealed TA families widespread in bacterial genomes, some of which deviate from the classical characteristics previously described for such modules. Introduction of these genes into E. coli validated that the toxin toxicity is mitigated by the antitoxin. Infection experiments with T7 phage showed that two of the new modules can provide resistance against phage. Moreover, our experiments revealed an 'anti-defense' protein in phage T7 that neutralizes phage resistance. Our results expose active fronts in the arms race between bacteria and phage.

  5. Comparative genomics of Shiga toxin encoding bacteriophages

    PubMed Central

    2012-01-01

    Background Stx bacteriophages are responsible for driving the dissemination of Stx toxin genes (stx) across their bacterial host range. Lysogens carrying Stx phages can cause severe, life-threatening disease and Stx toxin is an integral virulence factor. The Stx-bacteriophage vB_EcoP-24B, commonly referred to as Ф24B, is capable of multiply infecting a single bacterial host cell at a high frequency, with secondary infection increasing the rate at which subsequent bacteriophage infections can occur. This is biologically unusual, therefore determining the genomic content and context of Ф24B compared to other lambdoid Stx phages is important to understanding the factors controlling this phenomenon and determining whether they occur in other Stx phages. Results The genome of the Stx2 encoding phage, Ф24B was sequenced and annotated. The genomic organisation and general features are similar to other sequenced Stx bacteriophages induced from Enterohaemorrhagic Escherichia coli (EHEC), however Ф24B possesses significant regions of heterogeneity, with implications for phage biology and behaviour. The Ф24B genome was compared to other sequenced Stx phages and the archetypal lambdoid phage, lambda, using the Circos genome comparison tool and a PCR-based multi-loci comparison system. Conclusions The data support the hypothesis that Stx phages are mosaic, and recombination events between the host, phages and their remnants within the same infected bacterial cell will continue to drive the evolution of Stx phage variants and the subsequent dissemination of shigatoxigenic potential. PMID:22799768

  6. Sodium Channel Inhibiting Marine Toxins

    NASA Astrophysics Data System (ADS)

    Llewellyn, Lyndon E.

    Saxitoxin (STX), tetrodotoxin (TTX) and their many chemical relatives are part of our daily lives. From killing people who eat seafood containing these toxins, to being valuable research tools unveiling the invisible structures of their pharmacological receptor, their global impact is beyond measure. The pharmacological receptor for these toxins is the voltage-gated sodium channel which transports Na ions between the exterior to the interior of cells. The two structurally divergent families of STX and TTX analogues bind at the same location on these Na channels to stop the flow of ions. This can affect nerves, muscles and biological senses of most animals. It is through these and other toxins that we have developed much of our fundamental understanding of the Na channel and its part in generating action potentials in excitable cells.

  7. Botulinum Toxin in Migraine Treatment

    PubMed Central

    ILGAZ AYDINLAR, Elif; YALINAY DİKMEN, Pınar; SAĞDUYU KOCAMAN, Ayşe

    2013-01-01

    Since botulinum toxin might have a therapeutic effect on pain, many studies investigating the efficiency of botulinum toxin in headache treatment have been done. The most satisfying results were achieved by botulinum toxin type A (BoNT/A) in the treatment of chronic migraine. In this paper, we reviewed the clinical effectiveness of BoNT/A in migraine and included our clinical experience. In our ongoing pilot study, where we have repeated BoNT/A injections every 12 weeks, The difference in the Migraine Disability Assessment (MIDAS) scores between the first and the second injections was 61.1%; and between the first and the 3rd injections was found to be 65.72%.

  8. Inhibition of cholera toxin and other AB toxins by polyphenolic compounds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    All AB-type protein toxins have intracellular targets despite an initial extracellular location. These toxins use different methods to reach the cytosol and have different effects on the target cell. Broad-spectrum inhibitors against AB toxins are therefore hard to develop because the toxins use dif...

  9. Reassessment of the toxin profile of Cylindrospermopsis raciborskii T3 and function of putative sulfotransferases in synthesis of sulfated and sulfonated PSP toxins.

    PubMed

    Soto-Liebe, Katia; Murillo, Alejandro A; Krock, Bernd; Stucken, Karina; Fuentes-Valdés, Juan J; Trefault, Nicole; Cembella, Allan; Vásquez, Mónica

    2010-12-01

    The toxigenic freshwater cyanobacterium Cylindrospermopsis raciborskii T3 has been used as a model to study and elucidate the biosynthetic pathway of tetrahydropurine neurotoxins associated with paralytic shellfish poisoning (PSP). There are nevertheless several inconsistencies and contradictions in the toxin profile of this strain as published by different research groups, and claimed to include carbamoyl (STX, NEO, GTX2/3), decarbamoyl (dcSTX), and N-sulfocarbamoyl (C1/2, B1) derivatives. Our analysis of the complete genome of another PSP toxin-producing cyanobacterium, Raphidiopsis brookii D9, which is closely related to C. raciborskii T3, resolved many issues regarding the correlation between biosynthetic pathways, corresponding genes and the T3 toxin profile. The putative sxt gene cluster in R. brookii D9 has a high synteny with the T3 sxt cluster, with 100% nucleotide identity among the shared genes. We also compared the PSP toxin profile of the strains by liquid chromatography coupled to mass spectrometry (LC-MS/MS). In contrast to published reports, our reassessment of the PSP toxin profile of T3 confirmed production of only STX, NEO and dcNEO. We gained significant insights via correlation between specific sxt genes and their role in PSP toxin synthesis in both D9 and T3 strains. In particular, analysis of sulfotransferase functions for SxtN (N-sulfotransferase) and SxtSUL (O-sulfotransferase) enzymes allowed us to propose an extension of the PSP toxin biosynthetic pathway from STX to the production of the derivatives GTX2/3, C1/2 and B1. This is a significantly revised view of the genetic mechanisms underlying synthesis of sulfated and sulfonated STX analogues in toxigenic cyanobacteria.

  10. Comparative in vitro and in vivo assessment of toxin neutralization by anti-tetanus toxin monoclonal antibodies.

    PubMed

    Yousefi, Mehdi; Khosravi-Eghbal, Roya; Reza Mahmoudi, Ahmad; Jeddi-Tehrani, Mahmood; Rabbani, Hodjatallah; Shokri, Fazel

    2014-01-01

    Tetanus is caused by the tetanus neurotoxin (TeNT), a 150 kDa single polypeptide molecule which is cleaved into an active two-chain molecule composed of a 50 kDa N-terminal light (L) and a 100 kDa C-terminal heavy (H) chains. Recently, extensive effort has focused on characterization of TeNT binding receptors and toxin neutralization by monoclonal antibodies (mAbs). Toxin binding inhibition and neutralization is routinely assessed either in vitro by the ganglioside GT1b binding inhibition assay or in vivo using an animal model. These two assay systems have never been compared. In the present study, we report characterization of eleven mAbs against different parts of TeNT. The toxin inhibitory and neutralization activity of the mAbs was assessed in vitro and in vivo respectively. Our data demonstrated that seven mAbs bind to fragment C of the heavy chain, two mAbs react with the light chain, one mAb recognizes both chains and one mAb reacts with neither light chain nor fragment C. Six fragment C specific mAbs were able to inhibit TeNT binding to GT1b ganglioside in vitro but three failed to neutralize the toxin in vivo. One in vitro inhibitory mAb (1F3E3) was found to synergize with the in vivo neutralizing mAbs to reduce toxin lethal activity in vivo. Sequencing of the immunoglobulin heavy and light chain variable region genes revealed that the three in vivo neutralizing mAbs were derived from a common origin. Altogether, our data suggests that fragment C specific mAbs contribute to toxin neutralization in both systems, though some of the GT1b binding inhibitory mAbs may not be able to neutralize TeNT in vivo.

  11. Toxin yet not toxic: Botulinum toxin in dentistry.

    PubMed

    Archana, M S

    2016-04-01

    Paracelsus contrasted poisons from nonpoisons, stating that "All things are poisons, and there is nothing that is harmless; the dose alone decides that something is a poison". Living organisms, such as plants, animals, and microorganisms, constitute a huge source of pharmaceutically useful medicines and toxins. Depending on their source, toxins can be categorized as phytotoxins, mycotoxins, or zootoxins, which include venoms and bacterial toxins. Any toxin can be harmful or beneficial. Within the last 100 years, the perception of botulinum neurotoxin (BTX) has evolved from that of a poison to a versatile clinical agent with various uses. BTX plays a key role in the management of many orofacial and dental disorders. Its indications are rapidly expanding, with ongoing trials for further applications. However, despite its clinical use, what BTX specifically does in each condition is still not clear. The main aim of this review is to describe some of the unclear aspects of this potentially useful agent, with a focus on the current research in dentistry.

  12. Contact with enterocyte-like Caco-2 cells induces rapid upregulation of toxin production by Clostridium perfringens type C isolates

    PubMed Central

    Vidal, Jorge E.; Ohtani, Kaori; Shimizu, Tohru; McClane, Bruce A.

    2009-01-01

    Clostridium perfringens type C isolates cause necrotizing enteritis in humans and domestic animals. In vitro, type C isolates often produce beta toxin (CPB), beta2 toxin (CPB2), alpha toxin (CPA), perfringolysin O (PFO), and TpeL during (or after) late log-phase growth. In contrast, the current study found that many type C isolates respond to close contact with enterocyte-like Caco-2 cells by producing all toxins, except TpeL, much more rapidly than occurs during in vitro growth. This in vivo effect involves rapid transcriptional upregulation of the cpb, cpb2, pfoA and plc toxin genes. Rapid Caco-2 cell-induced upregulation of CPB and PFO production involves the VirS/VirR two-component system, since upregulated in vivo transcription of the pfoA and cpb genes was blocked by inactivating the virR gene and was reversible by complementation to restore VirR expression. However, the luxS quorum sensing system is not required for the rapid upregulation of type C toxin production induced by contact with Caco-2 cells. These results provide the first indication of host cell:pathogen cross-talk affecting toxin production kinetics by any pathogenic Clostridium spp., identify in vivo vs. in vitro differences in C. perfringens toxin expression, and implicate VirS/VirR as a possible contributor to some C. perfringens enteric diseases. PMID:19438515

  13. A System for Discovering Bioengineering Threats by Knowledge Base Driven Mining of Toxin Data

    DTIC Science & Technology

    2005-08-01

    Acid gene-sequence X62089 related-structures Belongs to the peptidase M27 family [view classification] metalcofactor Binds 1 zinc ion per subunit...inhibitors target abtoxin pore-former enzyme-catalyzed EC_number 3,.24.69 Botulinum toxin acts by inhibiting neurotransmitter release. It binds to...Ile- 181 bond in SNAP-25. Botulinum toxin acts by inhibiting neurotransmitter release. It binds to peripheral neuronal synapses, is internalized and

  14. Toxin content and cytotoxicity of algal dietary supplements

    SciTech Connect

    Heussner, A.H.; Mazija, L.; Fastner, J.; Dietrich, D.R.

    2012-12-01

    Blue-green algae (Spirulina sp., Aphanizomenon flos-aquae) and Chlorella sp. are commercially distributed as organic algae dietary supplements. Cyanobacterial dietary products in particular have raised serious concerns, as they appeared to be contaminated with toxins e.g. microcystins (MCs) and consumers repeatedly reported adverse health effects following consumption of these products. The aim of this study was to determine the toxin contamination and the in vitro cytotoxicity of algae dietary supplement products marketed in Germany. In thirteen products consisting of Aph. flos-aquae, Spirulina and Chlorella or mixtures thereof, MCs, nodularins, saxitoxins, anatoxin-a and cylindrospermopsin were analyzed. Five products tested in an earlier market study were re-analyzed for comparison. Product samples were extracted and analyzed for cytotoxicity in A549 cells as well as for toxin levels by (1) phosphatase inhibition assay (PPIA), (2) Adda-ELISA and (3) LC–MS/MS. In addition, all samples were analyzed by PCR for the presence of the mcyE gene, a part of the microcystin and nodularin synthetase gene cluster. Only Aph. flos-aquae products were tested positive for MCs as well as the presence of mcyE. The contamination levels of the MC-positive samples were ≤ 1 μg MC-LR equivalents g{sup −1} dw. None of the other toxins were found in any of the products. However, extracts from all products were cytotoxic. In light of the findings, the distribution and commercial sale of Aph. flos-aquae products, whether pure or mixed formulations, for human consumption appear highly questionable. -- Highlights: ► Marketed algae dietary supplements were analyzed for toxins. ► Methods: Phosphatase inhibition assay (PPIA), Adda-ELISA, LC-MS/MS. ► Aph. flos-aquae products all tested positive for microcystins. ► Products tested negative for nodularins, saxitoxins, anatoxin-a, cylindrospermopsin. ► Extracts from all products were cytotoxic.

  15. Molecular Evolutionary Constraints that Determine the Avirulence State of Clostridium botulinum C2 Toxin.

    PubMed

    Prisilla, A; Prathiviraj, R; Chellapandi, P

    2017-04-05

    Clostridium botulinum (group-III) is an anaerobic bacterium producing C2 toxin along with botulinum neurotoxins. C2 toxin is belonged to binary toxin A family in bacterial ADP-ribosylation superfamily. A structural and functional diversity of binary toxin A family was inferred from different evolutionary constraints to determine the avirulence state of C2 toxin. Evolutionary genetic analyses revealed evidence of C2 toxin cluster evolution through horizontal gene transfer from the phage or plasmid origins, site-specific insertion by gene divergence, and homologous recombination event. It has also described that residue in conserved NAD-binding core, family-specific domain structure, and functional motifs found to predetermine its virulence state. Any mutational changes in these residues destabilized its structure-function relationship. Avirulent mutants of C2 toxin were screened and selected from a crucial site required for catalytic function of C2I and pore-forming function of C2II. We found coevolved amino acid pairs contributing an essential role in stabilization of its local structural environment. Avirulent toxins selected in this study were evaluated by detecting evolutionary constraints in stability of protein backbone structure, folding and conformational dynamic space, and antigenic peptides. We found 4 avirulent mutants of C2I and 5 mutants of C2II showing more stability in their local structural environment and backbone structure with rapid fold rate, and low conformational flexibility at mutated sites. Since, evolutionary constraints-free mutants with lack of catalytic and pore-forming function suggested as potential immunogenic candidates for treating C. botulinum infected poultry and veterinary animals. Single amino acid substitution in C2 toxin thus provides a major importance to understand its structure-function link, not only of a molecule but also of the pathogenesis.

  16. Comparative Genomics Evidence That Only Protein Toxins are Tagging Bad Bugs

    PubMed Central

    Georgiades, Kalliopi; Raoult, Didier

    2011-01-01

    The term toxin was introduced by Roux and Yersin and describes macromolecular substances that, when produced during infection or when introduced parenterally or orally, cause an impairment of physiological functions that lead to disease or to the death of the infected organism. Long after the discovery of toxins, early genetic studies on bacterial virulence demonstrated that removing a certain number of genes from pathogenic bacteria decreases their capacity to infect hosts. Each of the removed factors was therefore referred to as a “virulence factor,” and it was speculated that non-pathogenic bacteria lack such supplementary factors. However, many recent comparative studies demonstrate that the specialization of bacteria to eukaryotic hosts is associated with massive gene loss. We recently demonstrated that the only features that seem to characterize 12 epidemic bacteria are toxin–antitoxin (TA) modules, which are addiction molecules in host bacteria. In this study, we investigated if protein toxins are indeed the only molecules specific to pathogenic bacteria by comparing 14 epidemic bacterial killers (“bad bugs”) with their 14 closest non-epidemic relatives (“controls”). We found protein toxins in significantly more elevated numbers in all of the “bad bugs.” For the first time, statistical principal components analysis, including genome size, GC%, TA modules, restriction enzymes, and toxins, revealed that toxins are the only proteins other than TA modules that are correlated with the pathogenic character of bacteria. Moreover, intracellular toxins appear to be more correlated with the pathogenic character of bacteria than secreted toxins. In conclusion, we hypothesize that the only truly identifiable phenomena, witnessing the convergent evolution of the most pathogenic bacteria for humans are the loss of metabolic activities, i.e., the outcome of the loss of regulatory and transcription factors and the presence of protein toxins, alone, or

  17. Polymer antidotes for toxin sequestration.

    PubMed

    Weisman, Adam; Chou, Beverly; O'Brien, Jeffrey; Shea, Kenneth J

    2015-08-01

    Toxins delivered by envenomation, secreted by microorganisms, or unintentionally ingested can pose an immediate threat to life. Rapid intervention coupled with the appropriate antidote is required to mitigate the threat. Many antidotes are biological products and their cost, methods of production, potential for eliciting immunogenic responses, the time needed to generate them, and stability issues contribute to their limited availability and effectiveness. These factors exacerbate a world-wide challenge for providing treatment. In this review we evaluate a number of polymer constructs that may serve as alternative antidotes. The range of toxins investigated includes those from sources such as plants, animals and bacteria. The development of polymeric heavy metal sequestrants for use as antidotes to heavy metal poisoning faces similar challenges, thus recent findings in this area have also been included. Two general strategies have emerged for the development of polymeric antidotes. In one, the polymer acts as a scaffold for the presentation of ligands with a known affinity for the toxin. A second strategy is to generate polymers with an intrinsic affinity, and in some cases selectivity, to a range of toxins. Importantly, in vivo efficacy has been demonstrated for each of these strategies, which suggests that these approaches hold promise as an alternative to biological or small molecule based treatments.

  18. MCEARD - CYANOBACTERIA AND THEIR TOXINS

    EPA Science Inventory

    Harmful algal blooms (HAB) of cyanobacteria, also known as blue-green algae, have recently become more spatially and temporally prevalent in the US and worldwide. Waterborne cyanobacteria and their highly potent toxins are a significant hazard for human health and the ecosystem....

  19. Risk Assessment of Shellfish Toxins

    PubMed Central

    Munday, Rex; Reeve, John

    2013-01-01

    Complex secondary metabolites, some of which are highly toxic to mammals, are produced by many marine organisms. Some of these organisms are important food sources for marine animals and, when ingested, the toxins that they produce may be absorbed and stored in the tissues of the predators, which then become toxic to animals higher up the food chain. This is a particular problem with shellfish, and many cases of poisoning are reported in shellfish consumers each year. At present, there is no practicable means of preventing uptake of the toxins by shellfish or of removing them after harvesting. Assessment of the risk posed by such toxins is therefore required in order to determine levels that are unlikely to cause adverse effects in humans and to permit the establishment of regulatory limits in shellfish for human consumption. In the present review, the basic principles of risk assessment are described, and the progress made toward robust risk assessment of seafood toxins is discussed. While good progress has been made, it is clear that further toxicological studies are required before this goal is fully achieved. PMID:24226039

  20. Transcriptional profiling analysis of Spodoptera litura larvae challenged with Vip3Aa toxin and possible involvement of trypsin in the toxin activation

    PubMed Central

    Song, Feifei; Chen, Chen; Wu, Songqing; Shao, Ensi; Li, Mengnan; Guan, Xiong; Huang, Zhipeng

    2016-01-01

    Vip proteins, a new group of insecticidal toxins produced by Bacillus thuringiensis, are effective against specific pests including Spodoptera litura. Here, we report construction of a transcriptome database of S. litura by de novo assembly along with detection of the transcriptional response of S. litura larvae to Vip3Aa toxin. In total, 56,498 unigenes with an N50 value of 1,853 bp were obtained. Results of transcriptome abundance showed that Vip3Aa toxin provoked a wide transcriptional response of the S. litura midgut. The differentially expressed genes were enriched for immunity-related, metabolic-related and Bt-related genes. Twenty-nine immunity-related genes, 102 metabolic-related genes and 62 Bt-related genes with differential expression were found. On the basis of transcriptional profiling analysis, we focus on the functional validation of trypsin which potentially participated in the activation of Vip3Aa protoxin. Zymogram analysis indicated that the presence of many proteases, including trypsin, in S. litura larvae midgut. Results of enzymolysis in vitro of Vip3Aa by trypsin, and bioassay and histopathology of the trypsin-digested Vip3Aa toxin showed that trypsin was possibly involved in the Vip3Aa activation. This study provides a transcriptome foundation for the identification and functional validation of the differentially expressed genes in an agricultural important pest, S. litura. PMID:27025647

  1. Transcriptional profiling analysis of Spodoptera litura larvae challenged with Vip3Aa toxin and possible involvement of trypsin in the toxin activation.

    PubMed

    Song, Feifei; Chen, Chen; Wu, Songqing; Shao, Ensi; Li, Mengnan; Guan, Xiong; Huang, Zhipeng

    2016-03-30

    Vip proteins, a new group of insecticidal toxins produced by Bacillus thuringiensis, are effective against specific pests including Spodoptera litura. Here, we report construction of a transcriptome database of S. litura by de novo assembly along with detection of the transcriptional response of S. litura larvae to Vip3Aa toxin. In total, 56,498 unigenes with an N50 value of 1,853 bp were obtained. Results of transcriptome abundance showed that Vip3Aa toxin provoked a wide transcriptional response of the S. litura midgut. The differentially expressed genes were enriched for immunity-related, metabolic-related and Bt-related genes. Twenty-nine immunity-related genes, 102 metabolic-related genes and 62 Bt-related genes with differential expression were found. On the basis of transcriptional profiling analysis, we focus on the functional validation of trypsin which potentially participated in the activation of Vip3Aa protoxin. Zymogram analysis indicated that the presence of many proteases, including trypsin, in S. litura larvae midgut. Results of enzymolysis in vitro of Vip3Aa by trypsin, and bioassay and histopathology of the trypsin-digested Vip3Aa toxin showed that trypsin was possibly involved in the Vip3Aa activation. This study provides a transcriptome foundation for the identification and functional validation of the differentially expressed genes in an agricultural important pest, S. litura.

  2. RNA toxins: mediators of stress adaptation and pathogen defense.

    PubMed

    Zhabokritsky, Alice; Kutky, Meherzad; Burns, Lydia A; Karran, Rajita A; Hudak, Katalin A

    2011-01-01

    RNA toxins are a group of enzymes primarily synthesized by bacteria, fungi, and plants that either cleave or depurinate RNA molecules. These proteins may be divided according to their RNA substrates: ribotoxins are nucleases that cleave ribosomal RNA (rRNA), ribosome inactivating proteins are glycosidases that remove a base from rRNA, messenger RNA (mRNA) interferases are nucleases that cleave mRNAs, and anticodon nucleases cleave transfer RNAs (tRNAs). These modifications to the RNAs may substantially alter gene expression and translation rates. Given that some of these enzymes cause cell death, it has been suggested that they function mainly in defense, either to kill competing cells or to elicit suicide and thereby limit pathogen spread from infected cells. Although good correlations have been drawn between their enzymatic functions and toxicity, recent work has shown that some RNA toxins cause apoptosis in the absence of damage to RNA and that defense against pathogens can be achieved without host cell death. Moreover, a decrease in cellular translation rate, insufficient to cause cell death, allows some organisms to adapt to stress and environmental change. Although ascribing effects observed in vitro to the roles of these toxins in nature has been challenging, recent results have expanded our understanding of their modes of action, and emphasized the importance of these toxins in development, adaptation to stress and defense against pathogens.

  3. Antibody microarrays for native toxin detection.

    PubMed

    Rucker, Victor C; Havenstrite, Karen L; Herr, Amy E

    2005-04-15

    We have developed antibody-based microarray techniques for the multiplexed detection of cholera toxin beta-subunit, diphtheria toxin, anthrax lethal factor and protective antigen, Staphylococcus aureus enterotoxin B, and tetanus toxin C fragment in spiked samples. Two detection schemes were investigated: (i) a direct assay in which fluorescently labeled toxins were captured directly by the antibody array and (ii) a competition assay that employed unlabeled toxins as reporters for the quantification of native toxin in solution. In the direct assay, fluorescence measured at each array element is correlated with labeled toxin concentration to yield baseline binding information (Langmuir isotherms and affinity constants). Extending from the direct assay, the competition assay yields information on the presence, identity, and concentration of toxins. A significant advantage of the competition assay over reported profiling assays is the minimal sample preparation required prior to analysis because the competition assay obviates the need to fluorescently label native proteins in the sample of interest. Sigmoidal calibration curves and detection limits were established for both assay formats. Although the sensitivity of the direct assay is superior to that of the competition assay, detection limits for unmodified toxins in the competition assay are comparable to values reported previously for sandwich-format immunoassays of antibodies arrayed on planar substrates. As a demonstration of the potential of the competition assay for unlabeled toxin detection, we conclude with a straightforward multiplexed assay for the differentiation and identification of both native S. aureus enterotoxin B and tetanus toxin C fragment in spiked dilute serum samples.

  4. Toxins as Weapons: A Historical Review.

    PubMed

    Pita, R; Romero, A

    2014-07-01

    This review article summarizes the use of toxins as weapons dating from the First World War until today, when there is a high concern of possible terrorist attacks with weapons of mass destruction. All through modern history, military programs and terrorist groups have favored toxins because of their high toxicity. However, difficulties of extraction or synthesis, as well as effective dissemination to cause a large number of casualties, have been the most important drawbacks. Special emphasis is focused on ricin and botulinum toxin, the most important toxins that have attracted the attention of military programs and terrorist groups. Other toxins like trichothecenes, saxitoxin, and Staphylococcal enterotoxin B (SEB) are also discussed. A short section about anthrax is also included: Although Bacillus anthracis is considered a biological weapon rather than a toxin weapon, it produces a toxin that is finally responsible for the anthrax disease.

  5. Antibody-based bacterial toxin detection

    NASA Astrophysics Data System (ADS)

    Menking, Darrell E.; Heitz, Jonathon M.; Anis, Nabil A.; Thompson, Roy G.

    1994-03-01

    Fiber optic evanescent fluorosensors are under investigation in our laboratory for the study of drug-receptor interactions for detection of threat agents and antibody-antigen interactions for detection of biological toxins. In a one step assay, antibodies against Cholera toxin or Staphylococcus Enterotoxin B were noncovalently immobilized on quartz fibers and probed with fluorescein-isothiocyanate (FITC)-labeled toxins. In the two-step assay, Cholera toxin or Botulinum toxoid A was immobilized onto the fiber, followed by incubation in an antiserum or partially purified antitoxin IgG. These were then probed with FITC-anti-IgG antibodies. Unlabeled toxins competed with labeled toxins or antitoxin IgG in a dose-dependent manner and the detection of the toxins was in the nanomolar range.

  6. Multicenter Evaluation of the BD Max Enteric Bacterial Panel PCR Assay for Rapid Detection of Salmonella spp., Shigella spp., Campylobacter spp. (C. jejuni and C. coli), and Shiga Toxin 1 and 2 Genes

    PubMed Central

    Doern, C.; Fader, R.; Ferraro, M. J.; Pillai, D. R.; Rychert, J.; Doyle, L.; Lainesse, A.; Karchmer, T.; Mortensen, J. E.

    2015-01-01

    Diarrhea due to enteric bacterial pathogens causes significant morbidity and mortality in the United States and worldwide. However, bacterial pathogens may be infrequently identified. Currently, culture and enzyme immunoassays (EIAs) are the primary methods used by clinical laboratories to detect enteric bacterial pathogens. We conducted a multicenter evaluation of the BD Max enteric bacterial panel (EBP) PCR assay in comparison to culture for the detection of Salmonella spp., Shigella spp., Campylobacter jejuni, and Campylobacter coli and an EIA for Shiga toxins 1 and 2. A total of 4,242 preserved or unpreserved stool specimens, including 3,457 specimens collected prospectively and 785 frozen, retrospective samples, were evaluated. Compared to culture or EIA, the positive percent agreement (PPA) and negative percent agreement (NPA) values for the BD Max EBP assay for all specimens combined were as follows: 97.1% and 99.2% for Salmonella spp., 99.1% and 99.7% for Shigella spp., 97.2% and 98.4% for C. jejuni and C. coli, and 97.4% and 99.3% for Shiga toxins, respectively. Discrepant results for prospective samples were resolved with alternate PCR assays and bidirectional sequencing of amplicons. Following discrepant analysis, PPA and NPA values were as follows: 97.3% and 99.8% for Salmonella spp., 99.2% and 100% for Shigella spp., 97.5% and 99.0% for C. jejuni and C. coli, and 100% and 99.7% for Shiga toxins, respectively. No differences in detection were observed for samples preserved in Cary-Blair medium and unpreserved samples. In this large, multicenter study, the BD Max EBP assay showed superior sensitivity compared to conventional methods and excellent specificity for the detection of enteric bacterial pathogens in stool specimens. PMID:25740779

  7. Multicenter evaluation of the BD max enteric bacterial panel PCR assay for rapid detection of Salmonella spp., Shigella spp., Campylobacter spp. (C. jejuni and C. coli), and Shiga toxin 1 and 2 genes.

    PubMed

    Harrington, S M; Buchan, B W; Doern, C; Fader, R; Ferraro, M J; Pillai, D R; Rychert, J; Doyle, L; Lainesse, A; Karchmer, T; Mortensen, J E

    2015-05-01

    Diarrhea due to enteric bacterial pathogens causes significant morbidity and mortality in the United States and worldwide. However, bacterial pathogens may be infrequently identified. Currently, culture and enzyme immunoassays (EIAs) are the primary methods used by clinical laboratories to detect enteric bacterial pathogens. We conducted a multicenter evaluation of the BD Max enteric bacterial panel (EBP) PCR assay in comparison to culture for the detection of Salmonella spp., Shigella spp., Campylobacter jejuni, and Campylobacter coli and an EIA for Shiga toxins 1 and 2. A total of 4,242 preserved or unpreserved stool specimens, including 3,457 specimens collected prospectively and 785 frozen, retrospective samples, were evaluated. Compared to culture or EIA, the positive percent agreement (PPA) and negative percent agreement (NPA) values for the BD Max EBP assay for all specimens combined were as follows: 97.1% and 99.2% for Salmonella spp., 99.1% and 99.7% for Shigella spp., 97.2% and 98.4% for C. jejuni and C. coli, and 97.4% and 99.3% for Shiga toxins, respectively. Discrepant results for prospective samples were resolved with alternate PCR assays and bidirectional sequencing of amplicons. Following discrepant analysis, PPA and NPA values were as follows: 97.3% and 99.8% for Salmonella spp., 99.2% and 100% for Shigella spp., 97.5% and 99.0% for C. jejuni and C. coli, and 100% and 99.7% for Shiga toxins, respectively. No differences in detection were observed for samples preserved in Cary-Blair medium and unpreserved samples. In this large, multicenter study, the BD Max EBP assay showed superior sensitivity compared to conventional methods and excellent specificity for the detection of enteric bacterial pathogens in stool specimens.

  8. The mitogen-activated protein kinase p38 is involved in insect defense against Cry toxins from Bacillus thuringiensis

    PubMed Central

    Cancino-Rodezno, Angeles; Alexander, Cynthia; Villaseñor, Roberto; Pacheco, Sabino; Porta, Helena; Pauchet, Yannick; Soberón, Mario; Gill, Sarjeet S.; Bravo, Alejandra

    2010-01-01

    The insecticidal Cry toxins are pore-forming toxins produced by the bacteria Bacillus thuringiensis that disrupt insect-midgut cells. In this work we analyzed the response of two different insect orders, the Lepidopteran Manduca sexta and Dipteran Aedes aegypti to highly specific Cry toxins, Cry1Ab and Cry11Aa, respectively. One pathway activated in different organisms in response to a variety of pore forming toxins is the mitogen activated protein kinase p38 pathway (MAPK p38) that activates a complex defense response. We analyzed the MAPK p38 activation by immunodetection of its phosphorylated isoform, and the induction of p38 by RT-PCR, real time-PCR quantitative assays and immunodection. We show that MAPK p38 is activated at postraductional level after Cry toxin intoxication in both insect orders. We detected the p38 induction at the transcriptional and traductional level, and observed a different response. In these three levels, we found that both insects respond to Cry toxin action but M sexta responses more strongly than A. aegypti. Gene silencing of MAPK p38 in vivo, resulted in both insect species becoming hypersensitive to Cry toxin action, suggesting that the MAPK p38 pathway is involved in insect defense against Bt Cry toxins. This finding may have biotechnological applications for enhancing the activity of some Bt Cry toxins against specific insect pests. PMID:20040372

  9. Structural overview of toxin-antitoxin systems in infectious bacteria: a target for developing antimicrobial agents.

    PubMed

    Park, Sung Jean; Son, Woo Sung; Lee, Bong-Jin

    2013-06-01

    The bacterial toxin-antitoxin (TA) system is a module that may play a role in cell survival under stress conditions. Generally, toxin molecules act as negative regulators in cell survival and antitoxin molecules as positive regulators. Thus, the expression levels and interactions between toxins and antitoxins should be systematically harmonized so that bacteria can escape such harmful conditions. Since TA systems are able to control the fate of bacteria, they are considered potent targets for the development of new antimicrobial agents. TA systems are widely prevalent with a variety of systems existing in bacteria: there are three types of bacterial TA systems depending on the property of the antitoxin which binds either the protein toxin or mRNA coding the toxin protein. Moreover, the multiplicity of TA genes has been observed even in species of bacteria. Therefore, knowledge on TA systems such as the individual characteristics of TA systems, integrative working mechanisms of various TA systems in bacteria, interactions between toxin molecules and cellular targets, and so on is currently limited due to their complexity. In this regard, it would be helpful to know the structural characteristics of TA modules for understanding TA systems in bacteria. Until now, 85 out of the total structures deposited in PDB have been bacterial TA system proteins including TA complexes or isolated toxins/antitoxins. Here, we summarized the structural information of TA systems and analyzed the structural characteristics of known TA modules from several bacteria, especially focusing on the TA modules of several infectious bacteria.

  10. The Pasteurella multocida toxin is encoded within a lysogenic bacteriophage.

    PubMed

    Pullinger, Gillian D; Bevir, Thomas; Lax, Alistair J

    2004-01-01

    Toxigenic strains of Pasteurella multocida produce a 146 kDa toxin (PMT) that acts as a potent mitogen. Sequence analysis of the structural gene for PMT, toxA, previously suggested it was horizontally acquired, because it had a low G + C content relative to the P. multocida genome. To address this, the sequence of DNA flanking toxA was determined. The sequence analysis showed the presence of homologues to bacteriophage tail protein genes and a bacteriophage antirepressor, suggesting that the toxin gene resides within a prophage. In addition to phage genes, the toxA flanking DNA contained a homologue of a restriction/modification system that was shown to be functional. The presence of a bacteriophage was demonstrated in spent medium from toxigenic P. multocida isolates. Its production was increased by mitomycin C addition, a treatment that is known to induce the lytic cycle of many temperate bacteriophages. The genomes of bacteriophages from three different toxigenic P. multocida strains had similar but not identical restriction profiles, and were approximately 45-50 kb in length. The prophages from two of these had integrated at the same site in the chromosome, in a tRNA gene. Southern blot analysis confirmed that these bacteriophages contained the toxA gene.

  11. Diversity and Impact of Prokaryotic Toxins on Aquatic Environments: A Review

    PubMed Central

    Valério, Elisabete; Chaves, Sandra; Tenreiro, Rogério

    2010-01-01

    . Clostridium members are also spore-forming bacteria and can persist in hostile environmental conditions for long periods of time, contributing to their hazard grade. Similarly, Pseudomonas species are widespread in the environment. Since P. aeruginosa is an emergent opportunistic pathogen, its toxins may represent new hazards for humans and animals. This review presents an overview of the diversity of toxins produced by prokaryotic microorganisms associated with aquatic habitats and their impact on environment, life and health of humans and other animals. Moreover, important issues like the availability of these toxins in the environment, contamination sources and pathways, genes involved in their biosynthesis and molecular mechanisms of some representative toxins are also discussed. PMID:22069558

  12. Regulating Toxin-Antitoxin Expression: Controlled Detonation of Intracellular Molecular Timebombs

    PubMed Central

    Hayes, Finbarr; Kędzierska, Barbara

    2014-01-01

    Genes for toxin-antitoxin (TA) complexes are widely disseminated in bacteria, including in pathogenic and antibiotic resistant species. The toxins are liberated from association with the cognate antitoxins by certain physiological triggers to impair vital cellular functions. TAs also are implicated in antibiotic persistence, biofilm formation, and bacteriophage resistance. Among the ever increasing number of TA modules that have been identified, the most numerous are complexes in which both toxin and antitoxin are proteins. Transcriptional autoregulation of the operons encoding these complexes is key to ensuring balanced TA production and to prevent inadvertent toxin release. Control typically is exerted by binding of the antitoxin to regulatory sequences upstream of the operons. The toxin protein commonly works as a transcriptional corepressor that remodels and stabilizes the antitoxin. However, there are notable exceptions to this paradigm. Moreover, it is becoming clear that TA complexes often form one strand in an interconnected web of stress responses suggesting that their transcriptional regulation may prove to be more intricate than currently understood. Furthermore, interference with TA gene transcriptional autoregulation holds considerable promise as a novel antibacterial strategy: artificial release of the toxin factor using designer drugs is a potential approach to induce bacterial suicide from within. PMID:24434949

  13. Why do we study animal toxins?

    PubMed Central

    ZHANG, Yun

    2015-01-01

    Venom (toxins) is an important trait evolved along the evolutionary tree of animals. Our knowledges on venoms, such as their origins and loss, the biological relevance and the coevolutionary patterns with other organisms are greatly helpful in understanding many fundamental biological questions, i.e., the environmental adaptation and survival competition, the evolution shaped development and balance of venoms, and the sophisticated correlations among venom, immunity, body power, intelligence, their genetic basis, inherent association, as well as the cost-benefit and trade-offs of biological economy. Lethal animal envenomation can be found worldwide. However, from foe to friend, toxin studies have led lots of important discoveries and exciting avenues in deciphering and fighting human diseases, including the works awarded the Nobel Prize and lots of key clinic therapeutics. According to our survey, so far, only less than 0.1% of the toxins of the venomous animals in China have been explored. We emphasize on the similarities shared by venom and immune systems, as well as the studies of toxin knowledge-based physiological toxin-like proteins/peptides (TLPs). We propose the natural pairing hypothesis. Evolution links toxins with humans. Our mission is to find out the right natural pairings and interactions of our body elements with toxins, and with endogenous toxin-like molecules. Although, in nature, toxins may endanger human lives, but from a philosophical point of view, knowing them well is an effective way to better understand ourselves. So, this is why we study toxins. PMID:26228472

  14. Exfoliative toxins of Staphylococcus aureus.

    PubMed

    Bukowski, Michal; Wladyka, Benedykt; Dubin, Grzegorz

    2010-05-01

    Staphylococcus aureus is an important pathogen of humans and livestock. It causes a diverse array of diseases, ranging from relatively harmless localized skin infections to life-threatening systemic conditions. Among multiple virulence factors, staphylococci secrete several exotoxins directly associated with particular disease symptoms. These include toxic shock syndrome toxin 1 (TSST-1), enterotoxins, and exfoliative toxins (ETs). The latter are particularly interesting as the sole agents responsible for staphylococcal scalded skin syndrome (SSSS), a disease predominantly affecting infants and characterized by the loss of superficial skin layers, dehydration, and secondary infections. The molecular basis of the clinical symptoms of SSSS is well understood. ETs are serine proteases with high substrate specificity, which selectively recognize and hydrolyze desmosomal proteins in the skin. The fascinating road leading to the discovery of ETs as the agents responsible for SSSS and the characterization of the molecular mechanism of their action, including recent advances in the field, are reviewed in this article.

  15. An update on uremic toxins.

    PubMed

    Neirynck, N; Vanholder, R; Schepers, E; Eloot, S; Pletinck, A; Glorieux, G

    2013-02-01

    In the last decade, uremic toxicity as a potential cause for the excess of cardiovascular disease and mortality observed in chronic kidney disease gained more and more interest. This review focuses on uremic toxins with known cardiovascular effects and their removal. For protein-bound solutes, for example, indoxylsulfate and the conjugates of p-cresol, and for small water-soluble solutes, for example, guanidines, such as ADMA and SDMA, there is a growing evidence for a role in cardiovascular toxicity in vitro (e.g., affecting leukocyte, endothelial, vascular smooth muscle cell function) and/or in vivo. Several middle molecules (e.g., beta-2-microglobulin, interleukin-6, TNF-alpha and FGF-23) were shown to be predictors for cardiovascular disease and/or mortality. Most of these solutes, however, are difficult to remove during dialysis, which is traditionally assessed by studying the removal of urea, which can be considered as a relatively inert uremic retention solute. However, even the effective removal of other small water-soluble toxins than urea can be hampered by their larger distribution volumes. Middle molecules (beta-2-microglobulin as prototype, but not necessarily representative for others) are cleared more efficiently when the pore size of the dialyzer membrane increases, convection is applied and dialysis time is prolonged. Only adding convection to diffusion improves the removal of protein-bound toxins. Therefore, alternative removal strategies, such as intestinal adsorption, drugs interfering with toxic biochemical pathways or decreasing toxin concentration, and extracorporeal plasma adsorption, as well as kinetic behavior during dialysis need further investigation. Even more importantly, randomized clinical studies are required to demonstrate a survival advantage through these strategies.

  16. Neutralising Antibodies against Ricin Toxin

    PubMed Central

    Prigent, Julie; Panigai, Laetitia; Lamourette, Patricia; Sauvaire, Didier; Devilliers, Karine; Plaisance, Marc; Volland, Hervé; Créminon, Christophe; Simon, Stéphanie

    2011-01-01

    The Centers for Disease Control and Prevention have listed the potential bioweapon ricin as a Category B Agent. Ricin is a so-called A/B toxin produced by plants and is one of the deadliest molecules known. It is easy to prepare and no curative treatment is available. An immunotherapeutic approach could be of interest to attenuate or neutralise the effects of the toxin. We sought to characterise neutralising monoclonal antibodies against ricin and to develop an effective therapy. For this purpose, mouse monoclonal antibodies (mAbs) were produced against the two chains of ricin toxin (RTA and RTB). Seven mAbs were selected for their capacity to neutralise the cytotoxic effects of ricin in vitro. Three of these, two anti-RTB (RB34 and RB37) and one anti-RTA (RA36), when used in combination improved neutralising capacity in vitro with an IC50 of 31 ng/ml. Passive administration of association of these three mixed mAbs (4.7 µg) protected mice from intranasal challenges with ricin (5 LD50). Among those three antibodies, anti-RTB antibodies protected mice more efficiently than the anti-RTA antibody. The combination of the three antibodies protected mice up to 7.5 hours after ricin challenge. The strong in vivo neutralising capacity of this three mAbs combination makes it potentially useful for immunotherapeutic purposes in the case of ricin poisoning or possibly for prevention. PMID:21633505

  17. Novel Class of Spider Toxin

    PubMed Central

    Vassilevski, Alexander A.; Fedorova, Irina M.; Maleeva, Ekaterina E.; Korolkova, Yuliya V.; Efimova, Svetlana S.; Samsonova, Olga V.; Schagina, Ludmila V.; Feofanov, Alexei V.; Magazanik, Lev G.; Grishin, Eugene V.

    2010-01-01

    Venom of the yellow sac spider Cheiracanthium punctorium (Miturgidae) was found unique in terms of molecular composition. Its principal toxic component CpTx 1 (15.1 kDa) was purified, and its full amino acid sequence (134 residues) was established by protein chemistry and mass spectrometry techniques. CpTx 1 represents a novel class of spider toxin with modular architecture. It consists of two different yet homologous domains (modules) each containing a putative inhibitor cystine knot motif, characteristic of the widespread single domain spider neurotoxins. Venom gland cDNA sequencing provided precursor protein (prepropeptide) structures of three CpTx 1 isoforms (a–c) that differ by single residue substitutions. The toxin possesses potent insecticidal (paralytic and lethal), cytotoxic, and membrane-damaging activities. In both fly and frog neuromuscular preparations, it causes stable and irreversible depolarization of muscle fibers leading to contracture. This effect appears to be receptor-independent and is inhibited by high concentrations of divalent cations. CpTx 1 lyses cell membranes, as visualized by confocal microscopy, and destabilizes artificial membranes in a manner reminiscent of other membrane-active peptides by causing numerous defects of variable conductance and leading to bilayer rupture. The newly discovered class of modular polypeptides enhances our knowledge of the toxin universe. PMID:20657014

  18. Emerging high throughput analyses of cyanobacterial toxins and toxic cyanobacteria.

    PubMed

    Sivonen, Kaarina

    2008-01-01

    The common occurrence of toxic cyanobacteria causes problems for health of animals and human beings. More research and good monitoring systems are needed to protect water users. It is important to have rapid, reliable and accurate analysis i.e. high throughput methods to identify the toxins as well as toxin producers in the environment. Excellent methods, such as ELISA already exist to analyse cyanobacterial hepatotoxins and saxitoxins, and PPIA for microcystins and nodularins. The LC/MS method can be fast in identifying the toxicants in the samples. Further development of this area should resolve the problems with sampling and sample preparation, which still are the bottlenecks of rapid analyses. In addition, the availability of reliable reference materials and standards should be resolved. Molecular detection methods are now routine in clinical and criminal laboratories and may also become important in environmental diagnostics. One prerequisite for the development of molecular analysis is that pure cultures of the producer organisms are available for identification of the biosynthetic genes responsible for toxin production and for proper testing of the diagnostic methods. Good methods are already available for the microcystin and nodularin-producing cyanobacteria such as conventional PCR, quantitative real-time PCR and microarrays/DNA chips. The DNA-chip technology offers an attractive monitoring system for toxic and non-toxic cyanobacteria. Only with these new technologies (PCR + DNA-chips) will we be able to study toxic cyanobacteria populations in situ and the effects of environmental factors on the occurrence and proliferation of especially toxic cyanobacteria. This is likely to yield important information for mitigation purposes. Further development of these methods should include all cyanobacterial biodiversity, including all toxin producers and primers/probes to detect producers of neurotoxins, cylindrospermopsins etc. (genes are unknown). The on

  19. Botulinum toxin to minimize facial scarring.

    PubMed

    Sherris, David A; Gassner, Holger G

    2002-02-01

    Botulinum toxin injection has been used for a variety of indications in humans, including blepharospasm and hyperfunctional facial lines. This article describes a novel formulation of botulinum toxin, which supplies immediate feedback to the injecting physician. Additionally, recent findings are described that indicate the immediate injection of botulinum toxin into the muscles underlying a wound can improve the cosmetic outcome of the facial cutaneous scar. Future applications of these findings are discussed.

  20. Medical Defense Against Protein Toxin Weapons

    DTIC Science & Technology

    2004-10-01

    greatly expanded the accessible delivery vehicles for protein toxins to include, for example, natural or genetically modified bacteria and engineered...01 OCT 2004 2. REPORT TYPE N/A 3. DATES COVERED - 4. TITLE AND SUBTITLE Medical defense against protein toxin weapons: review and perspective...SUPPLEMENTARY NOTES 14. ABSTRACT The term " toxin weapon" has been used to describe poisons, classically of natural origin but increasingly

  1. Dinoflagellate Toxins Responsible for Ciguatera Food Poisoning

    DTIC Science & Technology

    1987-12-10

    AD____ AD-A 194 466 DNOFLACU.ATh TOXINS RESIONSIBLE FOR CIGUATERA FOOD POISONING Annual Summary Report 0 Donald M. Miller 10 December 1987 Supported...21701-5012 62770A 162770A87] AA 7 7 A11. TITLE (Include Security Classification) DINOFLAGELLATE TOXINS RESPONSIBLE FOR CIGUATERA FOOD POISONING .12...occurring in humans who have become intoxicated from eating poison fish. Fish spontaneously accumulate the toxin through the food chain or directly from

  2. Anti-Idiotype Probes for Toxin Detection

    DTIC Science & Technology

    1991-09-13

    used for polyclonal and mono- clonal antibody production . We have identified proteins on the cell surface of thymocytes that bind to exfoliative Toxin A...of toxins represents the novel aspect of this proposal. It will rely upon the production of anti-idiotypic antibodies to the receptor molecules. Such...specifically Ser-197 results in loss of biological activity suggests that the toxins may autodigest. This has yet to be proven. AnOibody Production At about the

  3. Bioluminescent bioreporter sensing of foodborne toxins

    NASA Astrophysics Data System (ADS)

    Fraley, Amanda C.; Ripp, Steven; Sayler, Gary S.

    2004-06-01

    Histamine is the primary etiological agent in the foodborne disease scombrotoxicosis, one of the most common food toxicities related to fish consumption. Procedures for detecting histamine in fish products are available, but are often too expensive or too complex for routine use. As an alternative, a bacterial bioluminescent bioreporter has been constructed to develop a biosensor system that autonomously responds to low levels of histamine. The bioreporter contains a promoterless Photorhabdus luminescens lux operon (luxCDABE) fused with the Vibrio anguillarum angR regulatory gene promoter of the anguibactin biosynthetic operon. The bioreporter emitted 1.46 times more bioluminescence than background, 30 minutes after the addition of 100mM histamine. However, specificity was not optimal, as this biosensor generated significant bioluminescence in the presence of L-proline and L-histidine. As a means towards improving histamine specificity, the promoter region of a histamine oxidase gene from Arthrobacter globiformis was cloned upstream of the promotorless lux operon from Photorhabdus luminescens. This recently constructed whole-cell, lux-based bioluminescent bioreporter is currently being tested for optimal performance in the presence of histamine in order to provide a rapid, simple, and inexpensive model sensor for the detection of foodborne toxins.

  4. Cellular and Systemic Effects of Anthrax Lethal Toxin and Edema Toxin

    PubMed Central

    Moayeri, Mahtab; Leppla, Stephen H.

    2009-01-01

    Anthrax lethal toxin (LT) and edema toxin (ET) are the major virulence factors of anthrax and can replicate the lethality and symptoms associated with the disease. This review provides an overview of our current understanding of anthrax toxin effects in animal models and the cytotoxicity (necrosis and apoptosis) induced by LT in different cells. A brief reexamination of early historic findings on toxin in vivo effects in the context of our current knowledge is also presented. PMID:19638283

  5. Enhanced detection and identification of Shiga toxin 1 and 2 from pathogenic bacteria by MALDI-TOF-TOF-MS/MS-PSD and top-down proteomic analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin producing Escherichia coli (STEC) represent a continuing threat to the Nation’s food supply and public health. Shiga toxin genes (stx) are encoded in lambda-like bacteriophages whose genome is inserted into the bacterial DNA. Environmental stress can trigger bacteriophage replication a...

  6. Novel Yersinia Pestis Toxin that Resembles Bacillus Anthracis Edema Factor: Study of Activity and Structural Modeling

    SciTech Connect

    Motin, V; Garcia, E; Barsky, D; Zemla, A

    2003-02-05

    The goal of this project was to begin both experimental and computational studies of the novel plague toxin to establish its biological properties and create its 3D-model. The project was divided into two parts. (1) Experimental--This part was devoted to determine distribution of the genes encoding novel plague toxin among different isolates of Y.pestis. If the EF-like activity is important for Y.pestis pathogenicity, it is anticipated that all highly virulent strains will contain the toxin genes. Also, they proposed to initiate research to investigate the functionality of the novel Y.pestis toxin that they hypothesize is likely to significantly contribute to the virulence of this dangerous microbe. this research design consisted of amplification, cloning and expression in E.coli the toxin genes followed by affinity purification of the recombinant protein that can be further used for testing of enzymatic activity. (2) Computational--The structural modeling of the putative EF of Y.pestis was based on multiple sequence alignments, secondary structure predictions, and comparison with 3D models of the EF of B. anthracis. The x-ray structure of the last has been recently published [Nature. 2002. 415(Jan):396-402]. The final model was selected after detailed analysis to determine if the structure is consistent with the biological function.

  7. Isolation of an Escherichia coli O157:H7 strain producing Shiga toxin 1 but not Shiga toxin 2 from a patient with hemolytic uremic syndrome in Korea.

    PubMed

    Kim, Y B; Okuda, J; Matsumoto, C; Morigaki, T; Asai, N; Watanabe, H; Nishibuchi, M

    1998-09-01

    Escherichia coli strains isolated from patients with diarrhea or hemolytic uremic syndrome (HUS) at Pusan University Hospital, South Korea, between 1990 and 1996 were examined for traits of the O157:H7 serogroup. One strain isolated from a patient with HUS belonged to the O157:H7 serotype, possessed a 60-MDa plasmid, the eae gene, and ability to produce Shiga toxin 1 but not Shiga toxin 2. Arbitrarily primed PCR analysis suggested that this strain is genetically very close to a O157:H7 strain isolated in Japan.

  8. Identification and sequence determination of recombinant Clostridium perfringens alpha-toxin by use of electrospray ionization mass spectrometry.

    PubMed

    Saito, Hitoshi; Inoue, Masaharu; Tomiki, Masayoshi; Nemoto, Hiroshi; Komoriya, Tomoe; Kimata, Junko; Watanabe, Kunitomo; Kohno, Hideki

    2009-01-01

    Only a few methods exist for simple, sensitive and rapid detection of alpha-toxin in clinical and biological samples. The aim of our study was to establish a procedure for the production of an antibody against a recombinant antigen with confirmed sequence identity. We applied a noble approach based on proteomics using a mass spectrometer for the conclusive identification of the recombinant alpha-toxin that was subsequently used as an antigen. The recombinant alpha-toxin was produced in Escherichia coli. A clinical isolate of Clostridium perfringens GAI 94074 was amplified by polymerase chain reaction (PCR) and subsequently, cloning was performed. Three different fragments were cloned using a pET100/D-TOPO vector. These fragments coded for a ribosome binding site, a signal peptide and the alpha-toxin gene, respectively. Recombinant pET100 plasmids were cloned into TOP 10 cells and the isolated plasmids were transferred into BL21 Star (DE3) cells. Their expression was then induced with isopropyl-beta-D-thiogalactopyranoside (IPTG). Recombinant E. coli transformed with a plasmid encoding the alpha-toxin gene alone produced a biologically inactive protein. On the other hand, E. coli carrying the plasmid encoding the toxin sequence and its native signal peptide sequence, or the toxin sequence along with the ribosome binding sequence and the signal peptide sequence secreted an active alpha-toxin with phospholipase activity. Accordingly, the C. perfringens gene encoding the alpha-toxin protein along with its signal peptide was successfully cloned, expressed, and secreted by E. coli. Furthermore, without consideration of its activity, we used mass spectrometry to confirm that the expressed protein was indeed the alpha-toxin. Thus, the identification of alpha-toxin protein using both the biological activity testing and the mass spectrometry analysis is expected to verify the significant production of C. perfringens antibody. The study for the analysis of recombinant alpha-toxin

  9. Synthesis and biology of cyclic imine toxins, an emerging class of potent, globally distributed marine toxins.

    PubMed

    Stivala, Craig E; Benoit, Evelyne; Aráoz, Rómulo; Servent, Denis; Novikov, Alexei; Molgó, Jordi; Zakarian, Armen

    2015-03-01

    From a small group of exotic compounds isolated only two decades ago, Cyclic Imine (CI) toxins have become a major class of marine toxins with global distribution. Their distinct chemical structure, biological mechanism of action, and intricate chemistry ensures that CI toxins will continue to be the subject of fascinating fundamental studies in the broad fields of chemistry, chemical biology, and toxicology. The worldwide occurrence of potent CI toxins in marine environments, their accumulation in shellfish, and chemical stability are important considerations in assessing risk factors for human health. This review article aims to provide an account of chemistry, biology, and toxicology of CI toxins from their discovery to the present day.

  10. Botulinum toxin: The Midas touch

    PubMed Central

    Shilpa, P. S.; Kaul, Rachna; Sultana, Nishat; Bhat, Suraksha

    2014-01-01

    Botulinum Toxin (BT) is a natural molecule produced during growth and autolysis of bacterium called Clostridium botulinum. Use of BT for cosmetic purposes has gained popularity over past two decades, and recently, other therapeutic uses of BT has been extensively studied. BT is considered as a minimally invasive agent that can be used in the treatment of various orofacial disorders and improving the quality of life in such patients. The objective of this article is to review the nature, mechanism of action of BT, and its application in various head and neck diseases. PMID:24678189

  11. Burn sepsis and burn toxin

    PubMed Central

    Allgöwer, Martin; Städtler, Karl; Schoenenberger, Guido A

    1974-01-01

    The salient steps of a 20-year programme of research into the nature of burn disease are described. By burn disease we mean the late mortality and morbidity following burns. We have isolated a burn toxin which is derived from a thermal polymerization of cell membrane lipoproteins within the dermis and have studied its influence on the effects of sepsis. We have also used it in the development of active and passive immunization therapy of severe burns. ImagesFig. 2Fig. 5Fig. 6Fig. 7Fig. 8Fig. 9 PMID:4429330

  12. Designing Inhibitors of Anthrax Toxin

    PubMed Central

    Nestorovich, Ekaterina M.; Bezrukov, Sergey M.

    2014-01-01

    Introduction Present-day rational drug design approaches are based on exploiting unique features of the target biomolecules, small- or macromolecule drug candidates, and physical forces that govern their interactions. The 2013 Nobel Prize in chemistry awarded “for the development of multiscale models for complex chemical systems” once again demonstrated the importance of the tailored drug discovery that reduces the role of the trial and error approach to a minimum. The “rational drug design” term is rather comprehensive as it includes all contemporary methods of drug discovery where serendipity and screening are substituted by the information-guided search for new and existing compounds. Successful implementation of these innovative drug discovery approaches is inevitably preceded by learning the physics, chemistry, and physiology of functioning of biological structures under normal and pathological conditions. Areas covered This article provides an overview of the recent rational drug design approaches to discover inhibitors of anthrax toxin. Some of the examples include small-molecule and peptide-based post-exposure therapeutic agents as well as several polyvalent compounds. The review also directs the reader to the vast literature on the recognized advances and future possibilities in the field. Expert opinion Existing options to combat anthrax toxin lethality are limited. With the only anthrax toxin inhibiting therapy (PA-targeting with a monoclonal antibody, raxibacumab) approved to treat inhalational anthrax, in our view, the situation is still insecure. The FDA’s animal rule for drug approval, which clears compounds without validated efficacy studies on humans, creates a high level of uncertainty, especially when a well-characterized animal model does not exist. Besides, unlike PA, which is known to be unstable, LF remains active in cells and in animal tissues for days. Therefore, the effectiveness of the post-exposure treatment of the individuals

  13. The adherens junctions control susceptibility to Staphylococcus aureus α-toxin.

    PubMed

    Popov, Lauren M; Marceau, Caleb D; Starkl, Philipp M; Lumb, Jennifer H; Shah, Jimit; Guerrera, Diego; Cooper, Rachel L; Merakou, Christina; Bouley, Donna M; Meng, Wenxiang; Kiyonari, Hiroshi; Takeichi, Masatoshi; Galli, Stephen J; Bagnoli, Fabio; Citi, Sandra; Carette, Jan E; Amieva, Manuel R

    2015-11-17

    Staphylococcus aureus is both a transient skin colonizer and a formidable human pathogen, ranking among the leading causes of skin and soft tissue infections as well as severe pneumonia. The secreted bacterial α-toxin is essential for S. aureus virulence in these epithelial diseases. To discover host cellular factors required for α-toxin cytotoxicity, we conducted a genetic screen using mutagenized haploid human cells. Our screen identified a cytoplasmic member of the adherens junctions, plekstrin-homology domain containing protein 7 (PLEKHA7), as the second most significantly enriched gene after the known α-toxin receptor, a disintegrin and metalloprotease 10 (ADAM10). Here we report a new, unexpected role for PLEKHA7 and several components of cellular adherens junctions in controlling susceptibility to S. aureus α-toxin. We find that despite being injured by α-toxin pore formation, PLEKHA7 knockout cells recover after intoxication. By infecting PLEKHA7(-/-) mice with methicillin-resistant S. aureus USA300 LAC strain, we demonstrate that this junctional protein controls disease severity in both skin infection and lethal S. aureus pneumonia. Our results suggest that adherens junctions actively control cellular responses to a potent pore-forming bacterial toxin and identify PLEKHA7 as a potential nonessential host target to reduce S. aureus virulence during epithelial infections.

  14. SNAKE VENOMICS OF Crotalus tigris: THE MINIMALIST TOXIN ARSENAL OF THE DEADLIEST NEARTIC RATTLESNAKE VENOM

    PubMed Central

    CALVETE, Juan J.; PÉREZ, Alicia; LOMONTE, Bruno; SÁNCHEZ, Elda E.; SANZ, Libia

    2012-01-01

    We report the proteomic and antivenomic characterization of Crotalus tigris venom. This venom exhibits the highest lethality for mice among rattlesnakes and the simplest toxin proteome reported to date. The venom proteome of C. tigris comprises 7–8 gene products from 6 toxin families: the presynaptic β-neurotoxic heterodimeric PLA2, Mojave toxin, and two serine proteinases comprise, respectively, 66% and 27% of the C. tigris toxin arsenal, whereas a VEGF-like protein, a CRISP molecule, a medium-sized disintegrin, and 1–2 PIII-SVMPs, each represents 0.1–5% of the total venom proteome. This toxin profile really explains the systemic neuro- and myotoxic effects observed in envenomated animals. In addition, we found that venom lethality of C. tigris and other North American rattlesnake type II venoms correlates with the concentration of Mojave toxin A-subunit, supporting the view that the neurotoxic venom phenotype of crotalid type II venoms may be described as a single-allele adaptation. Our data suggest that the evolutionary trend towards neurotoxicity, which has been also reported for the South American rattlesnakes, may have resulted by paedomorphism. The ability of an experimental antivenom to effectively immunodeplete proteins from the type II venoms of C. tigris, C. horridus, C. oreganus helleri, C. scutulatus scutulatus, and S. catenatus catenatus, indicated the feasibility of generating a pan-American anti-Crotalus type II antivenom, suggested by the identification of shared evolutionary trends among South American and North American Crotalus. PMID:22181673

  15. The adherens junctions control susceptibility to Staphylococcus aureus α-toxin

    PubMed Central

    Popov, Lauren M.; Marceau, Caleb D.; Starkl, Philipp M.; Lumb, Jennifer H.; Shah, Jimit; Guerrera, Diego; Cooper, Rachel L.; Merakou, Christina; Bouley, Donna M.; Meng, Wenxiang; Kiyonari, Hiroshi; Takeichi, Masatoshi; Galli, Stephen J.; Bagnoli, Fabio; Citi, Sandra; Carette, Jan E.; Amieva, Manuel R.

    2015-01-01

    Staphylococcus aureus is both a transient skin colonizer and a formidable human pathogen, ranking among the leading causes of skin and soft tissue infections as well as severe pneumonia. The secreted bacterial α-toxin is essential for S. aureus virulence in these epithelial diseases. To discover host cellular factors required for α-toxin cytotoxicity, we conducted a genetic screen using mutagenized haploid human cells. Our screen identified a cytoplasmic member of the adherens junctions, plekstrin-homology domain containing protein 7 (PLEKHA7), as the second most significantly enriched gene after the known α-toxin receptor, a disintegrin and metalloprotease 10 (ADAM10). Here we report a new, unexpected role for PLEKHA7 and several components of cellular adherens junctions in controlling susceptibility to S. aureus α-toxin. We find that despite being injured by α-toxin pore formation, PLEKHA7 knockout cells recover after intoxication. By infecting PLEKHA7−/− mice with methicillin-resistant S. aureus USA300 LAC strain, we demonstrate that this junctional protein controls disease severity in both skin infection and lethal S. aureus pneumonia. Our results suggest that adherens junctions actively control cellular responses to a potent pore-forming bacterial toxin and identify PLEKHA7 as a potential nonessential host target to reduce S. aureus virulence during epithelial infections. PMID:26489655

  16. Yeast killer toxins, molecular mechanisms of their action and their applications.

    PubMed

    Liu, Guang-Lei; Chi, Zhe; Wang, Guang-Yuan; Wang, Zhi-Peng; Li, Yang; Chi, Zhen-Ming

    2015-06-01

    Killer toxins secreted by some yeast strains are the proteins that kill sensitive cells of the same or related yeast genera. In recent years, many new yeast species have been found to be able to produce killer toxins against the pathogenic yeasts, especially Candida albicans. Some of the killer toxins have been purified and characterized, and the genes encoding the killer toxins have been cloned and characterized. Many new targets including different components of cell wall, plasma membrane, tRNA, DNA and others in the sensitive cells for the killer toxin action have been identified so that the new molecular mechanisms of action have been elucidated. However, it is still unknown how some of the newly discovered killer toxins kill the sensitive cells. Studies on the killer phenomenon in yeasts have provided valuable insights into a number of fundamental aspects of eukaryotic cell biology and interactions of different eukaryotic cells. Elucidation of the molecular mechanisms of their action will be helpful to develop the strategies to fight more and more harmful yeasts.

  17. Multifunctional-autoprocessing repeats-in-toxin (MARTX) Toxins of Vibrios

    PubMed Central

    Satchell, Karla J. F.

    2015-01-01

    Multifunctional-autoprocessing repeats-in-toxin (MARTX) toxins are a heterogeneous group of toxins found in a number of Vibrio species and other Gram-negative bacteria. The toxins are composed of conserved repeat regions and an autoprocessing protease domain that together function as a delivery platform for transfer of cytotoxic and cytopathic domains into target eukaryotic cell cytosol. Within the cells, the effectors can alter biological processes such as signaling or cytoskeletal structure, presumably to the benefit of the bacterium. Ten effector domains are found in the various Vibrio MARTX toxins, although any one toxin carries only two to five effector domains. The specific toxin variant expressed by a species can be modified by homologous recombination to acquire or lose effector domains, such that different strains within the same species can express distinct variants of the toxins. This review examines the conserved structural elements of the MARTX toxins and details the different toxin arrangements carried by Vibrio species and strains. The catalytic function of domains and how the toxins are linked to pathogenesis of human and animals is described. PMID:26185092

  18. Emergence of Escherichia coli encoding Shiga toxin 2f in human Shiga toxin-producing E. coli (STEC) infections in the Netherlands, January 2008 to December 2011.

    PubMed

    Friesema, I; van der Zwaluw, K; Schuurman, T; Kooistra-Smid, M; Franz, E; van Duynhoven, Y; van Pelt, W

    2014-05-01

    The Shiga toxins of Shiga toxin-producing Escherichia coli (STEC) can be divided into Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) with several sub-variants. Variant Stx2f is one of the latest described, but has been rarely associated with symptomatic human infections. In the enhanced STEC surveillance in the Netherlands, 198 STEC O157 cases and 351 STEC non-O157 cases, including 87 stx2f STEC isolates, were reported between 2008 and 2011. Most stx2f strains belonged to the serogroups O63:H6 (n=47, 54%), O113:H6 (n=12, 14%) and O125:H6 (n=12, 14%). Of the 87 stx2f isolates, 84 (97%) harboured the E. coli attaching and effacing (eae) gene, but not the enterohaemorrhagic E. coli haemolysin (hly) gene. stx2f STEC infections show milder symptoms and a less severe clinical course than STEC O157 infections. Almost all infections with stx2f (n=83, 95%) occurred between June and December, compared to 170/198 (86%) of STEC O157 and 173/264 (66%) of other STEC non-O157. stx2f STEC infections in the Netherlands are more common than anticipated, and form a distinct group within STEC with regard to virulence genes and the relatively mild disease.

  19. Toxin-antitoxin systems in the mobile genome of Acidithiobacillus ferrooxidans.

    PubMed

    Bustamante, Paula; Tello, Mario; Orellana, Omar

    2014-01-01

    Toxin-antitoxin (TA) systems are genetic modules composed of a pair of genes encoding a stable toxin and an unstable antitoxin that inhibits toxin activity. They are widespread among plasmids and chromosomes of bacteria and archaea. TA systems are known to be involved in the stabilization of plasmids but there is no consensus about the function of chromosomal TA systems. To shed light on the role of chromosomally encoded TA systems we analyzed the distribution and functionality of type II TA systems in the chromosome of two strains from Acidithiobacillus ferrooxidans (ATCC 23270 and 53993), a Gram-negative, acidophilic, environmental bacterium that participates in the bioleaching of minerals. As in other environmental microorganisms, A. ferrooxidans has a high content of TA systems (28-29) and in twenty of them the toxin is a putative ribonuclease. According to the genetic context, some of these systems are encoded near or within mobile genetic elements. Although most TA systems are shared by both strains, four of them, which are encoded in the active mobile element ICEAfe1, are exclusive to the type strain ATCC 23270. We demonstrated that two TA systems from ICEAfe1 are functional in E. coli cells, since the toxins inhibit growth and the antitoxins counteract the effect of their cognate toxins. All the toxins from ICEAfe1, including a novel toxin, are RNases with different ion requirements. The data indicate that some of the chromosomally encoded TA systems are actually part of the A. ferrooxidans mobile genome and we propose that could be involved in the maintenance of these integrated mobile genetic elements.

  20. Stool Test: C. Difficile Toxin (For Parents)

    MedlinePlus

    ... Your 1- to 2-Year-Old Stool Test: C. Difficile Toxin KidsHealth > For Parents > Stool Test: C. Difficile Toxin A A A What's in this ... Questions en español Muestra de materia fecal: toxina C. difficile What It Is A stool (feces) sample ...

  1. The Ins and Outs of Anthrax Toxin

    PubMed Central

    Friebe, Sarah; van der Goot, F. Gisou; Bürgi, Jérôme

    2016-01-01

    Anthrax is a severe, although rather rare, infectious disease that is caused by the Gram-positive, spore-forming bacterium Bacillus anthracis. The infectious form is the spore and the major virulence factors of the bacterium are its poly-γ-D-glutamic acid capsule and the tripartite anthrax toxin. The discovery of the anthrax toxin receptors in the early 2000s has allowed in-depth studies on the mechanisms of anthrax toxin cellular entry and translocation from the endocytic compartment to the cytoplasm. The toxin generally hijacks the endocytic pathway of CMG2 and TEM8, the two anthrax toxin receptors, in order to reach the endosomes. From there, the pore-forming subunit of the toxin inserts into endosomal membranes and enables translocation of the two catalytic subunits. Insertion of the pore-forming unit preferentially occurs in intraluminal vesicles rather than the limiting membrane of the endosome, leading to the translocation of the enzymatic subunits in the lumen of these vesicles. This has important consequences that will be discussed. Ultimately, the toxins reach the cytosol where they act on their respective targets. Target modification has severe consequences on cell behavior, in particular on cells of the immune system, allowing the spread of the bacterium, in severe cases leading to host death. Here we will review the literature on anthrax disease with a focus on the structure of the toxin, how it enters cells and its immunological effects. PMID:26978402

  2. [Axillary hyperhidrosis, botulinium A toxin treatment: Review].

    PubMed

    Clerico, C; Fernandez, J; Camuzard, O; Chignon-Sicard, B; Ihrai, T

    2016-02-01

    Injection of type A botulinum toxin in the armpits is a temporary treatment for axillary hyperhidrosis. This technique described in 1996 by Bushara et al., is known to be efficient and safe. The purpose of this article was to review the data concerning the treatment of axillary hyperhidrosis with botulinum toxin type A, and discuss the other treatment modalities for this socially disabling entity.

  3. Dermatitis from purified sea algae toxin (debromoaplysiatoxin).

    PubMed

    Solomon, A E; Stoughton, R B

    1978-09-01

    Cutaneous inflammation was induced by debromoaplysiatoxin, a purified toxin extracted from Lyngbya majuscula Gomont. This alga causes a seaweed dermatitis that occurs in persons who have swum off the coast of Oahu in Hawaii. By topical application, the toxin was found to produce an irritant pustular folliculitis in humans and to cause a severe cutaneous inflammatory reaction in the rabbit and in hairless mice.

  4. Botulinum Toxin and Gastrointestinal Tract Disorders

    PubMed Central

    Weiser, Kirsten; Kennedy, Abigail

    2008-01-01

    The history of botulinum toxin is fascinating. First recognized as the cause of botulism nearly 200 years ago, it was originally feared as a deadly poison. Over the last 30 years, however, botulinum toxin has been transformed into a readily available medication used to treat a variety of medical disorders. Interest in the use of botulinum toxin has been particularly strong for patients with spastic smooth muscle disorders of the gastrointestinal tract. Patients with achalasia, diffuse esophageal spasm, gastroparesis, sphincter of Oddi dysfunction, and anal fissures have all been treated with botulinum toxin injections, often with impressive results. However, not all patients respond to botulinum toxin therapy, and large randomized controlled trials are lacking for many conditions commonly treated with botulinum toxin. This paper reviews the history, microbiology, and pharmacology of botulinum toxin, discusses its mechanism of action, and then presents recent evidence from the literature regarding the use of botulinum toxin for the treatment of a variety of gastrointestinal tract disorders. PMID:21960915

  5. Stool Test: C. Difficile Toxin (For Parents)

    MedlinePlus

    ... Your 1- to 2-Year-Old Stool Test: C. Difficile Toxin KidsHealth > For Parents > Stool Test: C. Difficile Toxin Print A A A What's in ... Questions en español Muestra de materia fecal: toxina C. difficile What It Is A stool (feces) sample ...

  6. Plant Insecticidal Toxins in Ecological Networks

    PubMed Central

    Ibanez, Sébastien; Gallet, Christiane; Després, Laurence

    2012-01-01

    Plant secondary metabolites play a key role in plant-insect interactions, whether constitutive or induced, C- or N-based. Anti-herbivore defences against insects can act as repellents, deterrents, growth inhibitors or cause direct mortality. In turn, insects have evolved a variety of strategies to act against plant toxins, e.g., avoidance, excretion, sequestration and degradation of the toxin, eventually leading to a co-evolutionary arms race between insects and plants and to co-diversification. Anti-herbivore defences also negatively impact mutualistic partners, possibly leading to an ecological cost of toxin production. However, in other cases toxins can also be used by plants involved in mutualistic interactions to exclude inadequate partners and to modify the cost/benefit ratio of mutualism to their advantage. When considering the whole community, toxins have an effect at many trophic levels. Aposematic insects sequester toxins to defend themselves against predators. Depending on the ecological context, toxins can either increase insects’ vulnerability to parasitoids and entomopathogens or protect them, eventually leading to self-medication. We conclude that studying the community-level impacts of plant toxins can provide new insights into the synthesis between community and evolutionary ecology. PMID:22606374

  7. Formation and Control of Cyanobacterial Toxins

    EPA Science Inventory

    This presentation will cover the formation of harmful algal blooms and the control of their toxins. Data will be presented from current ORD projects on the treatment of cyanobacterial toxins through drinking water treatment facilities. The results will demonstrate that current c...

  8. Brown spider dermonecrotic toxin directly induces nephrotoxicity

    SciTech Connect

    Chaim, Olga Meiri; Sade, Youssef Bacila; Bertoni da Silveira, Rafael; Toma, Leny; Kalapothakis, Evanguedes; Chavez-Olortegui, Carlos; Mangili, Oldemir Carlos; Gremski, Waldemiro; Dietrich, Carl Peter von; Nader, Helena B.; Sanches Veiga, Silvio . E-mail: veigass@ufpr.br

    2006-02-15

    Brown spider (Loxosceles genus) venom can induce dermonecrotic lesions at the bite site and systemic manifestations including fever, vomiting, convulsions, disseminated intravascular coagulation, hemolytic anemia and acute renal failure. The venom is composed of a mixture of proteins with several molecules biochemically and biologically well characterized. The mechanism by which the venom induces renal damage is unknown. By using mice exposed to Loxosceles intermedia recombinant dermonecrotic toxin (LiRecDT), we showed direct induction of renal injuries. Microscopic analysis of renal biopsies from dermonecrotic toxin-treated mice showed histological alterations including glomerular edema and tubular necrosis. Hyalinization of tubules with deposition of proteinaceous material in the tubule lumen, tubule epithelial cell vacuoles, tubular edema and epithelial cell lysis was also observed. Leukocytic infiltration was neither observed in the glomerulus nor the tubules. Renal vessels showed no sign of inflammatory response. Additionally, biochemical analyses showed such toxin-induced changes in renal function as urine alkalinization, hematuria and azotemia with elevation of blood urea nitrogen levels. Immunofluorescence with dermonecrotic toxin antibodies and confocal microscopy analysis showed deposition and direct binding of this toxin to renal intrinsic structures. By immunoblotting with a hyperimmune dermonecrotic toxin antiserum on renal lysates from toxin-treated mice, we detected a positive signal at the region of 33-35 kDa, which strengthens the idea that renal failure is directly induced by dermonecrotic toxin. Immunofluorescence reaction with dermonecrotic toxin antibodies revealed deposition and binding of this toxin directly in MDCK epithelial cells in culture. Similarly, dermonecrotic toxin treatment caused morphological alterations of MDCK cells including cytoplasmic vacuoles, blebs, evoked impaired spreading and detached cells from each other and from

  9. Biological methods for marine toxin detection.

    PubMed

    Vilariño, Natalia; Louzao, M Carmen; Vieytes, Mercedes R; Botana, Luis M

    2010-07-01

    The presence of marine toxins in seafood poses a health risk to human consumers which has prompted the regulation of the maximum content of marine toxins in seafood in the legislations of many countries. Most marine toxin groups are detected by animal bioassays worldwide. Although this method has well known ethical and technical drawbacks, it is the official detection method for all regulated phycotoxins except domoic acid. Much effort by the scientific and regulatory communities has been focused on the development of alternative techniques that enable the substitution or reduction of bioassays; some of these have recently been included in the official detection method list. During the last two decades several biological methods including use of biosensors have been adapted for detection of marine toxins. The main advances in marine toxin detection using this kind of technique are reviewed. Biological methods offer interesting possibilities for reduction of the number of biosassays and a very promising future of new developments.

  10. Crystallization of isoelectrically homogeneous cholera toxin

    SciTech Connect

    Spangler, B.D.; Westbrook, E.M. )

    1989-02-07

    Past difficulty in growing good crystals of cholera toxin has prevented the study of the crystal structure of this important protein. The authors have determined that failure of cholera toxin to crystallize well has been due to its heterogeneity. They have now succeeded in overcoming the problem by isolating a single isoelectric variant of this oligomeric protein (one A subunit and five B subunits). Cholera toxin purified by their procedure readily forms large single crystals. The crystal form has been described previously. They have recorded data from native crystals of cholera toxin to 3.0-{angstrom} resolution with our electronic area detectors. With these data, they have found the orientation of a 5-fold symmetry axis within these crystals, perpendicular to the screw dyad of the crystal. They are now determining the crystal structure of cholera toxin by a combination of multiple heavy-atom isomorphous replacement and density modification techniques, making use of rotational 5-fold averaging of the B subunits.

  11. Evaluation of the SHIGA TOXIN QUIK CHEK and ImmunoCard STAT! EHEC as screening tools for the detection of Shiga toxin in fecal specimens.

    PubMed

    Staples, Megan; Fang, Ning-Xia; Graham, Rikki Ma; Smith, Helen V; Jennison, Amy V

    2017-02-01

    In this study we evaluated the performance of the SHIGA TOXIN QUIK CHEK (Techlab®, Blacksburg, VA) and the ImmunoCard STAT! Enterohaemorrhagic E. coli (EHEC) (Meridian BioScience, Cincinnati, OH, USA) assays as methods for qualitatively detecting the presence of Shiga toxin in human fecal specimens. A multiplex PCR for the detection of stx1 and stx2 was used as the standard for comparison. The SHIGA TOXIN QUIK CHEK detected all known Shiga toxin subtypes with the exception of Stx2f, while the ImmunoCard STAT! EHEC was unable to identify four of the seven Stx2 subtypes, including Stx2b and Stx2d. When compared to multiplex PCR based on Shiga toxin gene presence alone both assays demonstrated 100% specificity, and gave sensitivity values of 50.0% and 41.2% respectively. Correlation between each assay and the multiplex PCR was calculated by the use of kappa, with both assays exhibiting a moderate level of agreement.

  12. Pdl1 is a putative lipase that enhances Photorhabdus toxin complex secretion.

    PubMed

    Yang, Guowei; Hernández-Rodríguez, Carmen Sara; Beeton, Michael L; Wilkinson, Paul; Ffrench-Constant, Richard H; Waterfield, Nicholas R

    2012-01-01

    The Toxin Complex (TC) is a large multi-subunit toxin first characterized in the insect pathogens Photorhabdus and Xenorhabdus, but now seen in a range of pathogens, including those of humans. These complexes comprise three protein subunits, A, B and C which in the Xenorhabdus toxin are found in a 4:1:1 stoichiometry. Some TCs have been demonstrated to exhibit oral toxicity to insects and have the potential to be developed as a pest control technology. The lack of recognisable signal sequences in the three large component proteins hinders an understanding of their mode of secretion. Nevertheless, we have shown the Photorhabdus luminescens (Pl) Tcd complex has been shown to associate with the bacteria's surface, although some strains can also release it into the surrounding milieu. The large number of tc gene homologues in Pl make study of the export process difficult and as such we have developed and validated a heterologous Escherichia coli expression model to study the release of these important toxins. In addition to this model, we have used comparative genomics between a strain that releases high levels of Tcd into the supernatant and one that retains the toxin on its surface, to identify a protein responsible for enhancing secretion and release of these toxins. This protein is a putative lipase (Pdl1) which is regulated by a small tightly linked antagonist protein (Orf53). The identification of homologues of these in other bacteria, linked to other virulence factor operons, such as type VI secretion systems, suggests that these genes represent a general and widespread mechanism for enhancing toxin release in gram negative pathogens.

  13. Toxins

    MedlinePlus

    ... trace metals and others. In: Goldman L, Schafer AI, eds. Goldman-Cecil Medicine . 25th ed. Philadelphia, PA: ... Ford MD. Acute poisoning. In: Goldman L, Schafer AI, eds. Goldman-Cecil Medicine . 25th ed. Philadelphia, PA: ...

  14. Bidirectional effect of Wnt signaling antagonist DKK1 on the modulation of anthrax toxin uptake.

    PubMed

    Qian, LiLi; Cai, ChangZu; Yuan, PengFei; Jeong, Sun-Young; Yang, XiaoZhou; Dealmeida, Venita; Ernst, James; Costa, Michael; Cohen, Stanley N; Wei, WenSheng

    2014-05-01

    LRP6, a co-receptor for the morphogen Wnt, aids endocytosis of anthrax complexes. Here we report that Dickkopf1 (DKK1) protein, a secreted LRP6 ligand and antagonist, is also a modulator of anthrax toxin sensitivity. shRNA-mediated gene silencing or TALEN-mediated gene knockout of DKK1 reduced sensitivity of cells to PA-dependent hybrid toxins. However, unlike the solely inhibitory effect on Wnt signaling, the effects of DKK1 overexpression on anthrax toxicity were bidirectional, depending on its endogenous expression and cell context. Fluorescence microscopy and biochemical analyses showed that DKK1 facilitates internalization of anthrax toxins and their receptors, an event mediated by DKK1-LRP6-Kremen2 complex. Monoclonal antibodies against DKK1 provided dose-dependent protection to macrophages from killing by anthrax lethal toxin (LT). Our discovery that DKK1 forms ternary structure with LRP6 and Kremen2 in promoting PA-mediated toxin internalization provides a paradigm for bacterial exploitation of mechanisms that host cells use to internalize signaling proteins.

  15. Recombinant production of bacterial toxins and their derivatives in the methylotrophic yeast Pichia pastoris

    PubMed Central

    Gurkan, Cemal; Ellar, David J

    2005-01-01

    The methylotrophic yeast Pichia pastoris is a popular heterologous expression host for the recombinant production of a variety of prokaryotic and eukaryotic proteins. The rapid emergence of P. pastoris as a robust heterologous expression host was facilitated by the ease with which it can be manipulated and propagated, which is comparable to that of Escherichia coli and Saccharomyces cerevisiae. P. pastoris offers further advantages such as the tightly-regulated alcohol oxidase promoter that is particularly suitable for heterologous expression of foreign genes. While recombinant production of bacterial toxins and their derivatives is highly desirable, attempts at their heterologous expression using the traditional E. coli expression system can be problematic due to the formation of inclusion bodies that often severely limit the final yields of biologically active products. However, recent literature now suggests that P. pastoris may be an attractive alternative host for the heterologous production of bacterial toxins, such as those from the genera Bacillus, Clostridium, and Corynebacterium, as well as their more complex derivatives. Here, we review the recombinant production of bacterial toxins and their derivatives in P. pastoris with special emphasis on their potential clinical applications. Considering that de novo design and construction of synthetic toxin genes have often been necessary to achieve optimal heterologous expression in P. pastoris, we also present general guidelines to this end based on our experience with the P. pastoris expression of the Bacillus thuringiensis Cyt2Aa1 toxin. PMID:16336647

  16. Nutrition affects insect susceptibility to Bt toxins

    NASA Astrophysics Data System (ADS)

    Deans, Carrie A.; Behmer, Spencer T.; Tessnow, Ashley E.; Tamez-Guerra, Patricia; Pusztai-Carey, Marianne; Sword, Gregory A.

    2017-01-01

    Pesticide resistance represents a major challenge to global food production. The spread of resistance alleles is the primary explanation for observations of reduced pesticide efficacy over time, but the potential for gene-by-environment interactions (plasticity) to mediate susceptibility has largely been overlooked. Here we show that nutrition is an environmental factor that affects susceptibility to Bt toxins. Protein and carbohydrates are two key macronutrients for insect herbivores, and the polyphagous pest Helicoverpa zea self-selects and performs best on diets that are protein-biased relative to carbohydrates. Despite this, most Bt bioassays employ carbohydrate-biased rearing diets. This study explored the effect of diet protein-carbohydrate content on H. zea susceptibility to Cry1Ac, a common Bt endotoxin. We detected a 100-fold increase in LC50 for larvae on optimal versus carbohydrate-biased diets, and significant diet-mediated variation in survival and performance when challenged with Cry1Ac. Our results suggest that Bt resistance bioassays that use ecologically- and physiologically-mismatched diets over-estimate susceptibility and under-estimate resistance.

  17. Nutrition affects insect susceptibility to Bt toxins

    PubMed Central

    Deans, Carrie A.; Behmer, Spencer T.; Tessnow, Ashley E.; Tamez-Guerra, Patricia; Pusztai-Carey, Marianne; Sword, Gregory A.

    2017-01-01

    Pesticide resistance represents a major challenge to global food production. The spread of resistance alleles is the primary explanation for observations of reduced pesticide efficacy over time, but the potential for gene-by-environment interactions (plasticity) to mediate susceptibility has largely been overlooked. Here we show that nutrition is an environmental factor that affects susceptibility to Bt toxins. Protein and carbohydrates are two key macronutrients for insect herbivores, and the polyphagous pest Helicoverpa zea self-selects and performs best on diets that are protein-biased relative to carbohydrates. Despite this, most Bt bioassays employ carbohydrate-biased rearing diets. This study explored the effect of diet protein-carbohydrate content on H. zea susceptibility to Cry1Ac, a common Bt endotoxin. We detected a 100-fold increase in LC50 for larvae on optimal versus carbohydrate-biased diets, and significant diet-mediated variation in survival and performance when challenged with Cry1Ac. Our results suggest that Bt resistance bioassays that use ecologically- and physiologically-mismatched diets over-estimate susceptibility and under-estimate resistance. PMID:28045087

  18. The occurrence of Photorhabdus-like toxin complexes in Bacillus thuringiensis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recently, genomic sequencing of a Bacillus thuringiensis (Bt) isolate from our collection revealed the presence of an apparent operon encoding an insecticidal toxin complex (Tca) similar to that first described from the entomopathogen Photorhabdus luminescens. To determine whether these genes are w...

  19. Diversity and distribution of cholix toxin, a novel ADP-ribosylating factor from Vibrio cholerae.

    PubMed

    Purdy, Alexandra E; Balch, Deborah; Lizárraga-Partida, Marcial Leonardo; Islam, Mohammad Sirajul; Martinez-Urtaza, Jaime; Huq, Anwar; Colwell, Rita R; Bartlett, Douglas H

    2010-02-01

    Non-toxigenic non-O1, non-O139 Vibrio cholerae strains isolated from both environmental and clinical settings carry a suite of virulence factors aside from cholera toxin. Among V. cholerae strains isolated from coastal waters of southern California, this includes cholix toxin, an ADP-ribosylating factor that is capable of halting protein synthesis in eukaryotic cells. The prevalence of the gene encoding cholix toxin, chxA, was assessed among a collection of 155 diverse V. cholerae strains originating from both clinical and environmental settings in Bangladesh and Mexico and other countries around the globe. The chxA gene was present in 47% of 83 non-O1, non-O139 strains and 16% of 72 O1/O139 strains screened as part of this study. A total of 86 chxA gene sequences were obtained, and phylogenetic analysis revealed that they fall into two distinct clades. These two clades were also observed in the phylogenies of several housekeeping genes, suggesting that the divergence observed in chxA extends to other regions of the V. cholerae genome, and most likely has arisen from vertical descent rather than horizontal transfer. Our results clearly indicate that ChxA is a major toxin of V. cholerae with a worldwide distribution that is preferentially associated with non-pandemic strains.

  20. Genomic sequencing identifies novel Bacillus thuringiensis Vip1/Vip2 binary and Cry8 toxins that have high toxicity to Scarabaeoidea larvae.

    PubMed

    Bi, Yang; Zhang, Yanrui; Shu, Changlong; Crickmore, Neil; Wang, Qinglei; Du, Lixin; Song, Fuping; Zhang, Jie

    2015-01-01

    The Bacillus thuringiensis strain HBF-18 (CGMCC 2070), which has previously been shown to encode the cry8Ga toxin gene, is active against both Holotrichia oblita and Holotrichia parallela. Recombinant Cry8Ga however is only weakly toxic to these insect pests suggesting the involvement of additional toxins in the native strain. We report that through the use of Illumina sequencing three additional, and novel, genes, namely vip1Ad1, vip2Ag1, and cry8-like, were identified in this strain. Although no protein corresponding to these genes could be identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the HBF-18 proteome, reverse transcription (RT)-PCR indicated that all three genes were transcribed in the native strain. The two vip genes were cloned and expressed and, as with other Vip1/2 toxins, appeared to function as a binary toxin and showed strong activity against H. oblita, H. parallela and Anomala corpulenta. This is the first report to demonstrate that the Vip1/Vip2 binary toxin is active against these Scarabaeoidea larvae. The cry8-like gene appeared to be a C-terminally truncated form of a typical cry8 gene and was not expressed in our usual recombinant Bt expression system. When however the missing C-terminal region was replaced with the corresponding sequence from cry8Ea, the resulting hybrid expressed well and the toxin was active against the three test insects.

  1. Roles of Anthrax Toxin Receptor 2 in Anthrax Toxin Membrane Insertion and Pore Formation.

    PubMed

    Sun, Jianjun; Jacquez, Pedro

    2016-01-22

    Interaction between bacterial toxins and cellular surface receptors is an important component of the host-pathogen interaction. Anthrax toxin protective antigen (PA) binds to the cell surface receptor, enters the cell through receptor-mediated endocytosis, and forms a pore on the endosomal membrane that translocates toxin enzymes into the cytosol of the host cell. As the major receptor for anthrax toxin in vivo, anthrax toxin receptor 2 (ANTXR2) plays an essential role in anthrax toxin action by providing the toxin with a high-affinity binding anchor on the cell membrane and a path of entry into the host cell. ANTXR2 also acts as a molecular clamp by shifting the pH threshold of PA pore formation to a more acidic pH range, which prevents premature pore formation at neutral pH before the toxin reaches the designated intracellular location. Most recent studies have suggested that the disulfide bond in the immunoglobulin (Ig)-like domain of ANTXR2 plays an essential role in anthrax toxin action. Here we will review the roles of ANTXR2 in anthrax toxin action, with an emphasis on newly updated knowledge.

  2. Polymorphic toxin systems: Comprehensive characterization of trafficking modes, processing, mechanisms of action, immunity and ecology using comparative genomics

    PubMed Central

    2012-01-01

    Background Proteinaceous toxins are observed across all levels of inter-organismal and intra-genomic conflicts. These include recently discovered prokaryotic polymorphic toxin systems implicated in intra-specific conflicts. They are characterized by a remarkable diversity of C-terminal toxin domains generated by recombination with standalone toxin-coding cassettes. Prior analysis revealed a striking diversity of nuclease and deaminase domains among the toxin modules. We systematically investigated polymorphic toxin systems using comparative genomics, sequence and structure analysis. Results Polymorphic toxin systems are distributed across all major bacterial lineages and are delivered by at least eight distinct secretory systems. In addition to type-II, these include type-V, VI, VII (ESX), and the poorly characterized “Photorhabdus virulence cassettes (PVC)”, PrsW-dependent and MuF phage-capsid-like systems. We present evidence that trafficking of these toxins is often accompanied by autoproteolytic processing catalyzed by HINT, ZU5, PrsW, caspase-like, papain-like, and a novel metallopeptidase associated with the PVC system. We identified over 150 distinct toxin domains in these systems. These span an extraordinary catalytic spectrum to include 23 distinct clades of peptidases, numerous previously unrecognized versions of nucleases and deaminases, ADP-ribosyltransferases, ADP ribosyl cyclases, RelA/SpoT-like nucleotidyltransferases, glycosyltranferases and other enzymes predicted to modify lipids and carbohydrates, and a pore-forming toxin domain. Several of these toxin domains are shared with host-directed effectors of pathogenic bacteria. Over 90 families of immunity proteins might neutralize anywhere between a single to at least 27 distinct types of toxin domains. In some organisms multiple tandem immunity genes or immunity protein domains are organized into polyimmunity loci or polyimmunity proteins. Gene-neighborhood-analysis of polymorphic toxin systems

  3. Fungi and fungal toxins as weapons.

    PubMed

    Paterson, R Russell M

    2006-09-01

    Recent aggressive attacks on innocent citizens have resulted in governments increasing security. However, there is a good case for prevention rather than reaction. Bioweapons, mycotoxins, fungal biocontrol agents (FBCA), and even pharmaceuticals contain, or are, toxins and need to be considered in the context of the new paradigm. Is it desirable to discuss such issues? None of the fungi are (a) as toxic as botulinum toxin from Clostridium botulinum, and (b) as dangerous as nuclear weapons. One toxin may be defined as a pharmaceutical and vice versa simply by a small change in concentration or a moiety. Mycotoxins are defined as naturally occurring toxic compounds obtained from fungi. They are the biggest chronic health risk when incorporated into the diet. The current list of fungal toxins as biochemical weapons is small, although awareness is growing of the threats they may pose. T-2 toxin is perhaps the biggest concern. A clear distinction is required between the biological (fungus) and chemical (toxin) aspects of the issue. There is an obvious requirement to be able to trace these fungi and compounds in the environment and to know when concentrations are abnormal. Many FBCA, produce toxins. This paper indicates how to treat mycotoxicosis and decontaminate mycotoxins. There is considerable confusion and inconsistency surrounding this topic which requires assessment in an impartial and scientific manner.

  4. Cyanobacterial toxins: risk management for health protection

    SciTech Connect

    Codd, Geoffrey A.; Morrison, Louise F.; Metcalf, James S

    2005-03-15

    This paper reviews the occurrence and properties of cyanobacterial toxins, with reference to the recognition and management of the human health risks which they may present. Mass populations of toxin-producing cyanobacteria in natural and controlled waterbodies include blooms and scums of planktonic species, and mats and biofilms of benthic species. Toxic cyanobacterial populations have been reported in freshwaters in over 45 countries, and in numerous brackish, coastal, and marine environments. The principal toxigenic genera are listed. Known sources of the families of cyanobacterial toxins (hepato-, neuro-, and cytotoxins, irritants, and gastrointestinal toxins) are briefly discussed. Key procedures in the risk management of cyanobacterial toxins and cells are reviewed, including derivations (where sufficient data are available) of tolerable daily intakes (TDIs) and guideline values (GVs) with reference to the toxins in drinking water, and guideline levels for toxigenic cyanobacteria in bathing waters. Uncertainties and some gaps in knowledge are also discussed, including the importance of exposure media (animal and plant foods), in addition to potable and recreational waters. Finally, we present an outline of steps to develop and implement risk management strategies for cyanobacterial cells and toxins in waterbodies, with recent applications and the integration of Hazard Assessment Critical Control Point (HACCP) principles.

  5. Clostridium difficile: its disease and toxins.

    PubMed Central

    Lyerly, D M; Krivan, H C; Wilkins, T D

    1988-01-01

    Clostridium difficile is the etiologic agent of pseudomembranous colitis, a severe, sometimes fatal disease that occurs in adults undergoing antimicrobial therapy. The disease, ironically, has been most effectively treated with antibiotics, although some of the newer methods of treatment such as the replacement of the bowel flora may prove more beneficial for patients who continue to relapse with pseudomembranous colitis. The organism produces two potent exotoxins designated toxin A and toxin B. Toxin A is an enterotoxin believed to be responsible for the diarrhea and mucosal tissue damage which occur during the disease. Toxin B is an extremely potent cytotoxin, but its role in the disease has not been as well studied. There appears to be a cascade of events which result in the expression of the activity of these toxins, and these events, ranging from the recognition of a trisaccharide receptor by toxin A to the synergistic action of the toxins and their possible dissemination in the body, are discussed in this review. The advantages and disadvantages of the various assays, including tissue culture assay, enzyme immunoassay, and latex agglutination, currently used in the clinical diagnosis of the disease also are discussed. PMID:3144429

  6. Single toxin dose-response models revisited

    PubMed Central

    Glaholt, SP; Kyker-Snowman, E; Shaw, JR; Chen, CY

    2016-01-01

    The goal of this paper is to offer a rigorous analysis of the sigmoid shape single toxin dose-response relationship. The toxin efficacy function is introduced and four special points, including maximum toxin efficacy and inflection points, on the dose-response curve are defined. The special points define three phases of the toxin effect on mortality: (1) toxin concentrations smaller than the first inflection point or (2) larger then the second inflection point imply low mortality rate, and (3) concentrations between the first and the second inflection points imply high mortality rate. Probabilistic interpretation and mathematical analysis for each of four models, Hill, logit, probit, and Weibull is provided. Two general model extensions are introduced: (1) the multi-target hit model that accounts for the existence of several vital receptors affected by the toxin, and (2) model with a nonzero mortalit