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Sample records for cerebratulus lacteus cytolysin

  1. Thr-E11 regulates O2 affinity in Cerebratulus lacteus mini-hemoglobin.

    PubMed

    Pesce, Alessandra; Nardini, Marco; Ascenzi, Paolo; Geuens, Eva; Dewilde, Sylvia; Moens, Luc; Bolognesi, Martino; Riggs, Austen F; Hale, Angela; Deng, Pengchi; Nienhaus, G Ulrich; Olson, John S; Nienhaus, Karin

    2004-08-01

    The mini-hemoglobin from Cerebratulus lacteus (CerHb) belongs to a class of globins containing the polar Tyr-B10/Gln-E7 amino acid pair that normally causes low rates of O2 dissociation and ultra-high O2 affinity, which suggest O2 sensing or NO scavenging functions. CerHb, however, has high rates of O2 dissociation (kO2 = 200-600 s(-1)) and moderate O2 affinity (KO2) approximately 1 microm(-1)) as a result of a third polar amino acid in its active site, Thr-E11. When Thr-E11 is replaced by Val, kO2 decreases 1000-fold and KO2 increases 130-fold at pH 7.0, 20 degrees C. The mutation also shifts the stretching frequencies of both heme-bound and photodissociated CO, indicating marked changes of the electrostatic field at the active site. The crystal structure of Thr-E11 --> Val CerHbO2 at 1.70 A resolution is almost identical to that of the wild-type protein (root mean square deviation of 0.12 A). The dramatic functional and spectral effects of the Thr-E11 --> Val mutation are due exclusively to changes in the hydrogen bonding network in the active site. Replacing Thr-E11 with Val "frees" the Tyr-B10 hydroxyl group to rotate toward and donate a strong hydrogen bond to the heme-bound ligand, causing a selective increase in O2 affinity, a decrease of the rate coefficient for O2 dissociation, a 40 cm(-1) decrease in nuCO of heme-bound CO, and an increase in ligand migration toward more remote intermediate sites. PMID:15161908

  2. Ligand Migration in the Apolar Tunnel of Cerebratulus lacteus Mini-Hemoglobin*

    PubMed Central

    Pesce, Alessandra; Nardini, Marco; Dewilde, Sylvia; Capece, Luciana; Martí, Marcelo A.; Congia, Sonia; Salter, Mallory D.; Blouin, George C.; Estrin, Darío A.; Ascenzi, Paolo; Moens, Luc; Bolognesi, Martino; Olson, John S.

    2011-01-01

    The large apolar tunnel traversing the mini-hemoglobin from Cerebratulus lacteus (CerHb) has been examined by x-ray crystallography, ligand binding kinetics, and molecular dynamic simulations. The addition of 10 atm of xenon causes loss of diffraction in wild-type (wt) CerHbO2 crystals, but Leu-86(G12)Ala CerHbO2, which has an increased tunnel volume, stably accommodates two discrete xenon atoms: one adjacent to Leu-86(G12) and another near Ala-55(E18). Molecular dynamics simulations of ligand migration in wt CerHb show a low energy pathway through the apolar tunnel when Leu or Ala, but not Phe or Trp, is present at the 86(G12) position. The addition of 10–15 atm of xenon to solutions of wt CerHbCO and L86A CerHbCO causes 2–3-fold increases in the fraction of geminate ligand recombination, indicating that the bound xenon blocks CO escape. This idea was confirmed by L86F and L86W mutations, which cause even larger increases in the fraction of geminate CO rebinding, 2–5-fold decreases in the bimolecular rate constants for ligand entry, and large increases in the computed energy barriers for ligand movement through the apolar tunnel. Both the addition of xenon to the L86A mutant and oxidation of wt CerHb heme iron cause the appearance of an out Gln-44(E7) conformer, in which the amide side chain points out toward the solvent and appears to lower the barrier for ligand escape through the E7 gate. However, the observed kinetics suggest little entry and escape (≤25%) through the E7 pathway, presumably because the in Gln-44(E7) conformer is thermodynamically favored. PMID:21147768

  3. DNA barcoding should accompany taxonomy - the case of Cerebratulus spp (Nemertea).

    PubMed

    Sundberg, P; Thuroczy Vodoti, E; Strand, M

    2010-03-01

    Many issues in DNA barcoding need to be solved before it can reach its goal to become a general database for species identification. While species delimitations are more or less well established in several taxa, there are still many groups where this is not the case. Without the proper taxonomic background/knowledge and corroboration with other kinds of data, the DNA barcoding approach may fail to identify species accurately. The classification and taxonomy of phylum Nemertea (nemerteans, ribbon worms) are traditionally based on morphology, but are not corroborated by an increasing amount of genetic data when it comes to classification either into species or into higher taxa. The taxonomy of the phylum needs to be improved before the full potential of DNA barcoding can be utilized to make sure that valid Linnean names accompany the barcode sequences. We illustrate the problematic situation in the phylum Nemertea by a case study from the genus Cerebratulus. PMID:21565022

  4. The Enterococcus faecalis cytolysin determinant and its relationship to those encoding lantibiotics.

    PubMed

    Bogie, C P; Hancock, L E; Gilmore, M S

    1995-01-01

    The E. faecalis cytolysin represents a new class of cytolytic agents which are related to a family of antibacterial peptides termed lantibiotics. Despite considerable similarity at the genetic level, the E. faecalis cytolysin differs from the lantibiotics in several respects. First, the E. faecalis cytolysin consists of two dissimilar precursors, both of which are required to effect target cell lysis. A second important difference is that the E. faecalis cytolysin is active against eukaryotic as well as Gram-positive prokaryotic cells. Originally identified as a haemolysin [32], the E. faecalis cytolysin has been shown to make a contribution to bacterial virulence in endocarditis [8, 33] and endophthalmitis [7] models, and the cytolytic phenotype is enriched among clinical isolates of the organism [2, 3]. Similarities between the E. faecalis cytolysin and lantibiotics such as nisin (which is used as a food preservative in several countries [34] and is the subject of continuing attempts at rational design of lantibiotic-based food preservatives [25]), and the observation of an association between the E. faecalis cytolysin and bacterial virulence indicate that it may be possible to engineer lantibiotics to a point where undesired toxic or cytolytic activities will arise. Further comparison of the chemical, structural and biological properties of the E. faecalis cytolysin and classical lantibiotics will define these limits. PMID:8586241

  5. Isotopic fingerprints of bacterial chemosymbiosis in the bivalve Loripes lacteus

    NASA Astrophysics Data System (ADS)

    Dreier, A.; Stannek, L.; Blumenberg, M.; Taviani, M.; Sigovini, M.; Wrede, C.; Thiel, V.; Hoppert, M.

    2012-04-01

    Metazoans with chemosynthetic bacterial endosymbionts are widespread in marine habitats and respective endosymbioses are known from seven recent animal phyla. However, little is known about endosymbioses in fossil settings and, hence, ecological significance in earth history. In the presented project, we investigate the ancient and recent bivalve fauna living at marine sedimentary oxic/anoxic interfaces. Two bivalve species collected from the same benthic environment - a Mediterranean lagoon - were studied in detail. The diet of Loripes lacteus is based on thiotrophic gill symbionts whereas Venerupis aureus is a filter feeding bivalve without symbionts. The presence of three key enzymes from sulfur oxidation (APS-reductase), carbon fixation (RubisCO) and assimilation of nitrogen (glutamine synthetase [GS]) were detected by immunofluorescence in symbionts of Loripes and/or by activity tests in living specimens. In search of biosignatures associated with thiotrophic chemosymbionts that might be suitable for detection of chemosymbiotic diets in recent and fossil bivalve shells, we analyzed the isotopic composition of shell lipids (δ13C) and the bulk organic matrix of the shell (δ13C, δ15N, δ34S). We could show that the combined δ15N and δ13C values from shell extracts are stable in subfossil (Pleistocene) bivalve specimens, as long as the isotopic data is "calibrated" with respective signatures from a filter feeding bivalve sampled from the same site or lithostratigraphic bed.

  6. The enterococcal cytolysin synthetase has an unanticipated lipid kinase fold

    PubMed Central

    Dong, Shi-Hui; Tang, Weixin; Lukk, Tiit; Yu, Yi; Nair, Satish K; van der Donk, Wilfred A

    2015-01-01

    The enterococcal cytolysin is a virulence factor consisting of two post-translationally modified peptides that synergistically kill human immune cells. Both peptides are made by CylM, a member of the LanM lanthipeptide synthetases. CylM catalyzes seven dehydrations of Ser and Thr residues and three cyclization reactions during the biosynthesis of the cytolysin large subunit. We present here the 2.2 Å resolution structure of CylM, the first structural information on a LanM. Unexpectedly, the structure reveals that the dehydratase domain of CylM resembles the catalytic core of eukaryotic lipid kinases, despite the absence of clear sequence homology. The kinase and phosphate elimination active sites that affect net dehydration are immediately adjacent to each other. Characterization of mutants provided insights into the mechanism of the dehydration process. The structure is also of interest because of the interactions of human homologs of lanthipeptide cyclases with kinases such as mammalian target of rapamycin. DOI: http://dx.doi.org/10.7554/eLife.07607.001 PMID:26226635

  7. Structural analysis and proteolytic activation of Enterococcus faecalis cytolysin, a novel lantibiotic.

    PubMed

    Booth, M C; Bogie, C P; Sahl, H G; Siezen, R J; Hatter, K L; Gilmore, M S

    1996-09-01

    Clinical isolates of Enterococcus faecalis more commonly produce a cytolysin than do commensal isolates. Epidemiologic evidence and animal-model studies have established a role for the cytolysin in the pathogenesis of enterococcal disease. The cytolysin consists of two structural subunits, CylLL and CylLS, that are activated by a third component, CylA. Genetic and biochemical characterization of CylA indicate that it is a serine protease, and that activation putatively results from cleavage of one or both cytolysin subunits. Genetic evidence also suggests that the cytolysin subunits are related to the rapidly growing class of bacteriocins termed lantibiotics. However, unlike lantibiotics, the cytolysin is lytic for eukaryotic as well as prokaryotic cells, and it consists of two structural subunits. This report describes the purification and characterization of the cytolysin subunits and detection of lanthionine-type post-translational modifications within their structures. Furthermore, the cleavage specificity of the CylA activator is reported and it is shown that proteolytic activation of both subunits is essential for activity. PMID:8898386

  8. Enterococcus faecalis cytolysin without effect on the intestinal growth of susceptible enterococci in mice.

    PubMed

    Huycke, M M; Joyce, W A; Gilmore, M S

    1995-07-01

    A murine model was developed to determine whether the Enterococcus faecalis cytolysin, through its bacteriolytic action on gram-positive bacteria, could promote intestinal overgrowth of cytolytic strains. Sets of E. faecalis strains with varying cytolytic production and susceptibility to cytolytic activity were mixed 1:1 and allowed to compete in vitro in broth or in vivo after orogastric administration in mice pretreated with antibiotics. In general, cytolytic strains outgrew, by as much as 2000-fold, competing cytolysin-susceptible or -hypersusceptible strains in vitro. In contrast, no growth advantage was observed in vivo, despite similar transient colonization of the murine intestinal tract by both cytolytic and cytolysin-susceptible strains. These data suggest that cytolysin plays little role in promoting intestinal overgrowth of enterococci through bacteriolytic activity. PMID:7797930

  9. The anti-membranous glomerulonephritic activity of purified polysaccharides from Irpex lacteus Fr.

    PubMed

    Wang, Juan; Song, Jingjing; Wang, Di; Zhang, Na; Lu, Jiahui; Meng, Qingfan; Zhou, Yulin; Wang, Ning; Liu, Yang; Wang, Di; Teng, Lesheng

    2016-03-01

    The present study aims to identify the major active component from mutant Irpex lacteus, which protects against cationic bovine serum albumin (C-BSA)-induced membranous glomerulonephropathy (MGN). The candidate component ILN3A (MW: 2264 kDa) was purified from mutant Irpex lacteus water extract. The backbone of ILN3A comprises (1→2) and (1→4) linkages, and (1)H NMR spectrum suggests the existence of α- and β-glycosidic anomeric carbon. In tissue culture study, ILN3A inhibits mesangial cell proliferation. In MGN rats, ILN3A reverses structural changes in kidney, suppresses abnormal high level of urine protein and restores concentration of serum albumin. ILN3A also reduces total cholesterol, triglycerides, and creatinine in serum, and 6-keto-PGF in kidney cortex. Further study shows ILN3A restores serum Interleukin 2, Interleukin 2 receptor, Interleukin 6, tumor necrosis factor α, and renal cortical nuclear factor kappa B. Our data shows ILN3A, the major active component of mutant Irpex lacteus, is a novel candidate anti-inflammatory medicine to treat MGN in clinics. PMID:26656595

  10. Differential proteomic analysis of the secretome of Irpex lacteus and other white-rot fungi during wheat straw pretreatment

    PubMed Central

    2013-01-01

    Background Identifying new high-performance enzymes or enzyme complexes to enhance biomass degradation is the key for the development of cost-effective processes for ethanol production. Irpex lacteus is an efficient microorganism for wheat straw pretreatment, yielding easily hydrolysable products with high sugar content. Thus, this fungus was selected to investigate the enzymatic system involved in lignocellulose decay, and its secretome was compared to those from Phanerochaete chrysosporium and Pleurotus ostreatus which produced different degradation patterns when growing on wheat straw. Extracellular enzymes were analyzed through 2D-PAGE, nanoLC/MS-MS, and homology searches against public databases. Results In wheat straw, I. lacteus secreted proteases, dye-decolorizing and manganese-oxidizing peroxidases, and H2O2 producing-enzymes but also a battery of cellulases and xylanases, excluding those implicated in cellulose and hemicellulose degradation to their monosaccharides, making these sugars poorly available for fungal consumption. In contrast, a significant increase of β-glucosidase production was observed when I. lacteus grew in liquid cultures. P. chrysosporium secreted more enzymes implicated in the total hydrolysis of the polysaccharides and P. ostreatus produced, in proportion, more oxidoreductases. Conclusion The protein pattern secreted during I. lacteus growth in wheat straw plus the differences observed among the different secretomes, justify the fitness of I. lacteus for biopretreatment processes in 2G-ethanol production. Furthermore, all these data give insight into the biological degradation of lignocellulose and suggest new enzyme mixtures interesting for its efficient hydrolysis. PMID:23937687

  11. Biodecolorization and biodegradation of reactive Levafix Blue E-RA granulate dye by the white rot fungus Irpex lacteus.

    PubMed

    Kalpana, Duraisamy; Velmurugan, Natarajan; Shim, Jae Hong; Oh, Byung-Taek; Senthil, Kalaiselvi; Lee, Yang Soo

    2012-11-30

    The treatment of effluents from textile industry with microorganisms, especially bacteria and fungi, has recently gained attention. The present study was conducted using white rot fungi Irpex lacteus, Trametes hirsuta, Trametes sp., and Lentinula edodes for the decolorization of reactive textile Levafix Blue E-RA granulate dye. I. lacteus resulted in the best decolorization and degradation of the dye within four days. Therefore, more detailed studies were carried out using I. lacteus. The decolorization was evaluated at various concentration, pH values, and temperatures. The activities of laccase, manganese peroxidase, and lignin peroxidase enzymes were estimated to reveal the roles of enzymes in decolorization. The colorless nature of the fungal cells revealed that decolorization occurred through degradation, and confirmed by analysis of the metabolites by UV-visible spectroscopy and High Performance Liquid Chromatography after decolorization. The metabolites were identified by Gas Chromatography-Mass Spectrometry, and functional group analysis was performed by Fourier Transform Infrared Spectroscopy. The degraded dye metabolites were assessed for phytotoxicity using Vigna radiata and Brassica juncea, which demonstrated nontoxic nature of the metabolites formed after degradation of dye. PMID:22846889

  12. Purification and characterization of two Listeria ivanovii cytolysins, a sphingomyelinase C and a thiol-activated toxin (ivanolysin O).

    PubMed Central

    Vazquez-Boland, J A; Dominguez, L; Rodriguez-Ferri, E F; Suarez, G

    1989-01-01

    The strong bizonal hemolysis on blood agar and the positive CAMP reaction with Rhodococcus equi denotes the production of two different cytolytic factors by Listeria ivanovii. One was characterized as a thiol-activated (SH) cytolysin of 61 kilodaltons and was termed ivanolysin O (ILO) since data suggested that it is different from listeriolysin O, the SH-cytolysin produced by Listeria monocytogenes. The other is a 27-kilodalton hemolytic sphingomyelinase C that was found to be the cytolytic factor responsible for the halo of incomplete hemolysis synergistically enhanced by R. equi exosubstances. When thiol-disulfide exchange affinity chromatography and gel filtration were applied to the purification of ILO from concentrated L. ivanovii culture supernatants, the copurification of the two cytolysins was observed. This phenomenon seems to be due to the formation of intermolecular disulfide bonds between ILO and the sphingomyelinase, since the latter was found to contain free SH groups, not essential for the activity. These SH groups could react with the single cysteine residue characteristically present in the SH-cytolysins, forming a dimeric cytolytic complex. The purification of ILO was achieved by a further gel filtration with a reducing agent (dithiothreitol) in the eluent. A method for the purification of the sphingomyelinase based on selective sequestration of ILO from the L. ivanovii concentrated culture supernatant by the SH cytolysin target molecule cholesterol and thiol-disulfide affinity chromatography is described. Images PMID:2553614

  13. Purification of thermostable α-galactosidase from Irpex lacteus and its use for hydrolysis of oligosaccharides.

    PubMed

    Guo, Yajie; Song, Yi; Qiu, Yi; Shao, Xiaoming; Wang, Hexiang; Song, Yuan

    2016-05-01

    A monomeric α-galactosidase (ILGI) from the mushroom Irpex lacteus was purified 94.19-fold to electrophoretic homogeneity. ILGI exhibited a specific activity of 18.36 U mg(-1) and demonstrated a molecular mass of 60 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). ILGI was optimally active at 80 °C and pH 5.0, and it was stable over a temperature range of 4-70 °C and a wide pH range of 2.0-12.0. ILGI was completely inactivated by Ag(+) and Hg(2+) ions and N-bromosuccinimide (NBS). Moreover, ILGI exhibited good resistance to proteases. Galactose acted as a noncompetitive inhibitor with Ki and Kis of 3.34 and 0.29 mM, respectively. The α-galactosidase presented a broad substrate specificity, which included p-nitrophenyl α-D-galactopyranoside (pNPGal), melibiose, stachyose, and raffinose with Km values of 1.27, 3.24, 7.1, and 22.12 mM, correspondingly. ILGI exhibited efficient and complete hydrolysis to raffinose and stachyose. The aforementioned features of this enzyme suggest its potential value in food and feed industries. PMID:26946959

  14. Ponticoccus lacteus sp. nov. of the family Rhodobacteraceae, isolated from surface seawater.

    PubMed

    Yang, Yujie; Sun, Jia; Tang, Kai; Lin, Dan; Li, Chenlan; Lin, Yingfang

    2015-04-01

    A Gram-stain-negative, rod-shaped, non-motile, aerobic bacterium, strain JL351(T), was isolated from the surface seawater of the South China Sea. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain had a close relationship with members of the genera Ponticoccus , Antarctobacter and Sagittula , and the closest relative was Ponticoccus litoralis CL-GR66(T) (with 96.56% 16S rRNA gene sequence similarity). The polar lipids of strain JL351(T) comprised diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, three unidentified aminolipids, three unidentified phospholipids and an unidentified glycolipid. The predominant isoprenoid quinone was Q-10. The major fatty acids were C(18 : 1)ω7c (60.9%), C(18 : 0) (13.7%), C(16 : 0) (9.4%), 11-methyl C(18 : 1)ω7c (4.5%), and C(12 : 1) 3-OH (4.4%). The DNA G+C content was 66.2 mol%. Based on phenotypic, phylogenetic and genotypic data, strain JL351(T) is considered to represent a novel species in the genus Ponticoccus , for which the name Ponticoccus lacteus sp. nov. is proposed. The type strain is JL351(T) ( = CGMCC 1.12986(T) = JCM 30379(T)). PMID:25634948

  15. Biobleaching of Acacia kraft pulp with extracellular enzymes secreted by Irpex lacteus KB-1.1 and Lentinus tigrinus LP-7 using low-cost media.

    PubMed

    Afrida, Sitompul; Tamai, Yutaka; Watanabe, Toshihiro; Osaki, Mitsuru

    2014-08-01

    The white-rot fungi Irpex lacteus KB-1.1 and Lentinus tigrinus LP-7 have been shown in previous studies to have high biobleaching activity in vivo. The aim of this study was to investigate the activities and stabilities of extracellular enzymes, prepared from I. lacteus and L. tigrinus culture grown in three types of economical media of agricultural and forestry wastes, for biobleaching of Acacia oxygen-delignified kraft pulp using kappa number reduction as an indicator of delignification. After 3 days of incubation, the extracellular enzymes preparations from I. lacteus and L. tigrinus cultures in media of Acacia mangium wood powder supplemented with rice bran and addition 1 % glucose (WRBG), resulted in significant decrease of 4.4 and 6.7 %, respectively. A slightly higher kappa number reduction (7.4 %) was achieved with the combine extracellular enzymes from I. lacteus and L. tigrinus. One of the strategies for reducing the cost of enzyme production for treatment processes in the pulp and paper industry is the utilization of agricultural and forestry waste. Thus, WRBG has potential as a culture medium for producing stable lignolytic enzymes simply and economically. PMID:24699808

  16. Characterization of a Novel Dye-Decolorizing Peroxidase (DyP)-Type Enzyme from Irpex lacteus and Its Application in Enzymatic Hydrolysis of Wheat Straw

    PubMed Central

    Salvachúa, Davinia; Prieto, Alicia

    2013-01-01

    Irpex lacteus is a white rot basidiomycete proposed for a wide spectrum of biotechnological applications which presents an interesting, but still scarcely known, enzymatic oxidative system. Among these enzymes, the production, purification, and identification of a new dye-decolorizing peroxidase (DyP)-type enzyme, as well as its physico-chemical, spectroscopic, and catalytic properties, are described in the current work. According to its N-terminal sequence and peptide mass fingerprinting analyses, I. lacteus DyP showed high homology (>95%) with the hypothetical (not isolated or characterized) protein cpop21 from an unidentified species of the family Polyporaceae. The enzyme had a low optimal pH, was very stable to acid pH and temperature, and showed improved activity and stability at high H2O2 concentrations compared to other peroxidases. Other attractive features of I. lacteus DyP were its high catalytic efficiency oxidizing the recalcitrant anthraquinone and azo dyes assayed (kcat/Km of 1.6 × 106 s-1 M-1) and its ability to oxidize nonphenolic aromatic compounds like veratryl alcohol. In addition, the effect of this DyP during the enzymatic hydrolysis of wheat straw was checked. The results suggest that I. lacteus DyP displayed a synergistic action with cellulases during the hydrolysis of wheat straw, increasing significantly the fermentable glucose recoveries from this substrate. These data show a promising biotechnological potential for this enzyme. PMID:23666335

  17. Immunocytochemical localization of hydroxyindole-o-methyltransferase (HIOMT) in the brain of Myoisophagos lacteus (Nemertea: Heteronemertea: Lineidae).

    PubMed

    Arnoult, F; Vernet, G

    2001-07-01

    In an attempt to identify the brain structures that synthesize melatonin and that probably mediate the photoperiodic response of the heteronemertean Myoisophagos lacteus, we utilized immunocytochemical techniques and employed immunoglobulins directed against hydroxyindole-O-methyltransferase (HIOMT, EC 2.1.1.4). This enzyme catalyzes the last step of melatonin biosynthesis. In immunocytochemically treated head sections of Myoisophagos lacteus, antibodies labelled a few cells in the dorsal region of the dorsal cerebral ganglia. Previous studies have shown that melatonin is present both in the brain and eyes of this nemertean species and that melatonin is involved in control of the worm reproduction. Other studies have demonstrated the presence of photoreceptor-like cells in the same region of the worm brain that showed HIOMT immunostaining. Therefore, anatomical findings of the present study, coupled with results of previous works, provide strong evidence that this region of the worm brain houses a photoperiodic receptor involved in melatonin biosynthesis. J. Exp. Zool. 290:156-162, 2001. PMID:11471145

  18. Crystal structure of an invertebrate cytolysin pore reveals unique properties and mechanism of assembly

    PubMed Central

    Podobnik, Marjetka; Savory, Peter; Rojko, Nejc; Kisovec, Matic; Wood, Neil; Hambley, Richard; Pugh, Jonathan; Wallace, E. Jayne; McNeill, Luke; Bruce, Mark; Liko, Idlir; Allison, Timothy M.; Mehmood, Shahid; Yilmaz, Neval; Kobayashi, Toshihide; Gilbert, Robert J. C.; Robinson, Carol V.; Jayasinghe, Lakmal; Anderluh, Gregor

    2016-01-01

    The invertebrate cytolysin lysenin is a member of the aerolysin family of pore-forming toxins that includes many representatives from pathogenic bacteria. Here we report the crystal structure of the lysenin pore and provide insights into its assembly mechanism. The lysenin pore is assembled from nine monomers via dramatic reorganization of almost half of the monomeric subunit structure leading to a β-barrel pore ∼10 nm long and 1.6–2.5 nm wide. The lysenin pore is devoid of additional luminal compartments as commonly found in other toxin pores. Mutagenic analysis and atomic force microscopy imaging, together with these structural insights, suggest a mechanism for pore assembly for lysenin. These insights are relevant to the understanding of pore formation by other aerolysin-like pore-forming toxins, which often represent crucial virulence factors in bacteria. PMID:27176125

  19. The membrane attack complex, perforin and cholesterol-dependent cytolysin superfamily of pore-forming proteins.

    PubMed

    Lukoyanova, Natalya; Hoogenboom, Bart W; Saibil, Helen R

    2016-06-01

    The membrane attack complex and perforin proteins (MACPFs) and bacterial cholesterol-dependent cytolysins (CDCs) are two branches of a large and diverse superfamily of pore-forming proteins that function in immunity and pathogenesis. During pore formation, soluble monomers assemble into large transmembrane pores through conformational transitions that involve extrusion and refolding of two α-helical regions into transmembrane β-hairpins. These transitions entail a dramatic refolding of the protein structure, and the resulting assemblies create large holes in cellular membranes, but they do not use any external source of energy. Structures of the membrane-bound assemblies are required to mechanistically understand and modulate these processes. In this Commentary, we discuss recent advances in the understanding of assembly mechanisms and molecular details of the conformational changes that occur during MACPF and CDC pore formation. PMID:27179071

  20. Crystal structure of an invertebrate cytolysin pore reveals unique properties and mechanism of assembly

    NASA Astrophysics Data System (ADS)

    Podobnik, Marjetka; Savory, Peter; Rojko, Nejc; Kisovec, Matic; Wood, Neil; Hambley, Richard; Pugh, Jonathan; Wallace, E. Jayne; McNeill, Luke; Bruce, Mark; Liko, Idlir; Allison, Timothy M.; Mehmood, Shahid; Yilmaz, Neval; Kobayashi, Toshihide; Gilbert, Robert J. C.; Robinson, Carol V.; Jayasinghe, Lakmal; Anderluh, Gregor

    2016-05-01

    The invertebrate cytolysin lysenin is a member of the aerolysin family of pore-forming toxins that includes many representatives from pathogenic bacteria. Here we report the crystal structure of the lysenin pore and provide insights into its assembly mechanism. The lysenin pore is assembled from nine monomers via dramatic reorganization of almost half of the monomeric subunit structure leading to a β-barrel pore ~10 nm long and 1.6-2.5 nm wide. The lysenin pore is devoid of additional luminal compartments as commonly found in other toxin pores. Mutagenic analysis and atomic force microscopy imaging, together with these structural insights, suggest a mechanism for pore assembly for lysenin. These insights are relevant to the understanding of pore formation by other aerolysin-like pore-forming toxins, which often represent crucial virulence factors in bacteria.

  1. Crystal structure of an invertebrate cytolysin pore reveals unique properties and mechanism of assembly.

    PubMed

    Podobnik, Marjetka; Savory, Peter; Rojko, Nejc; Kisovec, Matic; Wood, Neil; Hambley, Richard; Pugh, Jonathan; Wallace, E Jayne; McNeill, Luke; Bruce, Mark; Liko, Idlir; Allison, Timothy M; Mehmood, Shahid; Yilmaz, Neval; Kobayashi, Toshihide; Gilbert, Robert J C; Robinson, Carol V; Jayasinghe, Lakmal; Anderluh, Gregor

    2016-01-01

    The invertebrate cytolysin lysenin is a member of the aerolysin family of pore-forming toxins that includes many representatives from pathogenic bacteria. Here we report the crystal structure of the lysenin pore and provide insights into its assembly mechanism. The lysenin pore is assembled from nine monomers via dramatic reorganization of almost half of the monomeric subunit structure leading to a β-barrel pore ∼10 nm long and 1.6-2.5 nm wide. The lysenin pore is devoid of additional luminal compartments as commonly found in other toxin pores. Mutagenic analysis and atomic force microscopy imaging, together with these structural insights, suggest a mechanism for pore assembly for lysenin. These insights are relevant to the understanding of pore formation by other aerolysin-like pore-forming toxins, which often represent crucial virulence factors in bacteria. PMID:27176125

  2. Cardiovascular effects of Sp-CTx, a cytolysin from the scorpionfish (Scorpaena plumieri) venom.

    PubMed

    Gomes, Helena L; Menezes, Thiago N; Malacarne, Pedro F; Roman-Campos, Danilo; Gondim, Antonio N; Cruz, Jader S; Vassallo, Dalton V; Figueiredo, Suely G

    2016-08-01

    Fish venom cytolysins are multifunctional proteins that in addition to their cytolytic/hemolytic effects display neurotoxic, cardiotoxic and inflammatory activities, being described as "protein lethal factors". A pore-forming cytolysin called Sp-CTx (Scorpaena plumieriCytolytic Toxin) has been recently purified from the venom of the scorpionfish Scorpaena plumieri. It is a glycoprotein with dimeric constitution, comprising subunits of approximately 65 kDa. Previous studies have revealed that this toxin has a vasorelaxant activity that appears to involve the L-arginine-nitric oxide synthase pathway; however its cardiovascular effects have not been fully comprehended. The present study examined the cardiovascular effects of Sp-CTx in vivo and in vitro. In anesthetized rats Sp-CTx (70 μg/kg i.v) produced a biphasic response which consisted of an initial systolic and diastolic pressure increase followed by a sustained decrease of these parameters and the heart rate. In isolated rats hearts Sp-CTx (10(-9) to 5 × 10(-6) M) produced concentration-dependent and transient ventricular positive inotropic effect and vasoconstriction response on coronary bed. In papillary muscle, Sp-CTx (10(-7) M) also produced an increase in contractile isometric force, which was attenuated by the catecholamine releasing agent tyramine (100 μM) and the β-adrenergic antagonist propranolol (10 μM). On isolated ventricular cardiomyocytes Sp-CTx (1 nM) increased the L-type Ca(2+) current density. The results show that Sp-CTx induces disorders in the cardiovascular system through increase of sarcolemmal calcium influx, which in turn is partially caused by the release of endogenous noradrenaline. PMID:27155562

  3. Protective role of the dynamin inhibitor Dynasore against the cholesterol-dependent cytolysin of Trueperella pyogenes

    PubMed Central

    Preta, Giulio; Lotti, Virginia; Cronin, James G.; Sheldon, I. Martin

    2015-01-01

    The virulence of many Gram-positive bacteria depends on cholesterol-dependent cytolysins (CDCs), which form pores in eukaryotic cell plasma membranes. Pyolysin (PLO) from Trueperella pyogenes provided a unique opportunity to explore cellular responses to CDCs because it does not require thiol activation. Sublytic concentrations of PLO stimulated phosphorylation of MAPK ERK and p38 in primary stromal cells, and induced autophagy as determined by protein light-chain 3B cleavage. Although, inhibitors of MAPK or autophagy did not affect PLO-induced cytolysis. However, 10 μM 3-hydroxynaphthalene-2-carboxylic acid-(3,4-dihydroxybenzylidene)-hydrazide (Dynasore), a dynamin guanosine 5′-triphosphatase inhibitor, protected stromal cells against PLO-induced cytolysis as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (85 ± 17% versus 50 ± 9% cell viability), measuring extracellular ATP, and kinetic assays. This was a generalized mechanism because Dynasore also protected HeLa cells against streptolysin O. Furthermore, the effect was reversible, with stromal cell sensitivity to PLO restored within 30 minutes of Dynasore removal. The protective effect of Dynasore was not conferred by dynamin inhibition, induction of ERK phosphorylation, or Dynasore binding to PLO. Rather, Dynasore reduced cellular cholesterol and disrupted plasma membrane lipid rafts, similar to positive control methyl-β-cyclodextrin. Dynasore is a tractable tool to explore the complexity of cholesterol homeostasis in eukaryotic cells and to develop strategies to counter CDCs.—Preta, G., Lotti, V., Cronin, J. G., and Sheldon, I. M. Protective role of the dynamin inhibitor Dynasore against the cholesterol-dependent cytolysin of Trueperella pyogenes. PMID:25550455

  4. Identification of two novel cytolysins from the hydrozoan Olindias sambaquiensis (Cnidaria)

    PubMed Central

    2014-01-01

    Background Although the hydrozoan Olindias sambaquiensis is the most common jellyfish associated with human envenomation in southeastern and southern Brazil, information about the composition of its venom is rare. Thus, the present study aimed to analyze pharmacological aspects of O. sambaquiensis venom as well as clinical manifestations observed in affected patients. Crude protein extracts were prepared from the tentacles of animals; peptides and proteins were sequenced and submitted to circular dichroism spectroscopy. Creatine kinase, cytotoxicity and hemolytic activity were evaluated by specific methods. Results We identified two novel cytolysins denominated oshem 1 and oshem 2 from the tentacles of this jellyfish. The cytolysins presented the amino acid sequences NEGKAKCGNTAGSKLTFKSADECTKTGQK (oshem 1) and NNSKAKCGDLAGWSKLTFKSADECTKTGQKS (oshem 2) with respective molecular masses of 3.013 kDa and 3.375 kDa. Circular dichroism revealed that oshem 1 has random coils and small α-helix conformation as main secondary structure whereas oshem 2 presents mainly random coils as its main secondary structure probably due to the presence of W (13) in oshem 2. The hemolysis levels induced by oshem 1 and oshem 2 using a peptide concentration of 0.2 mg/mL were, respectively, 51.7 ± 6.5% and 32.9 ± 8.7% (n = 12 and p ≤ 0.05). Oshem 1 and oshem 2 showed significant myonecrotic activity, evaluated by respective CK level measurements of 1890.4 ± 89 and 1212.5 ± 103 (n = 4 and p ≤ 0.05). In addition, myonecrosis was also evaluated by cell survival, which was measured at 72.4 ± 8.6% and 83.5 ± 6.7% (n = 12 and p ≤ 0.05), respectively. The structural analysis showed that both oshem 1 and oshem 2 should be classified as a small basic hemolytic peptide. Conclusion The amino acid sequences of two peptides were highly similar while the primary amino acid sequence analysis revealed W (22th) as the most important mutation. Finally oshem 1 and oshem 2 are the first cytolytic

  5. Identification and Characterization of the First Cholesterol-Dependent Cytolysins from Gram-Negative Bacteria

    PubMed Central

    Hotze, Eileen M.; Le, Huynh M.; Sieber, Jessica R.; Bruxvoort, Christina; McInerney, Michael J.

    2013-01-01

    The cholesterol-dependent cytolysins (CDCs) are pore-forming toxins that have been exclusively associated with a wide variety of bacterial pathogens and opportunistic pathogens from the Firmicutes and Actinobacteria, which exhibit a Gram-positive type of cell structure. We have characterized the first CDCs from Gram-negative bacterial species, which include Desulfobulbus propionicus type species Widdel 1981 (DSM 2032) (desulfolysin [DLY]) and Enterobacter lignolyticus (formerly Enterobacter cloacae) SCF1 (enterolysin [ELY]). The DLY and ELY primary structures show that they maintain the signature motifs of the CDCs but lack an obvious secretion signal. Recombinant, purified DLY (rDLY) and ELY (rELY) exhibited cholesterol-dependent binding and cytolytic activity and formed the typical large CDC membrane oligomeric pore complex. Unlike the CDCs from Gram-positive species, which are human- and animal-opportunistic pathogens, neither D. propionicus nor E. lignolyticus is known to be a pathogen or commensal of humans or animals: the habitats of both organisms appear to be restricted to anaerobic soils and/or sediments. These studies reveal for the first time that the genes for functional CDCs are present in bacterial species that exhibit a Gram-negative cell structure. These are also the first bacterial species containing a CDC gene that are not known to inhabit or cause disease in humans or animals, which suggests a role of these CDCs in the defense against eukaryote bacterial predators. PMID:23115036

  6. Stepwise visualization of membrane pore formation by suilysin, a bacterial cholesterol-dependent cytolysin

    PubMed Central

    Lukoyanova, Natalya; Hodel, Adrian W; Farabella, Irene; Pandurangan, Arun P; Jahan, Nasrin; Pires Damaso, Mafalda; Osmanović, Dino; Reboul, Cyril F; Dunstone, Michelle A; Andrew, Peter W; Lonnen, Rana; Topf, Maya

    2014-01-01

    Membrane attack complex/perforin/cholesterol-dependent cytolysin (MACPF/CDC) proteins constitute a major superfamily of pore-forming proteins that act as bacterial virulence factors and effectors in immune defence. Upon binding to the membrane, they convert from the soluble monomeric form to oligomeric, membrane-inserted pores. Using real-time atomic force microscopy (AFM), electron microscopy (EM), and atomic structure fitting, we have mapped the structure and assembly pathways of a bacterial CDC in unprecedented detail and accuracy, focussing on suilysin from Streptococcus suis. We show that suilysin assembly is a noncooperative process that is terminated before the protein inserts into the membrane. The resulting ring-shaped pores and kinetically trapped arc-shaped assemblies are all seen to perforate the membrane, as also visible by the ejection of its lipids. Membrane insertion requires a concerted conformational change of the monomeric subunits, with a marked expansion in pore diameter due to large changes in subunit structure and packing. DOI: http://dx.doi.org/10.7554/eLife.04247.001 PMID:25457051

  7. More Than a Pore: The Cellular Response to Cholesterol-Dependent Cytolysins

    PubMed Central

    Cassidy, Sara K. B.; O’Riordan, Mary X. D.

    2013-01-01

    Targeted disruption of the plasma membrane is a ubiquitous form of attack used in all three domains of life. Many bacteria secrete pore-forming proteins during infection with broad implications for pathogenesis. The cholesterol-dependent cytolysins (CDC) are a family of pore-forming toxins expressed predominately by Gram-positive bacterial pathogens. The structure and assembly of some of these oligomeric toxins on the host membrane have been described, but how the targeted cell responds to intoxication by the CDCs is not as clearly understood. Many CDCs induce lysis of their target cell and can activate apoptotic cascades to promote cell death. However, the extent to which intoxication causes cell death is both CDC- and host cell-dependent, and at lower concentrations of toxin, survival of intoxicated host cells is well documented. Additionally, the effect of CDCs can be seen beyond the plasma membrane, and it is becoming increasingly clear that these toxins are potent regulators of signaling and immunity, beyond their role in intoxication. In this review, we discuss the cellular response to CDC intoxication with emphasis on the effects of pore formation on the host cell plasma membrane and subcellular organelles and whether subsequent cellular responses contribute to the survival of the affected cell. PMID:23584137

  8. The cholesterol-dependent cytolysin family of gram-positive bacterial toxins.

    PubMed

    Heuck, Alejandro P; Moe, Paul C; Johnson, Benjamin B

    2010-01-01

    The cholesterol-dependent cytolysins (CDCs) are a family of beta-barrel pore-forming toxins secreted by Gram-positive bacteria. These toxins are produced as water-soluble monomeric proteins that after binding to the target cell oligomerize on the membrane surface forming a ring-like pre-pore complex, and finally insert a large beta-barrel into the membrane (about 250 A in diameter). Formation of such a large transmembrane structure requires multiple and coordinated conformational changes. The presence of cholesterol in the target membrane is absolutely required for pore-formation, and therefore it was long thought that cholesterol was the cellular receptor for these toxins. However, not all the CDCs require cholesterol for binding. Intermedilysin, secreted by Streptoccocus intermedius only binds to membranes containing a protein receptor, but forms pores only if the membrane contains sufficient cholesterol. In contrast, perfringolysin O, secreted by Clostridium perfringens, only binds to membranes containing substantial amounts of cholesterol. The mechanisms by which cholesterol regulates the cytolytic activity of the CDCs are not understood at the molecular level. The C-terminus of perfringolysin O is involved in cholesterol recognition, and changes in the conformation of the loops located at the distal tip of this domain affect the toxin-membrane interactions. At the same time, the distribution of cholesterol in the membrane can modulate toxin binding. Recent studies support the concept that there is a dynamic interplay between the cholesterol-binding domain of the CDCs and the excess of cholesterol molecules in the target membrane. PMID:20213558

  9. Revisiting the oligomerization mechanism of Vibrio cholerae cytolysin, a beta-barrel pore-forming toxin.

    PubMed

    Rai, Anand Kumar; Chattopadhyay, Kausik

    2016-06-01

    Vibrio cholerae cytolysin (VCC) is a membrane-damaging beta-barrel pore-forming toxin (beta-PFT). VCC causes permeabilization of the target membranes by forming transmembrane oligomeric beta-barrel pores. Oligomerization is a key step in the mode of action of any beta-PFT, including that of VCC. Earlier studies have identified some of the key residues in VCC that are directly involved in the generation of the inter-protomer contacts, thus playing critical roles in the oligomerization of the membrane-bound toxin. Analysis of the VCC oligomeric pore structure reveals a potential hydrogen-bond network that appears to connect the sidechain of an asparagine residue (Asn582; located within an inter-domain linker sequence) from one protomer to the backbone CO- and NH-groups of the neighbouring protomer, indirectly through water molecules at most of the inter-protomer interfaces. In the present study, we show that the mutation of Asn582Ala affects the oligomerization and the pore-forming activity of VCC in the membrane lipid bilayer of the synthetic lipid vesicles, while the replacement of Asn582Gln results into the restoration of the oligomeric pore-forming ability of the toxin. Using a number of truncated variants of VCC, having deletion in the C-terminal region of the toxin starting from the Asn582 residue or beyond, we also show that the presence of Asn582 is critically required for the oligomerization of the truncated form of the protein. PMID:27150630

  10. Multifaceted Activity of Listeriolysin O, the Cholesterol-Dependent Cytolysin of Listeria monocytogenes

    PubMed Central

    2014-01-01

    The cholesterol-dependent cytolysins (CDCs) are a large family of pore-forming toxins that are produced by numerous Gram-positive bacterial pathogens. These toxins are released in the extracellular environment as water-soluble monomers or dimers that bind to cholesterol-rich membranes and assemble into large pore complexes. Depending upon their concentration, the nature of the host cell and membrane (cytoplasmic or intracellular) they target, the CDCs can elicit many different cellular responses. Among the CDCs, listeriolysin O (LLO), which is a major virulence factor of the facultative intracellular pathogen Listeria monocytogenes, is involved in several stages of the intracellular lifecycle of the bacterium and displays unique characteristics. It has long been known that following L. monocytogenes internalization into host cells, LLO disrupts the internalization vacuole, enabling the bacterium to replicate into the host cell cytosol. LLO is then used by cytosolic bacteria to spread from cell to cell, avoiding bacterial exposure to the extracellular environment. Although LLO is continuously produced during the intracellular lifecycle of L. monocytogenes, several processes limit its toxicity to ensure the survival of infected cells. It was previously thought that LLO activity was limited to mediating vacuolar escape during bacterial entry and cell to cell spreading. This concept has been challenged by compelling evidence suggesting that LLO secreted by extracellular L. monocytogenes perforates the host cell plasma membrane, triggering important host cell responses. This chapter provides an overview of the well-established intracellular activity of LLO and the multiple roles attributed to LLO secreted by extracellular L. monocytogenes. PMID:24798012

  11. Vibriocholerae cytolysin recognizes the heptasaccharide core of complex N-glycans with nanomolar affinity

    PubMed Central

    Levan, Sophia; De, Swastik; Olson, Rich

    2013-01-01

    Pathogens selectively target host cells using adhesion molecules and secreted virulence factors that may utilize protein, lipid, or carbohydrate ligands on the cell surface. The human intestinal pathogen Vibrio cholerae secretes a pore-forming toxin, Vibrio cholerae cytolysin (VCC), which contains two domains that are structurally similar to known carbohydrate-binding proteins. These tandem domains are attached to the carboxy-terminus of the cytolytic domain and contain a β-trefoil fold and a β-prism fold. VCC has been shown to bind glycosylated proteins, and removal of the β-prism domain leads to a large decrease in lytic activity against rabbit erythrocytes. Despite these clues, the identity of the glycan receptors of VCC and the role of glycan binding in toxin activity remains unknown. To better understand this specificity, we used a combination of structural and functional approaches to characterize the carbohydrate-binding activity of the VCC toxin. We first probed the monosaccharide-binding activity of VCC and demonstrated that the toxin exhibits millimolar affinity for aldohexoses. To understand this specificity, we solved the crystal structure of the VCC β-prism domain bound to methyl-α-mannose. Next, we utilized a mammalian glycan screen to determine that the β-prism domain preferentially binds complex N-glycans with a heptasaccharide GlcNAc4 Man3 core (NGA2). Fluorescence anisotropy and surface plasmon resonance indicated an approximately 100-nanomolar affinity of the β-prism domain for the heptasaccharide core. Our results suggest that carbohydrate-binding domains on the VCC toxin facilitate high-affinity targeting of mammalian cell membranes, which may contribute to the ability of VCC to lyse cells at picomolar concentrations. PMID:23274141

  12. Outer Membrane Vesicles Mediate Transport of Biologically Active Vibrio cholerae Cytolysin (VCC) from V. cholerae Strains

    PubMed Central

    Elluri, Sridhar; Enow, Constance; Vdovikova, Svitlana; Rompikuntal, Pramod K.; Dongre, Mitesh; Carlsson, Sven; Pal, Amit; Uhlin, Bernt Eric; Wai, Sun Nyunt

    2014-01-01

    Background Outer membrane vesicles (OMVs) released from Gram-negative bacteria can serve as vehicles for the translocation of virulence factors. Vibrio cholerae produce OMVs but their putative role in translocation of effectors involved in pathogenesis has not been well elucidated. The V. cholerae cytolysin (VCC), is a pore-forming toxin that lyses target eukaryotic cells by forming transmembrane oligomeric β-barrel channels. It is considered a potent toxin that contributes to V. cholerae pathogenesis. The mechanisms involved in the secretion and delivery of the VCC have not been extensively studied. Methodology/Principal Findings OMVs from V. cholerae strains were isolated and purified using a differential centrifugation procedure and Optiprep centrifugation. The ultrastructure and the contents of OMVs were examined under the electron microscope and by immunoblot analyses respectively. We demonstrated that VCC from V. cholerae strain V:5/04 was secreted in association with OMVs and the release of VCC via OMVs is a common feature among V. cholerae strains. The biological activity of OMV-associated VCC was investigated using contact hemolytic assay and epithelial cell cytotoxicity test. It showed toxic activity on both red blood cells and epithelial cells. Our results indicate that the OMVs architecture might play a role in stability of VCC and thereby can enhance its biological activities in comparison with the free secreted VCC. Furthermore, we tested the role of OMV-associated VCC in host cell autophagy signalling using confocal microscopy and immunoblot analysis. We observed that OMV-associated VCC triggered an autophagy response in the target cell and our findings demonstrated for the first time that autophagy may operate as a cellular defence mechanism against an OMV-associated bacterial virulence factor. Conclusion/Significance Biological assays of OMVs from the V. cholerae strain V:5/04 demonstrated that OMV-associated VCC is indeed biologically active and

  13. What planar lipid membranes tell us about the pore-forming activity of cholesterol-dependent cytolysins.

    PubMed

    Marchioretto, Marta; Podobnik, Marjetka; Dalla Serra, Mauro; Anderluh, Gregor

    2013-12-01

    Pore-forming toxins are an important group of natural molecules that damage cellular membranes by forming transmembrane pores. They are used by many organisms for attack or defense and similar proteins are employed in the immune system of vertebrates. Various biophysical approaches have been used to understand how these proteins act at the molecular level. One of the most useful, in terms of monitoring pore formation in real time, is a method that employs planar lipid membranes and involves ionic current measurements. Here we highlight the advantages and possibilities that this approach offers and show how it can advance understanding of the pore-forming mechanism and pore properties for one of the most important families of natural toxins, the cholesterol-dependent cytolysins. PMID:23876488

  14. Degradation and detoxification of the triphenylmethane dye malachite green catalyzed by crude manganese peroxidase from Irpex lacteus F17.

    PubMed

    Yang, Xueting; Zheng, Jinzhao; Lu, Yongming; Jia, Rong

    2016-05-01

    Malachite green (MG), a recalcitrant, carcinogenic, and mutagenic triphenylmethane dye, was decolorized and detoxified using crude manganese peroxidase (MnP) prepared from the white rot fungus Irpex lacteus F17. In this study, the key factors (pH, temperature, MG, Mn(2+), H2O2, MnP) in these processes were investigated. Under optimal conditions, 96 % of 200 mg L(-1) of MG was decolorized when 66.32 U L(-1) of MnP was added for 1 h. The K m, V max, and k cat values were 109.9 μmol L(-1), 152.8 μmol L(-1) min(-1), and 44.5 s(-1), respectively. The decolorization of MG by MnP followed first-order reaction kinetics with a kinetic rate constant of 0.0129 h(-1). UV-vis and UPLC analysis revealed degradation of MG. Furthermore, seven different intermediates formed during the MnP treatment of 0.5 h were identified by LC-TOF-MS. These degradation products were generated via two different routes by either N-demethylation of MG or the oxidative cleavage of the C-C double bond in MG. Based on ecotoxicity analyses performed on bacteria and algae, it was confirmed that MG metabolites produced by the MnP-catalyzed system were appreciably less toxic than the parent compound. These studies indicate the potential use of this enzyme system in the clean-up of aquatic and terrestrial environments. PMID:26846235

  15. Fungal bioremediation of the creosote-contaminated soil: influence of Pleurotus ostreatus and Irpex lacteus on polycyclic aromatic hydrocarbons removal and soil microbial community composition in the laboratory-scale study.

    PubMed

    Byss, Marius; Elhottová, Dana; Tříska, Jan; Baldrian, Petr

    2008-11-01

    The aim of this study was to determine the efficacy of selected basidiomycetes in the removing of polycyclic aromatic hydrocarbons (PAH) from the creosote-contaminated soil. Fungi Pleurotus ostreatus and Irpex lacteus were supplemented with creosote-contaminated (50-200 mg kg(-1) PAH) soil originating from a wood-preserving plant and incubated at 15 °C for 120 d. Either fungus degraded PAH with 4-6 aromatic rings more efficiently than the microbial community present initially in the soil. PAH removal was higher in P. ostreatus treatments (55-67%) than in I. lacteus treatments (27-36%) in general. P. ostreatus (respectively, I. lacteus) removed 86-96% (47-59%) of 2-rings PAH, 63-72% (33-45%) of 3-rings PAH, 32-49% (9-14%) of 4-rings PAH and 31-38% (11-13%) of 5-6-rings PAH. MIS (Microbial Identification System) Sherlock analysis of the bacterial community determined the presence of dominant Gram-negative bacteria (G-) Pseudomonas in the inoculated soil before the application of fungi. Complex soil microbial community was characterized by phospholipid fatty acids analysis followed by GC-MS/MS. Either fungus induced the decrease of bacterial biomass (G- bacteria in particular), but the soil microbial community was influenced by P. ostreatus in a different way than by I. lacteus. The bacterial community was stressed more by the presence of I. lacteus than P. ostreatus (as proved by the ratio of the fungal/bacterial markers and by the ratio of trans/cis mono-unsaturated fatty acids). Moreover, P. ostreatus stimulated the growth of Gram-positive bacteria (G+), especially actinobacteria and these results indicate the potential of the positive synergistic interaction of this fungus and actinobacteria in creosote biodegradation. PMID:18782639

  16. Induction, purification and characterization of a novel manganese peroxidase from Irpex lacteus CD2 and its application in the decolorization of different types of dye.

    PubMed

    Qin, Xing; Zhang, Jie; Zhang, Xiaoyu; Yang, Yang

    2014-01-01

    Manganese peroxidase (MnP) is the one of the important ligninolytic enzymes produced by lignin-degrading fungi which has the great application value in the field of environmental biotechnology. Searching for new MnP with stronger tolerance to metal ions and organic solvents is important for the maximization of potential of MnP in the biodegradation of recalcitrant xenobiotics. In this study, it was found that oxalic acid, veratryl alcohol and 2,6-Dimehoxyphenol could stimulate the synthesis of MnP in the white-rot fungus Irpex lacteus CD2. A novel manganese peroxidase named as CD2-MnP was purified and characterized from this fungus. CD2-MnP had a strong capability for tolerating different metal ions such as Ca2+, Cd2+, Co2+, Mg2+, Ni2+ and Zn2+ as well as organic solvents such as methanol, ethanol, DMSO, ethylene glycol, isopropyl alcohol, butanediol and glycerin. The different types of dyes including the azo dye (Remazol Brilliant Violet 5R, Direct Red 5B), anthraquinone dye (Remazol Brilliant Blue R), indigo dye (Indigo Carmine) and triphenylmethane dye (Methyl Green) as well as simulated textile wastewater could be efficiently decolorized by CD2-MnP. CD2-MnP also had a strong ability of decolorizing different dyes with the coexistence of metal ions and organic solvents. In summary, CD2-MnP from Irpex lacteus CD2 could effectively degrade a broad range of synthetic dyes and exhibit a great potential for environmental biotechnology. PMID:25412169

  17. Induction, Purification and Characterization of a Novel Manganese Peroxidase from Irpex lacteus CD2 and Its Application in the Decolorization of Different Types of Dye

    PubMed Central

    Qin, Xing; Zhang, Jie; Zhang, Xiaoyu; Yang, Yang

    2014-01-01

    Manganese peroxidase (MnP) is the one of the important ligninolytic enzymes produced by lignin-degrading fungi which has the great application value in the field of environmental biotechnology. Searching for new MnP with stronger tolerance to metal ions and organic solvents is important for the maximization of potential of MnP in the biodegradation of recalcitrant xenobiotics. In this study, it was found that oxalic acid, veratryl alcohol and 2,6-Dimehoxyphenol could stimulate the synthesis of MnP in the white-rot fungus Irpex lacteus CD2. A novel manganese peroxidase named as CD2-MnP was purified and characterized from this fungus. CD2-MnP had a strong capability for tolerating different metal ions such as Ca2+, Cd2+, Co2+, Mg2+, Ni2+ and Zn2+ as well as organic solvents such as methanol, ethanol, DMSO, ethylene glycol, isopropyl alcohol, butanediol and glycerin. The different types of dyes including the azo dye (Remazol Brilliant Violet 5R, Direct Red 5B), anthraquinone dye (Remazol Brilliant Blue R), indigo dye (Indigo Carmine) and triphenylmethane dye (Methyl Green) as well as simulated textile wastewater could be efficiently decolorized by CD2-MnP. CD2-MnP also had a strong ability of decolorizing different dyes with the coexistence of metal ions and organic solvents. In summary, CD2-MnP from Irpex lacteus CD2 could effectively degrade a broad range of synthetic dyes and exhibit a great potential for environmental biotechnology. PMID:25412169

  18. Cellular Functions and X-ray Structure of Anthrolysin O, a Cholesterol-dependent Cytolysin Secreted by Bacillus anthracis

    SciTech Connect

    Bourdeau, Raymond W.; Malito, Enrico; Chenal, Alexandre; Bishop, Brian L.; Musch, Mark W.; Villereal, Mitch L.; Chang, Eugene B.; Mosser, Elise M.; Rest, Richard F.; Tang, Wei-Jen

    2009-06-02

    Anthrolysin O (ALO) is a pore-forming, cholesterol-dependent cytolysin (CDC) secreted by Bacillus anthracis, the etiologic agent for anthrax. Growing evidence suggests the involvement of ALO in anthrax pathogenesis. Here, we show that the apical application of ALO decreases the barrier function of human polarized epithelial cells as well as increases intracellular calcium and the internalization of the tight junction protein occludin. Using pharmacological agents, we also found that barrier function disruption requires increased intracellular calcium and protein degradation. We also report a crystal structure of the soluble state of ALO. Based on our analytical ultracentrifugation and light scattering studies, ALO exists as a monomer. Our ALO structure provides the molecular basis as to how ALO is locked in a monomeric state, in contrast to other CDCs that undergo antiparallel dimerization or higher order oligomerization in solution. ALO has four domains and is globally similar to perfringolysin O (PFO) and intermedilysin (ILY), yet the highly conserved undecapeptide region in domain 4 (D4) adopts a completely different conformation in all three CDCs. Consistent with the differences within D4 and at the D2-D4 interface, we found that ALO D4 plays a key role in affecting the barrier function of C2BBE cells, whereas PFO domain 4 cannot substitute for this role. Novel structural elements and unique cellular functions of ALO revealed by our studies provide new insight into the molecular basis for the diverse nature of the CDC family.

  19. The Relationship between Glycan Binding and Direct Membrane Interactions in Vibrio cholerae Cytolysin, a Channel-forming Toxin.

    PubMed

    De, Swastik; Bubnys, Adele; Alonzo, Francis; Hyun, Jinsol; Lary, Jeffrey W; Cole, James L; Torres, Victor J; Olson, Rich

    2015-11-20

    Bacterial pore-forming toxins (PFTs) are structurally diverse pathogen-secreted proteins that form cell-damaging channels in the membranes of host cells. Most PFTs are released as water-soluble monomers that first oligomerize on the membrane before inserting a transmembrane channel. To modulate specificity and increase potency, many PFTs recognize specific cell surface receptors that increase the local toxin concentration on cell membranes, thereby facilitating channel formation. Vibrio cholerae cytolysin (VCC) is a toxin secreted by the human pathogen responsible for pandemic cholera disease and acts as a defensive agent against the host immune system. Although it has been shown that VCC utilizes specific glycan receptors on the cell surface, additional direct contacts with the membrane must also play a role in toxin binding. To better understand the nature of these interactions, we conducted a systematic investigation of the membrane-binding surface of VCC to identify additional membrane interactions important in cell targeting. Through cell-based assays on several human-derived cell lines, we show that VCC is unlikely to utilize high affinity protein receptors as do structurally similar toxins from Staphylococcus aureus. Next, we identified a number of specific amino acid residues that greatly diminish the VCC potency against cells and investigated the interplay between glycan binding and these direct lipid contacts. Finally, we used model membranes to parse the importance of these key residues in lipid and cholesterol binding. Our study provides a complete functional map of the VCC membrane-binding surface and insights into the integration of sugar, lipid, and cholesterol binding interactions. PMID:26416894

  20. The cholesterol-dependent cytolysins pneumolysin and streptolysin O require binding to red blood cell glycans for hemolytic activity

    PubMed Central

    Shewell, Lucy K.; Harvey, Richard M.; Higgins, Melanie A.; Day, Christopher J.; Hartley-Tassell, Lauren E.; Chen, Austen Y.; Gillen, Christine M.; James, David B. A.; Alonzo, Francis; Torres, Victor J.; Walker, Mark J.; Paton, Adrienne W.; Paton, James C.; Jennings, Michael P.

    2014-01-01

    The cholesterol-dependent cytolysin (CDC) pneumolysin (Ply) is a key virulence factor of Streptococcus pneumoniae. Membrane cholesterol is required for the cytolytic activity of this toxin, but it is not clear whether cholesterol is the only cellular receptor. Analysis of Ply binding to a glycan microarray revealed that Ply has lectin activity and binds glycans, including the Lewis histo-blood group antigens. Surface plasmon resonance analysis showed that Ply has the highest affinity for the sialyl LewisX (sLeX) structure, with a Kd of 1.88 × 10−5 M. Ply hemolytic activity against human RBCs showed dose-dependent inhibition by sLeX. Flow cytometric analysis and Western blots showed that blocking binding of Ply to the sLeX glycolipid on RBCs prevents deposition of the toxin in the membrane. The lectin domain responsible for sLeX binding is in domain 4 of Ply, which contains candidate carbohydrate-binding sites. Mutagenesis of these predicted carbohydrate-binding residues of Ply resulted in a decrease in hemolytic activity and a reduced affinity for sLeX. This study reveals that this archetypal CDC requires interaction with the sLeX glycolipid cellular receptor as an essential step before membrane insertion. A similar analysis conducted on streptolysin O from Streptococcus pyogenes revealed that this CDC also has glycan-binding properties and that hemolytic activity against RBCs can be blocked with the glycan lacto-N-neotetraose by inhibiting binding to the cell surface. Together, these data support the emerging paradigm shift that pore-forming toxins, including CDCs, have cellular receptors other than cholesterol that define target cell tropism. PMID:25422425

  1. Transmembrane oligomeric form of Vibrio cholerae cytolysin triggers TLR2/TLR6-dependent proinflammatory responses in monocytes and macrophages.

    PubMed

    Khilwani, Barkha; Mukhopadhaya, Arunika; Chattopadhyay, Kausik

    2015-02-15

    Vibrio cholerae cytolysin (VCC) kills target eukaryotic cells by forming transmembrane oligomeric β-barrel pores. Once irreversibly converted into the transmembrane oligomeric form, VCC acquires an unusual structural stability and loses its cytotoxic property. It is therefore possible that, on exertion of its cytotoxic activity, the oligomeric form of VCC retained in the disintegrated membrane fractions of the lysed cells would survive within the host cellular milieu for a long period, without causing any further cytotoxicity. Under such circumstances, VCC oligomers may potentially be recognized by the host immune cells. Based on such a hypothesis, in the present study we explored the interaction of the transmembrane oligomeric form of VCC with the monocytes and macrophages of the innate immune system. Our study shows that the VCC oligomers assembled in the liposome membranes elicit potent proinflammatory responses in monocytes and macrophages, via stimulation of the toll-like receptor (TLR)2/TLR6-dependent signalling cascades that involve myeloid differentiation factor 88 (MyD88)/interleukin-1-receptor-associated kinase (IRAK)1/tumour-necrosis-factor-receptor-associated factor (TRAF)6. VCC oligomer-mediated proinflammatory responses critically depend on the activation of the transcription factor nuclear factor-κB. Proinflammatory responses induced by the VCC oligomers also require activation of the mitogen-activated protein kinase (MAPK) family member c-Jun N-terminal kinase, which presumably acts via stimulation of the transcription factor activator protein-1. Notably, the role of the MAPK p38 could not be documented in the process. PMID:25431887

  2. Crystallization and preliminary crystallographic analysis of an Enterococcus faecalis repressor protein, CylR2, involved in regulating cytolysin production through quorum-sensing

    SciTech Connect

    Ni, Shuisong; McAteer, Kathleen; Bussiere, Dirksen E.; Kennedy, Michael A.

    2004-06-01

    CylR2 is one of the two regulatory proteins associated with the quorum-sensing-dependent synthesis of cytolysin for the common pathogen Enterococcus faecalis. The protein was expressed with a C-terminal 6-histidine tag and purified to homogeneity with a cobalt affinity column followed by another size exclusion column. Both native and SeMet proteins were crystallized. A complete X-ray diffraction data set from the native crystal was collected to 2.3 resolution. The crystal was tetragonal, belonging to space group P41/43, with unit-cell dimensions a=b=66.2 , c=40.9 and a=b=g=90. The asymmetric unit contained two molecules of CylR2.

  3. A family of antimicrobial and immunomodulatory peptides related to the frenatins from skin secretions of the Orinoco lime frog Sphaenorhynchus lacteus (Hylidae).

    PubMed

    Conlon, J Michael; Mechkarska, Milena; Radosavljevic, Gordana; Attoub, Samir; King, Jay D; Lukic, Miodrag L; McClean, Stephen

    2014-06-01

    Peptidomic analysis of norepinephrine-stimulated skin secretions of the Orinoco lime tree frog Sphaenorhynchus lacteus (Hylidae, Hylinae) revealed the presence of three structurally related host-defense peptides with limited sequence similarity to frenatin 2 from Litoria infrafrenata (Hylidae, Pelodryadinae) and frenatin 2D from Discoglossus sardus (Alytidae). Frenatin 2.1S (GLVGTLLGHIGKAILG.NH2) and frenatin 2.2S (GLVGTLLGHIGKAILS.NH2) are C-terminally α-amidated but frenatin 2.3S (GLVGTLLGHIGKAILG) is not. Frenatin 2.1S and 2.2S show potent bactericidal activity against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MIC ≤16μM) but are less active against a range of Gram-negative bacteria. Frenatin 2.1S (LC50=80±6 μM) and 2.2S (LC50=75±5 μM) are cytotoxic against non-small cell lung adenocarcinoma A549 cells but are less hemolytic against human erythrocytes (LC50=167±8 μM for frenatin 2.1S and 169±7 μM for 2.2S). Weak antimicrobial and cytotoxic potencies of frenatin 2.3S demonstrate the importance of C-terminal α-amidation for activity. Frenatin 2.1S and 2.2S significantly (P<0.05) increased production of proinflammatory cytokines IL-1β and IL-23 by lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages and frenatin 2.1S also enhanced production of TNF-α. Effects on IL-6 production were not significant. Frenatin 2.2S significantly downregulated production of the anti-inflammatory cytokine IL-10 by LPS-stimulated cells. The data support speculation that frenatins act on skin macrophages to produce a cytokine-mediated stimulation of the adaptive immune system in response to invasion by microorganisms. They may represent a template for the design of peptides with therapeutic applications as immunostimulatory agents. PMID:24704757

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

    PubMed

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

    2016-01-01

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

  5. 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

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

    PubMed

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

    2016-01-01

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

  7. Cellular Functions and X-ray Structure of Anthrolysin O, a Cholesterol-dependent Cytolysin Secreted by Bacillus anthracis*S⃞

    PubMed Central

    Bourdeau, Raymond W.; Malito, Enrico; Chenal, Alexandre; Bishop, Brian L.; Musch, Mark W.; Villereal, Mitch L.; Chang, Eugene B.; Mosser, Elise M.; Rest, Richard F.; Tang, Wei-Jen

    2009-01-01

    Anthrolysin O (ALO) is a pore-forming, cholesterol-dependent cytolysin (CDC) secreted by Bacillus anthracis, the etiologic agent for anthrax. Growing evidence suggests the involvement of ALO in anthrax pathogenesis. Here, we show that the apical application of ALO decreases the barrier function of human polarized epithelial cells as well as increases intracellular calcium and the internalization of the tight junction protein occludin. Using pharmacological agents, we also found that barrier function disruption requires increased intracellular calcium and protein degradation. We also report a crystal structure of the soluble state of ALO. Based on our analytical ultracentrifugation and light scattering studies, ALO exists as a monomer. Our ALO structure provides the molecular basis as to how ALO is locked in a monomeric state, in contrast to other CDCs that undergo antiparallel dimerization or higher order oligomerization in solution. ALO has four domains and is globally similar to perfringolysin O (PFO) and intermedilysin (ILY), yet the highly conserved undecapeptide region in domain 4 (D4) adopts a completely different conformation in all three CDCs. Consistent with the differences within D4 and at the D2-D4 interface, we found that ALO D4 plays a key role in affecting the barrier function of C2BBE cells, whereas PFO domain 4 cannot substitute for this role. Novel structural elements and unique cellular functions of ALO revealed by our studies provide new insight into the molecular basis for the diverse nature of the CDC family. PMID:19307185

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  10. Soluble Oligomers of the Pore-forming Toxin Cytolysin A from Escherichia coli Are Off-pathway Products of Pore Assembly.

    PubMed

    Roderer, Daniel; Benke, Stephan; Schuler, Benjamin; Glockshuber, Rudi

    2016-03-11

    The α-pore-forming toxin Cytolysin A (ClyA) is responsible for the hemolytic activity of various Escherichia coli and Salmonella enterica strains. Soluble ClyA monomers spontaneously assemble into annular dodecameric pore complexes upon contact with membranes or detergent. At ClyA monomer concentrations above ∼100 nm, the rate-limiting step in detergent- or membrane- induced pore assembly is the unimolecular reaction from the monomer to the assembly-competent protomer, which then oligomerizes rapidly to active pore complexes. In the absence of detergent, ClyA slowly forms soluble oligomers. Here we show that soluble ClyA oligomers cannot form dodecameric pore complexes after the addition of detergent and are hemolytically inactive. In addition, we demonstrate that the natural cysteine pair Cys-87/Cys-285 of ClyA forms a disulfide bond under oxidizing conditions and that both the oxidized and reduced ClyA monomers assemble to active pores via the same pathway in the presence of detergent, in which an unstructured, monomeric intermediate is transiently populated. The results show that the oxidized ClyA monomer assembles to pore complexes about one order of magnitude faster than the reduced monomer because the unstructured intermediate of oxidized ClyA is less stable and dissolves more rapidly than the reduced intermediate. Moreover, we show that oxidized ClyA forms soluble, inactive oligomers in the absence of detergent much faster than the reduced monomer, providing an explanation for several contradictory reports in which oxidized ClyA had been described as inactive. PMID:26757820

  11. Pore formation by actinoporins, cytolysins from sea anemones.

    PubMed

    Rojko, Nejc; Dalla Serra, Mauro; Maček, Peter; Anderluh, Gregor

    2016-03-01

    Actinoporins (APs) from sea anemones are ~20kDa pore forming toxins with a β-sandwich structure flanked by two α-helices. The molecular mechanism of APs pore formation is composed of several well-defined steps. APs bind to membrane by interfacial binding site composed of several aromatic amino acid residues that allow binding to phosphatidylcholine and specific recognition of sphingomyelin. Subsequently, the N-terminal α-helix from the β-sandwich has to be inserted into the lipid/water interphase in order to form a functional pore. Functional studies and single molecule imaging revealed that only several monomers, 3-4, oligomerise to form a functional pore. In this model the α-helices and surrounding lipid molecules build toroidal pore. In agreement, AP pores are transient and electrically heterogeneous. On the contrary, crystallized oligomers of actinoporin fragaceatoxin C were found to be composed of eight monomers with no lipids present between the adjacent α-helices. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Maur Dalla Serra and Franco Gambale. PMID:26351738

  12. Determination of Ligand Pathways in Globins

    PubMed Central

    Salter, Mallory D.; Blouin, George C.; Soman, Jayashree; Singleton, Eileen W.; Dewilde, Sylvia; Moens, Luc; Pesce, Alessandra; Nardini, Marco; Bolognesi, Martino; Olson, John S.

    2012-01-01

    Although molecular dynamics simulations suggest multiple interior pathways for O2 entry into and exit from globins, most experiments indicate well defined single pathways. In 2001, we highlighted the effects of large-to-small amino acid replacements on rates for ligand entry and exit onto the three-dimensional structure of sperm whale myoglobin. The resultant map argued strongly for ligand movement through a short channel from the heme iron to solvent that is gated by the distal histidine (His-64(E7)) near the solvent edge of the porphyrin ring. In this work, we have applied the same mutagenesis mapping strategy to the neuronal mini-hemoglobin from Cerebratulus lacteus (CerHb), which has a large internal tunnel from the heme iron to the C-terminal ends of the E and H helices, a direction that is 180° opposite to the E7 channel. Detailed comparisons of the new CerHb map with expanded results for Mb show unambiguously that the dominant (>90%) ligand pathway in CerHb is through the internal tunnel, and the major (>75%) ligand pathway in Mb is through the E7 gate. These results demonstrate that: 1) mutagenesis mapping can identify internal pathways when they exist; 2) molecular dynamics simulations need to be refined to address discrepancies with experimental observations; and 3) alternative pathways have evolved in globins to meet specific physiological demands. PMID:22859299

  13. DNA sequence organization in the genomes of five marine invertebrates.

    PubMed

    Goldberg, R B; Crain, W R; Ruderman, J V; Moore, G P; Barnett, T R; Higgins, R C; Gelfand, R A; Galau, G A; Britten, R J; Davidson, E H

    1975-07-21

    The arrangement of repetitive and non-repetitive sequence was studied in the genomic DNA of the oyster (Crassostrea virginica), the surf clam (Spisula solidissima), the horseshoe crab (Limulus polyphemus), a nemertean worm (Cerebratulus lacteus) and a jelly-fish (Aurelia aurita). Except for the jellyfish these animals belong to the protostomial branch of animal evolution, for which little information regarding DNA sequence organization has previously been available. The reassociation kinetics of short (250-300 nucleotide) and long (2,000-3,000 nucleotide) DNA fragments was studied by the hydroxyapatite method. It was shown that in each case a major fraction of the DNA consists of single copy sequences less than about 3,000 nucleotides in length, interspersed with short repetitive sequences. The lengths of the repetitive sequences were estimated by optical hyperchromicity and S1 nuclease measurements made on renaturation products. All the genomes studied include a prominent fraction of interspersed repetitive sequences about 300 nucleotides in length, as well as longer repetitive sequence regions. PMID:238802

  14. Determination of ligand pathways in globins: apolar tunnels versus polar gates.

    PubMed

    Salter, Mallory D; Blouin, George C; Soman, Jayashree; Singleton, Eileen W; Dewilde, Sylvia; Moens, Luc; Pesce, Alessandra; Nardini, Marco; Bolognesi, Martino; Olson, John S

    2012-09-28

    Although molecular dynamics simulations suggest multiple interior pathways for O(2) entry into and exit from globins, most experiments indicate well defined single pathways. In 2001, we highlighted the effects of large-to-small amino acid replacements on rates for ligand entry and exit onto the three-dimensional structure of sperm whale myoglobin. The resultant map argued strongly for ligand movement through a short channel from the heme iron to solvent that is gated by the distal histidine (His-64(E7)) near the solvent edge of the porphyrin ring. In this work, we have applied the same mutagenesis mapping strategy to the neuronal mini-hemoglobin from Cerebratulus lacteus (CerHb), which has a large internal tunnel from the heme iron to the C-terminal ends of the E and H helices, a direction that is 180° opposite to the E7 channel. Detailed comparisons of the new CerHb map with expanded results for Mb show unambiguously that the dominant (>90%) ligand pathway in CerHb is through the internal tunnel, and the major (>75%) ligand pathway in Mb is through the E7 gate. These results demonstrate that: 1) mutagenesis mapping can identify internal pathways when they exist; 2) molecular dynamics simulations need to be refined to address discrepancies with experimental observations; and 3) alternative pathways have evolved in globins to meet specific physiological demands. PMID:22859299

  15. Progress in nemertean biology: development and phylogeny.

    PubMed

    Turbeville, J M

    2002-07-01

    This paper reviews progress in developmental biology and phylogeny of the Nemertea, a common but poorly studied spiralian taxon of considerable ecological and evolutionary significance. Analyses of reproductive biology (including calcium dynamics during fertilization and oocyte maturation), larval morphology and development and developmental genetics have significantly extended our knowledge of spiralian developmental biology. Developmental genetics studies have in addition provided characters useful for reconstructing metazoan phylogeny. Reinvestigation of the cell lineage of Cerebratulus lacteus using fluorescent tracers revealed that endomesoderm forms from the 4d cell as in other spiralians and that ectomesoderm is derived from the 3a and 3b cells as in annelids, echiurans and molluscs. Studies examining blastomere specification show that cell fates are established precociously in direct developers and later in indirect developers. Morphological characters used to estimate the phylogenetic position of nemerteans are critically re-evaluated, and cladistic analyses of morphology reveal that conflicting hypotheses of nemertean relationships result because of different provisional homology statements. Analyses that include disputed homology statements (1, gliointerstitial cell system 2, coelomic circulatory system) suggest that nemerteans form the sister taxon to the coelomate spiralian taxa rather than the sister taxon to Platyhelminthes. Analyses of small subunit rRNA (18S rDNA) sequences alone or in combination with morphological characters support the inclusion of the nemerteans in a spiralian coelomate clade nested within a more inclusive lophotrochozoan clade. Ongoing evaluation of nemertean relationships with mitochondrial gene rearrangements and other molecular characters is discussed. PMID:21708766

  16. The cholesterol-dependent cytolysin listeriolysin O aggregates rafts via oligomerization.

    PubMed

    Gekara, Nelson O; Jacobs, Thomas; Chakraborty, Trinad; Weiss, Siegfried

    2005-09-01

    The pore-forming toxin listeriolysin O (LLO) is the main virulence factor of Listeria monocytogenes. LLO is known to act as a pseudo cytokine/chemokine, which induces a broad spectrum of host responses that ultimately influences the outcome of listeriosis. In the present study we demonstrate that LLO is a potent aggregator of lipid rafts. LLO was found to aggregate the raft associated molecules GM1, the GPI-anchored proteins CD14 and CD16 as well as the tyrosine kinase Lyn. Abrogation of the cytolytic activity of LLO by cholesterol pretreatment was found not to interfere with LLO's ability to aggregate rafts or trigger tyrosine phosphorylation in cells. However, a monoclonal antibody that blocks the oligomerization of LLO was found to inhibit rafts' aggregation as well as the induction of tyrosine phosphorylation. This implies that rafts aggregation by LLO which is independent of cytolytic activity, is due to the oligomerization of its membrane bound toxin monomers. Thus, LLO most likely induces signalling through the coaggregation of rafts' associated receptors, kinases and adaptors. PMID:16098221

  17. Fundamental properties of the spiralian developmental program are displayed by the basal nemertean Carinoma tremaphoros (Palaeonemertea, Nemertea).

    PubMed

    Maslakova, Svetlana A; Martindale, Mark Q; Norenburg, Jon L

    2004-03-15

    The first description of the cleavage program of the palaeonemertean Carinoma tremaphoros (a member of a basal clade of the Nemertea) is illustrated by confocal microscopy and microinjection and compared to development of more derived nemerteans and other eutrochozoans (Annelida, Mollusca, Sipunculida and Echiurida). Lineage tracers were injected into individual blastomeres of C. tremaphoros at the 2-, 4-, 8- and 16-cell stage. Subsequent development was followed to the formation of simple (so-called planuliform) planktonic larvae to establish the ultimate fates of the blastomeres. Results of labeling experiments demonstrate that the development of C. tremaphoros bears closer similarity to other Eutrochozoa than development of a previously studied hoplonemertean (Nemertopsis bivittata) and a heteronemertean (Cerebratulus lacteus) in that the first cleavage plane bears an invariant relationship to the plane of bilateral symmetry of the larval body. Additionally, our cell-labeling experiments support the earlier suggestion that the transitory pre-oral belt of cells in the larvae of C. tremaphoros corresponds to the prototroch of other Eutrochozoa. A unique feature of development of C. tremaphoros includes the oblique orientation of the trochal lineages with respect to the anterior-posterior axis of the larva. The significance and application of cleavage characters such as presence of molluscan vs. annelid cross for phylogenetic analyses is reviewed. We argue that molluscan or annelid cross, neither of which are present in nemerteans, are merely two out of much greater variety of patterns created by the differences in the relative size and timing of formation of micromere quartets and none can be considered, by itself, as evidence of close phylogenetic relationship between phyla. PMID:15013798

  18. An intermolecular electrostatic interaction controls the prepore-to-pore transition in a cholesterol-dependent cytolysin.

    PubMed

    Wade, Kristin R; Hotze, Eileen M; Kuiper, Michael J; Morton, Craig J; Parker, Michael W; Tweten, Rodney K

    2015-02-17

    β-Barrel pore-forming toxins (βPFTs) form an obligatory oligomeric prepore intermediate before the formation of the β-barrel pore. The molecular components that control the critical prepore-to-pore transition remain unknown for βPFTs. Using the archetype βPFT perfringolysin O, we show that E183 of each monomer within the prepore complex forms an intermolecular electrostatic interaction with K336 of the adjacent monomer on completion of the prepore complex. The signal generated throughout the prepore complex by this interaction irrevocably commits it to the formation of the membrane-inserted giant β-barrel pore. This interaction supplies the free energy to overcome the energy barrier (determined here to be ∼ 19 kcal/mol) to the prepore-to-pore transition by the coordinated disruption of a critical interface within each monomer. These studies provide the first insight to our knowledge into the molecular mechanism that controls the prepore-to-pore transition for a βPFT. PMID:25646411

  19. Prediction of B-Cell Epitopes in Listeriolysin O, a Cholesterol Dependent Cytolysin Secreted by Listeria monocytogenes

    PubMed Central

    Jones, Morris S.; Carter, J. Mark

    2014-01-01

    Listeria monocytogenes is a gram-positive, foodborne bacterium responsible for disease in humans and animals. Listeriolysin O (LLO) is a required virulence factor for the pathogenic effects of L. monocytogenes. Bioinformatics revealed conserved putative epitopes of LLO that could be used to develop monoclonal antibodies against LLO. Continuous and discontinuous epitopes were located by using four different B-cell prediction algorithms. Three-dimensional molecular models were generated to more precisely characterize the predicted antigenicity of LLO. Domain 4 was predicted to contain five of eleven continuous epitopes. A large portion of domain 4 was also predicted to comprise discontinuous immunogenic epitopes. Domain 4 of LLO may serve as an immunogen for eliciting monoclonal antibodies that can be used to study the pathogenesis of L. monocytogenes as well as develop an inexpensive assay. PMID:24523732

  20. New records of ribbon worms (Nemertea) from Ceará, Northeast Brazil.

    PubMed

    Mendes, Cecili B; Matthews-Cascon, Helena; Norenburg, Jon L

    2016-01-01

    Of 45 species of nemerteans reported for the Brazilian coast, only two were recorded from Brazil's Northeast coast. Here we report seven new records for the state of Ceará, in Northeast Brazil: Tubulanus rhabdotus Côrrea, 1954, Carinomella cf. lactea Coe, 1905, Baseodiscus delineatus (Delle-Chiaje 1825), Cerebratulus cf. lineolatus Coe, 1905, Cerebratulus sp. 1, Cerebratulus sp. 2 and Lineidae sp. 1. Specimens were collected at the following beaches: Praia dos Dois Coqueiros, Praia do Pacheco, Pecém harbor, Praia da Pedra Rachada and Praia do Guajiru. T. rhabdotus is a new record for Northeast Brazil, Carinomella cf. lactea and Cerebratulus cf. lineolatus are new records for the South Atlantic Ocean and both genera are new records for Brazil. PMID:27395488

  1. Genetic structure of the Enterococcus faecalis plasmid pAD1-encoded cytolytic toxin system and its relationship to lantibiotic determinants.

    PubMed Central

    Gilmore, M S; Segarra, R A; Booth, M C; Bogie, C P; Hall, L R; Clewell, D B

    1994-01-01

    Pheromone-responsive conjugative plasmids are unique to the species Enterococcus faecalis. Many pheromone-responsive plasmids, including those frequently isolated from sites of infection, express a novel cytolysin that possesses both hemolytic and bacteriocin activities. Further, this cytolysin has been shown to be a toxin in several disease models. In the present study, nucleotide sequence determination, mutagenesis, and complementation analysis were used to determine the organization of the E. faecalis plasmid pAD1 cytolysin determinant. Four open reading frames are required for expression of the cytolysin precursor (cylLL, cylLS, cylM, and cylB). The inferred products of two of these open reading frames, CyILL and CyILS, constitute the cytolysin precursor and bear structural resemblance to posttranslationally modified bacteriocins termed lantibiotics. Similarities between the organization of the E. faecalis cytolysin determinant and expression units for lantibiotics exist, indicating that the E. faecalis cytolysin represents a new branch of this class and is the first known to possess toxin activity. Images PMID:7961506

  2. Genetic structure of the Enterococcus faecalis plasmid pAD1-encoded cytolytic toxin system and its relationship to lantibiotic determinants.

    PubMed

    Gilmore, M S; Segarra, R A; Booth, M C; Bogie, C P; Hall, L R; Clewell, D B

    1994-12-01

    Pheromone-responsive conjugative plasmids are unique to the species Enterococcus faecalis. Many pheromone-responsive plasmids, including those frequently isolated from sites of infection, express a novel cytolysin that possesses both hemolytic and bacteriocin activities. Further, this cytolysin has been shown to be a toxin in several disease models. In the present study, nucleotide sequence determination, mutagenesis, and complementation analysis were used to determine the organization of the E. faecalis plasmid pAD1 cytolysin determinant. Four open reading frames are required for expression of the cytolysin precursor (cylLL, cylLS, cylM, and cylB). The inferred products of two of these open reading frames, CyILL and CyILS, constitute the cytolysin precursor and bear structural resemblance to posttranslationally modified bacteriocins termed lantibiotics. Similarities between the organization of the E. faecalis cytolysin determinant and expression units for lantibiotics exist, indicating that the E. faecalis cytolysin represents a new branch of this class and is the first known to possess toxin activity. PMID:7961506

  3. Immunocompetent cells requisite for graft rejection in Lineus (Invertebrata, Nemertea).

    PubMed

    Langlet, C; Bierne, J

    1984-01-01

    Antecerebral ends from donors of one Lineus species (L. sanguineus) were grafted onto bispecific recipients previously constructed from two other Lineus species (denoted L. ruber----L. lacteus because the anterior component of chimeras was from L. ruber and the posterior component was from L. lacteus) and onto monospecific controls. Histological examination of areas where the tissues from L. sanguineus and L. ruber had been brought into contact by grafting always showed, at early stages, (6 to 20 days postgrafting), a great deal of difference depending upon whether the recipients were monospecific L. ruber or bispecific L. ruber----L. lacteus: only in grafts onto the former was there lysis of gland cells, connective tissue, muscular fibers, and finally epidermis. We attribute this lytic process to a strongly and rapidly cytotoxic action of lymphocyte-like cells from the L. ruber intestinal segment and the absence of lysis during the same stage in grafts onto composite recipients and monospecific L. lacteus to weak, delayed actions of immunocytes from the L. lacteus intestinal segment. Subsequent phagocytosis of material from lysed cell of grafts in the process of being rejected was effected by wandering amebocytes usually involved in destruction of degenerating "self" components, as in oosorption and resorptive processes after fasting. This work supports the existence of immunocytes at an early phylogenetic level. PMID:6500134

  4. Listeriolysin O suppresses Phospholipase C-mediated activation of the microbicidal NADPH oxidase to promote Listeria monocytogenes infection

    PubMed Central

    Lam, Grace Y.; Fattouh, Ramzi; Muise, Aleixo M.; Grinstein, Sergio; Higgins, Darren E.; Brumell, John H.

    2012-01-01

    Summary The intracellular bacterial pathogen Listeria monocytogenes produces phospholipases C (PI-PLC and PC-PLC) and the pore-forming cytolysin listeriolysin O (LLO) to escape the phagosome and replicate within the host cytosol. We found that PLCs can also activate the phagocyte NADPH oxidase during L. monocytogenes infection, a response that would adversely affect pathogen survival. However, secretion of LLO inhibits the NADPH oxidase by preventing its localization to phagosomes. LLO-deficient bacteria can be complemented by perfringolysin O, a related cytolysin, suggesting that other pathogens may also use pore-forming cytolysins to inhibit the NADPH oxidase. Our studies demonstrate that while the PLCs induce antimicrobial NADPH oxidase activity, this effect is alleviated by the pore-forming activity of LLO. Therefore, the combined activities of PLCs and LLO on membrane lysis and the inhibitory effects of LLO on NADPH oxidase activity allows L. monocytogenes to efficiently escape the phagosome while avoiding the microbicidal respiratory burst. PMID:22177565

  5. In Vivo Assessment of Growth and Virulence Gene Expression during Commensal and Pathogenic Lifestyles of luxABCDE-Tagged Enterococcus faecalis Strains in Murine Gastrointestinal and Intravenous Infection Models

    PubMed Central

    Casey, Pat G.; Hill, Colin; Diep, Dzung B.; Nes, Ingolf F.

    2013-01-01

    Cytolysin and gelatinase are prominent pathogenicity determinants associated with highly virulent Enterococcus faecalis strains. In an effort to explore the expression profiles of these virulence traits in vivo, we have employed E. faecalis variants expressing the luxABCDE cassette under the control of either the P16S, cytolysin, or gelatinase promoter for infections of Galleria mellonella caterpillars and mice. Systemic infection of G. mellonella with bioluminescence-tagged E. faecalis MMH594 revealed temporal regulation of both gelatinase and cytolysin promoters and demonstrated that these traits were induced in response to the host environment. Gavage of mice pretreated perorally with antibiotics resulted in efficient colonization of the murine gastrointestinal tract (GIT) in a strain-dependent manner, where the commensal baby isolate EF62 was more persistent than the nosocomial isolate MMH594. A highly significant correlation (R2 > 0.94) was found between bioluminescence and the CFU counts in mouse fecal samples. Both strains showed similar preferences for growth and persistence in the ileum, cecum, and colon. Cytolysin expression was uniform in these compartments of the intestinal lumen. In spite of high numbers (109 CFU/g of intestinal matter) in the ileum, cecum, and colon, no evidence of translocation or systemic infection could be observed. In the murine intravenous infection model, cytolysin expression was readily detected in the liver, kidneys, and bladder. At 72 h postinfection, the highest bacterial loads were found in the liver, kidneys, and spleen, with organ-specific expression levels of cytolysin ∼400- and ∼900-fold higher in the spleen and heart, respectively, than in the liver and kidneys. Taken together, this system based on the bioluminescence imaging technology is established as a new, powerful method to monitor the differential regulation of E. faecalis virulence determinants and to study the spatiotemporal course of infection in living

  6. Downscaling the in vitro test of fungal bioremediation of polycyclic aromatic hydrocarbons: methodological approach.

    PubMed

    Drevinskas, Tomas; Mickienė, Rūta; Maruška, Audrius; Stankevičius, Mantas; Tiso, Nicola; Mikašauskaitė, Jurgita; Ragažinskienė, Ona; Levišauskas, Donatas; Bartkuvienė, Violeta; Snieškienė, Vilija; Stankevičienė, Antanina; Polcaro, Chiara; Galli, Emanuela; Donati, Enrica; Tekorius, Tomas; Kornyšova, Olga; Kaškonienė, Vilma

    2016-02-01

    The miniaturization and optimization of a white rot fungal bioremediation experiment is described in this paper. The optimized procedure allows determination of the degradation kinetics of anthracene. The miniaturized procedure requires only 2.5 ml of culture medium. The experiment is more precise, robust, and better controlled comparing it to classical tests in flasks. Using this technique, different parts, i.e., the culture medium, the fungi, and the cotton seal, can be analyzed. A simple sample preparation speeds up the analytical process. Experiments performed show degradation of anthracene up to approximately 60% by Irpex lacteus and up to approximately 40% by Pleurotus ostreatus in 25 days. Bioremediation of anthracene by the consortium of I. lacteus and P. ostreatus shows the biodegradation of anthracene up to approximately 56% in 23 days. At the end of the experiment, the surface tension of culture medium decreased comparing it to the blank, indicating generation of surfactant compounds. PMID:26660875

  7. Comparative examination of the olive mill wastewater biodegradation process by various wood-rot macrofungi.

    PubMed

    Koutrotsios, Georgios; Zervakis, Georgios I

    2014-01-01

    Olive mill wastewater (OMW) constitutes a major cause of environmental pollution in olive-oil producing regions. Sixty wood-rot macrofungi assigned in 43 species were evaluated for their efficacy to colonize solidified OMW media at initially established optimal growth temperatures. Subsequently eight strains of the following species were qualified: Abortiporus biennis, Ganoderma carnosum, Hapalopilus croceus, Hericium erinaceus, Irpex lacteus, Phanerochaete chrysosporium, Pleurotus djamor, and P. pulmonarius. Fungal growth in OMW (25%v/v in water) resulted in marked reduction of total phenolic content, which was significantly correlated with the effluent's decolorization. A. biennis was the best performing strain (it decreased phenolics by 92% and color by 64%) followed by P. djamor and I. lacteus. Increase of plant seeds germination was less pronounced evidencing that phenolics are only partly responsible for OMW's phytotoxicity. Laccase production was highly correlated with all three biodegradation parameters for H. croceus, Ph. chrysosporium, and Pleurotus spp., and so were manganese-independent and manganese dependent peroxidases for A. biennis and I. lacteus. Monitoring of enzymes with respect to biomass production indicated that Pleurotus spp., H. croceus, and Ph. chrysosporium shared common patterns for all three activities. Moreover, generation of enzymes at the early biodegradation stages enhanced the efficiency of OMW treatment. PMID:24987685

  8. Comparative Examination of the Olive Mill Wastewater Biodegradation Process by Various Wood-Rot Macrofungi

    PubMed Central

    Koutrotsios, Georgios; Zervakis, Georgios I.

    2014-01-01

    Olive mill wastewater (OMW) constitutes a major cause of environmental pollution in olive-oil producing regions. Sixty wood-rot macrofungi assigned in 43 species were evaluated for their efficacy to colonize solidified OMW media at initially established optimal growth temperatures. Subsequently eight strains of the following species were qualified: Abortiporus biennis, Ganoderma carnosum, Hapalopilus croceus, Hericium erinaceus, Irpex lacteus, Phanerochaete chrysosporium, Pleurotus djamor, and P. pulmonarius. Fungal growth in OMW (25%v/v in water) resulted in marked reduction of total phenolic content, which was significantly correlated with the effluent's decolorization. A. biennis was the best performing strain (it decreased phenolics by 92% and color by 64%) followed by P. djamor and I. lacteus. Increase of plant seeds germination was less pronounced evidencing that phenolics are only partly responsible for OMW's phytotoxicity. Laccase production was highly correlated with all three biodegradation parameters for H. croceus, Ph. chrysosporium, and Pleurotus spp., and so were manganese-independent and manganese dependent peroxidases for A. biennis and I. lacteus. Monitoring of enzymes with respect to biomass production indicated that Pleurotus spp., H. croceus, and Ph. chrysosporium shared common patterns for all three activities. Moreover, generation of enzymes at the early biodegradation stages enhanced the efficiency of OMW treatment. PMID:24987685

  9. Fungal biodegradation of anthracene-polluted cork: A comparative study.

    PubMed

    Jové, Patrícia; Olivella, Maria À; Camarero, Susana; Caixach, Josep; Planas, Carles; Cano, Laura; De Las Heras, Francesc X

    2016-01-01

    The efficiency of cork waste in adsorbing aqueous polycyclic aromatic hydrocarbons (PAHs) has been previously reported. Biodegradation of contaminated cork using filamentous fungi could be a good alternative for detoxifying cork to facilitate its final processing. For this purpose, the degradation efficiency of anthracene by three ligninolytic white-rot fungi (Phanerochaete chrysosporium, Irpex lacteus and Pleurotus ostreatus) and three non-ligninolytic fungi which are found in the cork itself (Aspergillus niger, Penicillium simplicissimum and Mucor racemosus) are compared. Anthracene degradation by all fungi was examined in solid-phase cultures after 0, 16, 30 and 61 days. The degradation products of anthracene by P. simplicissimum and I. lacteus were also identified by GC-MS and a metabolic pathway was proposed for P. simplicissimum. Results show that all the fungi tested degraded anthracene. After 61 days of incubation, approximately 86%, 40%, and 38% of the initial concentration of anthracene (i.e., 100 µM) was degraded by P. simplicissimum, P. chrysosporium and I. lacteus, respectively. The rest of the fungi degraded anthracene to a lesser extent (<30%). As a final remark, the results obtained in this study indicate that P. simplicissimum, a non-ligninolytic fungi characteristic of cork itself, could be used as an efficient degrader of PAH-contaminated cork. PMID:26540209

  10. Cytolytic pore-forming protein associated with the surface membrane of Naegleria fowleri

    SciTech Connect

    Lowrey, D.M.

    1985-01-01

    Whole cell homogenates of Naegleria fowleri were examined by hemolytic and /sup 51/Cr-release assays for the presence of cytolytic molecules which may participate in the cytopathogenic action of this amoeba. Two distinct cytolytic activities were found. A surface membrane cytolysin was identified which was found to be avidly associated with membranes possessing an equilibrium density of 1.135 g/cm/sup 3/ in isopycnic sucrose gradients. The activity of the surface membrane cytolysin was not affected by heating at 75/sup 0/C for 30 min. The second cytolytic activity was found in putative lysosomes possessing an equilibrium density of 1.162 g/cm/sup 3/ and was completely inactivated by heating at 75/sup 0/C for 30 min. Cytolysis produced in the presence of both cytolysins was consistently synergistic with respect to the activity of either cytolysin alone. The lesions produced on erythrocytes by this cooperative process were characterized by electron microscopy as transmembrane pores resembling a number of other cytolytic effector molecules including the ninth component of complement, perforins of cytolytic T lymphocytes, and the alphatoxin of Staphylococcus aureus.

  11. A taxonomic catalogue of Japanese nemerteans (phylum Nemertea).

    PubMed

    Kajihara, Hiroshi

    2007-04-01

    A literature-based taxonomic catalogue of the nemertean species (Phylum Nemertea) reported from Japanese waters is provided, listing 19 families, 45 genera, and 120 species as valid. Applications of the following species names to forms previously recorded from Japanese waters are regarded as uncertain: Amphiporus cervicalis, Amphiporus depressus, Amphiporus lactifloreus, Cephalothrix filiformis, Cephalothrix linearis, Cerebratulus fuscus, Lineus vegetus, Lineus bilineatus, Lineus gesserensis, Lineus grubei, Lineus longifissus, Lineus mcintoshii, Nipponnemertes pulchra, Oerstedia venusta, Prostoma graecense, and Prostoma grande. The identities of the taxa referred to by the following four nominal species require clarification through future investigations: Cosmocephala japonica, Dicelis rubra, Dichilus obscurus, and Nareda serpentina. The nominal species established from Japanese waters are tabulated. In addition, a brief history of taxonomic research on Japanese nemerteans is reviewed. PMID:17867829

  12. Activation of a heat-stable cytolytic protein associated with the surface membrane of Naegleria fowleri.

    PubMed Central

    Lowrey, D M; McLaughlin, J

    1985-01-01

    Surface membrane-enriched fractions of Naegleria fowleri obtained after isopycnic centrifugation experiments contain a potent cytolytic activity as determined by hemolysis and 51Cr release assays. This surface membrane cytolysin was unaffected by a treatment at 75 degrees C for 30 min and accounted for 70 to 90% of cytolysis by whole-cell lysates of amoebae. This heat resistance as well as intimate membrane association distinguished the surface membrane cytolytic activity from a second heat-labile cytolytic activity which appears to be latent within lysosomes. The surface membrane cytolysin was found to be specifically activated by diluted samples of lysosomal fractions. The possible role of this surface membrane cytotoxin in the pathogenicity of N. fowleri is discussed. PMID:4055029

  13. Vaccination Against Staphylococcus aureus Pneumonia

    PubMed Central

    Spaulding, Adam R.; Salgado-Pabón, Wilmara; Merriman, Joseph A.; Stach, Christopher S.; Ji, Yinduo; Gillman, Aaron N.; Peterson, Marnie L.; Schlievert, Patrick M.

    2014-01-01

    Background. Staphylococcus aureus causes serious infections in both hospital and community settings. Attempts have been made to prevent human infection through vaccination against bacterial cell-surface antigens; thus far all have failed. Here we show that superantigens and cytolysins, when used in vaccine cocktails, provide protection from S. aureus USA100–USA400 intrapulmonary challenge. Methods. Rabbits were actively vaccinated (wild-type toxins or toxoids) or passively immunized (hyperimmune serum) against combinations of superantigens (toxic shock syndrome toxin 1, enterotoxins B and C, and enterotoxin-like X) and cytolysins (α-, β-, and γ-toxins) and challenged intrapulmonarily with multiple strains of S. aureus, both methicillin-sensitive and methicillin-resistant. Results. Active vaccination against a cocktail containing bacterial cell-surface antigens enhanced disease severity as tested by infective endocarditis. Active vaccination against secreted superantigens and cytolysins resulted in protection of 86 of 88 rabbits when challenged intrapulmonarily with 9 different S. aureus strains, compared to only 1 of 88 nonvaccinated animals. Passive immunization studies demonstrated that production of neutralizing antibodies was an important mechanism of protection. Conclusions. The data suggest that vaccination against bacterial cell-surface antigens increases disease severity, but vaccination against secreted virulence factors provides protection against S. aureus. These results advance our understanding of S. aureus pathogenesis and have important implications in disease prevention. PMID:24357631

  14. Purification and Characterization of Gigantoxin-4, a New Actinoporin from the Sea Anemone Stichodactyla Gigantea

    PubMed Central

    Hu, Bo; Guo, Wei; Wang, Liang-hua; Wang, Jian-guang; Liu, Xiao-yu; Jiao, Bing-hua

    2011-01-01

    A new Cytolysin, termed as Gigantoxin-4, was isolated from the sea anemone Stichodactyla gigantea and found to be highly homologous with Cytolysin-3 (HMg III) from Heteractis magnifica, RTX-A from Radianthus macrodactylus, and Sticholysin-1 (St I) and Sticholysin-2 (St II) from Stichodactyla helianthus (homology 82%, 86%, 82% and 86% respectively). Its 20 N-terminal residues were identified and the full-length cDNA sequence was obtained by reverse transcription-polymerase chain reaction (RT-PCR). Multiple sequence alignments with other Cytolysins of the actinoporin family clearly indicated that Gigantoxin-4 belongs to this protein family. SDS-PAGE electrophoresis showed that this new actinoporin had a molecular mass of about 19 kDa, and possessed a high hemolytic activity to human erythrocytes (HA50= 40 ng/ml), which was inhibited by pre-incubation with sphingomyelin (SM) or SM-cholesterol mixtures. Our in vivo experiments showed that Gigantoxin-4 had wide toxicity to the rat cardiovascular system and the respiratory system. A concentration of 30μg/kg Gigantoxin-4, i.v. produced a positive inotropic effect on the rat heart although final cardiovascular failure was inevitable, and 60μg/kg Gigantoxin-4 caused respiratory arrest rapidly resulting in rat death. HE staining indicated pathological changes in various organs and tissues after i.v. administration of Gigantoxin-4. PMID:21697999

  15. Listeriolysin O suppresses phospholipase C-mediated activation of the microbicidal NADPH oxidase to promote Listeria monocytogenes infection.

    PubMed

    Lam, Grace Y; Fattouh, Ramzi; Muise, Aleixo M; Grinstein, Sergio; Higgins, Darren E; Brumell, John H

    2011-12-15

    The intracellular bacterial pathogen Listeria monocytogenes produces phospholipases C (PI-PLC and PC-PLC) and the pore-forming cytolysin listeriolysin O (LLO) to escape the phagosome and replicate within the host cytosol. We found that PLCs can also activate the phagocyte NADPH oxidase during L. monocytogenes infection, a response that would adversely affect pathogen survival. However, secretion of LLO inhibits the NADPH oxidase by preventing its localization to phagosomes. LLO-deficient bacteria can be complemented by perfringolysin O, a related cytolysin, suggesting that other pathogens may also use pore-forming cytolysins to inhibit the NADPH oxidase. Our studies demonstrate that while the PLCs induce antimicrobial NADPH oxidase activity, this effect is alleviated by the pore-forming activity of LLO. Therefore, the combined activities of PLCs and LLO on membrane lysis and the inhibitory effects of LLO on NADPH oxidase activity allow L. monocytogenes to efficiently escape the phagosome while avoiding the microbicidal respiratory burst. PMID:22177565

  16. The complete mitochondrial genome of Coptotermes testaceus (Isoptera: Rhinotermitidae).

    PubMed

    Li, Ya-Xiao; Wang, Xin-Guo; Ou, Jing; Yao, Fu-Jiao; Yang, Yan; Wei, Zhao-Ming

    2016-09-01

    The complete circular mitochondrial genome (mitogenome) of Coptotermes testaceus is 15 752bp in size, containing 37 typical genes and one non-coding AT-rich region. The AT content of the AT-rich region is 68.3%. All protein coding genes (PCGs) start with standard ATN initiation codons and end with complete termination codons TAA or TAG except for cox2, atp8, and nad5 genes using an incomplete stop codon T. tRNA genes are predicted with a characteristic cloverleaf secondary structure except for trnS1(()(AGN)()), whose dihydrouridine (DHU) arm is replaced by a simple loop. The size of the large and small ribosomal RNA genes are 1315 and 818 bp, respectively. Phylogenetic analysis found that (i) the C. testaceus clade formed the sister group with another clade containing Coptotermes lacteus and Coptotermes formosanus; and (ii) Coptotermes lacteus had a close relationship with Coptotermes clade, but with lower credibility than other clades, the bootstrap value was 97%. PMID:26242721

  17. Development of Fungal Inocula for Bioaugmentation of Contaminated Soils

    PubMed Central

    Lestan, D.; Lamar, R. T.

    1996-01-01

    This report describes novel fungal inocula for bioaugmentation of soils contaminated with hazardous organic compounds. The inocula are in the form of pelleted solid substrates coated with a sodium alginate suspension of fungal spores or mycelial fragments and incubated until overgrown with the mycelium of selected lignin-degrading fungi. The organisms evaluated were Phanerochaete chrysosporium (BKM F-1767, ATCC 42725), P. sordida (HHB-8922-Sp), Irpex lacteus (Mad-517, ATCC 11245), Bjerkandera adusta (FP-135160-Sp, ATCC 62023), and Trametes versicolor (MD-277). The pelleted fungal inocula resisted competition and proliferation from indigenous soil microbes, were lower in moisture content than current fungal inocula, and had sufficient mechanical strength to allow handling and introduction into the soil without a change in the mechanical consistency of the pellets. Inoculated at a rate of 3% in artificially contaminated nonsterile soil, I. lacteus, B. adusta, and T. versicolor removed 86, 82, and 90%, respectively, of the pentachlorophenol in 4 weeks. A mathematical model was developed to explain moisture distribution in a hydrogel-coated pelleted substrate. PMID:16535337

  18. The influence of termites on atmospheric trace gases: CH4, CO2, CHCl3, N2O, CO, H2, and light hydrocarbons

    NASA Astrophysics Data System (ADS)

    Khalil, M. A. K.; Rasmussen, R. A.; French, J. R. J.; Holt, J. A.

    1990-03-01

    Based on field studies of mounds of Australian termites we estimate that on a global scale termites emit about 12 × 1012 g/yr of methane (< 20 tg/yr) and about 4 ×1015 g CO2/yr (< 8 pg/yr). Most of the detailed results are based on studies of the species Coptotermes lacteus. We found that in mid-latitudes the emissions vary seasonally. As much methane is emitted in the summers as in all other seasons combined. The soils a few meters from the mounds consumed methane at an average rate of 40 μg/m2/h. We found no evidence of net emissions of CO and found that H2 is consistently consumed by the mounds and the soils near the mounds. All six species studied produced chloroform. The concentrations of chloroform inside the mounds of C. lacteus were a thousand times greater than ambient levels, but our calculations show that termites are not likely to be a significant global source of chloroform. Finally, we used the results of our study, and others before us, to construct a view of the role of termites in the global carbon cycle.

  19. The influence of termites on atmospheric trace gases: CH sub 4 , CO sub 2 , CHCl sub 3 , N sub 2 O, CO, H sub 2 , and light hydrocarbons

    SciTech Connect

    Khalil, M.A.K.; Rasmussen, R.A. ); French, J.R.J. ); Holt, J.A. )

    1990-03-20

    Based on field studies of mounds of Australian termites the authors estimate that on a global scale termites emit about 12 {times} 10{sup 12} g/yr of methane (< 20 tg/yr) and about 4 {times} 10{sup 15} g CO{sub 2}/yr (< 8 pg/yr). Most of the detailed results are based on studies of the species Coptotermes lacteus. They found that in mid-latitudes the emissions vary seasonally. As much methane is emitted in the summers as in all other seasons combined. The soils a few meters from the mounds consumed methane at an average rate of 40 {mu}g/m{sup 2}/h. They found no evidence of net emissions of CO and found that H{sub 2} is consistently consumed by the mounds and the soils near the mounds. All six species studied produced chloroform. The concentrations of chloroform inside the mounds of C. lacteus were a thousand times greater than ambient levels, but calculations show that termites are not likely to be a significant global source of chloroform. Finally, they used the results of this study, and others before them, to construct a view of the role of termites in the global carbon cycle.

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

    PubMed

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

    2000-01-01

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

  1. Identification of a Streptolysin S-Associated Gene Cluster and Its Role in the Pathogenesis of Streptococcus iniae Disease

    PubMed Central

    Fuller, Jeffrey D.; Camus, Alvin C.; Duncan, Carla L.; Nizet, Victor; Bast, Darrin J.; Thune, Ronald L.; Low, Donald E.; de Azavedo, Joyce C. S.

    2002-01-01

    Streptococcus iniae causes meningoencephalitis and death in cultured fish species and soft-tissue infection in humans. We recently reported that S. iniae is responsible for local tissue necrosis and bacteremia in a murine subcutaneous infection model. The ability to cause bacteremia in this model is associated with a genetic profile unique to strains responsible for disease in fish and humans (J. D. Fuller, D. J. Bast, V. Nizet, D. E. Low, and J. C. S. de Azavedo, Infect. Immun. 69:1994-2000, 2001). S. iniae produces a cytolysin that confers a hemolytic phenotype on blood agar media. In this study, we characterized the genomic region responsible for S. iniae cytolysin production and assessed its contribution to virulence. Transposon (Tn917) mutant libraries of commensal and disease-associated S. iniae strains were generated and screened for loss of hemolytic activity. Analysis of two nonhemolytic mutants identified a chromosomal locus comprising 9 genes with 73% homology to the group A streptococcus (GAS) sag operon for streptolysin S (SLS) biosynthesis. Confirmation that the S. iniae cytolysin is a functional homologue of SLS was achieved by PCR ligation mutagenesis, complementation of an SLS-negative GAS mutant, and use of the SLS inhibitor trypan blue. SLS-negative sagB mutants were compared to their wild-type S. iniae parent strains in the murine model and in human whole-blood killing assays. These studies demonstrated that S. iniae SLS expression is required for local tissue necrosis but does not contribute to the establishment of bacteremia or to resistance to phagocytic clearance. PMID:12228303

  2. Imaging the Lipid-Phase-Dependent Pore Formation of Equinatoxin II in Droplet Interface Bilayers

    PubMed Central

    Rojko, N.; Cronin, B.; Danial, J.S.H.; Baker, M.A.B.; Anderluh, G.; Wallace, M.I.

    2014-01-01

    Using phase-separated droplet interface bilayers, we observe membrane binding and pore formation of a eukaryotic cytolysin, Equinatoxin II (EqtII). EqtII activity is known to depend on the presence of sphingomyelin in the target membrane and is enhanced by lipid phase separation. By imaging the ionic flux through individual pores in vitro, we observe that EqtII pores form predominantly within the liquid-disordered phase. We observe preferential binding of labeled EqtII at liquid-ordered/liquid-disordered domain boundaries before it accumulates in the liquid-disordered phase. PMID:24739162

  3. ANTIBIOTIC ACTIVITY OF MYXOBACTERIA IN RELATION TO THEIR BACTERIOLYTIC CAPACITY

    PubMed Central

    Norén, Börge; Raper, Kenneth B.

    1962-01-01

    Norén, Börge (University of Wisconsin, Madison) and Kenneth B. Raper. Antibiotic activity of myxobacteria in relation to their bacteriolytic capacity. J. Bacteriol. 84:157–162. 1962—Myxococcus virescens, M. fulvus, M. stipitatus, M. lacteus, Chondrococcus blasticus, and Chondromyces crocatus were tested for antibacterial activity, and all were found to secrete products that inhibited the growth of gram-positive eubacteria. The amount of inhibition varied with the myxobacterium employed and with the eubacterium used as a test organism. The growth of gram-negative eubacteria was in no case visibly affected by the antibiotic products. The experiments performed failed to indicate any positive relationship between the antibacterial activity and the bacteriolytic capacity of the six myxobacteria investigated. PMID:14480333

  4. Occurrence of fungi and fungus-like organisms in the Horodnianka River in the vicinity of Białystok, Poland.

    PubMed

    Kiziewicz, Bozena; Zdrojkowska, Ewa; Gajo, Bernadetta; Godlewska, Anna; Muszyńska, Elzbieta; Mazalska, Bozenna

    2011-01-01

    Studies of fungi and fungus- like organisms in the northeastern Poland have mainly concentrated on running waters in the vicinity of Białystok, including the Horodnianka River. The main objective was to investigate biodiversity of fungi and fungus-like organisms which take part in decomposition of organic matter commonly found in inland waters. To obtain a complete picture of species composition of fungi and fungus-like organisms in running waters we decided to explore representative sites of the Horodnianka River such as Olmonty, Hryniewicze and Horodniany with close localization of landfill. Fungal species were isolated using baiting technique. Baits of onion skin (Alium cepa), hemp-seeds (Cannabis sativa), impregnated cellophane and snake skin (Natrix natrix) were applied to isolate fungi from water of the Horodnianka River. The fungal community consists of 26 species, 10 species of fungi belonging to class Chytridiomycetes (3), anamorphic fungi (6), and Zygomycetes (1). 16 species belong to fungus-like organisms from class Oomycetes. Most of the recognized species have already been found in other running waters. From all the examined habitats the fungi belonging to 26 species of 18 genera Achlya, Alternaria, Aphanomyces, Aspergillus, Catenophlyctis, Dictyuchus, Fusarium, Karlingia, Lagenidium, Leptomitus, Olpidiopsis, Penicillium, Phlyctochytrium, Pythium, Saprolegnia, Scoliognia, Thraustotheca and Zoophagus were obtained. Certain fungal species like Aphanomyces laevis, Fusarium aqueductum, F. moniliforme, F. oxysporum, Leptomitus lacteus, Saprolegnia feax and S. parasitica were found at all the study sites. Among fungi potentially pathogenic and allergogenic for humans the genera Alternaria, Aspergillus, Fusarium, Lagenidium and Penicillium have already been described. However, the species Lagenidium giganteum and Achlya androgyna are new in the fungal biota of Poland. The greatest number of fungal species occurred in Olmonty (24), the smallest in Horodniany

  5. Bioluminescence based biosensors for quantitative detection of enterococcal peptide–pheromone activity reveal inter-strain telesensing in vivo during polymicrobial systemic infection

    PubMed Central

    La Rosa, Sabina Leanti; Solheim, Margrete; Diep, Dzung B.; Nes, Ingolf F.; Brede, Dag Anders

    2015-01-01

    Enterococcus faecalis is a significant threat in the nosocomial setting due to the emergence of isolates that are multi-antibiotic resistant, refractory to the available therapies and equipped with a variety of pathogenicity determinants. This bacterium uses quorum-sensing systems to regulate its physiological processes, including the expression of virulence traits, to adapt and proliferate within a host. Here, we describe the construction and application of two bioluminescence-based reporter systems for the direct detection of the quorum-sensing regulated expression of (i) the gelatinase biosynthesis-activating pheromone (GBAP) and (ii) the cytolysin small subunit (CylLS) in natural samples. The two E. faecalis reporters conditionally expressed bioluminescence in the presence of GBAP and CylLS both in the supernatants of liquid cultures and in an agar-overlay assay in as little as three hours, with a high level of sensitivity. Biosensors employed to investigate the interaction between the fsr and cyl systems revealed that fsr impeded CylLS activity by 75%. Furthermore, we identified a clinical E. faecalis isolate that acted as a biological cheater, producing cytolysin only upon sensing CylLS-producers in its environment. This isolate enhanced its virulence during polymicrobial systemic infection of Galleria mellonella. PMID:25661457

  6. 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. PMID:26523409

  7. Staphylococcal superantigens interact with multiple host receptors to cause serious diseases.

    PubMed

    Stach, Christopher S; Herrera, Alfa; Schlievert, Patrick M

    2014-08-01

    Staphylococcus aureus strains that cause human diseases produce a large family of pyrogenic toxin superantigens (SAgs). These include toxic shock syndrome toxin-1 (TSST-1), the staphylococcal enterotoxins (SEs), and the SE-like proteins; to date, 23 staphylococcal SAgs have been described. Among the SAgs, three have been highly associated with human diseases (TSST-1, SEB, and SEC), likely because they are produced in high concentrations compared to other SAgs. Another major family of exotoxins produced by S. aureus is the cytolysins, particularly α-, β-, γ-, and δ-toxins, phenol soluble modulins, and leukocidins. This review discusses the association of SAgs with human diseases and particularly the "outside-in" signaling mechanism that leads to SAg-associated diseases. We discuss SAg interactions with three host immune cell receptors, including variable regions of the β-chain of the T cell receptor, MHC II α- and/or β-chains, and an epithelial/endothelial cell receptor that may include CD40. To a lesser extent, we discuss the role of cytolysins in facilitating disease production by SAgs. PMID:24838262

  8. Serine/Threonine Phosphatase Stp1 Mediates Post-transcriptional Regulation of Hemolysin, Autolysis, and Virulence of Group B Streptococcus*

    PubMed Central

    Burnside, Kellie; Lembo, Annalisa; Harrell, Maria Isabel; Gurney, Michael; Xue, Liang; BinhTran, Nguyen-Thao; Connelly, James E.; Jewell, Kelsea A.; Schmidt, Byron Z.; de los Reyes, Melissa; Tao, Weiguo Andy; Doran, Kelly S.; Rajagopal, Lakshmi

    2011-01-01

    Elucidating how serine/threonine phosphatases regulate kinase function and bacterial virulence is critical for our ability to combat these infections. Group B streptococci (GBS) are β-hemolytic Gram-positive bacteria that cause invasive infections in humans. To adapt to environmental changes, GBS encodes signaling mechanisms comprising two component systems and eukaryotic-like enzymes. We have previously described the importance of the serine/threonine kinase Stk1 to GBS pathogenesis. However, how the presence or absence of the cognate serine/threonine phosphatase Stp1 affects Stk1 function and GBS virulence is not known. Here, we show that GBS deficient only in Stp1 expression are markedly reduced for their ability to cause systemic infections, exhibit decreased β-hemolysin/cytolysin activity, and show increased sensitivity to autolysis. Although transcription of genes important for β-hemolysin/cytolysin expression and export is similar to the wild type (WT), 294 genes (excluding stp1) showed altered expression in the stp1 mutant and included autolysin genes. Furthermore, phosphopeptide enrichment analysis identified that 35 serine/threonine phosphopeptides, corresponding to 27 proteins, were unique to the stp1 mutant. This included phosphorylation of ATP synthase, DNA and RNA helicases, and proteins important for cell division and protein synthesis. Collectively, our results indicate that Stp1 is important for appropriate regulation of Stk1 function, hemolysin activity, autolysis, and GBS virulence. PMID:22081606

  9. Identification and expression of the Actinobacillus actinomycetemcomitans leukotoxin gene.

    PubMed

    Lally, E T; Kieba, I R; Demuth, D R; Rosenbloom, J; Golub, E E; Taichman, N S; Gibson, C W

    1989-02-28

    The leukotoxin produced by the oral bacterium Actinobacillus actinomycetemcomitans has been implicated in the pathogenesis of juvenile periodontitis. In order to elucidate the structure of the leukotoxin, molecular cloning of the leukotoxin gene was carried out. A DNA library of A. actinomycetemcomitans, strain JP2, was constructed by partial digestion of genomic DNA with Sau3AI and ligation of 0.5 to 5.0 kilobase pair fragments into the Bam HI site of the plasmid vector pENN-vrf. After transformation into E. coli RR1 (lambda cI857), the clones were screened for the production of A. actinomycetemcomitans leukotoxin with polyclonal antibody. Six immunoreactive clones were identified. The clones expressed proteins which ranged from 21-80 kilodaltons, and the clone designated pII-2, producing the largest protein was selected for further study. Antibodies eluted from immobilized pII-2 protein also recognized the native A. actinomycetemcomitans leukotoxin molecule indicating that both molecules shared at least one epitope. DNA sequence analysis demonstrated that there are regions of significant amino acid sequence homology between the cloned A. actinomycetemcomitans leukotoxin and two other cytolysins, Escherichia coli alpha-hemolysin and Pasteurella haemolytica leukotoxin, suggesting that a family of cytolysins may exist which share a common mechanism of killing but vary in their target cell specificity. PMID:2647082

  10. Characterization of a pore-forming cytotoxin expressed by Salmonella enterica serovars typhi and paratyphi A.

    PubMed

    Oscarsson, Jan; Westermark, Marie; Löfdahl, Sven; Olsen, Björn; Palmgren, Helena; Mizunoe, Yoshimitsu; Wai, Sun Nyunt; Uhlin, Bernt Eric

    2002-10-01

    Cytolysin A (ClyA) is a pore-forming cytotoxic protein encoded by the clyA gene that has been characterized so far only in Escherichia coli. Using DNA sequence analysis and PCR, we established that clyA is conserved in the human-specific typhoid Salmonella enterica serovars Typhi and Paratyphi A and that the entire clyA gene locus is absent in many other S. enterica serovars, including Typhimurium. The gene products, designated ClyA(STy) and ClyA(SPa), show >/=90% amino acid identity to E. coli cytolysin A, ClyA(EC), and they are immunogenically related. The Salmonella proteins showed a pore-forming activity and are hence functional homologues to ClyA(EC). The chromosomal clyA(STy) gene locus was expressed at detectable levels in the serovar Typhi strains S2369/96 and S1112/97. Furthermore, in the serovar Typhi vaccine strain Ty21a, expression of clyA(STy) reached phenotypic levels, as detected on blood agar plates. The hemolytic phenotype was abolished by the introduction of an in-frame deletion in the clyA(STy) chromosomal locus of Ty21a. Transcomplementation of the mutant with a cloned clyA(STy) gene restored the hemolytic phenotype. To our knowledge, Ty21a is the first reported phenotypically hemolytic Salmonella strain in which the genetic determinant has been identified. PMID:12228306

  11. Stimulators of AMP-activated kinase (AMPK) inhibit seawater- but not cAMP-induced oocyte maturation in a marine worm: Implications for interactions between cAMP and AMPK signaling.

    PubMed

    Stricker, Stephen A; Swiderek, Lee; Nguyen, Thanh

    2010-06-01

    Previous studies have shown that elevations in intraoocytic cAMP prevent mammalian oocytes from maturing, whereas cAMP degradation allows these oocytes to begin maturation, as evidenced by the onset of oocyte nuclear disassembly (="germinal vesicle breakdown", GVBD). Moreover, such cAMP degradation not only reduces cAMP levels but also generates AMP, which in turn can stimulate AMP-activated kinase (AMPK), a well-documented inducer of GVBD in mice. Alternatively, in some marine invertebrates, intraoocytic cAMP triggers, rather than blocks, GVBD, and whether AMPK up- or downregulates maturation in these species has not been tested. Thus, AMPK was monitored in the nemertean worm Cerebratulus during GVBD stimulated by seawater (SW) or cAMP elevators. In oocytes lacking surrounding follicle cells, AMPK activity was initially elevated in immature oocytes but subsequently reduced during SW- or cAMP-induced GVBD, given that the catalytic alpha-subunit of AMPK in maturing oocytes displayed a decreased stimulatory phosphorylation at T172 and an increased inhibitory phosphorylation at S485/491. Accordingly, AMPK-mediated phosphorylation of acetyl-CoA carboxylase, a known target of active AMPK, also declined during maturation. Moreover, treatments with either ice-cold calcium-free seawater (CaFSW) or AMPK agonists dissolved in SW maintained AMPK activity and inhibited GVBD. Conversely, adding cAMP elevators to CaFSW- or SW-solutions of AMPK activators restored GVBD while promoting S485/491 phosphorylation and AMPK deactivation. Collectively, such findings not only demonstrate for the first time that intraoocytic AMPK can block GVBD in the absence of surrounding follicle cells, but these results also provide evidence for a novel GVBD-regulating mechanism involving AMPK deactivation by cAMP-mediated S485/491 phosphorylation. PMID:20336704

  12. The Impact of Paralogy on Phylogenomic Studies – A Case Study on Annelid Relationships

    PubMed Central

    Struck, Torsten H.

    2013-01-01

    Phylogenomic studies based on hundreds of genes derived from expressed sequence tags libraries are increasingly used to reveal the phylogeny of taxa. A prerequisite for these studies is the assignment of genes into clusters of orthologous sequences. Sophisticated methods of orthology prediction are used in such analyses, but it is rarely assessed whether paralogous sequences have been erroneously grouped together as orthologous sequences after the prediction, and whether this had an impact on the phylogenetic reconstruction using a super-matrix approach. Herein, I tested the impact of paralogous sequences on the reconstruction of annelid relationships based on phylogenomic datasets. Using single-partition analyses, screening for bootstrap support, blast searches and pruning of sequences in the supermatrix, wrongly assigned paralogous sequences were found in eight partitions and the placement of five taxa (the annelids Owenia, Scoloplos, Sthenelais and Eurythoe and the nemertean Cerebratulus) including the robust bootstrap support could be attributed to the presence of paralogous sequences in two partitions. Excluding these sequences resulted in a different, weaker supported placement for these taxa. Moreover, the analyses revealed that paralogous sequences impacted the reconstruction when only a single taxon represented a previously supported higher taxon such as a polychaete family. One possibility of a priori detection of wrongly assigned paralogous sequences could combine 1) a screening of single-partition analyses based on criteria such as nodal support or internal branch length with 2) blast searches of suspicious cases as presented herein. Also possible are a posteriori approaches in which support for specific clades is investigated by comparing alternative hypotheses based on differences in per-site likelihoods. Increasing the sizes of EST libraries will also decrease the likelihood of wrongly assigned paralogous sequences, and in the case of orthology

  13. Subterfuge and Sabotage

    PubMed Central

    Okumura, Cheryl Y.M.; Nizet, Victor

    2015-01-01

    The development of a severe invasive bacterial infection in an otherwise healthy individual is one of the most striking and fascinating aspects of human medicine. A small cadre of gram-positive pathogens of the genera Streptococcus and Staphylococcus stand out for their unique invasive disease potential and sophisticated ability to counteract the multifaceted components of human innate defense. This review illustrates how these leading human disease agents evade host complement deposition and activation, impede phagocyte recruitment and activation, resist the microbicidal activities of host antimicrobial peptides and reactive oxygen species, escape neutrophil extracellular traps, and promote and accelerate phagocyte cell death through the action of pore-forming cytolysins. Understanding the molecular basis of bacterial innate immune resistance can open new avenues for therapeutic intervention geared to disabling specific virulence factors and resensitizing the pathogen to host innate immune clearance. PMID:25002085

  14. Membrane pore formation at protein-lipid interfaces.

    PubMed

    Gilbert, Robert J C; Dalla Serra, Mauro; Froelich, Christopher J; Wallace, Mark I; Anderluh, Gregor

    2014-11-01

    Pore-forming proteins (PFPs) interact with lipid bilayers to compromise membrane integrity. Many PFPs function by inserting a ring of oligomerized subunits into the bilayer to form a protein-lined hydrophilic channel. However, mounting evidence suggests that PFPs can also generate 'proteolipidic' pores by contributing to the fusion of inner and outer bilayer leaflets to form a toroidal structure. We discuss here toroidal pore formation by peptides including melittin, protegrin, and Alzheimer's Aβ1-41, as well as by PFPs from several evolutionarily unrelated families: the colicin/Bcl-2 grouping including the pro-apoptotic protein Bax, actinoporins derived from sea anemones, and the membrane attack complex-perforin/cholesterol dependent cytolysin (MACPF/CDC) set of proteins. We also explore how the structure and biological role of toroidal pores might be investigated further. PMID:25440714

  15. Crystal structure of Streptococcus pneumoniae pneumolysin provides key insights into early steps of pore formation

    PubMed Central

    Lawrence, Sara L.; Feil, Susanne C.; Morton, Craig J.; Farrand, Allison J.; Mulhern, Terrence D.; Gorman, Michael A.; Wade, Kristin R.; Tweten, Rodney K.; Parker, Michael W.

    2015-01-01

    Pore-forming proteins are weapons often used by bacterial pathogens to breach the membrane barrier of target cells. Despite their critical role in infection important structural aspects of the mechanism of how these proteins assemble into pores remain unknown. Streptococcus pneumoniae is the world’s leading cause of pneumonia, meningitis, bacteremia and otitis media. Pneumolysin (PLY) is a major virulence factor of S. pneumoniae and a target for both small molecule drug development and vaccines. PLY is a member of the cholesterol-dependent cytolysins (CDCs), a family of pore-forming toxins that form gigantic pores in cell membranes. Here we present the structure of PLY determined by X-ray crystallography and, in solution, by small-angle X-ray scattering. The crystal structure reveals PLY assembles as a linear oligomer that provides key structural insights into the poorly understood early monomer-monomer interactions of CDCs at the membrane surface. PMID:26403197

  16. Subterfuge and sabotage: evasion of host innate defenses by invasive gram-positive bacterial pathogens.

    PubMed

    Okumura, Cheryl Y M; Nizet, Victor

    2014-01-01

    The development of a severe invasive bacterial infection in an otherwise healthy individual is one of the most striking and fascinating aspects of human medicine. A small cadre of gram-positive pathogens of the genera Streptococcus and Staphylococcus stand out for their unique invasive disease potential and sophisticated ability to counteract the multifaceted components of human innate defense. This review illustrates how these leading human disease agents evade host complement deposition and activation, impede phagocyte recruitment and activation, resist the microbicidal activities of host antimicrobial peptides and reactive oxygen species, escape neutrophil extracellular traps, and promote and accelerate phagocyte cell death through the action of pore-forming cytolysins. Understanding the molecular basis of bacterial innate immune resistance can open new avenues for therapeutic intervention geared to disabling specific virulence factors and resensitizing the pathogen to host innate immune clearance. PMID:25002085

  17. A Common Fold Mediates Vertebrate Defense and Bacterial Attack

    SciTech Connect

    Rosado, Carlos J.; Buckle, Ashley M.; Law, Ruby H.P.; Butcher, Rebecca E.; Kan, Wan-Ting; Bird, Catherina H.; Ung, Kheng; Browne, Kylie A.; Baran, Katherine; Bashtannyk-Puhalovich, Tanya A.; Faux, Noel G.; Wong, Wilson; Porter, Corrine J.; Pike, Robert N.; Ellisdon, Andrew M.; Pearce, Mary C.; Bottomley, Stephen P.; Emsley, Jonas; Smith, A. Ian; Rossjohn, Jamie; Hartland, Elizabeth L.; Voskoboinik, Ilia; Trapani, Joseph A.; Bird, Phillip I.; Dunstone, Michelle A.; Whisstock, James C.

    2008-10-02

    Proteins containing membrane attack complex/perforin (MACPF) domains play important roles in vertebrate immunity, embryonic development, and neural-cell migration. In vertebrates, the ninth component of complement and perforin form oligomeric pores that lyse bacteria and kill virus-infected cells, respectively. However, the mechanism of MACPF function is unknown. We determined the crystal structure of a bacterial MACPF protein, Plu-MACPF from Photorhabdus luminescens, to 2.0 angstrom resolution. The MACPF domain reveals structural similarity with poreforming cholesterol-dependent cytolysins (CDCs) from Gram-positive bacteria. This suggests that lytic MACPF proteins may use a CDC-like mechanism to form pores and disrupt cell membranes. Sequence similarity between bacterial and vertebrate MACPF domains suggests that the fold of the CDCs, a family of proteins important for bacterial pathogenesis, is probably used by vertebrates for defense against infection.

  18. Crystal structure of listeriolysin O reveals molecular details of oligomerization and pore formation

    NASA Astrophysics Data System (ADS)

    Köster, Stefan; van Pee, Katharina; Hudel, Martina; Leustik, Martin; Rhinow, Daniel; Kühlbrandt, Werner; Chakraborty, Trinad; Yildiz, Özkan

    2014-04-01

    Listeriolysin O (LLO) is an essential virulence factor of Listeria monocytogenes that causes listeriosis. Listeria monocytogenes owes its ability to live within cells to the pH- and temperature-dependent pore-forming activity of LLO, which is unique among cholesterol-dependent cytolysins. LLO enables the bacteria to cross the phagosomal membrane and is also involved in activation of cellular processes, including the modulation of gene expression or intracellular Ca2+ oscillations. Neither the pore-forming mechanism nor the mechanisms triggering the signalling processes in the host cell are known in detail. Here, we report the crystal structure of LLO, in which we identified regions important for oligomerization and pore formation. Mutants were characterized by determining their haemolytic and Ca2+ uptake activity. We analysed the pore formation of LLO and its variants on erythrocyte ghosts by electron microscopy and show that pore formation requires precise interface interactions during toxin oligomerization on the membrane.

  19. Production of putative virulence factors by Renibacterium salmoninarum grown in cell culture.

    PubMed

    McIntosh, D; Flaño, E; Grayson, T H; Gilpin, M L; Austin, B; Villena, A J

    1997-10-01

    A cell culture system, employing the fish cell line Epithelioma papillosum cyprini (EPC), was developed to study the synthesis of intracellular antigen and the expression of putative virulence factors by Renibacterium salmoninarum. EPC cultures infected with R. salmoninarum could be maintained for 7 weeks, during which the pathogen multiplied intracellularly. Immunohistochemical examination of infected cultures revealed the production of the p57 antigen, haemolysin and cytolysin. The intracellular nature of the infection was confirmed by transmission electron microscopic examination of EPC monolayers. A comparison of the relative virulence of bacterial cells cultured in EPC cells and on agar plates revealed that the former were markedly more virulent in challenge experiments with juvenile rainbow trout (Oncorhynchus mykiss Walbaum). The EPC cell culture model provided a system for the study of R. salmoninarum under more natural conditions than those achieved with plate culture techniques. PMID:9353936

  20. Crystal structure of Streptococcus pneumoniae pneumolysin provides key insights into early steps of pore formation.

    PubMed

    Lawrence, Sara L; Feil, Susanne C; Morton, Craig J; Farrand, Allison J; Mulhern, Terrence D; Gorman, Michael A; Wade, Kristin R; Tweten, Rodney K; Parker, Michael W

    2015-01-01

    Pore-forming proteins are weapons often used by bacterial pathogens to breach the membrane barrier of target cells. Despite their critical role in infection important structural aspects of the mechanism of how these proteins assemble into pores remain unknown. Streptococcus pneumoniae is the world's leading cause of pneumonia, meningitis, bacteremia and otitis media. Pneumolysin (PLY) is a major virulence factor of S. pneumoniae and a target for both small molecule drug development and vaccines. PLY is a member of the cholesterol-dependent cytolysins (CDCs), a family of pore-forming toxins that form gigantic pores in cell membranes. Here we present the structure of PLY determined by X-ray crystallography and, in solution, by small-angle X-ray scattering. The crystal structure reveals PLY assembles as a linear oligomer that provides key structural insights into the poorly understood early monomer-monomer interactions of CDCs at the membrane surface. PMID:26403197

  1. The sensing of membrane microdomains based on pore-forming toxins.

    PubMed

    Skočaj, M; Bakrač, B; Križaj, I; Maček, P; Anderluh, G; Sepčić, K

    2013-01-01

    Membrane rafts are transient and unstable membrane microdomains that are enriched in sphingolipids, cholesterol, and specific proteins. They are involved in intracellular trafficking, signal transduction, pathogen entry, and attachment of various ligands. Increasing experimental evidence on the crucial biological roles of membrane rafts under normal and pathological conditions require new techniques for their structural and functional characterization. In particular, fluorescence-labeled cytolytic proteins that interact specifically with molecules enriched in rafts are of increasing interest. Cholera toxin subunit B interacts specifically with raft-residing ganglioside G(M1), and it has long been the lipid probe of choice for membrane rafts. Recently, four new pore-forming toxins have been proposed as selective raft markers: (i) equinatoxin II, a cytolysin from the sea anemone Actinia equina, which specifically recognizes free and membrane-embedded sphingomyelin; (ii) a truncated non-toxic mutant of a cytolytic protein, lysenin, from the earthworm Eisenia foetida, which specifically recognizes sphingomyelin-enriched membrane domains; (iii) a non-toxic derivative of the cholesterol-dependent cytolysin perfringolysin O, from the bacterium Clostridium perfringens, which selectively binds to membrane domains enriched in cholesterol; and (iv) ostreolysin, from the mushroom Pleurotus ostreatus, which does not bind to a single raft-enriched lipid component, but requires a specific combination of two of the most important raft-residing lipids: sphingomyelin and cholesterol. Nontoxic, raft-binding derivatives of cytolytic proteins have already been successfully used to explore both the structure and function of membrane rafts, and of raft-associated molecules. Here, we review these four new derivatives of pore-forming toxins as new putative markers of these membrane microdomains. PMID:23244522

  2. Phenotypic and molecular characterization of hyperpigmented group B Streptococci.

    PubMed

    Lupo, Agnese; Ruppen, Corinne; Hemphill, Andrew; Spellerberg, Barbara; Sendi, Parham

    2014-07-01

    Group B Streptococcus (GBS) causes invasive infections in neonates, older adults and patients with comorbidities. β-hemolysin/cytolysin is an important GBS virulence factor. It is encoded by the cyl operon and confers GBS hemolytic activity. Isolates displaying hyperpigmentation are typically hyperhemolytic. Comparison of clonally identical isolates displaying different levels of pigmentation has shown transcriptional dysregulation due to mutations in components of the control of the virulence S/R (CovS/R) regulatory system. In addition, hyperpigmented isolates show decreased CAMP factor and decreased capsule thickness. In analogy to findings in group A Streptococcus, a pivotal role of CovS/R has been proposed in the host-pathogen interaction of invasive GBS infection. However, corresponding investigations on multiple clinical GBS isolates have not been performed. We prospectively collected hyperpigmented isolates found in a diagnostic laboratory and performed phenotypic, molecular and transcriptional analyses. In the period from 2008 to 2012, we found 10 isolates obtained from 10 patients. The isolates reflected both invasive pathogens and colonizers. In three cases, clonally identical but phenotypically different variants were also found. Hence, the analyses included 13 isolates. No capsular serotype was found to be significantly more frequent. Bacterial pigments were analyzed via spectrophotometry and for their hemolytic activity. Data obtained for typical absorbance spectra peaks correlated significantly with hemolytic activity. Molecular analysis of the cyl operon showed that it was conserved in all isolates. The covR sequence displayed mutations in five isolates; in one isolate, the CovR binding site to cylX was abrogated. Our results on clinical isolates support previous findings on CovR-deficient isogenic mutants, but suggest that - at least in some clinical isolates - for β-hemolysin/cytolysin and CAMP factor production, other molecular pathways may be

  3. Expression of active streptolysin O in Escherichia coli as a maltose-binding-protein--streptolysin-O fusion protein. The N-terminal 70 amino acids are not required for hemolytic activity.

    PubMed

    Weller, U; Müller, L; Messner, M; Palmer, M; Valeva, A; Tranum-Jensen, J; Agrawal, P; Biermann, C; Döbereiner, A; Kehoe, M A; Bhakdi, S

    1996-02-15

    Streptolysin 0 (SLO) is the prototype of a family of cytolysins that consists of proteins which bind to cholesterol and form very large transmembrane pores. Structure/function studies on the pore-forming cytolysin SLO have been complicated by the proteolytic inactivation of a substantial portion of recombinant SLO (rSLO) expressed in Escherichia coli. To overcome this problem, translational fusions between the E. coli maltose-binding protein (MBP) gene and SLO were constructed, using the vectors pMAL-p2 and pMAL-c2. MBP-SLO fusion proteins were degraded if secreted into the E. coli periplasm, but intact, soluble MBP-SLO fusion proteins were produced at high levels in the cytoplasm. Active SLO with the expected N-terminus was separated from the MBP carrier by cleavage with factor Xa. Cleavage with plasmin or trypsin also yielded active, but slightly smaller forms of SLO. Surprisingly, uncleaved MBP-SLO was also hemolytic and cytotoxic to human fibroblasts and keratinocytes. The MBP-SLO fusion protein displayed equal activities to SLO. Sucrose density gradient analyses showed that the fusion protein assembled into polymers, and no difference in structure was discerned compared with polymers formed by native SLO. These studies show that the N-terminal 70 residues of mature (secreted) SLO are not required for pore formation and that the N-terminus of the molecule is probably not inserted into the bilayer. In addition, they provide a simple means for producing mutants for structure/function studies and highly purified SLO for use as a permeabilising reagent in cell biology research. PMID:8617283

  4. Regulation of Metalloprotease Gene Expression in Vibrio vulnificus by a Vibrio harveyi LuxR Homologue

    PubMed Central

    Shao, Chung-Ping; Hor, Lien-I

    2001-01-01

    Expression of the Vibrio vulnificus metalloprotease gene, vvp, was turned up rapidly when bacterial growth reached the late log phase. A similar pattern of expression has been found in the metalloprotease gene of Vibrio cholerae, and this has been shown to be regulated by a Vibrio harveyi LuxR-like transcriptional activator. To find out whether a LuxR homologue exists in V. vulnificus, a gene library of this organism was screened by colony hybridization using a probe derived from a sequence that is conserved in various luxR-like genes of vibrios. A gene containing a 618-bp open reading frame was identified and found to be identical to the smcR gene of V. vulnificus reported previously. An isogenic SmcR-deficient (RD) mutant was further constructed by an in vivo allelic exchange technique. This mutant exhibited an extremely low level of vvp transcription compared with that of the parent strain. On the other hand, the cytolysin gene, vvhA, was expressed at a higher level in the RD mutant than in the parent strain during the log phase of growth. These data suggested that SmcR might not only be a positive regulator of the protease gene but might also be involved in negative regulation of the cytolysin gene. Virulence of the RD mutant in either normal or iron-overloaded mice challenged by intraperitoneal injection was comparable to that of the parent strain, indicating that SmcR is not required for V. vulnificus virulence in mice. PMID:11157950

  5. Tethered bilayer membranes as a complementary tool for functional and structural studies: The pyolysin case.

    PubMed

    Preta, Giulio; Jankunec, Marija; Heinrich, Frank; Griffin, Sholeem; Sheldon, Iain Martin; Valincius, Gintaras

    2016-09-01

    We demonstrate the use of tethered bilayer lipid membranes (tBLMs) as an experimental platform for functional and structural studies of membrane associated proteins by electrochemical techniques. The reconstitution of the cholesterol-dependent cytolysin (CDC) pyolysin (PLO) from Trueperella pyogenes into tBLMs was followed in real-time by electrochemical impedance spectroscopy (EIS). Changes of the EIS parameters of the tBLMs upon exposure to PLO solutions were consistent with the dielectric barrier damage occurring through the formation of water-filled pores in membranes. Parallel experiments involving a mutant version of PLO, which is able to bind to the membranes but does not form oligomer pores, strengthen the reliability of this methodology, since no change in the electrochemical impedance was observed. Complementary atomic force microscopy (AFM) and neutron reflectometry (NR) measurements revealed structural details of the membrane bound PLO, consistent with the structural transformations of the membrane bound toxins found for other cholesterol dependent cytolysins. In this work, using the tBLMs platform we also observed a protective effect of the dynamin inhibitor Dynasore against pyolysin as well as pneumolysin. An effect of Dynasore in tBLMs, which was earlier observed in experiments with live cells, confirms the biological relevance of the tBLMs models, as well as demonstrates the potential of the electrochemical impedance spectroscopy to quantify membrane damage by the pore forming toxins. In conclusion, tBLMs are a reliable and complementary method to explore the activity of CDCs in eukaryotic cells and to develop strategies to limit the toxic effects of CDCs. PMID:27211243

  6. A novel and potent ribonuclease from fruiting bodies of the mushroom Pleurotus pulmonarius.

    PubMed

    Ye, X Y; Ng, T B

    2002-05-01

    A ribonuclease (RNase), with an N-terminal sequence different from those of ribonucleases from the mushrooms Irpex lacteus, Lentinus edodes, Pleurotus ostreatus, Pleurotus tuber-regium, and Volvariella volvacea, was purified from fruiting bodies of the edible mushroom Pleurotus pulmonarius. The N-terminal sequence of P. pulmonarius RNase manifested homology to a portion of the sequences of ribosome inactivating protein abrin-b, abrin-c, and abrin-d, and Bacillus subtilis transcriptional regulator. The ribonuclease was adsorbed on Affi-gel blue gel, CM-Sepharose, and Mono S. It displayed a molecular mass of 14.4 kDa in both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration on Superdex 75. The ribonuclease exhibited an activity of 25 114 U/mg on yeast tRNA. The highest ribonucleolytic activity was demonstrated toward poly C, followed by poly A, and then by poly G. There was no activity toward poly U. The optimal pH for its activity was 7 and the optimal temperature was 55 degrees C. It inhibited cell-free translation in a rabbit reticulocyte lysate with an IC50 of 0.33 nM. PMID:12054550

  7. Isolation of a new ribonuclease from fruiting bodies of the silver plate mushroom Clitocybe maxima.

    PubMed

    Wang, Hexiang; Ng, T B

    2004-06-01

    A ribonuclease, with an N-terminal sequence exhibiting some homology to ribonuclease from Pleurotus ostreatus (Family Pleurotaceae), has been purified from fruiting bodies of the silver plate mushroom Clitocybe maxima (Family Tricholomataceae). However, there is little resemblance between the N-terminal sequences of ribonucleases from various Pleurotus species, and a lesser extent of resemblance between ribonucleases from C. maxima and Pleurotus tuber-regium. No structural relationship exists between ribonuclease from C. maxima, and those from Volvariella volvacea, Lentinus edodes and Irpex lacteus. The purification protocol involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-Sepharose, and fast protein liquid chromatography on Superdex 75. The ribonuclease was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and CM-Sepharose. It exhibited a molecular mass of 17.5 kDa in both gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It manifested roughly the same ribonucleolytic potency toward poly A and poly G followed by poly U. Its activity toward poly C was, by comparison, meager. The temperature and pH required for its optimal activity were, respectively, 70 degrees C and 6.5-7.0. PMID:15203239

  8. Population genomics of sexual and asexual lineages in fissiparous ribbon worms (Lineus, Nemertea): hybridization, polyploidy and the Meselson effect.

    PubMed

    Ament-Velásquez, S L; Figuet, E; Ballenghien, M; Zattara, E E; Norenburg, J L; Fernández-Álvarez, F A; Bierne, J; Bierne, N; Galtier, N

    2016-07-01

    Comparative population genetics in asexual vs. sexual species offers the opportunity to investigate the impact of asexuality on genome evolution. Here, we analyse coding sequence polymorphism and divergence patterns in the fascinating Lineus ribbon worms, a group of marine, carnivorous nemerteans with unusual regeneration abilities, and in which asexual reproduction by fissiparity is documented. The population genomics of the fissiparous L. pseudolacteus is characterized by an extremely high level of heterozygosity and unexpectedly elevated πN /πS ratio, in apparent agreement with theoretical expectations under clonal evolution. Analysis of among-species allele sharing and read-count distribution, however, reveals that L. pseudolacteus is a triploid hybrid between Atlantic populations of L. sanguineus and L. lacteus. We model and quantify the relative impact of hybridity, polyploidy and asexuality on molecular variation patterns in L. pseudolacteus and conclude that (i) the peculiarities of L. pseudolacteus population genomics result in the first place from hybridization and (ii) the accumulation of new mutations through the Meselson effect is more than compensated by processes of heterozygosity erosion, such as gene conversion or gene copy loss. This study illustrates the complexity of the evolutionary processes associated with asexuality and identifies L. pseudolacteus as a promising model to study the first steps of polyploid genome evolution in an asexual context. PMID:27286413

  9. Genomic and molecular mechanisms for efficient biodegradation of aromatic dye.

    PubMed

    Sun, Su; Xie, Shangxian; Chen, Hu; Cheng, Yanbing; Shi, Yan; Qin, Xing; Dai, Susie Y; Zhang, Xiaoyu; Yuan, Joshua S

    2016-01-25

    Understanding the molecular mechanisms for aromatic compound degradation is crucial for the development of effective bioremediation strategies. We report the discovery of a novel phenomenon for improved degradation of Direct Red 5B azo dye by Irpex lacteus CD2 with lignin as a co-substrate. Transcriptomics analysis was performed to elucidate the molecular mechanisms of aromatic degradation in white rot fungus by comparing dye, lignin, and dye/lignin combined treatments. A full spectrum of lignin degradation peroxidases, oxidases, radical producing enzymes, and other relevant components were up-regulated under DR5B and lignin treatments. Lignin induced genes complemented the DR5B induced genes to provide essential enzymes and redox conditions for aromatic compound degradation. The transcriptomics analysis was further verified by manganese peroxidase (MnP) protein over-expression, as revealed by proteomics, dye decolorization assay by purified MnP and increased hydroxyl radical levels, as indicated by an iron reducing activity assay. Overall, the molecular and genomic mechanisms indicated that effective aromatic polymer degradation requires synergistic enzymes and radical-mediated oxidative reactions to form an effective network of chemical processes. This study will help to guide the development of effective bioremediation and biomass degradation strategies. PMID:26476316

  10. Chaetochromones A and B, two new polyketides from the fungus Chaetomium indicum (CBS.860.68).

    PubMed

    Lu, Keyang; Zhang, Yisheng; Li, Li; Wang, Xuewei; Ding, Gang

    2013-01-01

    Chaetochromones A (1) and B (2), two novel polyketides, were isolated from the crude extract of fungus Chaetomium indicum (CBS.860.68) together with three known analogues PI-3(3), PI-4 (4) and SB236050 (5). The structures of these compounds were determined by HRESI-MS and NMR experiments. Chaetochromones A (1) and B (2) are a member of the polyketides family, which might originate from a similar biogenetic pathway as the known compounds PI-3 (3), PI-4 (4) and SB236050 (5). The biological activities of these secondary metabolites were evaluated against eight plant pathogens, including Alternaria alternata, Ilyonectria radicicola, Trichoderma viride pers, Aspergillus niger, Fusarium verticillioide, Irpex lacteus (Fr.), Poria placenta (Fr.) Cooke and Coriolus versicolor (L.) Quél. Compound 1 displayed moderate inhibitory rate (>60%) against the brown rot fungus Poria placenta (Fr.) Cooke, which causes significant wood decay. In addition, the cytotoxic activities against three cancer cell lines A549, MDA-MB-231, PANC-1 were also tested, without any inhibitory activities being detected. PMID:24013408

  11. Improving the conversion of biomass in catalytic fast pyrolysis via white-rot fungal pretreatment.

    PubMed

    Yu, Yanqing; Zeng, Yelin; Zuo, Jiane; Ma, Fuying; Yang, Xuewei; Zhang, Xiaoyu; Wang, Yujue

    2013-04-01

    This study investigated the effect of white-rot fungal pretreatment on corn stover conversion in catalytic fast pyrolysis (CFP). Corn stover pretreated by white-rot fungus Irpex lacteus CD2 was fast pyrolyzed alone (non-CFP) and with ZSM-5 zeolite (CFP) in a semi-batch pyroprobe reactor. The fungal pretreatment considerably increased the volatile product yields (predominantly oxygenated compounds) in non-CFP, indicating that fungal pretreatment enhances the corn stover conversion in fast pyrolysis. In the presence of ZSM-5 zeolite, these oxygenated volatiles were further catalytically converted to aromatic hydrocarbons, whose yield increased from 10.03 wt.% for the untreated corn stover to 11.49 wt.% for the pretreated sample. In contrast, the coke yield decreased from 14.29 to 11.93 wt.% in CFP following the fungal pretreatment. These results indicate that fungal pretreatment can enhance the production of valuable aromatics and decrease the amount of undesired coke, and thus has a beneficial effect on biomass conversion in CFP. PMID:23506976

  12. Synthesis of rebaudioside A from stevioside and their interaction model with hTAS2R4 bitter taste receptor.

    PubMed

    Singla, Ramit; Jaitak, Vikas

    2016-05-01

    Steviol glycosides (SG's) from Stevia rebaudiana (Bertoni) have been used as a natural low-calorie sweeteners. Its aftertaste bitterness restricts its use for human consumption and limits its application in food and pharmaceutical products. In present study, we have performed computational analysis in order to investigate the interaction of two major constituents of SG's against homology model of the hTAS2R4 receptor. Molecular simulation study was performed using stevioside and rebaudioside A revealed that, sugar moiety at the C-3'' position in rebaudioside A causes restriction of its entry into the receptor site thereby unable to trigger the bitter reception signaling cascade. Encouraged by the current finding, we have also developed a greener route using β-1,3-glucanase from Irpex lacteus for the synthesis of de-bittered rebaudioside A from stevioside. The rebaudioside A obtained was of high quality with percent conversion of 62.5%. The results here reported could be used for the synthesis of rebaudioside A which have large application in food and pharmaceutical industry. PMID:26976334

  13. Degradation of Bunker C Fuel Oil by White-Rot Fungi in Sawdust Cultures Suggests Potential Applications in Bioremediation

    PubMed Central

    Young, Darcy; Rice, James; Martin, Rachael; Lindquist, Erika; Lipzen, Anna; Grigoriev, Igor; Hibbett, David

    2015-01-01

    Fungal lignocellulolytic enzymes are promising agents for oxidizing pollutants. This study investigated degradation of Number 6 “Bunker C” fuel oil compounds by the white-rot fungi Irpex lacteus, Trichaptum biforme, Phlebia radiata, Trametes versicolor, and Pleurotus ostreatus (Basidiomycota, Agaricomycetes). Averaging across all studied species, 98.1%, 48.6%, and 76.4% of the initial Bunker C C10 alkane, C14 alkane, and phenanthrene, respectively were degraded after 180 days of fungal growth on pine media. This study also investigated whether Bunker C oil induces changes in gene expression in the white-rot fungus Punctularia strigosozonata, for which a complete reference genome is available. After 20 days of growth, a monokaryon P. strigosozonata strain degraded 99% of the initial C10 alkane in both pine and aspen media but did not affect the amounts of the C14 alkane or phenanthrene. Differential gene expression analysis identified 119 genes with ≥ log2(2-fold) greater expression in one or more treatment comparisons. Six genes were significantly upregulated in media containing oil; these genes included three enzymes with potential roles in xenobiotic biotransformation. Carbohydrate metabolism genes showing differential expression significantly accumulated transcripts on aspen vs. pine substrates, perhaps reflecting white-rot adaptations to growth on hardwood substrates. The mechanisms by which P. strigosozonata may degrade complex oil compounds remain obscure, but degradation results of the 180-day cultures suggest that diverse white-rot fungi have promise for bioremediation of petroleum fuels. PMID:26111162

  14. Field and laboratory transmission studies of haemic neoplasia in the soft-shell clam, Mya arenaria, from Atlantic Canada.

    PubMed

    Mateo, D R; MacCallum, G S; Davidson, J

    2016-08-01

    A two-year laboratory and field study was initiated in 2001 in response to mass mortalities associated with haemic neoplasia (HN) in 1999 in Prince Edward Island (PEI) soft-shell clams, Mya arenaria. A laboratory proximity experiment (cohabitation) and an inoculation challenge were conducted with clams and mussels (Mytilus edulis). Three field exposure experiments were also conducted, in which naive clams were held in sediment (in trays) or out of sediment (in mesh bags) at three high HN prevalence sites on PEI. There was a conversion to HN positive in clams in the proximity experiment and in clams injected with whole blood and cell-free homogenate, but not at statistically significant levels. No mussels or control clams became HN positive. There was a significant conversion to HN positive in as little as 24 and 58 days after transfer with clams held out of sediment and in sediment, respectively. The laboratory and field experiments' results suggest that HN-infected clams are spreading the disease through water from infected clams to naïve individuals and via transplantation from affected to unaffected sites. Some environmental conditions (e.g. abnormally high water temperature and hypoxia-induced sea lettuce [Ulva lacteus] invasion) may make clams susceptible to infections or exacerbate the proliferation of HN. PMID:26687447

  15. Species Diversity of Ramphogordius sanguineus/Lineus ruber-Like Nemerteans (Nemertea: Heteronemertea) and Geographic Distribution of R. sanguineus.

    PubMed

    Kang, Xing-Xing; Fernández-Álvarez, Fernando Ángel; Alfaya, José E F; Machordom, Annie; Strand, Malin; Sundberg, Per; Sun, Shi-Chun

    2015-12-01

    Heteronemerteans, such as Lineus ruber, L. viridis, Ramphogordius sanguineus, R. lacteus, Riseriellus occultus, and Micrura varicolor, share many similar external characters. Although several internal characters useful for distinguishing these nemertean species have been documented, their identification is based mostly on coloration, the shape of the head, and how they contract, which may not be always reliable. We sequenced the mitochondrial COI gene for 160 specimens recently collected from 27 locations around the world (provisionally identified as the above species, according to external characters and contraction patterns, with most of them as R. sanguineus). Based on these specimens, together with sequences of 16 specimens from GenBank, we conducted a DNA-based species delimitation/identification by means of statistical parsimony and phylogenetic analyses. Our results show that the analyzed specimens may contain nine species, which can be separated by large genetic gaps; heteronemerteans with an external appearance similar to R. sanguineus/Lineus ruber/L. viridis have high species diversity in European waters from where eight species can be discriminated. Our 42 individuals from Vancouver Island (Canada) are revealed to be R. sanguineus, which supports an earlier argument that nemerteans reported as L. ruber or L. viridis from the Pacific Northwest may refer to this species. We report R. sanguineus from Chile, southern China, and the species is also distributed on the Atlantic coast of South America (Argentina). In addition, present analyses reveal the occurrence of L. viridis in Qingdao, which is the first record of the species from Chinese waters. PMID:26654041

  16. Eco-taxonomic insights into actinomycete symbionts of termites for discovery of novel bioactive compounds.

    PubMed

    Kurtböke, D Ipek; French, John R J; Hayes, R Andrew; Quinn, Ronald J

    2015-01-01

    Termites play a major role in foraging and degradation of plant biomass as well as cultivating bioactive microorganisms for their defense. Current advances in "omics" sciences are revealing insights into function-related presence of these symbionts, and their related biosynthetic activities and genes identified in gut symbiotic bacteria might offer a significant potential for biotechnology and biodiscovery. Actinomycetes have been the major producers of bioactive compounds with an extraordinary range of biological activities. These metabolites have been in use as anticancer agents, immune suppressants, and most notably, as antibiotics. Insect-associated actinomycetes have also been reported to produce a range of antibiotics such as dentigerumycin and mycangimycin. Advances in genomics targeting a single species of the unculturable microbial members are currently aiding an improved understanding of the symbiotic interrelationships among the gut microorganisms as well as revealing the taxonomical identity and functions of the complex multilayered symbiotic actinofloral layers. If combined with target-directed approaches, these molecular advances can provide guidance towards the design of highly selective culturing methods to generate further information related to the physiology and growth requirements of these bioactive actinomycetes associated with the termite guts. This chapter provides an overview on the termite gut symbiotic actinoflora in the light of current advances in the "omics" science, with examples of their detection and selective isolation from the guts of the Sunshine Coast regional termite Coptotermes lacteus in Queensland, Australia. PMID:24817085

  17. Degradation of Bunker C Fuel Oil by White-Rot Fungi in Sawdust Cultures Suggests Potential Applications in Bioremediation.

    PubMed

    Young, Darcy; Rice, James; Martin, Rachael; Lindquist, Erika; Lipzen, Anna; Grigoriev, Igor; Hibbett, David

    2015-01-01

    Fungal lignocellulolytic enzymes are promising agents for oxidizing pollutants. This study investigated degradation of Number 6 "Bunker C" fuel oil compounds by the white-rot fungi Irpex lacteus, Trichaptum biforme, Phlebia radiata, Trametes versicolor, and Pleurotus ostreatus (Basidiomycota, Agaricomycetes). Averaging across all studied species, 98.1%, 48.6%, and 76.4% of the initial Bunker C C10 alkane, C14 alkane, and phenanthrene, respectively were degraded after 180 days of fungal growth on pine media. This study also investigated whether Bunker C oil induces changes in gene expression in the white-rot fungus Punctularia strigosozonata, for which a complete reference genome is available. After 20 days of growth, a monokaryon P. strigosozonata strain degraded 99% of the initial C10 alkane in both pine and aspen media but did not affect the amounts of the C14 alkane or phenanthrene. Differential gene expression analysis identified 119 genes with ≥ log2(2-fold) greater expression in one or more treatment comparisons. Six genes were significantly upregulated in media containing oil; these genes included three enzymes with potential roles in xenobiotic biotransformation. Carbohydrate metabolism genes showing differential expression significantly accumulated transcripts on aspen vs. pine substrates, perhaps reflecting white-rot adaptations to growth on hardwood substrates. The mechanisms by which P. strigosozonata may degrade complex oil compounds remain obscure, but degradation results of the 180-day cultures suggest that diverse white-rot fungi have promise for bioremediation of petroleum fuels. PMID:26111162

  18. Enhanced saccharification of biologically pretreated wheat straw for ethanol production.

    PubMed

    López-Abelairas, M; Lu-Chau, T A; Lema, J M

    2013-02-01

    The biological pretreatment of lignocellulosic biomass with white-rot fungi for the production of bioethanol is an alternative to the most used physico-chemical processes. After biological treatment, a solid composed of cellulose, hemicellulose, and lignin-this latter is with a composition lower than that found in the initial substrate-is obtained. On the contrary, after applying physico-chemical methods, most of the hemicellulose fraction is solubilized, while cellulose and lignin fractions remain in the solid. The optimization of the combination of cellulases and hemicellulases required to saccharify wheat straw pretreated with the white-rot fungus Irpex lacteus was carried out in this work. The application of the optimal dosage made possible the increase of the sugar yield from 33 to 54 %, and at the same time the reduction of the quantity of enzymatic mixture in 40 %, with respect to the initial dosage. The application of a pre-hydrolysis step with xylanases was also studied. PMID:23306886

  19. In silico exploration of novel phytoligands against probable drug target of Clostridium tetani.

    PubMed

    Skariyachan, Sinosh; Prakash, Nisha; Bharadwaj, Navya

    2012-12-01

    Though tetanus is an old disease with well known medicines, its complications are still a serious issue worldwide. Tetanus is mainly due to a powerful neurotoxin, tetanolysin-O, produced by a Gram positive anaerobic bacterium, Clostridium tetani. The toxin has a thiol-activated cytolysin which causes lysis of human platelets, lysosomes and a variety of subcellular membranes. The existing therapy seems to have challenged as available vaccines are not so effective and the bacteria developed resistance to many drugs. Computer aided approach is a novel platform to screen drug targets and design potential inhibitors. The three dimensional structure of the toxin is essential for structure based drug design. But the structure of tetanolysin-O is not available in its native form. Moreover, the interaction and pharmacological activities of current drugs against tetanolysin-O is not clear. Hence, there is need for three dimensional model of the toxin. The model was generated by homology modeling using crystal structure of perfringolysin-O, chain-A (PDB ID: 1PFO) as the template. The modeled structure has 22.7% α helices, 27.51% β sheets and 41.75% random coils. A thiol-activated cytolysin was predicted in the region of 105 to 1579, which acts as a functional domain of the toxin. The hypothetical model showed the backbone root mean square deviation (RMSD) value of 0.6 Å and the model was validated by ProCheck. The Ramachandran plot of the model accounts for 92.3% residues in the most allowed region. The model was further refined by various tools and deposited to Protein Model Database (PMDB ID: PM0077550). The model was used as the drug target and the interaction of various lead molecules with protein was studied by molecular docking. We have selected phytoligands based on literatures and pharmacophoric studies. The efficiency of herbal compounds and chemical leads was compared. Our study concluded that herbal derivatives such as berberine (7, 8, 13, 13a-tetradehydro-9

  20. An initial examination of the potential role of T-cell immunity in protection against feline immunodeficiency virus (FIV) infection.

    PubMed

    Aranyos, Alek M; Roff, Shannon R; Pu, Ruiyu; Owen, Jennifer L; Coleman, James K; Yamamoto, Janet K

    2016-03-14

    The importance of vaccine-induced T-cell immunity in conferring protection with prototype and commercial FIV vaccines is still unclear. Current studies performed adoptive transfer of T cells from prototype FIV-vaccinated cats to partial-to-complete feline leukocyte antigen (FLA)-matched cats a day before either homologous FIVPet or heterologous-subtype pathogenic FIVFC1 challenge. Adoptive-transfer (A-T) conferred a protection rate of 87% (13 of 15, p < 0.001) against FIVPet using the FLA-matched T cells, whereas all 12 control cats were unprotected. Furthermore, A-T conferred protection rate of 50% (6 of 12, p<0.023) against FIVFC1 using FLA-matched T cells, whereas all 8 control cats were unprotected. Transfer of FLA-matched T and B cells demonstrated that T cells are needed to confer A-T protection. In addition, complete FLA-matching and addition of T-cell numbers > 13 × 10(6) cells were required for A-T protection against FIVFC1 strain, reported to be a highly pathogenic virus resistant to vaccine-induced neutralizing-antibodies. The addition of FLA-matched B cells alone was not protective. The poor quality of the anti-FIV T-cell immunity induced by the vaccine likely contributed to the lack of protection in an FLA-matched recipient against FIVFC1. The quality of the immune response was determined by the presence of high mRNA levels of cytolysin (perforin) and cytotoxins (granzymes A, B, and H) and T helper-1 cytokines (interferon-γ [IFNγ] and IL2). Increased cytokine, cytolysin and cytotoxin production was detected in the donors which conferred protection in A-T studies. In addition, the CD4(+) and CD8(+) T-cell proliferation and/or IFNγ responses to FIV p24 and reverse transcriptase increased with each year in cats receiving 1X-3X vaccine boosts over 4 years. These studies demonstrate that anti-FIV T-cell immunity induced by vaccination with a dual-subtype FIV vaccine is essential for prophylactic protection against AIDS lentiviruses such as FIV and

  1. Comparison of Staphylococcus aureus strains for ability to cause infective endocarditis and lethal sepsis in rabbits.

    PubMed

    Spaulding, Adam R; Satterwhite, Erin A; Lin, Ying-Chi; Chuang-Smith, Olivia N; Frank, Kristi L; Merriman, Joseph A; Schaefers, Matthew M; Yarwood, Jeremy M; Peterson, Marnie L; Schlievert, Patrick M

    2012-01-01

    Staphylococcus aureus is a major cause of infective endocarditis (IE) and sepsis. Both methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) strains cause these illnesses. Common S. aureus strains include pulsed-field gel electrophoresis (PFGE) types USA200, 300, and 400 types where we hypothesize that secreted virulence factors contribute to both IE and sepsis. Rabbit cardiac physiology is considered similar to humans, and rabbits exhibit susceptibility to S. aureus superantigens (SAgs) and cytolysins. As such, rabbits are an excellent model for studying IE and sepsis, which over the course of four days develop IE vegetations and/or fatal septicemia. We examined the ability of MRSA and MSSA strains (4 USA200, 2 USA300, 2 USA400, and three additional common strains, FRI1169, Newman, and COL) to cause vegetations and lethal sepsis in rabbits. USA200, TSST-1(+) strains that produce only low amounts of α-toxin, exhibited modest LD(50) in sepsis (1 × 10(8) - 5 × 10(8)) colony-forming units (CFUs), and 3/4 caused significant IE. USA200 strain MNPE, which produces high-levels of α-toxin, was both highly lethal (LD(50) 5 × 10(6) CFUs) and effective in causing IE. In contrast, USA300 strains were highly effective in causing lethal sepsis (LD(50)s 1 × 10(6) and 5 × 10(7) CFUs) but were minimally capable of causing IE. Strain Newman, which is phylogenetically related to USA300 strains, was not highly lethal (LD(50) of 2 × 10(9) CFUs) and was effective in causing IE. USA400 strains were both highly lethal (LD(50)s of 1 × 10(7) and 5 × 10(7) CFUs) and highly effective causes of IE. The menstrual TSS isolate FRI1169, that is TSST-1(+), produces high-levels of α-toxin, but is not USA200, was both highly lethal and effective in causing IE. Additional studies showed that phenol soluble modulins (PSMs) produced by FRI1169 were important for sepsis but did not contribute to IE. Our studies show that these clonal groups of S. aureus differ in abilities to cause IE

  2. Recognizing filamentous basidiomycetes as agents of human disease: A review.

    PubMed

    Chowdhary, Anuradha; Kathuria, Shallu; Agarwal, Kshitij; Meis, Jacques F

    2014-11-01

    Filamentous basidiomycetes (BM) are common environmental fungi that have recently emerged as important human pathogens, inciting a wide array of clinical manifestations that include allergic and invasive diseases. We reviewed 218 reported global cases of BM fungi. The most common etiologic agent was Schizophyllum commune in 52.3% (114/218) of the cases followed by Hormographiella aspergillata (n = 13; 5.9%), Ceriporia lacerata (n = 11; 5%), and, rarely, Volvariella volvacea, Inonotus tropicalis, Irpex lacteus, Phellinus undulates, Perenniporia species, Bjerkandera adusta, Sporotrichum pruinosum, Phanerochaete steroids, and Cyclomyces tabacinus. These fungi are present in the environment as gilled mushrooms, shelf fungi, and bracket fungi. However, in clinical settings, they usually present as nonsporulating white moulds that are difficult to identify. Moreover, the GenBank database of these fungi is limited. Regarding the country-wise distribution of cases, Japan topped the list with about 43% (n = 94) of globally reported cases, followed by India (57; 26%), the United States (4%), Austria (3.2%), Iran (3.2%), France (2.8%), and the remaining one-third from 16 other countries. The respiratory tract was the most commonly afflicted site (n = 71), with the majority of the cases (42; 59.1%) being allergic in etiology and comprising 34 cases of allergic bronchopulmonary mycosis. Also, B. adusta has been implicated in a recently described clinical entity, that is, fungus associated chronic cough, reported exclusively from Japan. BM fungi-incited diseases are currently underdiagnosed due to lack of awareness and expertise, warranting comprehensive epidemiological and susceptibility studies to determine their prevalence and to predict a more appropriate therapy. PMID:25202126

  3. Basidiomycete DyPs: Genomic diversity, structural-functional aspects, reaction mechanism and environmental significance.

    PubMed

    Linde, Dolores; Ruiz-Dueñas, Francisco J; Fernández-Fueyo, Elena; Guallar, Victor; Hammel, Kenneth E; Pogni, Rebecca; Martínez, Angel T

    2015-05-15

    The first enzyme with dye-decolorizing peroxidase (DyP) activity was described in 1999 from an arthroconidial culture of the fungus Bjerkandera adusta. However, the first DyP sequence had been deposited three years before, as a peroxidase gene from a culture of an unidentified fungus of the family Polyporaceae (probably Irpex lacteus). Since the first description, fewer than ten basidiomycete DyPs have been purified and characterized, but a large number of sequences are available from genomes. DyPs share a general fold and heme location with chlorite dismutases and other DyP-type related proteins (such as Escherichia coli EfeB), forming the CDE superfamily. Taking into account the lack of an evolutionary relationship with the catalase-peroxidase superfamily, the observed heme pocket similarities must be considered as a convergent type of evolution to provide similar reactivity to the enzyme cofactor. Studies on the Auricularia auricula-judae DyP showed that high-turnover oxidation of anthraquinone type and other DyP substrates occurs via long-range electron transfer from an exposed tryptophan (Trp377, conserved in most basidiomycete DyPs), whose catalytic radical was identified in the H2O2-activated enzyme. The existence of accessory oxidation sites in DyP is suggested by the residual activity observed after site-directed mutagenesis of the above tryptophan. DyP degradation of substituted anthraquinone dyes (such as Reactive Blue 5) most probably proceeds via typical one-electron peroxidase oxidations and product breakdown without a DyP-catalyzed hydrolase reaction. Although various DyPs are able to break down phenolic lignin model dimers, and basidiomycete DyPs also present marginal activity on nonphenolic dimers, a significant contribution to lignin degradation is unlikely because of the low activity on high redox-potential substrates. PMID:25637654

  4. In vivo administration of the frog skin peptide frenatin 2.1S induces immunostimulatory phenotypes of mouse mononuclear cells.

    PubMed

    Pantic, Jelena M; Radosavljevic, Gordana D; Jovanovic, Ivan P; Arsenijevic, Nebojsa N; Conlon, J Michael; Lukic, Miodrag L

    2015-09-01

    Host-defense peptides secreted by epithelial cells exhibit cytotoxic and immunoregulatory effects in order to protect the organism against invading microorganisms. Antimicrobial peptides derived from frog skin display both immunostimulatory and immunosuppressive actions as demonstrated by in vitro cytokine production by macrophages. Frenatin 2.1S, first isolated from skin secretions of the frog, Sphaenorhynchus lacteus (Hylidae), enhances the in vitro production of pro-inflammatory IL-1β, TNF-α and IL-23 by mouse peritoneal cells. In order to test whether the immunostimulatory action of frenatin 2.1S may be reproduced in vivo, effects of intraperitoneal injections of this peptide on mononuclear cells in the peritoneum and spleen were determined 24h after administration. The data indicate that frenatin 2.1S enhances the activation state and homing capacity of Th1 type lymphocytes and NKT cells in the mouse peritoneal cavity, as evaluated by increased expression of early activation marker CD69 among T and NKT cells and chemokine receptor CXCR3 among T cells. Frenatin 2.1S significantly increases the percentage of (F4/80(+)CD11c(+)CD206(+)) pro-inflammatory M1 macrophages and enhances the expression of MHC class II molecules on F4/80(+)CD11c(+) macrophages in the mouse peritoneal cavity. Additionally, injection of frenatin 2.1S, in the presence or absence of lipopolysaccharide, increases the percentage of peritoneal B cells of the (CD19(+)CD11b(+)CD5(+)) B1a phenotype thus contributing to an inflammatory milieu. We suggest that the immunostimulatory effect of frenatin 2.1S may have therapeutic relevance in disease states, such as certain types of cancer, in which an enhanced inflammatory response may be beneficial. PMID:25861850

  5. Influence of Hyphal Inoculum potential on the Competitive Success of Fungi Colonizing Wood.

    PubMed

    Song, Zewei; Vail, Andrew; Sadowsky, Michael J; Schilling, Jonathan S

    2015-05-01

    The relative amounts of hyphal inoculum in forest soils may determine the capacity for fungi to compete with and replace early colonizers of wood in ground contact. Our aim in this study was to test the flexibility of priority effects (colonization timing) by varying the timing of inoculum introduction (i.e., precolonization) and amount of inoculum (i.e., inoculum potential). We controlled these variables in soil-block microcosms using fungi with known competitive outcomes in similar conditions, tracking isolate-specific fungal biomass, and residue physiochemistry over time. In the precolonization trial (experiment I), a brown rot fungus Gloeophyllum trabeum was given 1, 3, or 5 weeks to precolonize wood blocks (oak, birch, pine, and spruce) prior the introduction of a white rot fungus, Irpex lacteus, a more aggressive colonizer in this set-up. In the inoculum potential trial (experiment II), the fungi were inoculated simultaneously, but with eightfold higher brown rot inoculum than that of experiment I. As expected, longer precolonization duration increased the chance for the less-competitive brown rot fungus to outcompete its white rot opponent. Higher brown rot fungal inoculum outside of the wood matrix also resulted in competitive success for the brown rot isolate in most cases. These temporal shifts in fungal dominance were detectable in a 'community snapshot' as isolate-specific quantitative PCR, but also as functionally-relevant consequences of wood rot type, including carbohydrate depolymerization and pH. These results from a controlled system reinforce fungal-fungal interaction and suggest that relative inoculum availability beyond the wood matrix (i.e., soils) might regulate the duration of priority effects and shift the functional trajectory of wood decomposition. PMID:25750000

  6. Presence of extracellular NAD(+) and NADH in cultures of wood-degrading fungi.

    PubMed

    Kido, Ryuta; Takeeda, Midori; Manabe, Mitsuhiro; Miyagawa, Yutaka; Itakura, Shuji; Tanaka, Hiromi

    2015-01-01

    Our previous studies indicated that extracellular glycoproteins produced by some white-rot and brown-rot basidiomycetous fungi reduce Fe(III) to Fe(II) and O2 to H2O2 and produce hydroxyl radicals. The continuous generation of hydroxyl radicals requires a constant supply of O2 and an electron donor for the reduction of oxidized forms of the glycoproteins to the reduced forms. However, electron donors for this reaction, such as NADH, have not been identified. In this study, the amounts of the extracellular pyridine coenzymes, NAD(+) and NADH, were measured in agar cultures of four white-rot fungi, one brown-rot fungus, and three soft-rot fungi. The sums of NAD(+) and NADH detected in wood-containing cultures of all five basidiomycetes were greater than those in glucose cultures. The amounts of NAD(+) were higher than those of NADH in all wood-containing cultures except that of Irpex lacteus, and NAD(+) was greater than NADH in all glucose cultures except that of Fomitopsis palustris. Significant amounts of pyridine coenzymes were present in glucose and wood-containing cultures of the three soft-rot fungi. The non-wood-degrading fungus, Penicillium funiculosum, did not produce NAD(+) or NADH in either glucose or wood-containing cultures. The extracellular pyridine coenzyme levels were relatively high compared to the rates of extracellular hydroxyl radical generation in wood-degrading fungal cultures. Thus, white-, brown-, and soft-rot fungi produce pyridine coenzymes that could serve as electron donors for the production of hydroxyl radicals during wood degradation. PMID:26133508

  7. Enterococcus faecalis from Food, Clinical Specimens, and Oral Sites: Prevalence of Virulence Factors in Association with Biofilm Formation

    PubMed Central

    Anderson, Annette C.; Jonas, Daniel; Huber, Ingrid; Karygianni, Lamprini; Wölber, Johan; Hellwig, Elmar; Arweiler, Nicole; Vach, Kirstin; Wittmer, Annette; Al-Ahmad, Ali

    2016-01-01

    Enterococci have gained significance as the cause of nosocomial infections; they occur as food contaminants and have also been linked to dental diseases. E. faecalis has a great potential to spread virulence as well as antibiotic resistance genes via horizontal gene transfer. The integration of food-borne enterococci into the oral biofilm in-vivo has been observed. Therefore, we investigated the virulence determinants and antibiotic resistance of 97 E. faecalis isolates from the oral cavity, food, and clinical specimens. In addition, phenotypic expression of gelatinase and cytolysin were tested, in-vitro biofilm formation was quantified and isolates were compared for strain relatedness via pulsed field gel electrophoresis (PFGE). Each isolate was found to possess two or more virulence genes, most frequently gelE, efaA, and asa1. Notably, plaque/saliva isolates possessed the highest abundance of virulence genes, the highest levels of phenotypic gelatinase and hemolysin activity and concurrently a high ability to form biofilm. The presence of asa1 was associated with biofilm formation. The biofilm formation capacity of clinical and plaque/saliva isolates was considerably higher than that of food isolates and they also showed similar antibiotic resistance patterns. These results indicate that the oral cavity can constitute a reservoir for virulent E. faecalis strains possessing antibiotic resistance traits and at the same time distinct biofilm formation capabilities facilitating exchange of genetic material. PMID:26793174

  8. A toxin-based probe reveals cytoplasmic exposure of Golgi sphingomyelin.

    PubMed

    Bakrac, Biserka; Kladnik, Ales; Macek, Peter; McHaffie, Gavin; Werner, Andreas; Lakey, Jeremy H; Anderluh, Gregor

    2010-07-16

    Although sphingomyelin is an important cellular lipid, its subcellular distribution is not precisely known. Here we use a sea anemone cytolysin, equinatoxin II (EqtII), which specifically binds sphingomyelin, as a new marker to detect cellular sphingomyelin. A purified fusion protein composed of EqtII and green fluorescent protein (EqtII-GFP) binds to the SM rich apical membrane of Madin-Darby canine kidney (MDCK) II cells when added exogenously, but not to the SM-free basolateral membrane. When expressed intracellularly within MDCK II cells, EqtII-GFP colocalizes with markers for Golgi apparatus and not with those for nucleus, mitochondria, endoplasmic reticulum or plasma membrane. Colocalization with the Golgi apparatus was confirmed by also using NIH 3T3 fibroblasts. Moreover, EqtII-GFP was enriched in cis-Golgi compartments isolated by gradient ultracentrifugation. The data reveal that EqtII-GFP is a sensitive probe for membrane sphingomyelin, which provides new information on cytosolic exposure, essential to understand its diverse physiological roles. PMID:20463009

  9. The Influence of Natural Lipid Asymmetry upon the Conformation of a Membrane-inserted Protein (Perfringolysin O)*

    PubMed Central

    Lin, Qingqing; London, Erwin

    2014-01-01

    Eukaryotic membrane proteins generally reside in membrane bilayers that have lipid asymmetry. However, in vitro studies of the impact of lipids upon membrane proteins are generally carried out in model membrane vesicles that lack lipid asymmetry. Our recently developed method to prepare lipid vesicles with asymmetry similar to that in plasma membranes and with controlled amounts of cholesterol was used to investigate the influence of lipid composition and lipid asymmetry upon the conformational behavior of the pore-forming, cholesterol-dependent cytolysin perfringolysin O (PFO). PFO conformational behavior in asymmetric vesicles was found to be distinct both from that in symmetric vesicles with the same lipid composition as the asymmetric vesicles and from that in vesicles containing either only the inner leaflet lipids from the asymmetric vesicles or only the outer leaflet lipids from the asymmetric vesicles. The presence of phosphatidylcholine in the outer leaflet increased the cholesterol concentration required to induce PFO binding, whereas phosphatidylethanolamine and phosphatidylserine in the inner leaflet of asymmetric vesicles stabilized the formation of a novel deeply inserted conformation that does not form pores, even though it contains transmembrane segments. This conformation may represent an important intermediate stage in PFO pore formation. These studies show that lipid asymmetry can strongly influence the behavior of membrane-inserted proteins. PMID:24398685

  10. Intermedilysin, a novel cytotoxin specific for human cells secreted by Streptococcus intermedius UNS46 isolated from a human liver abscess.

    PubMed Central

    Nagamune, H; Ohnishi, C; Katsuura, A; Fushitani, K; Whiley, R A; Tsuji, A; Matsuda, Y

    1996-01-01

    A novel cytotoxin (intermedilysin) specific for human cells was identified as a cytolytic factor of Streptococcus intermedius UNS46 isolated from a human liver abscess. Intermedilysin caused human cell death with membrane blebs. Intermedilysin was purified from UNS46 culture medium by means of gel filtration and hydrophobic chromatography. The purified toxin was resolved into major and minor bands of 54 and 53 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These proteins reacted with an antibody against intermedilysin. Five internal peptide fragments of intermedilysin were sequenced and found to have 42 to 71% homology with the thiol-activated cytotoxin pneumolysin. However, the action of intermedilysin differed from that of thiol-activated cytotoxins, especially in terms of a lack of activation by dithiothreitol and resistance to treatments with N-ethylmaleimide and 5,5'-dithio-bis-(2-nitrobenzoic acid), although cholesterol inhibited the toxin activity. Intermedilysin was potently hemolytic on human erythrocytes but was 100-fold less effective on chimpanzee and cynomolgus monkey erythrocytes. Intermedilysin was not hemolytic in nine other animal species tested. Since human erythrocytes treated with trypsin were far less sensitive to intermedilysin than were the intact cells, a cell membrane protein(s) may participate in the intermedilysin action. These data demonstrated that intermedilysin is distinguishable from all known bacterial cytolysins. PMID:8757839

  11. Stonefish toxin defines an ancient branch of the perforin-like superfamily

    PubMed Central

    Ellisdon, Andrew M.; Reboul, Cyril F.; Huynh, Kitmun; Oellig, Christine A.; Winter, Kelly L.; Hodgson, Wayne C.; Seymour, Jamie; Dearden, Peter K.; Tweten, Rodney K.; Whisstock, James C.; McGowan, Sheena

    2015-01-01

    The lethal factor in stonefish venom is stonustoxin (SNTX), a heterodimeric cytolytic protein that induces cardiovascular collapse in humans and native predators. Here, using X-ray crystallography, we make the unexpected finding that SNTX is a pore-forming member of an ancient branch of the Membrane Attack Complex-Perforin/Cholesterol-Dependent Cytolysin (MACPF/CDC) superfamily. SNTX comprises two homologous subunits (α and β), each of which comprises an N-terminal pore-forming MACPF/CDC domain, a central focal adhesion-targeting domain, a thioredoxin domain, and a C-terminal tripartite motif family-like PRY SPla and the RYanodine Receptor immune recognition domain. Crucially, the structure reveals that the two MACPF domains are in complex with one another and arranged into a stable early prepore-like assembly. These data provide long sought after near-atomic resolution insights into how MACPF/CDC proteins assemble into prepores on the surface of membranes. Furthermore, our analyses reveal that SNTX-like MACPF/CDCs are distributed throughout eukaryotic life and play a broader, possibly immune-related function outside venom. PMID:26627714

  12. Safety assessment and probiotic evaluation of Enterococcus faecium YF5 isolated from sourdough.

    PubMed

    Tan, Qianglai; Xu, Hengyi; Aguilar, Zoraida P; Peng, Shanshan; Dong, Suqin; Wang, Baogui; Li, Ping; Chen, Tingtao; Xu, Feng; Wei, Hua

    2013-04-01

    Enterococcus faecium YF5, a strain previously isolated from sourdough, was assessed for safety and probiotic potential. Its virulence and antibiotic resistant phenotypes (cytolysin and gelatinase production, antibiotic susceptibility) and genes (cylA, gelE, ace, agg, esp, and vanA) were surveyed. Results indicated that the tested virulence determinants were nontoxic. In addition, E. faecium YF5 was sensitive to 3 antibiotics such as amoxicillin, vancomycin, and chloramphenicol. Furthermore, results of in vivo animal acute oral toxicity of E. faecium YF5 studies were similar to the control group that indicated no abnormalities. In addition, E. faecium YF5 stably survived in low pH, bile salts, gastric, and intestinal fluids in vitro. Moreover, E. faecium YF5 was found to adhere to human colon cancer cell line HT-29 at 3.39 (±0.67) × 10(5) CFU/mL. When cocultured with pathogenic organisms (Enterobacter sakazakii CMCC45402, Escherichia coli CMCC44102, enterohemorrhage Escherichia coli O157: H7 CMCC44828, Salmonella Typhimurium CMCC50071, Shigella flexneri 301, and Shigella sonnei ATCC 29930) and 2 gram-positive strains (Listeria monocytogenes CMCC54001 and Staphylococcus aureus CMCC 26003), it inhibited these foodborne pathogens with exception of S. aureus. Therefore, E. faecium YF5 can be regarded as a safe strain and it may be used as a probiotic preparation or for microecologics. PMID:23488799

  13. Crystal structure of enterococcus faecalis sly A-like transcriptional factor.

    SciTech Connect

    Wu, R.; Zhang, R.; Zagnitko, O.; Dementieva, I.; Maltsev, N.; Watson, J. D.; Laskowski, R.; Gornicki, P.; Joachimiak, A.; Univ. of Chicago; European Bioinformatics Inst.

    2003-05-30

    The crystal structure of a SlyA transcriptional regulator at 1.6 {angstrom} resolution is presented, and structural relationships between members of the MarR/SlyA family are discussed. The SlyA family, which includes SlyA, Rap, Hor, and RovA proteins, is widely distributed in bacterial and archaeal genomes. Current evidence suggests that SlyA-like factors act as repressors, activators, and modulators of gene transcription. These proteins have been shown to up-regulate the expression of molecular chaperones, acid-resistance proteins, and cytolysin, and down-regulate several biosynthetic enzymes. The structure of SlyA from Enterococcus faecalis, determined as a part of an ongoing structural genomics initiative (www.mcsg.anl.gov), revealed the same winged helix DNA-binding motif that was recently found in the MarR repressor from Escherichia coli and the MexR repressor from Pseudomonas aeruginosa, a sequence homologue of MarR. Phylogenetic analysis of the MarR/SlyA family suggests that Sly is placed between the SlyA and MarR subfamilies and shows significant sequence similarity to members of both subfamilies.

  14. Structural basis of complement membrane attack complex formation.

    PubMed

    Serna, Marina; Giles, Joanna L; Morgan, B Paul; Bubeck, Doryen

    2016-01-01

    In response to complement activation, the membrane attack complex (MAC) assembles from fluid-phase proteins to form pores in lipid bilayers. MAC directly lyses pathogens by a 'multi-hit' mechanism; however, sublytic MAC pores on host cells activate signalling pathways. Previous studies have described the structures of individual MAC components and subcomplexes; however, the molecular details of its assembly and mechanism of action remain unresolved. Here we report the electron cryo-microscopy structure of human MAC at subnanometre resolution. Structural analyses define the stoichiometry of the complete pore and identify a network of interaction interfaces that determine its assembly mechanism. MAC adopts a 'split-washer' configuration, in contrast to the predicted closed ring observed for perforin and cholesterol-dependent cytolysins. Assembly precursors partially penetrate the lipid bilayer, resulting in an irregular β-barrel pore. Our results demonstrate how differences in symmetric and asymmetric components of the MAC underpin a molecular basis for pore formation and suggest a mechanism of action that extends beyond membrane penetration. PMID:26841837

  15. Measuring kinetic drivers of pneumolysin pore structure.

    PubMed

    Gilbert, Robert J C; Sonnen, Andreas F-P

    2016-05-01

    Most membrane attack complex-perforin/cholesterol-dependent cytolysin (MACPF/CDC) proteins are thought to form pores in target membranes by assembling into pre-pore oligomers before undergoing a pre-pore to pore transition. Assembly during pore formation is into both full rings of subunits and incomplete rings (arcs). The balance between arcs and full rings is determined by a mechanism dependent on protein concentration in which arc pores arise due to kinetic trapping of the pre-pore forms by the depletion of free protein subunits during oligomerization. Here we describe the use of a kinetic assay to study pore formation in red blood cells by the MACPF/CDC pneumolysin from Streptococcus pneumoniae. We show that cell lysis displays two kinds of dependence on protein concentration. At lower concentrations, it is dependent on the pre-pore to pore transition of arc oligomers, which we show to be a cooperative process. At higher concentrations, it is dependent on the amount of pneumolysin bound to the membrane and reflects the affinity of the protein for its receptor, cholesterol. A lag occurs before cell lysis begins; this is dependent on oligomerization of pneumolysin. Kinetic dissection of cell lysis by pneumolysin demonstrates the capacity of MACPF/CDCs to generate pore-forming oligomeric structures of variable size with, most likely, different functional roles in biology. PMID:26906727

  16. The 2003 ASBMB-Avanti Award in Lipids Address: Applications of novel synthetic lipids to biological problems.

    PubMed

    Bittman, Robert

    2004-05-01

    This paper is an overview of the 2003 Avanti Award in Lipids address that was presented by Robert Bittman at the American Society for Biochemistry and Molecular Biology (ASBMB) Annual Meeting held in San Diego, CA in conjunction with meetings of five other FASEB Societies, April 15, 2003. The theme of the lecture is: "How can the chemical synthesis of unnatural lipids provide insights into problems ranging from cell biology to biophysics?" The following examples are presented: (1) novel ceramide analogs as experimental anticancer agents, (2) photoactivatable sphingosine 1-phosphate analogs as probes of protein targets of this bioactive lipid, (3) a 13C-enriched cerebroside as a quantitative probe of glycosphingolipid (GSL) transbilayer distribution in bilayers with and without sphingomyelin, (4) cis and trans unsaturated sphingomyelin analogs as modulators of the existence of cholesterol-enriched microdomains (rafts) that may facilitate fusion of alphaviruses with target membranes, (5) ceramide as an indirect enhancer of the permeabilization of membranes induced by cholesterol-specific cytolysins, (6) fluorescent GSL analogs of widely disparate structure as probes of the molecular features responsible for the selective internalization of GSLs in caveolae of living mammalian cells, (7) enantiomeric lysophosphatidic acid (LPA) analogs as probes of receptor subtypes that mediate LPA signaling, and (8) phosphonocholine analogs of the antitumor ether lipid ET-18-OCH3 as tools for discerning the primary targets that are critical for cytotoxic activity in tumor cells. PMID:15081855

  17. Preclinical evaluation of VAX-IP, a novel bacterial minicell-based biopharmaceutical for nonmuscle invasive bladder cancer.

    PubMed

    Tsuji, Shingo; Chen, Xuguang; Hancock, Bryan; Hernandez, Veronica; Visentin, Barbara; Reil, Katherine; Sabbadini, Roger; Giacalone, Matthew; Godbey, W T

    2016-01-01

    The development of new therapies that can prevent recurrence and progression of nonmuscle invasive bladder cancer remains an unmet clinical need. The continued cost of monitoring and treatment of recurrent disease, along with its high prevalence and incidence rate, is a strain on healthcare economics worldwide. The current work describes the characterization and pharmacological evaluation of VAX-IP as a novel bacterial minicell-based biopharmaceutical agent undergoing development for the treatment of nonmuscle invasive bladder cancer and other oncology indications. VAX-IP minicells selectively target two oncology-associated integrin heterodimer subtypes to deliver a unique bacterial cytolysin protein toxin, perfringolysin O, specifically to cancer cells, rapidly killing integrin-expressing murine and human urothelial cell carcinoma cells with a unique tumorlytic mechanism. The in vivo pharmacological evaluation of VAX-IP minicells as a single agent administered intravesically in two clinically relevant variations of a syngeneic orthotopic model of superficial bladder cancer results in a significant survival advantage with 28.6% (P = 0.001) and 16.7% (P = 0.003) of animals surviving after early or late treatment initiation, respectively. The results of these preclinical studies warrant further nonclinical and eventual clinical investigation in underserved nonmuscle invasive bladder cancer patient populations where complete cures are achievable. PMID:27119118

  18. Giant MACPF/CDC pore forming toxins: A class of their own.

    PubMed

    Reboul, Cyril F; Whisstock, James C; Dunstone, Michelle A

    2016-03-01

    Pore Forming Toxins (PFTs) represent a key mechanism for permitting the passage of proteins and small molecules across the lipid membrane. These proteins are typically produced as soluble monomers that self-assemble into ring-like oligomeric structures on the membrane surface. Following such assembly PFTs undergo a remarkable conformational change to insert into the lipid membrane. While many different protein families have independently evolved such ability, members of the Membrane Attack Complex PerForin/Cholesterol Dependent Cytolysin (MACPF/CDC) superfamily form distinctive giant β-barrel pores comprised of up to 50 monomers and up to 300Å in diameter. In this review we focus on recent advances in understanding the structure of these giant MACPF/CDC pores as well as the underlying molecular mechanisms leading to their formation. Commonalities and evolved variations of the pore forming mechanism across the superfamily are discussed. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale. PMID:26607011

  19. Incomplete pneumolysin oligomers form membrane pores.

    PubMed

    Sonnen, Andreas F-P; Plitzko, Jürgen M; Gilbert, Robert J C

    2014-01-01

    Pneumolysin is a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming proteins that are produced as water-soluble monomers or dimers, bind to target membranes and oligomerize into large ring-shaped assemblies comprising approximately 40 subunits and approximately 30 nm across. This pre-pore assembly then refolds to punch a large hole in the lipid bilayer. However, in addition to forming large pores, pneumolysin and other CDCs form smaller lesions characterized by low electrical conductance. Owing to the observation of arc-like (rather than full-ring) oligomers by electron microscopy, it has been hypothesized that smaller oligomers explain smaller functional pores. To investigate whether this is the case, we performed cryo-electron tomography of pneumolysin oligomers on model lipid membranes. We then used sub-tomogram classification and averaging to determine representative membrane-bound low-resolution structures and identified pre-pores versus pores by the presence of membrane within the oligomeric curve. We found pre-pore and pore forms of both complete (ring) and incomplete (arc) oligomers and conclude that arc-shaped oligomeric assemblies of pneumolysin can form pores. As the CDCs are evolutionarily related to the membrane attack complex/perforin family of proteins, which also form variably sized pores, our findings are of relevance to that class of proteins as well. PMID:24759615

  20. Lipidome and Transcriptome Profiling of Pneumolysin Intoxication Identifies Networks Involved in Statin-Conferred Protection of Airway Epithelial Cells

    PubMed Central

    Statt, Sarah; Ruan, Jhen-Wei; Huang, Chih-Ting; Wu, Reen; Kao, Cheng-Yuan

    2015-01-01

    Pneumonia remains one of the leading causes of death in both adults and children worldwide. Despite the adoption of a wide variety of therapeutics, the mortality from community-acquired pneumonia has remained relatively constant. Although viral and fungal acute airway infections can result in pneumonia, bacteria are the most common cause of community-acquired pneumonia, with Streptococcus pneumoniae isolated in nearly 50% of cases. Pneumolysin is a cholesterol-dependent cytolysin or pore-forming toxin produced by Streptococcus pneumonia and has been shown to play a critical role in bacterial pathogenesis. Airway epithelium is the initial site of many bacterial contacts and its barrier and mucosal immunity functions are central to infectious lung diseases. In our studies, we have shown that the prior exposure to statins confers significant resistance of airway epithelial cells to the cytotoxicity of pneumolysin. We decided to take this study one step further, assessing changes in both the transcriptome and lipidome of human airway epithelial cells exposed to toxin, statin or both. Our current work provides the first global view in human airway epithelial cells of both the transcriptome and the lipid interactions that result in cellular protection from pneumolysin. PMID:26023727

  1. New Cyt-like δ-endotoxins from Dickeya dadantii: structure and aphicidal activity.

    PubMed

    Loth, Karine; Costechareyre, Denis; Effantin, Géraldine; Rahbé, Yvan; Condemine, Guy; Landon, Céline; da Silva, Pedro

    2015-01-01

    In the track of new biopesticides, four genes namely cytA, cytB, cytC and cytD encoding proteins homologous to Bacillus thuringiensis (Bt) Cyt toxins have been identified in the plant pathogenic bacteria Dickeya dadantii genome. Here we show that three Cyt-like δ-endotoxins from D. dadantii (CytA, CytB and CytC) are toxic to the pathogen of the pea aphid Acyrthosiphon pisum in terms of both mortality and growth rate. The phylogenetic analysis of the comprehensive set of Cyt toxins available in genomic databases shows that the whole family is of limited taxonomic occurrence, though in quite diverse microbial taxa. From a structure-function perspective the 3D structure of CytC and its backbone dynamics in solution have been determined by NMR. CytC adopts a cytolysin fold, structurally classified as a Cyt2-like protein. Moreover, the identification of a putative lipid binding pocket in CytC structure, which has been probably maintained in most members of the Cyt-toxin family, could support the importance of this lipid binding cavity for the mechanism of action of the whole family. This integrative approach provided significant insights into the evolutionary and functional history of D. dadantii Cyt toxins, which appears to be interesting leads for biopesticides. PMID:25740111

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

    PubMed

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

    2015-11-01

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

  3. Pneumococcal Pneumolysin Induces DNA Damage and Cell Cycle Arrest.

    PubMed

    Rai, Prashant; He, Fang; Kwang, Jimmy; Engelward, Bevin P; Chow, Vincent T K

    2016-01-01

    Streptococcus pneumoniae produces pneumolysin toxin as a key virulence factor against host cells. Pneumolysin is a cholesterol-dependent cytolysin (CDC) toxin that forms lytic pores in host membranes and mediates pneumococcal disease pathogenesis by modulating inflammatory responses. Here, we show that pneumolysin, which is released during bacterial lysis, induces DNA double strand breaks (DSBs), as indicated by ataxia telangiectasia mutated (ATM)-mediated H2AX phosphorylation (γH2AX). Pneumolysin-induced γH2AX foci recruit mediator of DNA damage checkpoint 1 (MDC1) and p53 binding protein 1 (53BP1), to sites of DSBs. Importantly, results show that toxin-induced DNA damage precedes cell cycle arrest and causes apoptosis when DNA-dependent protein kinase (DNA-PK)-mediated non-homologous end joining is inhibited. Further, we observe that cells that were undergoing DNA replication harbored DSBs in greater frequency during pneumolysin treatment. This observation raises the possibility that DSBs might be arising as a result of replication fork breakdown. Additionally, neutralizing the oligomerization domain of pneumolysin with monoclonal antibody suppresses DNA damage and also cell cycle arrest, indicating that pneumolysin oligomerization is important for causing DNA damage. Taken together, this study reveals a previously unidentified ability of pneumolysin to induce cytotoxicity via DNA damage, with implications in the pathophysiology of S. pneumoniae infection. PMID:27026501

  4. Passive administration of monoclonal antibodies to Anthrolysin O prolong survival in mice lethally infected with Bacillus anthracis

    PubMed Central

    Nakouzi, Antonio; Rivera, Johanna; Rest, Richard F; Casadevall, Arturo

    2008-01-01

    Background Bacillus anthracis has two major virulence factors: a tripartite toxin that produces lethal and edema toxins and a polyglutamic acid capsule. A recent report suggested that a toxin belonging to the cholesterol dependant cytolysin (CDC) family, anthrolysin O (ALO) was a new virulence factor for B. anthracis but subsequent studies have questioned its relevance in pathogenesis. In this study, we examined the immunogenicity of recombinant anthrolysin O (rALO) in mice. Results BALB/c mice immunized with rALO and boosted after two weeks, produce sera with strong Ab responses with a predominance of IgG1 and IgG2a. Five hybridomas to rALO were recovered representing the IgM, IgG1, and IgG2b isotypes. Passive administration of 3 of the five monoclonal antibodies (mAbs) to rALO prior to infection with lethal intravenous (i.v.) B. anthracis Sterne strain infection in mice was associated with enhanced average survival and a greater likelihood of surviving infection. A combination of two mAbs to ALO was more effective than either mAb separately. One mAb (64F8) slowed the toxicity of rALO for J774.16 macrophage-like cells. Conclusion Our results suggest that ALO contributes to the virulence of B. anthracis Sterne strain in this infection model and that Ab response to ALO may contribute to protection in certain circumstances. PMID:18811967

  5. Regulation of CovR expression in Group B Streptococcus impacts blood-brain barrier penetration.

    PubMed

    Lembo, Annalisa; Gurney, Michael A; Burnside, Kellie; Banerjee, Anirban; de los Reyes, Melissa; Connelly, James E; Lin, Wan-Jung; Jewell, Kelsea A; Vo, Anthony; Renken, Christian W; Doran, Kelly S; Rajagopal, Lakshmi

    2010-07-01

    Group B Streptococcus (GBS) is an important cause of invasive infections in humans. The pathogen encodes a number of virulence factors including the pluripotent beta-haemolysin/cytolysin (beta-H/C). As GBS has the disposition of both a commensal organism and an invasive pathogen, it is important for the organism to appropriately regulate beta-H/C and other virulence factors in response to the environment. GBS can repress transcription of beta-H/C using the two-component system, CovR/CovS. Recently, we described that the serine/threonine kinase Stk1 can phosphorylate CovR at threonine 65 to relieve repression of beta-H/C. In this study, we show that infection with CovR-deficient GBS strains resulted in increased sepsis. Although CovR-deficient GBS showed decreased ability to invade the brain endothelium in vitro, they were more proficient in induction of permeability and pro-inflammatory signalling pathways in brain endothelium and penetration of the blood-brain barrier (BBB) in vivo. Microarray analysis revealed that CovR positively regulates its own expression and regulates the expression of 153 genes. Collectively, our results suggest that the positive feedback loop which regulates CovR transcription modulates host cell interaction and immune defence and may facilitate the transition of GBS from a commensal organism to a virulent meningeal pathogen. PMID:20497331

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

    PubMed Central

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

    2015-01-01

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

  7. Extracellular group A Streptococcus induces keratinocyte apoptosis by dysregulating calcium signalling.

    PubMed

    Cywes Bentley, Colette; Hakansson, Anders; Christianson, Jennifer; Wessels, Michael R

    2005-07-01

    Group A Streptococcus (GAS) colonizes the oropharynx and damaged skin. To cause local infection or severe invasive syndromes the bacteria must gain access into deeper tissues. Host cell death may facilitate this process. GAS internalization has been identified to induce apoptosis. We now report an alternate mechanism of GAS-mediated apoptosis of primary human keratinocytes, initiated by extracellular GAS and involving dysregulation of intracellular calcium to produce endoplasmic reticulum stress. Two bacterial virulence factors are required for effective induction of apoptosis by extracellular GAS: (i) hyaluronic acid capsule that inhibits bacterial internalization and (ii) secreted cytolysin, streptolysin O (SLO), that forms transmembrane pores that permit extracellular calcium influx into the cytosol. Induction of keratinocyte apoptosis by wild-type GAS was accompanied by cell detachment and loss of epithelial integrity, a phenomenon not observed with GAS deficient in capsule or SLO. We propose that cell signalling initiated by extracellular GAS compromises the epithelial barrier by inducing premature keratinocyte differentiation and apoptosis, thereby facilitating GAS invasion of deeper tissues. PMID:15953027

  8. Preclinical evaluation of VAX-IP, a novel bacterial minicell-based biopharmaceutical for nonmuscle invasive bladder cancer

    PubMed Central

    Tsuji, Shingo; Chen, Xuguang; Hancock, Bryan; Hernandez, Veronica; Visentin, Barbara; Reil, Katherine; Sabbadini, Roger; Giacalone, Matthew; Godbey, WT

    2016-01-01

    The development of new therapies that can prevent recurrence and progression of nonmuscle invasive bladder cancer remains an unmet clinical need. The continued cost of monitoring and treatment of recurrent disease, along with its high prevalence and incidence rate, is a strain on healthcare economics worldwide. The current work describes the characterization and pharmacological evaluation of VAX-IP as a novel bacterial minicell-based biopharmaceutical agent undergoing development for the treatment of nonmuscle invasive bladder cancer and other oncology indications. VAX-IP minicells selectively target two oncology-associated integrin heterodimer subtypes to deliver a unique bacterial cytolysin protein toxin, perfringolysin O, specifically to cancer cells, rapidly killing integrin-expressing murine and human urothelial cell carcinoma cells with a unique tumorlytic mechanism. The in vivo pharmacological evaluation of VAX-IP minicells as a single agent administered intravesically in two clinically relevant variations of a syngeneic orthotopic model of superficial bladder cancer results in a significant survival advantage with 28.6% (P = 0.001) and 16.7% (P = 0.003) of animals surviving after early or late treatment initiation, respectively. The results of these preclinical studies warrant further nonclinical and eventual clinical investigation in underserved nonmuscle invasive bladder cancer patient populations where complete cures are achievable. PMID:27119118

  9. Characterization of variants of the pore-forming toxin ClyA from Escherichia coli controlled by a redox switch.

    PubMed

    Roderer, Daniel; Benke, Stephan; Müller, Marcus; Fäh-Rechsteiner, Helene; Ban, Nenad; Schuler, Benjamin; Glockshuber, Rudi

    2014-10-14

    The α-pore-forming toxin Cytolysin A (ClyA) is responsible for the hemolytic phenotype of several Escherichia coli and Salmonella enterica strains. ClyA is a soluble, 34 kDa monomer that assembles into a dodecameric pore complex in the presence of membranes or detergent. The comparison of the X-ray structures of monomeric ClyA and the ClyA protomer in the pore complex revealed one of the largest conformational transitions observed so far in proteins, involving the structural rearrangement of more than half of all residues, which is consistent with the finding that conversion from the monomer to the assembly competent protomer is rate-limiting for pore assembly. Here, we introduced artificial disulfide bonds at two distinct sites into the ClyA monomer that both prevent a specific structural rearrangement required for protomer formation. Using electron microscopy and hemolytic activity assays, we show that the engineered disulfides indeed trap these ClyA variants in an assembly incompetent state. Assembly of the variants into functional pore complexes can be completely recovered by disulfide reduction. The assembly kinetics of the ClyA variants recorded with circular dichroism and fluorescence spectroscopy revealed the same mechanism of protomer formation that was observed for wild-type ClyA, proceeding via an intermediate with decreased secondary structure content. PMID:25222267

  10. The Intraperitoneal Transcriptome of the Opportunistic Pathogen Enterococcus faecalis in Mice

    PubMed Central

    Muller, Cécile; Cacaci, Margherita; Sauvageot, Nicolas; Sanguinetti, Maurizio; Rattei, Thomas; Eder, Thomas; Giard, Jean-Christophe; Kalinowski, Jörn; Hain, Torsten; Hartke, Axel

    2015-01-01

    Enterococcus faecalis is a Gram-positive lactic acid intestinal opportunistic bacterium with virulence potential. For a better understanding of the adapation of this bacterium to the host conditions, we performed a transcriptome analysis of bacteria isolated from an infection site (mouse peritonitis) by RNA-sequencing. We identified a total of 211 genes with significantly higher transcript levels and 157 repressed genes. Our in vivo gene expression database reflects well the infection process since genes encoding important virulence factors like cytolysin, gelatinase or aggregation substance as well as stress response proteins, are significantly induced. Genes encoding metabolic activities are the second most abundant in vivo induced genes demonstrating that the bacteria are metabolically active and adapt to the special nutrient conditions of the host. α- and β- glucosides seem to be important substrates for E. faecalis inside the host. Compared to laboratory conditions, the flux through the upper part of glycolysis seems to be reduced and more carbon may enter the pentose phosphate pathway. This may reflect the need of the bacteria under infection conditions to produce more reducing power for biosynthesis. Another important substrate is certainly glycerol since both pathways of glycerol catabolism are strongly induced. Strongly in vivo induced genes should be important for the infection process. This assumption has been verified in a virulence test using well characterized mutants affected in glycerol metabolism. This showed indeed that mutants unable to metabolize this sugar alcohol are affected in organ colonisation in a mouse model. PMID:25978463

  11. Molecular Characterization of Nonhemolytic and Nonpigmented Group B Streptococci Responsible for Human Invasive Infections

    PubMed Central

    Six, Anne; Firon, Arnaud; Plainvert, Céline; Caplain, Camille; Touak, Gérald; Dmytruk, Nicolas; Longo, Magalie; Letourneur, Franck; Fouet, Agnès; Trieu-Cuot, Patrick

    2015-01-01

    Group B Streptococcus (GBS) is a common commensal bacterium in adults, but is also the leading cause of invasive bacterial infections in neonates in developed countries. The β-hemolysin/cytolysin (β-h/c), which is always associated with the production of an orange-to-red pigment, is a major virulence factor that is also used for GBS diagnosis. A collection of 1,776 independent clinical GBS strains isolated in France between 2006 and 2013 was evaluated on specific medium for β-h/c activity and pigment production. The genomic sequences of nonhemolytic and nonpigmented (NH/NP) strains were analyzed to identify the molecular basis of this phenotype. Gene deletions or complementations were carried out to confirm the genotype-phenotype association. Sixty-three GBS strains (3.5%) were NH/NP, and 47 of these (74.6%) originated from invasive infections, including bacteremia and meningitis, in neonates or adults. The mutations are localized predominantly in the cyl operon, encoding the β-h/c pigment biosynthetic pathway and, in the abx1 gene, encoding a CovSR regulator partner. In conclusion, although usually associated with GBS virulence, β-h/c pigment production is not absolutely required to cause human invasive infections. Caution should therefore be taken in the use of hemolysis and pigmentation as criteria for GBS diagnosis in routine clinical laboratory settings. PMID:26491182

  12. Characterization of Enterococcus faecalis and Enterococcus faecium from wild flowers.

    PubMed

    Sánchez Valenzuela, Antonio; Benomar, Nabil; Abriouel, Hikmate; Pérez Pulido, Rubén; Martínez Cañamero, Magdalena; Gálvez, Antonio

    2012-05-01

    Wild flowers in the South of Spain were screened for Enterococcus faecalis and Enterococcus faecium. Enterococci were frequently associated with prickypear and fieldpoppy flowers. Forty-six isolates, from 8 different flower species, were identified as E. faecalis (28 isolates) or E. faecium (18 isolates) and clustered in well-defined groups by ERIC-PCR fingerprinting. A high incidence of antibiotic resistance was detected among the E. faecalis isolates, especially to quinupristin/dalfopristin (75%), rifampicin (68%) and ciprofloxacin (57%), and to a lesser extent to levofloxacin (35.7%), erythromycin (28.5%), tetracycline (3.5%), chloramphenicol (3.5%) and streptomycin (3.5%). Similar results were observed for E. faecium isolates, except for a higher incidence of resistance to tetracycline (17%) and lower to erythromycin (11%) or quinupristin/dalfopristin (22%). Vancomycin or teicoplanin resistances were not detected. Most isolates (especially E. faecalis) were proteolytic and carried the gelatinase gene gelE. Genes encoding other potential virulence factors (ace, efaA (fs), ccf and cpd) were frequently detected. Cytolysin genes were mainly detected in a few haemolytic E. faecium isolates, three of which also carried the collagen adhesin acm gene. Hyaluronidase gene (hyl ( Efm )) was detected in two isolates. Many isolates produced bacteriocins and carried genes for enterocins A, B, and L50 mainly. The similarities found between enterococci from wild flowers and those from animal and food sources raise new questions about the puzzling lifestyle of these commensals and opportunistic pathogens. PMID:22183298

  13. Dual modes of membrane binding direct pore formation by Streptolysin O.

    PubMed

    Mozola, Cara C; Caparon, Michael G

    2015-09-01

    Effector translocation is central to the virulence of many bacterial pathogens, including Streptococcus pyogenes, which utilizes the cholesterol-dependent cytolysin Streptolysin O (SLO) to translocate the NAD(+) glycohydrolase SPN into host cells during infection. SLO's translocation activity does not require host cell membrane cholesterol or pore formation by SLO, yet SLO does form pores during infection via a cholesterol-dependent mechanism. Although cholesterol was considered the primary receptor for SLO, SLO's membrane-binding domain also encodes a putative carbohydrate-binding site, implicating a potential glycan receptor in binding and pore formation. Analysis of carbohydrate-binding site SLO mutants and carbohydrate-defective cell lines revealed that glycan recognition is involved in SLO's pore formation pathway and is an essential step when SLO is secreted by non-adherent bacteria, as occurs during lysis of erythrocytes. However, SLO also recognizes host cell membranes via a second mechanism when secreted from adherent bacteria, which requires co-secretion of SPN but not glycan binding by SLO. This SPN-mediated membrane binding of SLO correlates with SPN translocation, and requires SPN's non-enzymatic domain, which is predicted to adopt the structure of a carbohydrate-binding module. SPN-dependent membrane binding also promotes pore formation by SLO, demonstrating that pore formation can occur by distinct pathways during infection. PMID:26059530

  14. The Photobacterium damselae subsp. damselae Hemolysins Damselysin and HlyA Are Encoded within a New Virulence Plasmid ▿

    PubMed Central

    Rivas, Amable J.; Balado, Miguel; Lemos, Manuel L.; Osorio, Carlos R.

    2011-01-01

    Photobacterium damselae subsp. damselae (formerly Vibrio damsela) is a marine bacterium that causes infections and fatal disease in a wide range of marine animals and in humans. Highly hemolytic strains produce damselysin (Dly), a cytolysin encoded by the dly gene that is lethal for mice and has hemolytic activity. We found that Dly is encoded in the highly hemolytic strain RM-71 within a 153,429-bp conjugative plasmid that we dubbed pPHDD1. In addition to Dly, pPHDD1 also encodes a homologue of the pore-forming toxin HlyA. We found a direct correlation between presence of pPHDD1 and a strong hemolytic phenotype in a collection of P. damselae subsp. damselae isolates. Hemolysis was strongly reduced in a double dly hlyA mutant, demonstrating the role of the two pPHDD1-encoded genes in hemolysis. Interestingly, although single hlyA and dly mutants showed different levels of hemolysis reduction depending on the erythrocyte source, hemolysis was not abolished in any of the single mutants, suggesting that the hemolytic phenotype is the result of the additive effect of Dly and HlyA. We found that pPHDD1-encoded dly and hlyA genes are necessary for full virulence for mice and fish. Our results suggest that pPHDD1 can be considered as a driving force for the emergence of a highly hemolytic lineage of P. damselae subsp. damselae. PMID:21875966

  15. Structural basis of complement membrane attack complex formation

    NASA Astrophysics Data System (ADS)

    Serna, Marina; Giles, Joanna L.; Morgan, B. Paul; Bubeck, Doryen

    2016-02-01

    In response to complement activation, the membrane attack complex (MAC) assembles from fluid-phase proteins to form pores in lipid bilayers. MAC directly lyses pathogens by a `multi-hit' mechanism; however, sublytic MAC pores on host cells activate signalling pathways. Previous studies have described the structures of individual MAC components and subcomplexes; however, the molecular details of its assembly and mechanism of action remain unresolved. Here we report the electron cryo-microscopy structure of human MAC at subnanometre resolution. Structural analyses define the stoichiometry of the complete pore and identify a network of interaction interfaces that determine its assembly mechanism. MAC adopts a `split-washer' configuration, in contrast to the predicted closed ring observed for perforin and cholesterol-dependent cytolysins. Assembly precursors partially penetrate the lipid bilayer, resulting in an irregular β-barrel pore. Our results demonstrate how differences in symmetric and asymmetric components of the MAC underpin a molecular basis for pore formation and suggest a mechanism of action that extends beyond membrane penetration.

  16. β-sitosterol interacts with pneumolysin to prevent Streptococcus pneumoniae infection

    PubMed Central

    Li, Hongen; Zhao, Xiaoran; Wang, Jianfeng; Dong, Yu; Meng, Song; Li, Rui; Niu, Xiaodi; Deng, Xuming

    2015-01-01

    Pneumolysin is one of the major virulence factors elaborated by Streptococcus pneumoniae; this toxin is a member of the cholesterol-dependent cytolysins. Engagement of cholesterol induces the formation of a multi-subunit complex by pneumolysin that lyses host cells by forming pores on the membrane. Because pneumolysin released by bacteria which have been killed by conventional antibiotics is still active, agents capable of directly attacking the toxin are considered advantageous against antimicrobials in the treatment of S. pneumoniae infections. Here we found that the phytosterol, β-sitosterol, effectively protects against cell lysis caused by pneumolysin. This compound interacts with the toxin at Thr459 and Leu460, two sites important for being recognized by its natural ligand, cholesterol. Similar to cholesterol, β-sitosterol induces pneumolysin oligomerization. This compound also protects cells from damage by other cholesterol-dependent toxins. Finally, this compound protects mice against S. pneumoniae infection. Thus, β-sitosterol is a candidate for the development of anti-virulence agents against pathogens that rely on cholesterol-dependent toxins for successful infections. PMID:26631364

  17. Prevalence of sorbitol non-fermenting Shiga toxin-producing Escherichia coli in Black Bengal goats on smallholdings.

    PubMed

    Gupta, M DAS; DAS, A; Islam, M Z; Biswas, P K

    2016-09-01

    A cross-sectional survey was carried out in Bangladesh with the sampling of 514 Black Bengal goats on smallholdings to determine the presence of sorbitol non-fermenting (SNF) Shiga toxin-producing E. coli (STEC). Swab samples collected from the recto-anal junction were plated onto cefixime and potassium tellurite added sorbitol MacConkey (CT-SMAC) agar, a selective medium for STEC O157 serogroup, where this serogroup and other SNF STEC produce colourless colonies. The SNF E. coli (SNF EC) isolates obtained from the survey were investigated by PCR for the presence of Shiga toxin-producing genes, stx1 and stx2, and two other virulence genes, eae and hlyA that code for adherence factor (intimin protein) and pore-forming cytolysin, respectively. The SNF EC isolates were also assessed for the presence of the rfbO157 gene to verify their identity to O157 serogroup. The results revealed that the proportions of goats carrying SNF EC isolates and stx1 and stx2 genes were 6·2% (32/514) [95% confidence interval (CI) 4·4-8·7)], 1·2% (95% CI 0·5-2·6) and 1·2% (95% CI 0·5-2·6), respectively. All the SNF STEC tested negative for rfbO157, hlyA and eae genes. The risk for transmission of STEC from Black Bengal goats to humans is low. PMID:27267779

  18. Native Microbial Colonization of Drosophila melanogaster and Its Use as a Model of Enterococcus faecalis Pathogenesis▿ †

    PubMed Central

    Cox, Christopher R.; Gilmore, Michael S.

    2007-01-01

    Enterococci are commensal organisms of the gastrointestinal (GI) tracts of a broad range of mammalian and insect hosts, but they are also leading causes of nosocomial infection. Little is known about the ecological role of enterococci in the GI tract consortia. To develop a tractable model for studying the roles of these organisms as commensals and pathogens, we characterized the Drosophila melanogaster microflora and examined the occurrence of enterococci in the gastrointestinal consortium of Drosophila. In a survey of laboratory-reared Drosophila and wild-captured flies, we found that Drosophila was naturally colonized by representatives of five bacterial phyla. Among these organisms were several species of enterococci, including Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinaraum, and Enterococcus durans, as well as a previously detected but uncultured Enterococcus species. Drosophila could be cured of enterococcal carriage by antibiotic treatment and could be reassociated with laboratory strains. High-level colonization by a well-characterized strain expressing the enterococcal cytolysin was found to be detrimental to Drosophila compared to the effect of an isogenic, noncytolytic control. The anatomical distribution of enterococci in the Drosophila GI tract was determined by immunohistochemical staining of thin sections of naturally colonized and reassociated flies. PMID:17220307

  19. The pore-forming haemolysins of bacillus cereus: a review.

    PubMed

    Ramarao, Nalini; Sanchis, Vincent

    2013-06-01

    The Bacillus cereus sensu lato group contains diverse Gram-positive spore-forming bacteria that can cause gastrointestinal diseases and severe eye infections in humans. They have also been incriminated in a multitude of other severe, and frequently fatal, clinical infections, such as osteomyelitis, septicaemia, pneumonia, liver abscess and meningitis, particularly in immuno-compromised patients and preterm neonates. The pathogenic properties of this organism are mediated by the synergistic effects of a number of virulence products that promote intestinal cell destruction and/or resistance to the host immune system. This review focuses on the pore-forming haemolysins produced by B. cereus: haemolysin I (cereolysin O), haemolysin II, haemolysin III and haemolysin IV (CytK). Haemolysin I belongs to the cholesterol-dependent cytolysin (CDC) family whose best known members are listeriolysin O and perfringolysin O, produced by L. monocytogenes and C. perfringens respectively. HlyII and CytK are oligomeric ß-barrel pore-forming toxins related to the α-toxin of S. aureus or the ß-toxin of C. perfringens. The structure of haemolysin III, the least characterized haemolytic toxin from the B. cereus, group has not yet been determined. PMID:23748204

  20. A pore-forming toxin requires a specific residue for its activity in membranes with particular physicochemical properties.

    PubMed

    Morante, Koldo; Caaveiro, Jose M M; Tanaka, Koji; González-Mañas, Juan Manuel; Tsumoto, Kouhei

    2015-04-24

    The physicochemical landscape of the bilayer modulates membrane protein function. Actinoporins are a family of potent hemolytic proteins from sea anemones acting at the membrane level. This family of cytolysins preferentially binds to target membranes containing sphingomyelin, where they form lytic pores giving rise to cell death. Although the cytolytic activity of the actinoporin fragaceatoxin C (FraC) is sensitive to vesicles made of various lipid compositions, it is far from clear how this toxin adjusts its mechanism of action to a broad range of physiochemical landscapes. Herein, we show that the conserved residue Phe-16 of FraC is critical for pore formation in cholesterol-rich membranes such as those of red blood cells. The interaction of a panel of muteins of Phe-16 with model membranes composed of raft-like lipid domains is inactivated in cholesterol-rich membranes but not in cholesterol-depleted membranes. These results indicate that actinoporins recognize different membrane environments, resulting in a wider repertoire of susceptible target membranes (and preys) for sea anemones. In addition, this study has unveiled promising candidates for the development of protein-based biosensors highly sensitive to the concentration of cholesterol within the membrane. PMID:25759390

  1. A Pore-Forming Toxin Requires a Specific Residue for Its Activity in Membranes with Particular Physicochemical Properties*

    PubMed Central

    Morante, Koldo; Caaveiro, Jose M. M.; Tanaka, Koji; González-Mañas, Juan Manuel; Tsumoto, Kouhei

    2015-01-01

    The physicochemical landscape of the bilayer modulates membrane protein function. Actinoporins are a family of potent hemolytic proteins from sea anemones acting at the membrane level. This family of cytolysins preferentially binds to target membranes containing sphingomyelin, where they form lytic pores giving rise to cell death. Although the cytolytic activity of the actinoporin fragaceatoxin C (FraC) is sensitive to vesicles made of various lipid compositions, it is far from clear how this toxin adjusts its mechanism of action to a broad range of physiochemical landscapes. Herein, we show that the conserved residue Phe-16 of FraC is critical for pore formation in cholesterol-rich membranes such as those of red blood cells. The interaction of a panel of muteins of Phe-16 with model membranes composed of raft-like lipid domains is inactivated in cholesterol-rich membranes but not in cholesterol-depleted membranes. These results indicate that actinoporins recognize different membrane environments, resulting in a wider repertoire of susceptible target membranes (and preys) for sea anemones. In addition, this study has unveiled promising candidates for the development of protein-based biosensors highly sensitive to the concentration of cholesterol within the membrane. PMID:25759390

  2. Mechanisms linking bacterial infections of the bovine endometrium to disease and infertility.

    PubMed

    Carneiro, Luísa Cunha; Cronin, James Graham; Sheldon, Iain Martin

    2016-03-01

    Bacterial infections of the endometrium after parturition commonly cause metritis and endometritis in dairy cattle, and these diseases are important because they compromise animal welfare and incur economic costs, as well as delaying or preventing conception. Here we highlight that uterine infections cause infertility, discuss which bacteria cause uterine disease, and review the evidence for mechanisms of inflammation and tissue damage in the endometrium. Bacteria cultured from the uterus of diseased animals include Escherichia coli, Trueperella pyogenes, and several anaerobic species, but their causative role in disease is challenged by the discovery of many other bacteria in the uterine disease microbiome. Irrespective of the species of bacteria, endometrial cell inflammatory responses to infection initially depend on innate immunity, with Toll-like receptors binding pathogen-associated molecular patterns, such as lipopolysaccharide and bacterial lipopeptides. In addition to tissue damage associated with parturition and inflammation, endometrial cell death is caused by a cholesterol-dependent cytolysin secreted by T. pyogenes, called pyolysin, which forms pores in plasma membranes of endometrial cells. However, endometrial cells surprisingly do not sense damage-associated molecular patterns, but a combination of infections followed by cell damage leads to release of the intracellular cytokine interleukin (IL)-1 alpha from endometrial cells, which then acts to scale inflammatory responses. To develop strategies to limit the impact of uterine disease on fertility, future work should focus on determining which bacteria and virulence factors cause endometritis, and understanding how the host response to infection is regulated in the endometrium. PMID:26952747

  3. Structural basis of complement membrane attack complex formation

    PubMed Central

    Serna, Marina; Giles, Joanna L.; Morgan, B. Paul; Bubeck, Doryen

    2016-01-01

    In response to complement activation, the membrane attack complex (MAC) assembles from fluid-phase proteins to form pores in lipid bilayers. MAC directly lyses pathogens by a ‘multi-hit' mechanism; however, sublytic MAC pores on host cells activate signalling pathways. Previous studies have described the structures of individual MAC components and subcomplexes; however, the molecular details of its assembly and mechanism of action remain unresolved. Here we report the electron cryo-microscopy structure of human MAC at subnanometre resolution. Structural analyses define the stoichiometry of the complete pore and identify a network of interaction interfaces that determine its assembly mechanism. MAC adopts a ‘split-washer' configuration, in contrast to the predicted closed ring observed for perforin and cholesterol-dependent cytolysins. Assembly precursors partially penetrate the lipid bilayer, resulting in an irregular β-barrel pore. Our results demonstrate how differences in symmetric and asymmetric components of the MAC underpin a molecular basis for pore formation and suggest a mechanism of action that extends beyond membrane penetration. PMID:26841837

  4. Structure of the poly-C9 component of the complement membrane attack complex

    NASA Astrophysics Data System (ADS)

    Dudkina, Natalya V.; Spicer, Bradley A.; Reboul, Cyril F.; Conroy, Paul J.; Lukoyanova, Natalya; Elmlund, Hans; Law, Ruby H. P.; Ekkel, Susan M.; Kondos, Stephanie C.; Goode, Robert J. A.; Ramm, Georg; Whisstock, James C.; Saibil, Helen R.; Dunstone, Michelle A.

    2016-02-01

    The membrane attack complex (MAC)/perforin-like protein complement component 9 (C9) is the major component of the MAC, a multi-protein complex that forms pores in the membrane of target pathogens. In contrast to homologous proteins such as perforin and the cholesterol-dependent cytolysins (CDCs), all of which require the membrane for oligomerisation, C9 assembles directly onto the nascent MAC from solution. However, the molecular mechanism of MAC assembly remains to be understood. Here we present the 8 Å cryo-EM structure of a soluble form of the poly-C9 component of the MAC. These data reveal a 22-fold symmetrical arrangement of C9 molecules that yield an 88-strand pore-forming β-barrel. The N-terminal thrombospondin-1 (TSP1) domain forms an unexpectedly extensive part of the oligomerisation interface, thus likely facilitating solution-based assembly. These TSP1 interactions may also explain how additional C9 subunits can be recruited to the growing MAC subsequent to membrane insertion.

  5. Virulence of enterococci.

    PubMed Central

    Jett, B D; Huycke, M M; Gilmore, M S

    1994-01-01

    Enterococci are commensal organisms well suited to survival in intestinal and vaginal tracts and the oral cavity. However, as for most bacteria described as causing human disease, enterococci also possess properties that can be ascribed roles in pathogenesis. The natural ability of enterococci to readily acquire, accumulate, and share extrachromosomal elements encoding virulence traits or antibiotic resistance genes lends advantages to their survival under unusual environmental stresses and in part explains their increasing importance as nosocomial pathogens. This review discusses the current understanding of enterococcal virulence relating to (i) adherence to host tissues, (ii) invasion and abscess formation, (iii) factors potentially relevant to modulation of host inflammatory responses, and (iv) potentially toxic secreted products. Aggregation substance, surface carbohydrates, or fibronectin-binding moieties may facilitate adherence to host tissues. Enterococcus faecalis appears to have the capacity to translocate across intact intestinal mucosa in models of antibiotic-induced superinfection. Extracellular toxins such as cytolysin can induce tissue damage as shown in an endophthalmitis model, increase mortality in combination with aggregation substance in an endocarditis model, and cause systemic toxicity in a murine peritonitis model. Finally, lipoteichoic acid, superoxide production, or pheromones and corresponding peptide inhibitors each may modulate local inflammatory reactions. Images PMID:7834601

  6. Red Blood Cell Susceptibility to Pneumolysin: CORRELATION WITH MEMBRANE BIOCHEMICAL AND PHYSICAL PROPERTIES.

    PubMed

    Bokori-Brown, Monika; Petrov, Peter G; Khafaji, Mawya A; Mughal, Muhammad K; Naylor, Claire E; Shore, Angela C; Gooding, Kim M; Casanova, Francesco; Mitchell, Tim J; Titball, Richard W; Winlove, C Peter

    2016-05-01

    This study investigated the effect of the biochemical and biophysical properties of the plasma membrane as well as membrane morphology on the susceptibility of human red blood cells to the cholesterol-dependent cytolysin pneumolysin, a key virulence factor of Streptococcus pneumoniae, using single cell studies. We show a correlation between the physical properties of the membrane (bending rigidity and surface and dipole electrostatic potentials) and the susceptibility of red blood cells to pneumolysin-induced hemolysis. We demonstrate that biochemical modifications of the membrane induced by oxidative stress, lipid scrambling, and artificial cell aging modulate the cell response to the toxin. We provide evidence that the diversity of response to pneumolysin in diabetic red blood cells correlates with levels of glycated hemoglobin and that the mechanical properties of the red blood cell plasma membrane are altered in diabetes. Finally, we show that diabetic red blood cells are more resistant to pneumolysin and the related toxin perfringolysin O relative to healthy red blood cells. Taken together, these studies indicate that the diversity of cell response to pneumolysin within a population of human red blood cells is influenced by the biophysical and biochemical status of the plasma membrane and the chemical and/or oxidative stress pre-history of the cell. PMID:26984406

  7. Stonefish toxin defines an ancient branch of the perforin-like superfamily.

    PubMed

    Ellisdon, Andrew M; Reboul, Cyril F; Panjikar, Santosh; Huynh, Kitmun; Oellig, Christine A; Winter, Kelly L; Dunstone, Michelle A; Hodgson, Wayne C; Seymour, Jamie; Dearden, Peter K; Tweten, Rodney K; Whisstock, James C; McGowan, Sheena

    2015-12-15

    The lethal factor in stonefish venom is stonustoxin (SNTX), a heterodimeric cytolytic protein that induces cardiovascular collapse in humans and native predators. Here, using X-ray crystallography, we make the unexpected finding that SNTX is a pore-forming member of an ancient branch of the Membrane Attack Complex-Perforin/Cholesterol-Dependent Cytolysin (MACPF/CDC) superfamily. SNTX comprises two homologous subunits (α and β), each of which comprises an N-terminal pore-forming MACPF/CDC domain, a central focal adhesion-targeting domain, a thioredoxin domain, and a C-terminal tripartite motif family-like PRY SPla and the RYanodine Receptor immune recognition domain. Crucially, the structure reveals that the two MACPF domains are in complex with one another and arranged into a stable early prepore-like assembly. These data provide long sought after near-atomic resolution insights into how MACPF/CDC proteins assemble into prepores on the surface of membranes. Furthermore, our analyses reveal that SNTX-like MACPF/CDCs are distributed throughout eukaryotic life and play a broader, possibly immune-related function outside venom. PMID:26627714

  8. Efficient suilysin-mediated invasion and apoptosis in porcine respiratory epithelial cells after streptococcal infection under air-liquid interface conditions.

    PubMed

    Meng, Fandan; Wu, Nai-Huei; Seitz, Maren; Herrler, Georg; Valentin-Weigand, Peter

    2016-01-01

    Streptococci may colonize the epithelium in the airways and other entry sites. While local infection often remains asymptomatic, severe or even fatal diseases occur when streptococci become invasive and spread to different sites in the infected host. We have established porcine respiratory air-liquid interface cultures (ALI) from the porcine lung to analyze the interaction of streptococci with their primary target cells. As representative of the streptococcal family we chose Streptococcus suis (S. suis) that is not only a major swine respiratory pathogen but can also infect humans. Suilysin, a cholesterol-dependent cytolysin (CDC), is an important virulence factor. By comparing a S. suis wt strain with a suilysin-deficient mutant, we demonstrate that suilysin contributes to (i) adherence to airway cells (ii) loss of ciliated cells (iii) apoptosis, and (iv) invasion. Furthermore, we show that cytolytic activity of suilysin is crucial for these effects. A striking result of our analysis was the high efficiency of S. suis-induced apoptosis and invasion upon infection under ALI conditions. These properties have been reported to be less efficient when analyzed with immortalized cells. We hypothesize that soluble effectors such as suilysin are present at higher concentrations in cells kept at ALI conditions and thus more effective. These results should be relevant also for infection of the respiratory tract by other respiratory pathogens. PMID:27229328

  9. Efficient suilysin-mediated invasion and apoptosis in porcine respiratory epithelial cells after streptococcal infection under air-liquid interface conditions

    PubMed Central

    Meng, Fandan; Wu, Nai-Huei; Seitz, Maren; Herrler, Georg; Valentin-Weigand, Peter

    2016-01-01

    Streptococci may colonize the epithelium in the airways and other entry sites. While local infection often remains asymptomatic, severe or even fatal diseases occur when streptococci become invasive and spread to different sites in the infected host. We have established porcine respiratory air-liquid interface cultures (ALI) from the porcine lung to analyze the interaction of streptococci with their primary target cells. As representative of the streptococcal family we chose Streptococcus suis (S. suis) that is not only a major swine respiratory pathogen but can also infect humans. Suilysin, a cholesterol-dependent cytolysin (CDC), is an important virulence factor. By comparing a S. suis wt strain with a suilysin-deficient mutant, we demonstrate that suilysin contributes to (i) adherence to airway cells (ii) loss of ciliated cells (iii) apoptosis, and (iv) invasion. Furthermore, we show that cytolytic activity of suilysin is crucial for these effects. A striking result of our analysis was the high efficiency of S. suis-induced apoptosis and invasion upon infection under ALI conditions. These properties have been reported to be less efficient when analyzed with immortalized cells. We hypothesize that soluble effectors such as suilysin are present at higher concentrations in cells kept at ALI conditions and thus more effective. These results should be relevant also for infection of the respiratory tract by other respiratory pathogens. PMID:27229328

  10. Structural Studies of Streptococcus pyogenes Streptolysin O Provide Insights into the Early Steps of Membrane Penetration

    PubMed Central

    Feil, Susanne C.; Ascher, David B.; Kuiper, Michael J.; Tweten, Rodney K.; Parker, Michael W.

    2015-01-01

    Cholesterol-dependent cytolysins (CDCs) are a large family of bacterial toxins that exhibit a dependence on the presence of membrane cholesterol in forming large pores in cell membranes. Significant changes in the three-dimensional structure of these toxins are necessary to convert the soluble monomeric protein into a membrane pore. We have determined the crystal structure of the archetypical member of the CDC family, streptolysin O (SLO), a virulence factor from Streptococcus pyogenes. The overall fold is similar to previously reported CDC structures, although the C-terminal domain is in a different orientation with respect to the rest of the molecule. Surprisingly, a signature stretch of CDC sequence called the undecapeptide motif, a key region involved in membrane recognition, adopts a very different structure in SLO to that of the well-characterized CDC perfringolysin O (PFO), although the sequences in this region are identical. An analysis reveals that, in PFO, there are complementary interactions between the motif and the rest of domain 4 that are lost in SLO. Molecular dynamics simulations suggest that the loss of a salt bridge in SLO and a cation–pi interaction are determining factors in the extended conformation of the motif, which in turn appears to result in a greater flexibility of the neighboring L1 loop that houses a cholesterol-sensing motif. These differences may explain the differing abilities of SLO and PFO to efficiently penetrate target cell membranes in the first step of toxin insertion into the membrane. PMID:24316049

  11. Gigantoxin-4-4D5 scFv is a novel recombinant immunotoxin with specific toxicity against HER2/neu-positive ovarian carcinoma cells.

    PubMed

    Lv, Xinxin; Zhang, Jian; Xu, Rui; Dong, Yuguo; Sun, Aiyou; Shen, Yaling; Wei, Dongzhi

    2016-07-01

    Immunotoxins are a new class of antibody-targeted therapy in clinical development. Traditional immunotoxins that are constructed from the toxins of plants or bacteria need to be internalized to the cytoplasm and thus have limited antitumor efficacy. In the present study, we combined a recently reported sea anemone cytolysin Gigantoxin-4 with an anti-HER2/neu single-chain variable fragment 4D5 scFv to construct a novel immunotoxin. We fused a SUMO tag to the N-terminus of Gigantoxin-4-4D5 scFv and it was successfully expressed in Escherichia coli strain BL21 (DE3) in a soluble form. After purification, the purity of Gigantoxin-4-4D5 scFv reached 96 % and the yield was 14.3 mg/L. Our results demonstrated that Gigantoxin-4-4D5 scFv exerted a highly cytotoxic effect on the HER2/neu-positive ovarian carcinoma SK-OV-3 cell line. And the hemolytic activity was weaker, making it safe for normal cells. The results of immunofluorescence analysis showed that this novel immunotoxin could specifically bind to SK-OV-3 cells with no recognition of human embryonic kidney 293 cells. Scanning electron microscope observations and extracellular lactate dehydrogenase activity indicated that it could induce necrosis in SK-OV-3 cells by disrupting the cell membrane. Moreover, it could also mediate apoptosis of SK-OV-3 cells. PMID:27063011

  12. Increasing of temperature induces pathogenicity of Streptococcus agalactiae and the up-regulation of inflammatory related genes in infected Nile tilapia (Oreochromis niloticus).

    PubMed

    Kayansamruaj, Pattanapon; Pirarat, Nopadon; Hirono, Ikuo; Rodkhum, Channarong

    2014-08-01

    Temperature strongly affects the health of aquatic poikilotherms. In Nile tilapia (Oreochromis niloticus), elevated water temperatures increase the severity of streptococcosis. Here we investigated the effects of temperature on the vulnerability and inflammatory response of Nile tilapia to Streptococcus agalactiae (Group B streptococci; GBS). At 35 and 28 °C, GBS took 4 and 7h, respectively to reach the log-phase and, when incubated with tilapia whole blood, experienced survival rates of 97% and 2%, respectively. The hemolysis activity of GBS grown at 35 °C was five times higher than that of GBS grown at 28 °C. GBS expressed cylE (β-hemolysin/cytolysin), cfb (CAMP factor) and PI-2b (pili-backbone) much more strongly at 35 °C than at 28 °C. Challenging Nile tilapia reared at 35 and 28 °C with GBS resulted in accumulated mortalities of about 85% and 45%, respectively. At 35 °C, infected tilapia exhibited tremendous inflammatory responses due to a dramatic up-regulation (30-40-fold) of inflammatory-related genes (cyclooxygenase-2, IL-1β and TNF-α) between 6 and 96 h-post infection. These results suggest that the increase of GBS pathogenicity to Nile tilapia induced by elevated temperature is associated with massive inflammatory responses, which may lead to acute mortality. PMID:24856132

  13. Cationic Antimicrobial Peptides Disrupt the Streptococcus pyogenes ExPortal

    PubMed Central

    Vega, Luis Alberto; Caparon, Michael G.

    2012-01-01

    Summary Although they possess a well-characterized ability to porate the bacterial membrane, emerging research suggests that cationic antimicrobial peptides (CAPs) can influence pathogen behavior at levels that are sub-lethal. In this study, we investigated the interaction of polymyxin B and human neutrophil peptide (HNP-1) with the human pathogen Streptococcus pyogenes. At sub-lethal concentrations, these CAPs preferentially targeted the ExPortal, a unique microdomain of the S. pyogenes membrane, specialized for protein secretion and processing. A consequence of this interaction was the disruption of ExPortal organization and a redistribution of ExPortal components into the peripheral membrane. Redistribution was associated with inhibition of secretion of certain toxins, including the SpeB cysteine protease and the Streptolysin O (SLO) cytolysin, but not SIC, a protein that protects S. pyogenes from CAPs. These data suggest a novel function for CAPs in targeting the ExPortal and interfering with secretion of factors required for infection and survival. This mechanism may prove valuable for the design of new types of antimicrobial agents to combat the emergence of antibiotic-resistant pathogens. PMID:22780862

  14. Cholesterol Exposure at the Membrane Surface Is Necessary and Sufficient to Trigger Perfringolysin O Binding†

    PubMed Central

    Flanagan, John J.; Tweten, Rodney K.; Johnson, Arthur E.; Heuck, Alejandro P.

    2010-01-01

    Perfringolysin O (PFO) is the prototype for the cholesterol-dependent cytolysins, a family of bacterial pore-forming toxins that act on eukaryotic membranes. The pore-forming mechanism of PFO exhibits an absolute requirement for membrane cholesterol, but the complex interplay between the structural arrangement of the PFO C-terminal domain and the distribution of cholesterol in the target membrane is poorly understood. Herein we show that PFO binding to the bilayer and the initiation of the sequence of events that culminate in the formation of a transmembrane pore depend on the availability of free cholesterol at the membrane surface, while changes in the acyl chain packing of the phospholipids and cholesterol in the membrane core, or the presence or absence of detergent-resistant domains do not correlate with PFO binding. Moreover, PFO association with the membrane was inhibited by the addition of sphingomyelin, a typical component of membrane rafts in cell membranes. Finally, addition of molecules that do not interact with PFO, but intercalate into the membrane and displace cholesterol from its association with phospholipids (e.g., epicholesterol), reduced the amount of cholesterol required to trigger PFO binding. Taken together, our studies reveal that PFO binding to membranes is triggered when the concentration of cholesterol exceeds the association capacity of the phospholipids, and this cholesterol excess is then free to associate with the toxin. PMID:19292457

  15. Genomes and Virulence Factors of Novel Bacterial Pathogens Causing Bleaching Disease in the Marine Red Alga Delisea pulchra

    PubMed Central

    Fernandes, Neil; Case, Rebecca J.; Longford, Sharon R.; Seyedsayamdost, Mohammad R.; Steinberg, Peter D.; Kjelleberg, Staffan; Thomas, Torsten

    2011-01-01

    Nautella sp. R11, a member of the marine Roseobacter clade, causes a bleaching disease in the temperate-marine red macroalga, Delisea pulchra. To begin to elucidate the molecular mechanisms underpinning the ability of Nautella sp. R11 to colonize, invade and induce bleaching of D. pulchra, we sequenced and analyzed its genome. The genome encodes several factors such as adhesion mechanisms, systems for the transport of algal metabolites, enzymes that confer resistance to oxidative stress, cytolysins, and global regulatory mechanisms that may allow for the switch of Nautella sp. R11 to a pathogenic lifestyle. Many virulence effectors common in phytopathogenic bacteria are also found in the R11 genome, such as the plant hormone indole acetic acid, cellulose fibrils, succinoglycan and nodulation protein L. Comparative genomics with non-pathogenic Roseobacter strains and a newly identified pathogen, Phaeobacter sp. LSS9, revealed a patchy distribution of putative virulence factors in all genomes, but also led to the identification of a quorum sensing (QS) dependent transcriptional regulator that was unique to pathogenic Roseobacter strains. This observation supports the model that a combination of virulence factors and QS-dependent regulatory mechanisms enables indigenous members of the host alga's epiphytic microbial community to switch to a pathogenic lifestyle, especially under environmental conditions when innate host defence mechanisms are compromised. PMID:22162749

  16. Pneumococcal Pneumolysin Induces DNA Damage and Cell Cycle Arrest

    PubMed Central

    Rai, Prashant; He, Fang; Kwang, Jimmy; Engelward, Bevin P.; Chow, Vincent T.K.

    2016-01-01

    Streptococcus pneumoniae produces pneumolysin toxin as a key virulence factor against host cells. Pneumolysin is a cholesterol-dependent cytolysin (CDC) toxin that forms lytic pores in host membranes and mediates pneumococcal disease pathogenesis by modulating inflammatory responses. Here, we show that pneumolysin, which is released during bacterial lysis, induces DNA double strand breaks (DSBs), as indicated by ataxia telangiectasia mutated (ATM)-mediated H2AX phosphorylation (γH2AX). Pneumolysin-induced γH2AX foci recruit mediator of DNA damage checkpoint 1 (MDC1) and p53 binding protein 1 (53BP1), to sites of DSBs. Importantly, results show that toxin-induced DNA damage precedes cell cycle arrest and causes apoptosis when DNA-dependent protein kinase (DNA-PK)-mediated non-homologous end joining is inhibited. Further, we observe that cells that were undergoing DNA replication harbored DSBs in greater frequency during pneumolysin treatment. This observation raises the possibility that DSBs might be arising as a result of replication fork breakdown. Additionally, neutralizing the oligomerization domain of pneumolysin with monoclonal antibody suppresses DNA damage and also cell cycle arrest, indicating that pneumolysin oligomerization is important for causing DNA damage. Taken together, this study reveals a previously unidentified ability of pneumolysin to induce cytotoxicity via DNA damage, with implications in the pathophysiology of S. pneumoniae infection. PMID:27026501

  17. Structure of the poly-C9 component of the complement membrane attack complex

    PubMed Central

    Dudkina, Natalya V.; Spicer, Bradley A.; Reboul, Cyril F.; Conroy, Paul J.; Lukoyanova, Natalya; Elmlund, Hans; Law, Ruby H. P.; Ekkel, Susan M.; Kondos, Stephanie C.; Goode, Robert J. A.; Ramm, Georg; Whisstock, James C.; Saibil, Helen R.; Dunstone, Michelle A.

    2016-01-01

    The membrane attack complex (MAC)/perforin-like protein complement component 9 (C9) is the major component of the MAC, a multi-protein complex that forms pores in the membrane of target pathogens. In contrast to homologous proteins such as perforin and the cholesterol-dependent cytolysins (CDCs), all of which require the membrane for oligomerisation, C9 assembles directly onto the nascent MAC from solution. However, the molecular mechanism of MAC assembly remains to be understood. Here we present the 8 Å cryo-EM structure of a soluble form of the poly-C9 component of the MAC. These data reveal a 22-fold symmetrical arrangement of C9 molecules that yield an 88-strand pore-forming β-barrel. The N-terminal thrombospondin-1 (TSP1) domain forms an unexpectedly extensive part of the oligomerisation interface, thus likely facilitating solution-based assembly. These TSP1 interactions may also explain how additional C9 subunits can be recruited to the growing MAC subsequent to membrane insertion. PMID:26841934

  18. Perfringolysin O: The Underrated Clostridium perfringens Toxin?

    PubMed Central

    Verherstraeten, Stefanie; Goossens, Evy; Valgaeren, Bonnie; Pardon, Bart; Timbermont, Leen; Haesebrouck, Freddy; Ducatelle, Richard; Deprez, Piet; Wade, Kristin R.; Tweten, Rodney; Van Immerseel, Filip

    2015-01-01

    The anaerobic bacterium Clostridium perfringens expresses multiple toxins that promote disease development in both humans and animals. One such toxin is perfringolysin O (PFO, classically referred to as θ toxin), a pore-forming cholesterol-dependent cytolysin (CDC). PFO is secreted as a water-soluble monomer that recognizes and binds membranes via cholesterol. Membrane-bound monomers undergo structural changes that culminate in the formation of an oligomerized prepore complex on the membrane surface. The prepore then undergoes conversion into the bilayer-spanning pore measuring approximately 250–300 Å in diameter. PFO is expressed in nearly all identified C. perfringens strains and harbors interesting traits that suggest a potential undefined role for PFO in disease development. Research has demonstrated a role for PFO in gas gangrene progression and bovine necrohemorrhagic enteritis, but there is limited data available to determine if PFO also functions in additional disease presentations caused by C. perfringens. This review summarizes the known structural and functional characteristics of PFO, while highlighting recent insights into the potential contributions of PFO to disease pathogenesis. PMID:26008232

  19. Group B streptococcal haemolysin and pigment, a tale of twins

    PubMed Central

    Rosa-Fraile, Manuel; Dramsi, Shaynoor; Spellerberg, Barbara

    2014-01-01

    Group B streptococcus [(GBS or Streptococcus agalactiae)] is a leading cause of neonatal meningitis and septicaemia. Most clinical isolates express simultaneously a β-haemolysin/cytolysin and a red polyenic pigment, two phenotypic traits important for GBS identification in medical microbiology. The genetic determinants encoding the GBS haemolysin and pigment have been elucidated and the molecular structure of the pigment has been determined. The cyl operon involved in haemolysin and pigment production is regulated by the major two-component system CovS/R, which coordinates the expression of multiple virulence factors of GBS. Genetic analyses indicated strongly that the haemolysin activity was due to a cytolytic toxin encoded by cylE. However, the biochemical nature of the GBS haemolysin has remained elusive for almost a century because of its instability during purification procedures. Recently, it has been suggested that the haemolytic and cytolytic activity of GBS is due to the ornithine rhamnopolyenic pigment and not to the CylE protein. Here we review and summarize our current knowledge of the genetics, regulation and biochemistry of these twin GBS phenotypic traits, including their functions as GBS virulence factors. PMID:24617549

  20. pH controlled gating of toxic protein pores by dendrimers

    NASA Astrophysics Data System (ADS)

    Mandal, Taraknath; Kanchi, Subbarao; Ayappa, K. G.; Maiti, Prabal K.

    2016-06-01

    Designing effective nanoscale blockers for membrane inserted pores formed by pore forming toxins, which are expressed by several virulent bacterial strains, on a target cell membrane is a challenging and active area of research. Here we demonstrate that PAMAM dendrimers can act as effective pH controlled gating devices once the pore has been formed. We have used fully atomistic molecular dynamics (MD) simulations to characterize the cytolysin A (ClyA) protein pores modified with fifth generation (G5) PAMAM dendrimers. Our results show that the PAMAM dendrimer, in either its protonated (P) or non-protonated (NP) states can spontaneously enter the protein lumen. Protonated dendrimers interact strongly with the negatively charged protein pore lumen. As a consequence, P dendrimers assume a more expanded configuration efficiently blocking the pore when compared with the more compact configuration adopted by the neutral NP dendrimers creating a greater void space for the passage of water and ions. To quantify the effective blockage of the protein pore, we have calculated the pore conductance as well as the residence times by applying a weak force on the ions/water. Ionic currents are reduced by 91% for the P dendrimers and 31% for the NP dendrimers. The preferential binding of Cl- counter ions to the P dendrimer creates a zone of high Cl- concentration in the vicinity of the internalized dendrimer and a high concentration of K+ ions in the transmembrane region of the pore lumen. In addition to steric effects, this induced charge segregation for the P dendrimer effectively blocks ionic transport through the pore. Our investigation shows that the bio-compatible PAMAM dendrimers can potentially be used to develop therapeutic protocols based on the pH sensitive gating of pores formed by pore forming toxins to mitigate bacterial infections.Designing effective nanoscale blockers for membrane inserted pores formed by pore forming toxins, which are expressed by several virulent

  1. Intermedilysin induces EGR-1 expression through calcineurin/NFAT pathway in human cholangiocellular carcinoma cells

    SciTech Connect

    Susilowati, Heni; Okamura, Hirohiko; Hirota, Katsuhiko; Shono, Masayuki; Yoshida, Kaya; Murakami, Keiji; Tabata, Atsushi; Nagamune, Hideaki; Haneji, Tatsuji; Miyake, Yoichiro

    2011-01-07

    Research highlights: {yields} ILY leads to the accumulation of [Ca{sup 2+}]i in the nucleus in HuCCT1 cells. {yields} ILY induced activation of NFAT1 through a calcineurin-dependent pathway. {yields} Calcineuri/NFAT pathway is involved in EGR-1 expression in response to ILY treatment. -- Abstract: Intermedilysin (ILY) is a cholesterol-dependent cytolysin produced by Streptococcus intermedius, which is associated with human brain and liver abscesses. Although intrahepatic bile duct cells play a valuable role in the pathogenesis of liver abscess, the molecular mechanism of ILY-treated intrahepatic bile duct cells remains unknown. In this study, we report that ILY induced a nuclear accumulation of intracellular calcium ([Ca{sup 2+}]i) in human cholangiocellular cells HuCCT1. We also demonstrate that 10 ng/ml ILY induced NFAT1 dephosphorylation and its nuclear translocation in HuCCT1 cells. In contrast to the result that ILY induced NF-{kappa}B translocation in human hepatic HepG2 cells, ILY did not affect NF-{kappa}B localization in HuCCT1 cells. Dephosphorylation and nuclear translocation of NFAT1 caused by ILY were prevented by [Ca{sup 2+}]i calcium chelator, BAPTA/AM, and calcineurin inhibitors, cyclosporine A and tacrolimus. ILY induced early growth response-1 (EGR-1) expression and it was inhibited by the pre-treatment with cyclosporine A, indicating that the calcineurin/NFAT pathway was involved in EGR-1 expression in response to ILY. ILY-induced calcineurin/NFAT1 activation and sequential EGR-1 expression might be related to the pathogenesis of S. intermedius in human bile duct cells.

  2. Altering Hydrophobic Sequence Lengths Shows That Hydrophobic Mismatch Controls Affinity for Ordered Lipid Domains (Rafts) in the Multitransmembrane Strand Protein Perfringolysin O*

    PubMed Central

    Lin, Qingqing; London, Erwin

    2013-01-01

    The hypothesis that mismatch between transmembrane (TM) length and bilayer width controls TM protein affinity for ordered lipid domains (rafts) was tested using perfringolysin O (PFO), a pore-forming cholesterol-dependent cytolysin. PFO forms a multimeric barrel with many TM segments. The properties of PFO mutants with lengthened or shortened TM segments were compared with that of PFO with wild type TM sequences. Both mutant and wild type length PFO exhibited cholesterol-dependent membrane insertion. Maximal PFO-induced pore formation occurred in vesicles with wider bilayers for lengthened TM segments and in thinner bilayers for shortened TM segments. In diC18:0 phosphatidylcholine (PC)/diC14:1 PC/cholesterol vesicles, which form ordered domains with a relatively thick bilayer and disordered domains with a relatively thin bilayer, affinity for ordered domains was greatest with lengthened TM segments and least with shortened TM segments as judged by FRET. Similar results were observed by microscopy in giant vesicles containing sphingomyelin in place of diC18:0 PC. In contrast, in diC16:0 PC/diC14:0 PC/diC20:1 PC/cholesterol vesicles, which should form ordered domains with a relatively thin bilayer and disordered domains with a relatively thick bilayer, relative affinity for ordered domains was greatest with shortened TM segments and least with lengthened TM segments. The inability of multi-TM segment proteins (unlike single TM segment proteins) to adapt to mismatch by tilting may explain the sensitivity of raft affinity to mismatch. The difference in width sensitivity for single and multi-TM helix proteins may link raft affinity to multimeric state and thus control the assembly of multimeric TM complexes in rafts. PMID:23150664

  3. Detection of lytA, pspC, and rrgA genes in Streptococcus pneumoniae isolated from healthy children

    PubMed Central

    Gholamhosseini-Moghaddam, Tahereh; Rad, Mehrnaz; Mousavi, Seyed Fazlollah; Ghazvini, Kiarash

    2015-01-01

    Background and Objectives: Many surface proteins are implicated in nasopharyngeal colonization and pathogenesis of Streptococcus pneumoniae. Some of these factors are candidate antigens for protein based vaccines. New vaccine designs focus on the surface proteins (e. g., pspA and pspC) and also cytolysin, and pneumolysin. In this study, 3 key virulence genes, lytA, pspC, and rrgA, which encoded surface proteins, were detected among S. pneumoniae isolates. Materials and Methods: A total of 260 nasopharyngeal swabs were collected from healthy children under 6 years old attending day care centers in Mashhad, Iran. Isolates of S. pneumoniae were confirmed by optochin susceptibility and colony appearance and also by PCR for cpsA gene. The presence of lytA, pspC, and rrgA genes were also detected by PCR. Results: A total of 59 isolates were confirmed as S. pneumoniae. Among these isolates, 50 (84.74%), 19 (32.20%), and 2 (3.38%) were positive for lytA, rrgA, and pspC genes respectively. The presence of these genes among S.pneumoniae isolates were as follows: 1) rrgA, lytA, pspC (1 isolate), 2) rrgA, lytA(17isolates), 3) pspC (2 isolate), 4) lytA (50 isolates). Conclusion: cpsA gene was specific for detection of S. pneumoniae isolates which were colonized in nasopharynx. The lytA gene was the most frequent gene among the S. pneumoniae isolates, and combination of rrgA, lytA was the most observed pattern. Thus, it is important for future monitoring of vaccine formulation in our country. PMID:26668703

  4. Membrane cholesterol and sphingomyelin, and ostreolysin A are obligatory for pore-formation by a MACPF/CDC-like pore-forming protein, pleurotolysin B.

    PubMed

    Ota, Katja; Leonardi, Adrijana; Mikelj, Miha; Skočaj, Matej; Wohlschlager, Therese; Künzler, Markus; Aebi, Markus; Narat, Mojca; Križaj, Igor; Anderluh, Gregor; Sepčić, Kristina; Maček, Peter

    2013-10-01

    The mushroom Pleurotus ostreatus has been reported to produce the hemolytic proteins ostreolysin (OlyA), pleurotolysin A (PlyA) and pleurotolysin B (PlyB). The present study of the native and recombinant proteins dissects out their lipid-binding characteristics and their roles in lipid binding and membrane permeabilization. Using lipid-binding studies, permeabilization of erythrocytes, large unilamellar vesicles of various lipid compositions, and electron microscopy, we show that OlyA, a PlyA homolog, preferentially binds to membranes rich in sterol and sphingomyelin, but it does not permeabilize them. The N-terminally truncated Δ48PlyB corresponds to the mature and active form of native PlyB, and it has a membrane attack complex-perforin (MACPF) domain. Δ48PlyB spontaneously oligomerizes in solution, and binds weakly to various lipid membranes but is not able to perforate them. However, binding of Δ48PlyB to the cholesterol and sphingomyelin membranes, and consequently, their permeabilization is dramatically promoted in the presence of OlyA. On these membranes, Δ48PlyB and OlyA form predominantly 13-meric oligomers. These are rosette-like structures with a thickness of ∼9 nm from the membrane surface, with 19.7 nm and 4.9 nm outer and inner diameters, respectively. When present on opposing vesicle membranes, these oligomers can dimerize and thus promote aggregation of vesicles. Based on the structural and functional characteristics of Δ48PlyB, we suggest that it shares some features with MACPF/cholesterol-dependent cytolysin (CDC) proteins. OlyA is obligatory for the Δ48PlyB permeabilization of membranes rich in cholesterol and sphingomyelin. PMID:23806422

  5. Human hyperimmune globulin protects against the cytotoxic action of staphylococcal alpha-toxin in vitro and in vivo.

    PubMed Central

    Bhakdi, S; Mannhardt, U; Muhly, M; Hugo, F; Ronneberger, H; Hungerer, K D

    1989-01-01

    Alpha-toxin, the major cytolysin of Staphylococcus aureus, preferentially attacks human platelets and cultured monocytes, thereby promoting coagulation and the release of interleukin-1 and tumor necrosis factor. Titers of naturally occurring antibodies in human blood are not high enough to substantially inhibit these pathological reactions. In the present study, F(ab')2 fragment preparations from hyperimmune globulin obtained from immunized volunteers were tested for their capacity to inhibit the cytotoxic action of alpha-toxin in vitro and in vivo. These antibody preparations exhibited neutralizing anti-alpha-toxin titers of 80 to 120 IU/ml, whereas titers in commercial immunoglobulin preparations were 1 to 4 IU/ml. In vitro, the presence of 2 to 4 mg of hyperimmune globulin per ml protected human platelets against the action of 1 to 2 micrograms of alpha-toxin per ml. Similarly, these antibodies fully protected human monocytes against the ATP-depleting and cytokine-liberating effects of 0.1 to 1 microgram of alpha-toxin per ml. Intravenous application of 0.5 mg (85 to 120 micrograms/kg of body weight) of alpha-toxin in cynomolgus monkeys elicited acute pathophysiological reactions which were heralded by a selective drop in blood platelet counts. Toxin doses of 1 to 2 mg (170 to 425 micrograms/kg) had a rapid lethal effect, the animals presenting with signs of cardiovascular collapse and pulmonary edema. Prior intravenous application of 4 ml of hyperimmune globulins per kg inhibited the systemic toxic and lethal effects of 1 mg (200 micrograms/kg) of alpha-toxin. In contrast, normal human immunoglobulins exhibited no substantial protective efficacy in vitro and only marginal effects in vivo. It is concluded that high-titered anti-alpha-toxin antibodies effectively protect against the cytotoxic actions of alpha-toxin. PMID:2777380

  6. Sea Anemone (Cnidaria, Anthozoa, Actiniaria) Toxins: An Overview

    PubMed Central

    Frazão, Bárbara; Vasconcelos, Vitor; Antunes, Agostinho

    2012-01-01

    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. PMID:23015776

  7. Functional and topological studies with Trp-containing analogs of the peptide StII(1-30) derived from the N-terminus of the pore forming toxin sticholysin II: contribution to understand its orientation in membrane.

    PubMed

    Ros, Uris; Souto, Ana Lucia C F; de Oliveira, Felipe J; Crusca, Edson; Pazos, Fabiola; Cilli, Eduardo M; Lanio, Maria E; Schreier, Shirley; Alvarez, Carlos

    2013-07-01

    Sticholysin II (St II) is the most potent cytolysin produced by the sea anemone Stichodactyla helianthus, exerting hemolytic activity via pore formation in membranes. The toxin's N-terminus contains an amphipathic α-helix that is very likely involved in pore formation. We have previously demonstrated that the synthetic peptide StII(1-30) encompassing the 1-30 segment of St II forms pores of similar radius to that of the protein (around 1 nm), being a good model of toxin functionality. Here we have studied the functional and conformational properties of fluorescent analogs of StII(1-30) in lipid membranes. The analogs were obtained by replacing Leu residues at positions 2, 12, 17, and 24 with the intrinsically fluorescent amino acid Trp (StII(1-30L2W), StII(1-30L12W), StII(1-30L17W), or StII(1-30L24W), respectively). The exchange by Trp did not significantly modify the activity and conformation of the parent peptide. The blue-shift and intensity enhancement of fluorescence in the presence of membrane indicated that Trp at position 2 is more deeply buried in the hydrophobic region of the bilayer. These experiments, as well as assays with water-soluble or spin-labeled lipid-soluble fluorescence quenchers suggest an orientation of StII(1-30) with its N-terminus oriented towards the hydrophobic core of the bilayer while the rest of the peptide is more exposed to the aqueous environment, as hypothesized for sticholysins. PMID:23868208

  8. Functional characterization of sticholysin I and W111C mutant reveals the sequence of the actinoporin's pore assembly.

    PubMed

    Antonini, Valeria; Pérez-Barzaga, Victor; Bampi, Silvia; Pentón, David; Martínez, Diana; Dalla Serra, Mauro; Tejuca, Mayra

    2014-01-01

    The use of pore-forming toxins in the construction of immunotoxins against tumour cells is an alternative for cancer therapy. In this protein family one of the most potent toxins are the actinoporins, cytolysins from sea anemones. We work on the construction of tumour proteinase-activated immunotoxins using sticholysin I (StI), an actinoporin isolated from the sea anemone Stichodactyla helianthus. To accomplish this objective, recombinant StI (StIr) with a mutation in the membrane binding region has been employed. In this work, it was evaluated the impact of mutating tryptophan 111 to cysteine on the toxin pore forming capability. StI W111C is still able to permeabilize erythrocytes and liposomes, but at ten-fold higher concentration than StI. This is due to its lower affinity for the membrane, which corroborates the importance of residue 111 for the binding of actinoporins to the lipid bilayer. In agreement, other functional characteristics not directly associated to the binding, are essentially the same for both variants, that is, pores have oligomeric structures with similar radii, conductance, cation-selectivity, and instantaneous current-voltage behavior. In addition, this work provides experimental evidence sustaining the toroidal protein-lipid actinoporins lytic structures, since the toxins provoke the trans-bilayer movement (flip-flop) of a pyrene-labeled analogue of phosphatidylcholine in liposomes, indicating the existence of continuity between the outer and the inner membrane leaflet. Finally, our planar lipid membranes results have also contributed to a better understanding of the actinoporin's pore assembly mechanism. After the toxin binding and the N-terminal insertion in the lipid membrane, the pore assembly occurs by passing through different transient sub-conductance states. These states, usually 3 or 4, are due to the successive incorporation of N-terminal α-helices and lipid heads to the growing pores until a stable toroidal oligomeric structure

  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. PMID:23015776

  10. Protein-lipid interactions and non-lamellar lipidic structures in membrane pore formation and membrane fusion.

    PubMed

    Gilbert, Robert J C

    2016-03-01

    Pore-forming proteins and peptides act on their targeted lipid bilayer membranes to increase permeability. This approach to the modulation of biological function is relevant to a great number of living processes, including; infection, parasitism, immunity, apoptosis, development and neurodegeneration. While some pore-forming proteins/peptides assemble into rings of subunits to generate discrete, well-defined pore-forming structures, an increasing number is recognised to form pores via mechanisms which co-opt membrane lipids themselves. Among these, membrane attack complex-perforin/cholesterol-dependent cytolysin (MACPF/CDC) family proteins, Bax/colicin family proteins and actinoporins are especially prominent and among the mechanisms believed to apply are the formation of non-lamellar (semi-toroidal or toroidal) lipidic structures. In this review I focus on the ways in which lipids contribute to pore formation and contrast this with the ways in which lipids are co-opted also in membrane fusion and fission events. A variety of mechanisms for pore formation that involve lipids exists, but they consistently result in stable hybrid proteolipidic structures. These structures are stabilised by mechanisms in which pore-forming proteins modify the innate capacity of lipid membranes to respond to their environment, changing shape and/or phase and binding individual lipid molecules directly. In contrast, and despite the diversity in fusion protein types, mechanisms for membrane fusion are rather similar to each other, mapping out a pathway from pairs of separated compartments to fully confluent fused membranes. Fusion proteins generate metastable structures along the way which, like long-lived proteolipidic pore-forming complexes, rely on the basic physical properties of lipid bilayers. Membrane fission involves similar intermediates, in the reverse order. I conclude by considering the possibility that at least some pore-forming and fusion proteins are evolutionarily related

  11. Detoxified pneumolysin derivative Plym2 directly protects against pneumococcal infection via induction of inflammatory cytokines.

    PubMed

    Lu, Jingcai; Sun, Tianxu; Hou, Hongjia; Xu, Man; Gu, Tiejun; Dong, Yunliang; Wang, Dandan; Chen, Pinxu; Wu, Chunlai; Liang, Chunshu; Sun, Shiyang; Jiang, Chunlai; Kong, Wei; Wu, Yongge

    2014-01-01

    Streptococcus pneumoniae is a major cause of infectious disease and complications worldwide, such as pneumonia, otitis media, bacteremia and meningitis. New generation protein-based pneumococcal vaccines are recognized as alternative vaccine candidates. Pneumolysin (Ply) is a cholesterol-dependent cytolysin produced by all clinical isolates of S. pneumoniae. Our research group previously developed a highly detoxified Ply mutant designated Plym2 by replacement of two animo acids (C428G and W433F). Exhibiting undetectable levels of cytotoxicity, Plym2 could still elicit high titer neutralizing antibodies against the native toxin. However, evaluation of the active immunoprotective effects of Plym2 by subcutaneous immunization and lethal challenge with S. pneumoniae in mice did not yield favorable results. In the present work, we confirmed the previous observations by using passive immunization and systemic challenge. Results of the passive immunization were consistent with those of active immunization. Further experiments were conducted to explain the inability of high titer neutralizing antibodies against Ply to protect mice from S. pneumoniae challenge. Pneumococcal Ply is known to be the major factor responsible for the induction of inflammation that benefits the host. Proinflammatory cytokines facilitate the clearance of invaders by the recruitment and activation of leukocytes at the early infection stage. We demonstrated that Plym2 could induce proinflammatory cytokines similarly to wild-type Ply. A systemic infection model was used to clarify that Plym2 lacking cytolytic activity could protect mice from intraperitoneal challenge directly, while antibodies to the mutant had no effect. Therefore, the protective function of Plym2 may be due to its induction of proinflammatory cytokines. When used in the systemic infection model, Plym2 antibodies may block the induction of proinflammatory cytokines by Ply. These findings demonstrate that a Ply-based vaccine would

  12. The impact of pneumolysin on the macrophage response to Streptococcus pneumoniae is strain-dependent.

    PubMed

    Harvey, Richard M; Hughes, Catherine E; Paton, Adrienne W; Trappetti, Claudia; Tweten, Rodney K; Paton, James C

    2014-01-01

    Streptococcus pneumoniae is the world's leading cause of pneumonia, bacteremia, meningitis and otitis media. A major pneumococcal virulence factor is the cholesterol-dependent cytolysin, which has the defining property of forming pores in cholesterol-containing membranes. In recent times a clinically significant and internationally successful serotype 1 ST306 clone has been found to express a non-cytolytic variant of Ply (Ply306). However, while the pneumococcus is a naturally transformable organism, strains of the ST306 clonal group have to date been virtually impossible to transform, severely restricting efforts to understand the role of non-cytolytic Ply in the success of this clone. In this study isogenic Ply mutants were constructed in the D39 background and for the first time in the ST306 background (A0229467) to enable direct comparisons between Ply variants for their impact on the immune response in a macrophage-like cell line. Strains that expressed cytolytic Ply were found to induce a significant increase in IL-1β release from macrophage-like cells compared to the non-cytolytic and Ply-deficient strains in a background-independent manner, confirming the requirement for pore formation in the Ply-dependent activation of the NLRP3 inflammasome. However, cytolytic activity in the D39 background was found to induce increased expression of the genes encoding GM-CSF (CSF2), p19 subunit of IL-23 (IL23A) and IFNβ (IFNB1) compared to non-cytolytic and Ply-deficient D39 mutants, but had no effect in the A0229467 background. The impact of Ply on the immune response to the pneumococcus is highly dependent on the strain background, thus emphasising the importance of the interaction between specific virulence factors and other components of the genetic background of this organism. PMID:25105894

  13. Ecology of Antibiotic Resistance Genes: Characterization of Enterococci from Houseflies Collected in Food Settings†

    PubMed Central

    Macovei, Lilia; Zurek, Ludek

    2006-01-01

    In this project, enterococci from the digestive tracts of 260 houseflies (Musca domestica L.) collected from five restaurants were characterized. Houseflies frequently (97% of the flies were positive) carried enterococci (mean, 3.1 × 103 CFU/fly). Using multiplex PCR, 205 of 355 randomly selected enterococcal isolates were identified and characterized. The majority of these isolates were Enterococcus faecalis (88.2%); in addition, 6.8% were E. faecium, and 4.9% were E. casseliflavus. E. faecalis isolates were phenotypically resistant to tetracycline (66.3%), erythromycin (23.8%), streptomycin (11.6%), ciprofloxacin (9.9%), and kanamycin (8.3%). Tetracycline resistance in E. faecalis was encoded by tet(M) (65.8%), tet(O) (1.7%), and tet(W) (0.8%). The majority (78.3%) of the erythromycin-resistant E. faecalis isolates carried erm(B). The conjugative transposon Tn916 and members of the Tn916/Tn1545 family were detected in 30.2% and 34.6% of the identified isolates, respectively. E. faecalis carried virulence genes, including a gelatinase gene (gelE; 70.7%), an aggregation substance gene (asa1; 33.2%), an enterococcus surface protein gene (esp; 8.8%), and a cytolysin gene (cylA; 8.8%). Phenotypic assays showed that 91.4% of the isolates with the gelE gene were gelatinolytic and that 46.7% of the isolates with the asa1 gene aggregated. All isolates with the cylA gene were hemolytic on human blood. This study showed that houseflies in food-handling and -serving facilities carry antibiotic-resistant and potentially virulent enterococci that have the capacity for horizontal transfer of antibiotic resistance genes to other bacteria. PMID:16751512

  14. Novel recombinant BCG expressing perfringolysin O and the over-expression of key immunodominant antigens; pre-clinical characterization, safety and protection against challenge with Mycobacterium tuberculosis.

    PubMed

    Sun, Ronggai; Skeiky, Yasir A W; Izzo, Angelo; Dheenadhayalan, Veerabadran; Imam, Zakaria; Penn, Erica; Stagliano, Katherine; Haddock, Scott; Mueller, Stefanie; Fulkerson, John; Scanga, Charles; Grover, Ajay; Derrick, Steven C; Morris, Sheldon; Hone, David M; Horwitz, Marcus A; Kaufmann, Stefan H E; Sadoff, Jerald C

    2009-07-16

    Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), has infected approximately two billion individuals worldwide with approximately 9.2 million new cases and 1.6 million deaths annually. Current efforts are focused on making better BCG priming vaccines designed to induce a comprehensive and balanced immunity followed by booster(s) targeting a specific set of relevant antigens in common with the BCG prime. We describe the generation and immunological characterization of recombinant BCG strains with properties associated with lysis of the endosome compartment and over-expression of key Mtb antigens. The endosome lysis strain, a derivative of BCG SSI-1331 (BCG(1331)) expresses a mutant form of perfringolysin O (PfoA(G137Q)), a cytolysin normally secreted by Clostridium perfringens. Integration of the PfoA(G137Q) gene into the BCG genome was accomplished using an allelic exchange plasmid to replace ureC with pfoA(G137Q) under the control of the Ag85B promoter. The resultant BCG construct, designated AERAS-401 (BCG(1331) DeltaureC::OmegapfoA(G137Q)) secreted biologically active Pfo, was well tolerated with a good safety profile in immunocompromised SCID mice. A second rBCG strain, designated AFRO-1, was generated by incorporating an expression plasmid encoding three mycobacterial antigens, Ag85A, Ag85B and TB10.4, into AERAS-401. Compared to the parental BCG strain, vaccination of mice and guinea pigs with AFRO-1 resulted in enhanced immune responses. Mice vaccinated with AFRO-1 and challenged with the hypervirulent Mtb strain HN878 also survived longer than mice vaccinated with the parental BCG. Thus, we have generated improved rBCG vaccine candidates that address many of the shortcomings of the currently licensed BCG vaccine strains. PMID:19500523

  15. Role of house flies in the ecology of Enterococcus faecalis from wastewater treatment facilities.

    PubMed

    Doud, C W; Scott, H M; Zurek, L

    2014-02-01

    Enterococci are important nosocomial pathogens, with Enterococcus faecalis most commonly responsible for human infections. In this study, we used several measures to test the hypothesis that house flies, Musca domestica (L.), acquire and disseminate antibiotic-resistant and potentially virulent E. faecalis from wastewater treatment facilities (WWTF) to the surrounding urban environment. House flies and sludge from four WWTF (1-4) as well as house flies from three urban sites close to WWTF-1 were collected and cultured for enterococci. Enterococci were identified, quantified, screened for antibiotic resistance and virulence traits, and assessed for clonality. Of the 11 antibiotics tested, E. faecalis was most commonly resistant to tetracycline, doxycycline, streptomycin, gentamicin, and erythromycin, and these traits were intra-species horizontally transferrable by in vitro conjugation. Profiles of E. faecalis (prevalence, antibiotic resistance, and virulence traits) from each of WWTF sludge and associated house flies were similar, indicating that flies successfully acquired these bacteria from this substrate. The greatest number of E. faecalis with antibiotic resistance and virulence factors (i.e., gelatinase, cytolysin, enterococcus surface protein, and aggregation substance) originated from WWTF-1 that processed meat waste from a nearby commercial meat-processing plant, suggesting an agricultural rather than human clinical source of these isolates. E. faecalis from house flies collected from three sites 0.7-1.5 km away from WWTF-1 were also similar in their antibiotic resistance profiles; however, antibiotic resistance was significantly less frequent. Clonal diversity assessment using pulsed-field gel electrophoresis revealed the same clones of E. faecalis from sludge and house flies from WWTF-1 but not from the three urban sites close to WWTF-1. This study demonstrates that house flies acquire antibiotic-resistant enterococci from WWTF and potentially

  16. Molecular detection of virulence factors among food and clinical Enterococcus faecalis strains in South Brazil

    PubMed Central

    Medeiros, A.W.; Pereira, R.I.; Oliveira, D.V.; Martins, P.D.; d’Azevedo, P.A.; Van der Sand, S.; Frazzon, J.; Frazzon, A.P.G

    2014-01-01

    The present report aimed to perform a molecular epidemiological survey by investigating the presence of virulence factors in E. faecalis isolated from different human clinical (n = 57) and food samples (n = 55) in Porto Alegre, Brazil, collected from 2006 to 2009. In addition, the ability to form biofilm in vitro on polystyrene and the β-haemolytic and gelatinase activities were determined. Clinical strains presented a higher prevalence of aggregation substance (agg), enterococcal surface protein (esp) and cytolysin (cylA) genes when compared with food isolates. The esp gene was found only in clinical strains. On the other hand, the gelatinase (gelE) and adherence factor (ace) genes had similar prevalence among the strains, showing the widespread occurrence of these virulence factors among food and clinical E. faecalis strains in South Brazil. More than three virulence factor genes were detected in 77.2% and 18.2% of clinical and food strains, respectively. Gelatinase and β-haemolysin activities were not associated with the presence of gelE and cylA genes. The ability to produce biofilm was detected in 100% of clinical and 94.6% of food isolates, and clinical strains were more able to form biofilm than the food isolates (Student’s t-test, p < 0.01). Results from the statistical analysis showed significant associations between strong biofilm formation and ace (p = 0.015) and gelE (p = 0.007) genes in clinical strains. In conclusion, our data indicate that E. faecalis strains isolated from clinical and food samples possess distinctive patterns of virulence factors, with a larger number of genes that encode virulence factors detected in clinical strains. PMID:24948952

  17. Colostrum of Healthy Slovenian Mothers: Microbiota Composition and Bacteriocin Gene Prevalence

    PubMed Central

    Obermajer, Tanja; Lipoglavšek, Luka; Tompa, Gorazd; Treven, Primož; Lorbeg, Petra Mohar; Matijašić, Bojana Bogovič; Rogelj, Irena

    2015-01-01

    Microbial communities inhabiting the breast milk microenvironment are essential in supporting mammary gland health in lactating women and in providing gut-colonizing bacterial 'inoculum' for their infants’ gastro-intestinal development. Bacterial DNA was extracted from colostrum samples of 45 healthy Slovenian mothers. Characteristics of the communities in the samples were assessed by polymerase chain reaction (PCR) coupled with denaturing gradient gel electrophoresis (DGGE) and by quantitative real-time PCR (qPCR). PCR screening for the prevalence of bacteriocin genes was performed on DNA of culturable and total colostrum bacteria. DGGE profiling revealed the presence of Staphylococcus and Gemella in most of the samples and exposed 4 clusters based on the abundance of 3 bands: Staphylococcus epidermidis/Gemella, Streptococcus oralis/pneumonia and Streptococcus salivarius. Bacilli represented the largest proportion of the communities. High prevalence in samples at relatively low quantities was confirmed by qPCR for enterobacteria (100%), Clostridia (95.6%), Bacteroides-Prevotella group (62.2%) and bifidobacteria (53.3%). Bacterial quantities (genome equivalents ml-1) varied greatly among the samples; Staphylococcus epidermidis and staphylococci varied in the range of 4 logs, streptococci and all bacteria varied in the range of 2 logs, and other researched groups varied in the range of 1 log. The quantity of most bacterial groups was correlated with the amount of all bacteria. The majority of the genus Staphylococcus was represented by the species Staphylococcus epidermidis (on average 61%), and their abundances were linearly correlated. Determinants of salivaricin A, salivaricin B, streptin and cytolysin were found in single samples. This work provides knowledge on the colostrum microbial community composition of healthy lactating Slovenian mothers and reports bacteriocin gene prevalence. PMID:25919457

  18. The Role of Autophagy during Group B Streptococcus Infection of Blood-Brain Barrier Endothelium*

    PubMed Central

    Cutting, Andrew S.; Del Rosario, Yvette; Mu, Rong; Rodriguez, Anthony; Till, Andreas; Subramani, Suresh; Gottlieb, Roberta A.; Doran, Kelly S.

    2014-01-01

    Bacterial meningitis occurs when bloodborne pathogens invade and penetrate the blood-brain barrier (BBB), provoking inflammation and disease. Group B Streptococcus (GBS), the leading cause of neonatal meningitis, can enter human brain microvascular endothelial cells (hBMECs), but the host response to intracellular GBS has not been characterized. Here we sought to determine whether antibacterial autophagy, which involves selective recognition of intracellular organisms and their targeting to autophagosomes for degradation, is activated in BBB endothelium during bacterial infection. GBS infection resulted in increased punctate distribution of GFP-microtubule-associated protein 1 light chain 3 (LC3) and increased levels of endogenous LC3-II and p62 turnover, two hallmark indicators of active autophagic flux. Infection with GBS mutants revealed that bacterial invasion and the GBS pore-forming β-hemolysin/cytolysin (β-h/c) trigger autophagic activation. Cell-free bacterial extracts containing β-h/c activity induced LC3-II conversion, identifying this toxin as a principal provocative factor for autophagy activation. These results were confirmed in vivo using a mouse model of GBS meningitis as infection with WT GBS induced autophagy in brain tissue more frequently than a β-h/c-deficient mutant. Elimination of autophagy using Atg5-deficient fibroblasts or siRNA-mediated impairment of autophagy in hBMECs led to increased recovery of intracellular GBS. However, electron microscopy revealed that GBS was rarely found within double membrane autophagic structures even though we observed GBS-LC3 co-localization. These results suggest that although autophagy may act as a BBB cellular defense mechanism in response to invading and toxin-producing bacteria, GBS may actively thwart the autophagic pathway. PMID:25371213

  19. Regulation of cytotoxin expression by converging eukaryotic-type and two-component signalling mechanisms in Streptococcus agalactiae.

    PubMed

    Rajagopal, Lakshmi; Vo, Anthony; Silvestroni, Aurelio; Rubens, Craig E

    2006-11-01

    Signal transducing mechanisms are essential for regulation of gene expression in both prokaryotic and eukaryotic organisms. Regulation of gene expression in eukaryotes is accomplished by serine/threonine and tyrosine kinases and cognate phosphatases. In contrast, gene expression in prokaryotes is controlled by two-component systems that comprise a sensor histidine kinase and a cognate DNA binding response regulator. Pathogenic bacteria utilize two-component systems to regulate expression of their virulence factors and for adaptive responses to the external environment. We have previously shown that the human pathogen Streptococcus agalactiae (Group B Streptococci, GBS) encodes a single eukaryotic-type serine/threonine kinase Stk1, which is important for virulence of the organism. In this study, we aimed to understand how Stk1 contributes to virulence of GBS. Our results indicate that Stk1 expression is important for resistance of GBS to human blood, neutrophils and oxidative stress. Consistent with these observations, Stk1 positively regulates transcription of a cytotoxin, beta-haemolysin/cytolysin (beta-H/C) that is critical for survival of GBS in the bloodstream and for resistance to oxidative stress. Interestingly, positive regulation of beta-H/C by Stk1 requires the two-component regulator CovR. Further, we show that Stk1 can negatively regulate transcription of CAMP factor in a CovR-dependent manner. As Stk1 phosphorylates CovR in vitro, these data suggest that serine/threonine phosphorylation impacts CovR-mediated regulation of GBS gene expression. In summary, our studies provide novel information that a eukaryotic-type serine/threonine kinase regulates two-component-mediated expression of GBS cytotoxins. PMID:17005013

  20. Avoidance of Autophagy Mediated by PlcA or ActA Is Required for Listeria monocytogenes Growth in Macrophages

    PubMed Central

    Mitchell, Gabriel; Ge, Liang; Huang, Qiongying; Chen, Chen; Kianian, Sara; Roberts, Mary F.; Schekman, Randy

    2015-01-01

    Listeria monocytogenes is a facultative intracellular pathogen that escapes from phagosomes and grows in the cytosol of infected host cells. Most of the determinants that govern its intracellular life cycle are controlled by the transcription factor PrfA, including the pore-forming cytolysin listeriolysin O (LLO), two phospholipases C (PlcA and PlcB), and ActA. We constructed a strain that lacked PrfA but expressed LLO from a PrfA-independent promoter, thereby allowing the bacteria to gain access to the host cytosol. This strain did not grow efficiently in wild-type macrophages but grew normally in macrophages that lacked ATG5, a component of the autophagy LC3 conjugation system. This strain colocalized more with the autophagy marker LC3 (42% ± 7%) at 2 h postinfection, which constituted a 5-fold increase over the colocalization exhibited by the wild-type strain (8% ± 6%). While mutants lacking the PrfA-dependent virulence factor PlcA, PlcB, or ActA grew normally, a double mutant lacking both PlcA and ActA failed to grow in wild-type macrophages and colocalized more with LC3 (38% ± 5%). Coexpression of LLO and PlcA in a PrfA-negative strain was sufficient to restore intracellular growth and decrease the colocalization of the bacteria with LC3. In a cell-free assay, purified PlcA protein blocked LC3 lipidation, a key step in early autophagosome biogenesis, presumably by preventing the formation of phosphatidylinositol 3-phosphate (PI3P). The results of this study showed that avoidance of autophagy by L. monocytogenes primarily involves PlcA and ActA and that either one of these factors must be present for L. monocytogenes growth in macrophages. PMID:25776746

  1. Colostrum of healthy Slovenian mothers: microbiota composition and bacteriocin gene prevalence.

    PubMed

    Obermajer, Tanja; Lipoglavšek, Luka; Tompa, Gorazd; Treven, Primož; Lorbeg, Petra Mohar; Matijašić, Bojana Bogovič; Rogelj, Irena

    2014-01-01

    Microbial communities inhabiting the breast milk microenvironment are essential in supporting mammary gland health in lactating women and in providing gut-colonizing bacterial 'inoculum' for their infants' gastro-intestinal development. Bacterial DNA was extracted from colostrum samples of 45 healthy Slovenian mothers. Characteristics of the communities in the samples were assessed by polymerase chain reaction (PCR) coupled with denaturing gradient gel electrophoresis (DGGE) and by quantitative real-time PCR (qPCR). PCR screening for the prevalence of bacteriocin genes was performed on DNA of culturable and total colostrum bacteria. DGGE profiling revealed the presence of Staphylococcus and Gemella in most of the samples and exposed 4 clusters based on the abundance of 3 bands: Staphylococcus epidermidis/Gemella, Streptococcus oralis/pneumonia and Streptococcus salivarius. Bacilli represented the largest proportion of the communities. High prevalence in samples at relatively low quantities was confirmed by qPCR for enterobacteria (100%), Clostridia (95.6%), Bacteroides-Prevotella group (62.2%) and bifidobacteria (53.3%). Bacterial quantities (genome equivalents ml-1) varied greatly among the samples; Staphylococcus epidermidis and staphylococci varied in the range of 4 logs, streptococci and all bacteria varied in the range of 2 logs, and other researched groups varied in the range of 1 log. The quantity of most bacterial groups was correlated with the amount of all bacteria. The majority of the genus Staphylococcus was represented by the species Staphylococcus epidermidis (on average 61%), and their abundances were linearly correlated. Determinants of salivaricin A, salivaricin B, streptin and cytolysin were found in single samples. This work provides knowledge on the colostrum microbial community composition of healthy lactating Slovenian mothers and reports bacteriocin gene prevalence. PMID:25919457

  2. Streptolysin S Promotes Programmed Cell Death and Enhances Inflammatory Signaling in Epithelial Keratinocytes during Group A Streptococcus Infection

    PubMed Central

    Flaherty, Rebecca A.; Puricelli, Jessica M.; Higashi, Dustin L.; Park, Claudia J.

    2015-01-01

    Streptococcus pyogenes, or group A Streptococcus (GAS), is a pathogen that causes a multitude of human diseases from pharyngitis to severe infections such as toxic shock syndrome and necrotizing fasciitis. One of the primary virulence factors produced by GAS is the peptide toxin streptolysin S (SLS). In addition to its well-recognized role as a cytolysin, recent evidence has indicated that SLS may influence host cell signaling pathways at sublytic concentrations during infection. We employed an antibody array-based approach to comprehensively identify global host cell changes in human epithelial keratinocytes in response to the SLS toxin. We identified key SLS-dependent host responses, including the initiation of specific programmed cell death and inflammatory cascades with concomitant downregulation of Akt-mediated cytoprotection. Significant signaling responses identified by our array analysis were confirmed using biochemical and protein identification methods. To further demonstrate that the observed SLS-dependent host signaling changes were mediated primarily by the secreted toxin, we designed a Transwell infection system in which direct bacterial attachment to host cells was prevented, while secreted factors were allowed access to host cells. The results using this approach were consistent with our direct infection studies and reveal that SLS is a bacterial toxin that does not require bacterial attachment to host cells for activity. In light of these findings, we propose that the production of SLS by GAS during skin infection promotes invasive outcomes by triggering programmed cell death and inflammatory cascades in host cells to breach the keratinocyte barrier for dissemination into deeper tissues. PMID:26238711

  3. Isolation and identification of Enterococcus faecium from seafoods: antimicrobial resistance and production of bacteriocin-like substances.

    PubMed

    Valenzuela, Antonio Sánchez; Benomar, Nabil; Abriouel, Hikmate; Cañamero, Magdalena Martínez; Gálvez, Antonio

    2010-10-01

    A collection of isolates from uncooked seafoods (molluscs, fish, and fish fillets) were identified as Enterococcus faecium species and studied in further detail. Isolates were clustered in well-defined genomic groups according to food origin after ERIC-PCR analysis. Four isolates (FR 1-2, FB 1-3-B, FB 3-1, FTA 1-2) decarboxylated lysine, ornithine, and tyrosine. Isolate FR 1-2 also decarboxylated histidine. Most isolates were sensitive to antibiotics of clinical use, but resistance was detected more frequently towards nitrofurantoin (50%), erythromycin (33.33%) or rifampicin (33.33%) to quinupristin/dalfopristin (12.5%). Resistance to beta-lactams or vancomycin was not detected. The enterococcal antigen A was the presumed virulence trait detected most frequently. None of isolates carried haemolysin/cytolysin genes. Twelve isolates produced anti-listerial activity. Among them, seven isolates also produced bacteriocin-like inhibitory substances against other enterococci, and one isolate was also able to inhibit Staphylococcus aureus. Three isolates only were active against Listeria monocytogenes, and two only were active against enterococci. One bacteriocinogenic isolate carried the enterocin A structural gene, but genes corresponding to other enterocins (EntB, EntP, EntQ, Ent1071, EntL50A/EntL50B, and Ent31) were not detected. Bacteriocin-producing enterococci lacking undesirable traits (such as antibiotic resistance or biogenic amine production) or their produced bacteriocins could be potential candidates to aid in preservation of seafoods and other food products as well. PMID:20688238

  4. pH controlled gating of toxic protein pores by dendrimers.

    PubMed

    Mandal, Taraknath; Kanchi, Subbarao; Ayappa, K G; Maiti, Prabal K

    2016-07-14

    Designing effective nanoscale blockers for membrane inserted pores formed by pore forming toxins, which are expressed by several virulent bacterial strains, on a target cell membrane is a challenging and active area of research. Here we demonstrate that PAMAM dendrimers can act as effective pH controlled gating devices once the pore has been formed. We have used fully atomistic molecular dynamics (MD) simulations to characterize the cytolysin A (ClyA) protein pores modified with fifth generation (G5) PAMAM dendrimers. Our results show that the PAMAM dendrimer, in either its protonated (P) or non-protonated (NP) states can spontaneously enter the protein lumen. Protonated dendrimers interact strongly with the negatively charged protein pore lumen. As a consequence, P dendrimers assume a more expanded configuration efficiently blocking the pore when compared with the more compact configuration adopted by the neutral NP dendrimers creating a greater void space for the passage of water and ions. To quantify the effective blockage of the protein pore, we have calculated the pore conductance as well as the residence times by applying a weak force on the ions/water. Ionic currents are reduced by 91% for the P dendrimers and 31% for the NP dendrimers. The preferential binding of Cl(-) counter ions to the P dendrimer creates a zone of high Cl(-) concentration in the vicinity of the internalized dendrimer and a high concentration of K(+) ions in the transmembrane region of the pore lumen. In addition to steric effects, this induced charge segregation for the P dendrimer effectively blocks ionic transport through the pore. Our investigation shows that the bio-compatible PAMAM dendrimers can potentially be used to develop therapeutic protocols based on the pH sensitive gating of pores formed by pore forming toxins to mitigate bacterial infections. PMID:27328315

  5. Staphylococcus epidermidis strategies to avoid killing by human neutrophils.

    PubMed

    Cheung, Gordon Y C; Rigby, Kevin; Wang, Rong; Queck, Shu Y; Braughton, Kevin R; Whitney, Adeline R; Teintze, Martin; DeLeo, Frank R; Otto, Michael

    2010-01-01

    Staphylococcus epidermidis is a leading nosocomial pathogen. In contrast to its more aggressive relative S. aureus, it causes chronic rather than acute infections. In highly virulent S. aureus, phenol-soluble modulins (PSMs) contribute significantly to immune evasion and aggressive virulence by their strong ability to lyse human neutrophils. Members of the PSM family are also produced by S. epidermidis, but their role in immune evasion is not known. Notably, strong cytolytic capacity of S. epidermidis PSMs would be at odds with the notion that S. epidermidis is a less aggressive pathogen than S. aureus, prompting us to examine the biological activities of S. epidermidis PSMs. Surprisingly, we found that S. epidermidis has the capacity to produce PSMδ, a potent leukocyte toxin, representing the first potent cytolysin to be identified in that pathogen. However, production of strongly cytolytic PSMs was low in S. epidermidis, explaining its low cytolytic potency. Interestingly, the different approaches of S. epidermidis and S. aureus to causing human disease are thus reflected by the adaptation of biological activities within one family of virulence determinants, the PSMs. Nevertheless, S. epidermidis has the capacity to evade neutrophil killing, a phenomenon we found is partly mediated by resistance mechanisms to antimicrobial peptides (AMPs), including the protease SepA, which degrades AMPs, and the AMP sensor/resistance regulator, Aps (GraRS). These findings establish a significant function of SepA and Aps in S. epidermidis immune evasion and explain in part why S. epidermidis may evade elimination by innate host defense despite the lack of cytolytic toxin expression. Our study shows that the strategy of S. epidermidis to evade elimination by human neutrophils is characterized by a passive defense approach and provides molecular evidence to support the notion that S. epidermidis is a less aggressive pathogen than S. aureus. PMID:20949069

  6. Characterization of functional properties of Enterococcus faecium strains isolated from human gut.

    PubMed

    İspirli, Hümeyra; Demirbaş, Fatmanur; Dertli, Enes

    2015-11-01

    The aim of this work was to characterize the functional properties of Enterococcus faecium strains identified after isolation from human faeces. Of these isolates, strain R13 showed the best resistance to low pH, bile salts, and survival in the simulated in vitro digestion assay, and demonstrated an important level of adhesion to hexadecane as a potential probiotic candidate. Analysis of the antibiotic resistance of E. faecium strains indicated that in general these isolates were sensitive to the tested antibiotics and no strain appeared to be resistant to vancomycin. Examination of the virulence determinants for E. faecium strains demonstrated that all strains contained the virulence genes common in gut- and food-originated enterococci, and strain R13 harboured the lowest number of virulence genes. Additionally, no strain contained the genes related to cytolysin metabolism and showed hemolytic activity. The antimicrobial role of E. faecium strains was tested against several pathogens, in which different levels of inhibitory effects were observed, and strain R13 was inhibitory to all tested pathogens. PCR screening of genes encoding enterocin A and B indicated the presence of these genes in E. faecium strains. Preliminary characterization of bacteriocins revealed that their activity was lost after proteolytic enzyme treatments, but no alteration in antimicrobial activity was observed at different pHs (3.5 to 9.5) and after heat treatments. In conclusion, this study revealed the functional characteristics of E. faecium R13 as a gut isolate, and this strain could be developed as a new probiotic after further tests. PMID:26485327

  7. Functional Characterization of Sticholysin I and W111C Mutant Reveals the Sequence of the Actinoporin’s Pore Assembly

    PubMed Central

    Antonini, Valeria; Pérez-Barzaga, Victor; Bampi, Silvia; Pentón, David; Martínez, Diana; Serra, Mauro Dalla; Tejuca, Mayra

    2014-01-01

    The use of pore-forming toxins in the construction of immunotoxins against tumour cells is an alternative for cancer therapy. In this protein family one of the most potent toxins are the actinoporins, cytolysins from sea anemones. We work on the construction of tumour proteinase-activated immunotoxins using sticholysin I (StI), an actinoporin isolated from the sea anemone Stichodactyla helianthus. To accomplish this objective, recombinant StI (StIr) with a mutation in the membrane binding region has been employed. In this work, it was evaluated the impact of mutating tryptophan 111 to cysteine on the toxin pore forming capability. StI W111C is still able to permeabilize erythrocytes and liposomes, but at ten-fold higher concentration than StI. This is due to its lower affinity for the membrane, which corroborates the importance of residue 111 for the binding of actinoporins to the lipid bilayer. In agreement, other functional characteristics not directly associated to the binding, are essentially the same for both variants, that is, pores have oligomeric structures with similar radii, conductance, cation-selectivity, and instantaneous current-voltage behavior. In addition, this work provides experimental evidence sustaining the toroidal protein-lipid actinoporins lytic structures, since the toxins provoke the trans-bilayer movement (flip–flop) of a pyrene-labeled analogue of phosphatidylcholine in liposomes, indicating the existence of continuity between the outer and the inner membrane leaflet. Finally, our planar lipid membranes results have also contributed to a better understanding of the actinoporin’s pore assembly mechanism. After the toxin binding and the N-terminal insertion in the lipid membrane, the pore assembly occurs by passing through different transient sub-conductance states. These states, usually 3 or 4, are due to the successive incorporation of N-terminal α-helices and lipid heads to the growing pores until a stable toroidal oligomeric

  8. Alpha-toxin promotes Staphylococcus aureus mucosal biofilm formation.

    PubMed

    Anderson, Michele J; Lin, Ying-Chi; Gillman, Aaron N; Parks, Patrick J; Schlievert, Patrick M; Peterson, Marnie L

    2012-01-01

    Staphylococcus aureus causes many diseases in humans, ranging from mild skin infections to serious, life-threatening, superantigen-mediated Toxic Shock Syndrome (TSS). S. aureus may be asymptomatically carried in the anterior nares or vagina or on the skin, serving as a reservoir for infection. Pulsed-field gel electrophoresis clonal type USA200 is the most widely disseminated colonizer and the leading cause of TSS. The cytolysin α-toxin (also known as α-hemolysin or Hla) is the major epithelial proinflammatory exotoxin produced by TSS S. aureus USA200 isolates. The current study aims to characterize the differences between TSS USA200 strains [high (hla(+)) and low (hla(-)) α-toxin producers] in their ability to disrupt vaginal mucosal tissue and to characterize the subsequent infection. Tissue viability post-infection and biofilm formation of TSS USA200 isolates CDC587 and MN8, which contain the α-toxin pseudogene (hla(-)), MNPE (hla(+)), and MNPE isogenic hla knockout (hlaKO), were observed via LIVE/DEAD® staining and confocal microscopy. All TSS strains grew to similar bacterial densities (1-5 × 10(8) CFU) on the mucosa and were proinflammatory over 3 days. However, MNPE formed biofilms with significant reductions in the mucosal viability whereas neither CDC587 (hla(-)), MN8 (hla(-)), nor MNPE hlaKO formed biofilms. The latter strains were also less cytotoxic than wild-type MNPE. The addition of exogenous, purified α-toxin to MNPE hlaKO restored the biofilm phenotype. We speculate that α-toxin affects S. aureus phenotypic growth on vaginal mucosa by promoting tissue disruption and biofilm formation. Further, α-toxin mutants (hla(-)) are not benign colonizers, but rather form a different type of infection, which we have termed high density pathogenic variants (HDPV). PMID:22919655

  9. Regulation of virulence by a two-component system in group B streptococcus.

    PubMed

    Jiang, Sheng-Mei; Cieslewicz, Michael J; Kasper, Dennis L; Wessels, Michael R

    2005-02-01

    Group B Streptococcus (GBS) is frequently carried in the gastrointestinal or genitourinary tract as a commensal organism, yet it has the potential to cause life-threatening infection in newborn infants, pregnant women, and individuals with chronic illness. Regulation of virulence factor expression may affect whether GBS behaves as an asymptomatic colonizer or an invasive pathogen, but little is known about how such factors are controlled in GBS. We now report the characterization of a GBS locus that encodes a two-component regulatory system similar to CsrRS (or CovRS) in Streptococcus pyogenes. Inactivation of csrR, encoding the putative response regulator, in two unrelated wild-type strains of GBS resulted in a marked increase in production of beta-hemolysin/cytolysin and a striking decrease in production of CAMP factor, an unrelated cytolytic toxin. Quantitative RNA hybridization experiments revealed that these two phenotypes were associated with a marked increase and decrease in expression of the corresponding genes, cylE and cfb, respectively. The CsrR mutant strains also displayed increased expression of scpB encoding C5a peptidase. Similar, but less marked, changes in gene expression were observed in CsrS (putative sensor component) mutants, evidence that CsrR and CsrS constitute a functional two-component system. Experimental infection studies in mice demonstrated reduced virulence of both CsrR and CsrS mutant strains relative to the wild type. Together, these results indicate that CsrRS regulates expression of multiple GBS virulence determinants and is likely to play an important role in GBS pathogenesis. PMID:15659687

  10. Virulence factors and antibiotic susceptibility in enterococci isolated from oral mucosal and deep infections

    PubMed Central

    Dahlén, Gunnar; Blomqvist, Susanne; Almståhl, Annica; Carlén, Anette

    2012-01-01

    Objective This study evaluates the presence of virulence factors and antibiotic susceptibility among enterococcal isolates from oral mucosal and deep infections. Methods Forty-three enterococcal strains from oral mucosal lesions and 18 from deep infections were isolated from 830 samples that were sent during 2 years to Oral Microbiology, University of Gothenburg, for analysis. The 61 strains were identified by 16S rDNA, and characterized by the presence of the virulence genes efa A (endocarditis gene), gel E (gelatinase gene), ace (collagen binding antigen gene), asa (aggregation substance gene), cyl A (cytolysin activator gene) and esp (surface adhesin gene), tested for the production of bacteriocins and presence of plasmids. MIC determination was performed using the E-test method against the most commonly used antibiotics in dentistry, for example, penicillin V, amoxicillin and clindamycin. Vancomycin was included in order to detect vancomycin-resistant enterococci (VRE) strains. Results Sixty strains were identified as Enterococcus faecalis and one as Enterococcus faecium. All the virulence genes were detected in more than 93.3% (efa A and esp) of the E. faecalis strains, while the presence of phenotypic characteristics was much lower (gelatinase 10% and hemolysin 16.7%). Forty-six strains produced bacteriocins and one to six plasmids were detected in half of the isolates. Conclusions Enterococcal strains from oral infections had a high virulence capacity, showed bacteriocin production and had numerous plasmids. They were generally susceptible to ampicillins but were resistant to clindamycin, commonly used in dentistry, and no VRE-strain was found. PMID:22368771

  11. Ecology of antibiotic resistance genes: characterization of enterococci from houseflies collected in food settings.

    PubMed

    Macovei, Lilia; Zurek, Ludek

    2006-06-01

    In this project, enterococci from the digestive tracts of 260 houseflies (Musca domestica L.) collected from five restaurants were characterized. Houseflies frequently (97% of the flies were positive) carried enterococci (mean, 3.1 x 10(3) CFU/fly). Using multiplex PCR, 205 of 355 randomly selected enterococcal isolates were identified and characterized. The majority of these isolates were Enterococcus faecalis (88.2%); in addition, 6.8% were E. faecium, and 4.9% were E. casseliflavus. E. faecalis isolates were phenotypically resistant to tetracycline (66.3%), erythromycin (23.8%), streptomycin (11.6%), ciprofloxacin (9.9%), and kanamycin (8.3%). Tetracycline resistance in E. faecalis was encoded by tet(M) (65.8%), tet(O) (1.7%), and tet(W) (0.8%). The majority (78.3%) of the erythromycin-resistant E. faecalis isolates carried erm(B). The conjugative transposon Tn916 and members of the Tn916/Tn1545 family were detected in 30.2% and 34.6% of the identified isolates, respectively. E. faecalis carried virulence genes, including a gelatinase gene (gelE; 70.7%), an aggregation substance gene (asa1; 33.2%), an enterococcus surface protein gene (esp; 8.8%), and a cytolysin gene (cylA; 8.8%). Phenotypic assays showed that 91.4% of the isolates with the gelE gene were gelatinolytic and that 46.7% of the isolates with the asa1 gene aggregated. All isolates with the cylA gene were hemolytic on human blood. This study showed that houseflies in food-handling and -serving facilities carry antibiotic-resistant and potentially virulent enterococci that have the capacity for horizontal transfer of antibiotic resistance genes to other bacteria. PMID:16751512

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

    PubMed

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

    2015-01-01

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

  13. Structure of Complement C6 Suggests a Mechanism for Initiation and Unidirectional, Sequential Assembly of Membrane Attack Complex (MAC)*♦

    PubMed Central

    Aleshin, Alexander E.; Schraufstatter, Ingrid U.; Stec, Boguslaw; Bankston, Laurie A.; Liddington, Robert C.; DiScipio, Richard G.

    2012-01-01

    The complement membrane attack complex (MAC) is formed by the sequential assembly of C5b with four homologous proteins as follows: one copy each of C6, C7, and C8 and 12–14 copies of C9. Together these form a lytic pore in bacterial membranes. C6 through C9 comprise a MAC-perforin domain flanked by 4–9 “auxiliary” domains. Here, we report the crystal structure of C6, the first and longest of the pore proteins to be recruited by C5b. Comparisons with the structures of the C8αβγ heterodimer and perforin show that the central domain of C6 adopts a “closed” (perforin-like) state that is distinct from the “open” conformations in C8. We further show that C6, C8α, and C8β contain three homologous subdomains (“upper,” “lower,” and “regulatory”) related by rotations about two hinge points. In C6, the regulatory segment includes four auxiliary domains that stabilize the closed conformation, inhibiting release of membrane-inserting elements. In C8β, rotation of the regulatory segment is linked to an opening of the central β-sheet of its clockwise partner, C8α. Based on these observations, we propose a model for initiation and unidirectional propagation of the MAC in which the auxiliary domains play key roles: in the assembly of the C5b-8 initiation complex; in driving and regulating the opening of the β-sheet of the MAC-performin domain of each new recruit as it adds to the growing pore; and in stabilizing the final pore. Our model of the assembled pore resembles those of the cholesterol-dependent cytolysins but is distinct from that recently proposed for perforin. PMID:22267737

  14. eIF2α Confers Cellular Tolerance to S. aureus α-Toxin.

    PubMed

    von Hoven, Gisela; Neukirch, Claudia; Meyenburg, Martina; Füser, Sabine; Petrivna, Maria Bidna; Rivas, Amable J; Ryazanov, Alexey; Kaufman, Randal J; Aroian, Raffi V; Husmann, Matthias

    2015-01-01

    We report on the role of conserved stress-response pathways for cellular tolerance to a pore forming toxin. First, we observed that small molecular weight inhibitors including of eIF2α-phosphatase, jun-N-terminal kinase (JNK), and PI3-kinase sensitized normal mouse embryonal fibroblasts (MEFs) to the small pore forming S. aureus α-toxin. Sensitization depended on expression of mADAM10, the murine ortholog of a proposed high-affinity receptor for α-toxin in human cells. Similarly, eIF2α (S51A/S51A) MEFs, which harbor an Ala knock-in mutation at the regulated Ser51 phosphorylation site of eukaryotic translation initiation factor 2α, were hyper-sensitive to α-toxin. Inhibition of translation with cycloheximide did not mimic the tolerogenic effect of eIF2α-phosphorylation. Notably, eIF2α-dependent tolerance of MEFs was toxin-selective, as wild-type MEFs and eIF2α (S51A/S51A) MEFs exhibited virtually equal sensitivity to Vibrio cholerae cytolysin. Binding of S. aureus α-toxin to eIF2α (S51A/S51A) MEFs and toxicity in these cells were enhanced as compared to wild-type cells. This led to the unexpected finding that the mutant cells carried more ADAM10. Because basal phosphorylation of eIF2α in MEFs required amino acid deprivation-activated eIF2α-kinase 4/GCN2, the data reveal that basal activity of this kinase mediates tolerance of MEFs to α-toxin. Further, they suggest that modulation of ADAM10 is involved. During infection, bacterial growth may cause nutrient shortage in tissues, which might activate this response. Tolerance to α-toxin was robust in macrophages and did not depend on GCN2. However, JNKs appeared to play a role, suggesting differential cell type and toxin selectivity of tolerogenic stress responses. Understanding their function or failure will be important to comprehend anti-bacterial immune responses. PMID:26284068

  15. [Enterohemorrhagic Escherichia coli and hemolytic-uremic syndrome].

    PubMed

    Allerberger, F; Sölder, B; Caprioli, A; Karch, H

    1997-09-19

    Enterohemorrhagic Escherichia coli (EHEC) are increasingly identified as the cause of diarrhea and hemorrhagic colitis in countries with highly developed livestock. In 5-10% of patients, full-blown hemolytic uremic syndrome (HUS) occurs as a postinfectious life-threatening complication. Up to 1996, 5 out of 39 patients (12.8%) with EHEC O157 infections in Austria developed HUS. Acute complications of HUS such as brain edema may also lead to death; one fatal outcome has been observed so far in Austrian patients. Aside from the cytotoxic Shiga toxins, other different pathogenic factors are often found in clinical EHEC isolates. These include a cytolysin termed EHEC-hemolysin and a low molecular heat-stabile enterotoxin. Furthermore, most EHEC strains express an important surface protein, intimin, which is important for adherence to intestinal epithelial cells. EHEC are heterogeneous in their antigenic structure (O-, H-antigens). In Austria O157:H7 and O157:H- are the dominating serogroups; in 1997 the first Austrian case of HUS due to EHEC O26:H11 was documented. Because there are no known reliable phenotypical markers for EHEC, diagnostic strategies should focus on the demonstration of Shiga toxins or Shiga toxin genes. For epidemiological purposes it is also important to attempt to isolate the causative agent. Cows and other ruminants are reservoirs for EHEC. In the Tyrol 3% of unpasteurised milk samples, up to 10% of minced beef samples, and 6% of calves yield EHEC O157. Aside from transmission via contaminated food, direct transmission from person to person also plays a major role in the chain of EHEC infection. In contrast to Italy and Bavaria, Austria has not experienced a major outbreak due to this organism so far. A nationwide surveillance system of HUS has shown an incidence of 0.37 HUS cases per 100,000 residents in the age group 0-14 years for 1995 (Italy: 0.2 cases per 100,000; Bavaria: approx. 1.5 cases per 100,000). PMID:9381722

  16. Enterocins L50A and L50B, Two Novel Bacteriocins from Enterococcus faecium L50, Are Related to Staphylococcal Hemolysins

    PubMed Central

    Cintas, Luis M.; Casaus, Pilar; Holo, Helge; Hernandez, Pablo E.; Nes, Ingolf F.; Håvarstein, Leiv Sigve

    1998-01-01

    Enterocin L50 (EntL50), initially referred to as pediocin L50 (L. M. Cintas, J. M. Rodríguez, M. F. Fernández, K. Sletten, I. F. Nes, P. E. Hernández, and H. Holo, Appl. Environ. Microbiol. 61:2643–2648, 1995), is a plasmid-encoded broad-spectrum bacteriocin produced by Enterococcus faecium L50. It has previously been purified from the culture supernatant and partly sequenced by Edman degradation. In the present work, the nucleotide sequence of the EntL50 locus was determined, and several putative open reading frames (ORFs) were identified. Unexpectedly, two ORFs were found to encode EntL50-like peptides. These peptides, termed enterocin L50A (EntL50A) and enterocin L50B (EntL50B), have 72% sequence identity and consist of 44 and 43 amino acids, respectively. Interestingly, a comparison of the deduced sequences of EntL50A and EntL50B with the corresponding sequences obtained by Edman degradation shows that these bacteriocins, in contrast to other peptide bacteriocins, are secreted without an N-terminal leader sequence or signal peptide. Expression in vivo and in vitro transcription/translation experiments demonstrated that entL50A and entL50B are the only genes required to obtain antimicrobial activity, strongly indicating that their bacteriocin products are not posttranslationally modified. Both bacteriocins possess antimicrobial activity on their own, with EntL50A being the most active. In addition, when the two bacteriocins were combined, a considerable synergism was observed, especially with some indicator strains. Even though the enterocins in some respects are similar to class II bacteriocins, several conserved features common to class II bacteriocins are absent from the EntL50 system. The enterocins have more in common with members of a small group of cytolytic peptides secreted by certain staphylococci. We therefore propose that the enterocins L50A and L50B and the staphylococcal cytolysins together constitute a new family of peptide toxins, unrelated

  17. Dogs leaving the ICU carry a very large multi-drug resistant enterococcal population with capacity for biofilm formation and horizontal gene transfer.

    PubMed

    Ghosh, Anuradha; Dowd, Scot E; Zurek, Ludek

    2011-01-01

    The enterococcal community from feces of seven dogs treated with antibiotics for 2-9 days in the veterinary intensive care unit (ICU) was characterized. Both, culture-based approach and culture-independent 16S rDNA amplicon 454 pyrosequencing, revealed an abnormally large enterococcal community: 1.4±0.8×10(8) CFU gram(-1) of feces and 48.9±11.5% of the total 16,228 sequences, respectively. The diversity of the overall microbial community was very low which likely reflects a high selective antibiotic pressure. The enterococcal diversity based on 210 isolates was also low as represented by Enterococcus faecium (54.6%) and Enterococcus faecalis (45.4%). E. faecium was frequently resistant to enrofloxacin (97.3%), ampicillin (96.5%), tetracycline (84.1%), doxycycline (60.2%), erythromycin (53.1%), gentamicin (48.7%), streptomycin (42.5%), and nitrofurantoin (26.5%). In E. faecalis, resistance was common to tetracycline (59.6%), erythromycin (56.4%), doxycycline (53.2%), and enrofloxacin (31.9%). No resistance was detected to vancomycin, tigecycline, linezolid, and quinupristin/dalfopristin in either species. Many isolates carried virulence traits including gelatinase, aggregation substance, cytolysin, and enterococcal surface protein. All E. faecalis strains were biofilm formers in vitro and this phenotype correlated with the presence of gelE and/or esp. In vitro intra-species conjugation assays demonstrated that E. faecium were capable of transferring tetracycline, doxycycline, streptomycin, gentamicin, and erythromycin resistance traits to human clinical strains. Multi-locus variable number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE) of E. faecium strains showed very low genotypic diversity. Interestingly, three E. faecium clones were shared among four dogs suggesting their nosocomial origin. Furthermore, multi-locus sequence typing (MLST) of nine representative MLVA types revealed that six sequence types (STs) originating from five

  18. Characterization of Enterococcus faecium isolates and first report of vanB phenotype-vanA genotype incongruence in the Middle East.

    PubMed

    Al-Ahdal, M N; Abozaid, S M; Al-Shammary, H F; Bohol, M F; Al-Thawadi, S I; Al-Jaberi, A A; Senok, A C; Shibl, A M; Al-Qahtani, A A

    2012-11-01

    We aimed to characterize the vancomycin genotype/phenotype, carriage of putative virulence genes, and genetic relatedness of Enterococcus faecium isolates in Saudi Arabia. E. faecium isolated from inpatients at our medical center were studied. Sensitivity to ampicillin, linezolid, teicoplanin, quinupristin/dalfopristin, tetracycline, and ciprofloxacin was determined. The presence of van genes and virulence genes for aggregation substance (Asa-1), enterococcal surface proteins (esp), cytolysin (cylA, cylL, cylM), gelatinase (gelE), E. faecium endocarditis antigen (EfaA( fm )), hyaluronidase (hyl), and collagen adhesion (Ace) was assessed. Genetic relatedness was determined by pulsed-field gel electrophoresis (PFGE). Twenty-nine E. faecium isolates were obtained and the majority of isolates (n/N = 22/29) were from stool specimens. PFGE analysis identified eight pulsotypes (A-H) based on 80 % similarities. Isolates were represented in five major pulsotypes: type A (n = 5), type B (n = 3), type D (n = 6), type E (n = 5), and type F (n = 7). All isolates were vanA gene-positive. Thirteen isolates had vanA(+)/vanB(+) genotype. Of these, ten exhibited a vanB phenotype and three had a vanA phenotype. Eight isolates with vanA(+)/vanB(-) genotype exhibited vanB phenotype. Six of these eight isolates belonged to the same pulsotype. All isolates were positive for gelE, esp, and EfaA( fm ) genes. Five were CylA-positive and 24 had the hyl genes. Of the eight isolates harboring a combination of gelE, esp, EfaA( fm ), and hyl genes, five showed vanB phenotype-vanA genotype incongruence. This is the first report of vanB phenotype-vanA genotype incongruent E. faecium in the Middle East region. Molecular typing indicates clonal spread and high occurrence of virulence genes, especially esp genes, associated with epidemic clones. PMID:22790538

  19. The engineered Salmonella typhimurium inhibits tumorigenesis in advanced glioma

    PubMed Central

    Chen, Jian-qiang; Zhan, Yue-fu; Wang, Wei; Jiang, Sheng-nan; Li, Xiang-ying

    2015-01-01

    Objective To explore the antitumor role of the attenuated Salmonella typhimurium ΔppGpp with inducible cytolysin A (ClyA) in advanced stage of glioma. Materials and methods The C6 rat glioma cells were orthotopically implanted by surgery into the caudate nucleus of rat brains. The rats were then randomly divided into the treatment group (SL + ClyA) (n=12), negative control group (SL) (n=12), and control group (phosphate-buffered saline [PBS]) (n=12). In the treatment group, the attenuated S. typhimurium were transformed with the plasmid-encoded antitumor gene ClyA. The expression of ClyA was controlled by the TetR-regulated promoter in response to extracellular doxycycline. The plasmid also contained an imaging gene lux to allow illumination of the tumor infected by the bacteria. The rat glioma C6 cells were implanted into the caudate nucleus of all rats. The engineered S. typhimurium and respective controls were injected intravenously into the rats 21 days after initial tumor implantation. The pathological analysis of the glioma tumor was performed at 21 days and 28 days (7 days after doxycycline treatment) postimplantation. All rats underwent MRI (magnetic resonance imaging) and bioluminescence study at 21 days and 28 days postimplantation to detect tumor volume. The differences between the three groups in tumor volume and survival time were analyzed. Results Advanced stage glioma was detected at 21 days postimplantation. Bioluminescence showed that the engineered S. typhimurium accumulated in glioma tumors and disappeared in the normal reticuloendothelial tissues 3 days after intravenous injection. MRI showed that the tumor volume in the S. typhimurium with ClyA group were significantly reduced compared to the bacteria alone and no bacteria groups 7 days post-doxycycline treatment (P<0.05), while the necrotic tumor volume in the S. typhimurium with ClyA group and S. typhimurium alone group increased significantly compared to the control group (P<0.01). In

  20. Molecular Characterization of Virulence Genes in Vancomycin-Resistant and Vancomycin-Sensitive Enterococci

    PubMed Central

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

    2016-01-01

    Background: The aim of this study was to find out the correlation between presence of virulence (gelatinase [gel E], enterococcal surface protein [esp], cytolysin A [cyl A], hyaluronidase [hyl], and aggregation substance [asa1]) and vancomycin-resistant genes (van A and van B) in enterococci, with their phenotypic expression. Materials and Methods: A total of 500 isolates (250 each clinical and fecal) were processed. Enterococci were isolated from various clinical samples and from fecal specimens of colonized patients. Various virulence determinants namely asa1, esp, hyl, gel E, and cyl were detected by phenotypic methods. Minimum inhibitory concentration (MIC) of vancomycin was determined by agar dilution method. Multiplex polymerase chain reaction (PCR) was used to detect the presence of virulence and van genes. Results: Out of all the samples processed, 12.0% (60/500) isolates carried van A or van B genes as confirmed by MIC test and PCR methods. Genes responsible for virulence were detected by multiplex PCR and at least one of the five was detected in all the clinical vancomycin-resistant enterococci (VRE) and vancomycin-sensitive enterococci (VSE). gel E, esp, and hyl genes were found to be significantly higher in clinical VRE. Of the fecal isolates, presence of gel E, esp, and asa1 was significantly higher in VRE as compared to VSE. The presence of hyl gene in the clinical VRE was found to be statistically significant (P = 0.043) as against the fecal VRE. Correlation between the presence of virulence genes and their expression as detected by phenotypic tests showed that while biofilm production was seen in 61.1% (22/36) of clinical VRE, the corresponding genes, i.e., asa1 and esp were detected in 30.5% (11/36) and 27.8% (10/36) of strains only. Conclusion: Enterococcus faecium isolates were found to carry esp gene, a phenomenon that has been described previously only for Enterococcus faecalis, but we were unable to correlate the presence of esp with their

  1. Dogs Leaving the ICU Carry a Very Large Multi-Drug Resistant Enterococcal Population with Capacity for Biofilm Formation and Horizontal Gene Transfer

    PubMed Central

    Ghosh, Anuradha; Dowd, Scot E.; Zurek, Ludek

    2011-01-01

    The enterococcal community from feces of seven dogs treated with antibiotics for 2–9 days in the veterinary intensive care unit (ICU) was characterized. Both, culture-based approach and culture-independent 16S rDNA amplicon 454 pyrosequencing, revealed an abnormally large enterococcal community: 1.4±0.8×108 CFU gram−1 of feces and 48.9±11.5% of the total 16,228 sequences, respectively. The diversity of the overall microbial community was very low which likely reflects a high selective antibiotic pressure. The enterococcal diversity based on 210 isolates was also low as represented by Enterococcus faecium (54.6%) and Enterococcus faecalis (45.4%). E. faecium was frequently resistant to enrofloxacin (97.3%), ampicillin (96.5%), tetracycline (84.1%), doxycycline (60.2%), erythromycin (53.1%), gentamicin (48.7%), streptomycin (42.5%), and nitrofurantoin (26.5%). In E. faecalis, resistance was common to tetracycline (59.6%), erythromycin (56.4%), doxycycline (53.2%), and enrofloxacin (31.9%). No resistance was detected to vancomycin, tigecycline, linezolid, and quinupristin/dalfopristin in either species. Many isolates carried virulence traits including gelatinase, aggregation substance, cytolysin, and enterococcal surface protein. All E. faecalis strains were biofilm formers in vitro and this phenotype correlated with the presence of gelE and/or esp. In vitro intra-species conjugation assays demonstrated that E. faecium were capable of transferring tetracycline, doxycycline, streptomycin, gentamicin, and erythromycin resistance traits to human clinical strains. Multi-locus variable number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE) of E. faecium strains showed very low genotypic diversity. Interestingly, three E. faecium clones were shared among four dogs suggesting their nosocomial origin. Furthermore, multi-locus sequence typing (MLST) of nine representative MLVA types revealed that six sequence types (STs) originating from five