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Infection and Immunity Sep 2013Photobacterium damselae subsp. damselae causes infections and fatal disease in marine animals and in humans. Highly hemolytic strains produce damselysin (Dly) and...
Photobacterium damselae subsp. damselae causes infections and fatal disease in marine animals and in humans. Highly hemolytic strains produce damselysin (Dly) and plasmid-encoded HlyA (HlyA(pl)). These hemolysins are encoded by plasmid pPHDD1 and contribute to hemolysis and virulence for fish and mice. In this study, we report that all the hemolytic strains produce a hitherto uncharacterized chromosome-encoded HlyA (HlyAch). Hemolysis was completely abolished in a single hlyAch mutant of a plasmidless strain and in a dly hlyApl hlyAch triple mutant. We found that Dly, HlyA(pl), and HlyAch are needed for full hemolytic values in strains harboring pPHDD1, and these values are the result of the additive effects between HlyApl and HlyAch, on the one hand, and of the synergistic effect of Dly with HlyApl and HlyAch, on the other hand. Interestingly, Dly-producing strains produced synergistic effects with strains lacking Dly production but secreting HlyA, constituting a case of the CAMP (Christie, Atkins, and Munch-Petersen) reaction. Environmental factors such as iron starvation and salt concentration were found to regulate the expression of the three hemolysins. We found that the contributions, in terms of the individual and combined effects, of the three hemolysins to hemolysis and virulence varied depending on the animal species tested. While Dly and HlyApl were found to be main contributors in the virulence for mice, we observed that the contribution of hemolysins to virulence for fish was mainly based on the synergistic effects between Dly and either of the two HlyA hemolysins rather than on their individual effects.
Topics: Animals; Chromosomes; Escherichia coli; Fish Diseases; Fishes; Hemolysin Proteins; Hemolysis; Iron; Mice; Mice, Inbred BALB C; Photobacterium; Plasmids; Sodium Chloride; Transcription, Genetic; Virulence
PubMed: 23798530
DOI: 10.1128/IAI.00155-13 -
Acta Biochimica Polonica 2015A total of 104 coagulase negative staphylococci, belonging to S. capitis, S. hominis, S. haemolyticus and S. warneri, originating from the collection of the Department...
A total of 104 coagulase negative staphylococci, belonging to S. capitis, S. hominis, S. haemolyticus and S. warneri, originating from the collection of the Department of Pharmaceutical Microbiology (ZMF), Medical University of Lodz, Poland, were tested for their synergistic hemolytic activity. 83% of strains produced δ-hemolysin, however, the percentage of positive strains of S. haemolyticus, S. warneri, S. capitis and S. hominis was different - 98%, 78%, 75% and 68%, respectively. Highly pure hemolysins were obtained from culture supernatants by protein precipitation with ammonium sulphate (0-70% of saturation) and extraction by using a mixture of organic solvents. The purity and molecular mass of hemolysins was determined by TRIS/Tricine PAGE. All CoNS hemolysins were small peptides with a molar mass of about 3.5 kDa; they possessed cytotoxic activity against the line of human foreskin fibroblasts ATCC Hs27 and lysed red cells from different mammalian species, however, the highest activity was observed when guinea pig, dog and human red blood cells were used. The cytotoxic effect on fibroblasts occurred within 30 minutes. The S. cohnii ssp. urealyticus strain was used as a control. The antimicrobial activity was examined using hemolysins of S. capitis, S. hominis, S. cohnii ssp. cohnii and S. cohnii ssp. urealyticus. Hemolysins of the two S. cohnii subspecies did not demonstrate antimicrobial activity. Cytolysins of S. capitis and S. hominis had a very narrow spectrum of action; out of 37 examined strains, the growth of only Micrococcus luteus, Corynebacterium diphtheriae and Pasteurella multocida was inhibited.
Topics: Coagulase; Electrophoresis, Polyacrylamide Gel; Fibroblasts; Hemolysin Proteins; Humans; Species Specificity; Staphylococcus
PubMed: 26634231
DOI: 10.18388/abp.2015_1126 -
Journal of Clinical Microbiology Jun 1989Reference strains of serotypes 1 to 12 of Actinobacillus (Haemophilus) pleuropneumoniae were cultured in Eagle minimal essential medium with 10% Serum Plus. Culture...
Reference strains of serotypes 1 to 12 of Actinobacillus (Haemophilus) pleuropneumoniae were cultured in Eagle minimal essential medium with 10% Serum Plus. Culture supernatants were examined for cytotoxicity to alveolar macrophages and for the ability to hemolyze sheep erythrocytes. All strains except the reference strain of serotype 6 produced cytotoxin, whereas only serotypes 1, 5, 9, 10, and 11 produced hemolysin. Both cytotoxin and hemolysin appeared to be heat labile. Antisera were raised against cytotoxin- and hemolysin-containing culture supernatants of serotypes 1 to 11. Cross-neutralization studies revealed that the hemolysins were serologically homogeneous. In contrast, four serologically different cytotoxins were distinguished. One cytotoxin was produced by serotypes 1, 5, 9, and 11, and a second was produced by serotypes 2, 3, 4, and 8. A third cytotoxin was produced by serotypes 7 and 12; this cytotoxin was related to the cytotoxins of serotypes 1, 2, 4, 5, 9, and 11. A fourth cytotoxin, produced by serotype 10, was related to the cytotoxin of serotypes 1, 5, 9, and 11. Seventy field strains belonging to serotypes 2, 3, 7, 8, 9, and 11 were also tested for production of cytotoxin and hemolysin. All strains belonging to serotypes 9 and 11 produced hemolysin and cytotoxin, whereas all strains of serotypes 2, 3, 7, and 8 produced only cytotoxin. Hemolysins and cytotoxins of both the field strains and the corresponding serotype reference strains were comparably neutralized. These findings strongly suggest that the observed differences in production and type of hemolysin and cytotoxin were related to serotype and not to strain.
Topics: Actinobacillus; Animals; Cross Reactions; Cytotoxins; Haemophilus; Hemolysin Proteins; Hot Temperature; Immune Sera; Neutralization Tests; Pleuropneumonia, Contagious; Serotyping; Specific Pathogen-Free Organisms; Swine; Swine Diseases
PubMed: 2526820
DOI: 10.1128/jcm.27.6.1187-1191.1989 -
Toxins Oct 2020Hemolysin BL is a tripartite toxin responsible for a diarrheal type of food poisoning. Open questions remain regarding its mode of action, including the extent to which...
Hemolysin BL is a tripartite toxin responsible for a diarrheal type of food poisoning. Open questions remain regarding its mode of action, including the extent to which complex formation prior to cell binding contributes to pore-forming activity, how these complexes are composed, and the properties of the pores formed in the target cell membrane. Distinct complexes of up to 600 kDa were found on native gels, whose structure and size were primarily defined by Hbl B. Hbl L1 and L2 were also identified in these complexes using Western blotting and an LC-MS approach. LC-MS also revealed that many other proteins secreted by exist in complexes. Further, a decrease of toxic activity at temperatures ≥60 °C was shown, which was unexpectedly restored at higher temperatures. This could be attributed to a release of Hbl B monomers from tight complexation, resulting in enhanced cell binding. In contrast, Hbl L1 was rather susceptible to heat, while heat treatment of Hbl L2 seemed not to be crucial. Furthermore, Hbl-induced pores had a rather small single-channel conductance of around 200 pS and a probable channel diameter of at least 1 nm on planar lipid bilayers. These were highly instable and had a limited lifetime, and were also slightly cation-selective. Altogether, this study provides astonishing new insights into the complex mechanism of Hbl pore formation, as well as the properties of the pores.
Topics: Animals; Bacillus cereus; Bacterial Proteins; Cell Survival; Chlorocebus aethiops; Hemolysin Proteins; Lipid Bilayers; Porosity; Vero Cells
PubMed: 33114414
DOI: 10.3390/toxins12110672 -
Infection and Immunity Jan 1994Simultaneous production of a thermostable direct hemolysin (TDH)-like toxin (TDHx) and a TDH-related hemolysin (TRH)-like toxin (TRHx) by a clinical isolate (strain... (Comparative Study)
Comparative Study
Demonstration and characterization of simultaneous production of a thermostable direct hemolysin (TDH/I) and a TDH-related hemolysin (TRHx) by a clinically isolated Vibrio parahaemolyticus strain, TH3766.
Simultaneous production of a thermostable direct hemolysin (TDH)-like toxin (TDHx) and a TDH-related hemolysin (TRH)-like toxin (TRHx) by a clinical isolate (strain TH3766) of Kanagawa phenomenon-positive Vibrio parahaemolyticus was demonstrated and characterized. The two hemolysins were differentially purified by column chromatography on hydroxyapatite and immunoaffinity columns. The molecular weight of the two hemolysins were estimated to be 23,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE). The purified TDHx was indistinguishable from the previously reported TDH/I (from strain TH012) but was different from the authentic TDH of a Kanagawa phenomenon-positive strain (T4750) physicochemically. The mobility of TRHx in nondenaturing PAGE differed from all the known TDHs and TRHs. The genes (tdhX and trhX) coding for TDHx and TRHx were cloned and sequenced. Homologies of nucleotide sequences of the coding regions between tdhX and tdhA (a gene for the authentic TDH) and between trhX and trh (a gene for the authentic TRH) were 98.1 and 99.1%, respectively, and homology between tdhX and trhX was 68.1%. At the amino acid level, TdhX was completely identical to TDH/I, although two base differences were found in the nucleotide sequences between tdhX and tdh/I. Two amino acid differences were observed between TrhX and Trh. Thus, these findings suggest that the TH3766 strain produces two types of hemolysins simultaneously. This is the first evidence that a strain of V. parahaemolyticus produces two types of toxins of the TDH-TRH family at the same time.
Topics: Amino Acid Sequence; Bacterial Toxins; Base Sequence; Cloning, Molecular; Genes, Bacterial; Hemolysin Proteins; Immunodiffusion; Molecular Sequence Data; Recombinant Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Vibrio parahaemolyticus
PubMed: 8262624
DOI: 10.1128/iai.62.1.166-171.1994 -
PloS One 2015Vibrio parahaemolyticus is a recognized human enteropathogen. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) as well as the type III secretion...
BACKGROUND
Vibrio parahaemolyticus is a recognized human enteropathogen. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) as well as the type III secretion system 2 (T3SS2) are considered as major virulence factors. As tdh positive strains are not detected in coastal waters of Germany, we focused on the characterization of trh positive strains, which were isolated from mussels, seawater and patients in Germany.
RESULTS
Ten trh harbouring V. parahaemolyticus strains from Germany were compared to twenty-one trh positive strains from other countries. The complete trh sequences revealed clustering into three different types: trh1 and trh2 genes and a pseudogene Ψtrh. All German isolates possessed alleles of the trh2 gene. MLST analysis indicated a close relationship to Norwegian isolates suggesting that these strains belong to the autochthonous microflora of Northern Europe seawaters. Strains carrying the pseudogene Ψtrh were negative for T3SS2β effector vopC. Transcription of trh and vopC genes was analyzed under different growth conditions. Trh2 gene expression was not altered by bile while trh1 genes were inducible. VopC could be induced by urea in trh2 bearing strains. Most trh1 carrying strains were hemolytic against sheep erythrocytes while all trh2 positive strains did not show any hemolytic activity. TRH variants were synthesized in a prokaryotic cell-free system and their hemolytic activity was analyzed. TRH1 was active against sheep erythrocytes while TRH2 variants were not active at all.
CONCLUSION
Our study reveals a high diversity among trh positive V. parahaemolyticus strains. The function of TRH2 hemolysins and the role of the pseudogene Ψtrh as pathogenicity factors are questionable. To assess the pathogenic potential of V. parahaemolyticus strains a differentiation of trh variants and the detection of T3SS2β components like vopC would improve the V. parahaemolyticus diagnostics and could lead to a refinement of the risk assessment in food analyses and clinical diagnostics.
Topics: Animals; Bacterial Proteins; Cell-Free System; Erythrocytes; Fishes; Genotype; Germany; Hemolysin Proteins; Hemolysis; Humans; Multiplex Polymerase Chain Reaction; Norway; Sheep; Vibrio parahaemolyticus; Virulence Factors
PubMed: 25799574
DOI: 10.1371/journal.pone.0118559 -
Toxins Dec 2020This Special Issue, on and its toxins, seems to be the right place to pay tribute to one of the most influential scientists in the field of research into this peculiar...
This Special Issue, on and its toxins, seems to be the right place to pay tribute to one of the most influential scientists in the field of research into this peculiar bacterium [...].
Topics: Bacillus thuringiensis; Bacillus thuringiensis Toxins; Endotoxins; Hemolysin Proteins; History, 20th Century; History, 21st Century; Insect Proteins; Microbiology; Receptors, Cell Surface
PubMed: 33287128
DOI: 10.3390/toxins12120764 -
Diseases of Aquatic Organisms Jun 2010Hemolysin is a putative pathogenicity factor in many bacterial pathogens. In this study, a DNA fragment containing the open reading frame (1254 bp) of the thermolabile...
Hemolysin is a putative pathogenicity factor in many bacterial pathogens. In this study, a DNA fragment containing the open reading frame (1254 bp) of the thermolabile hemolysin gene (tlh) from Vibrio alginolyticus V05 was amplified and cloned into the expression plasmid pET-24d(+). The deduced amino acid sequence of the thermolabile hemolysin (TLH) shared 94 and 83% identity with the lecithin-dependent hemolysin (LDH)/TLH of V. parahaemolyticus and V. harveyi thermolabile hemolysin (VHH), respectively. The sequence analysis also indicated that it contained a GDSL lipase domain like VHH. The recombinant protein with a predicted molecular mass of 47.2 kDa was expressed in the Escherichia coli strain BL21 (DE3) as a His-tag fused protein. TLH purified by the nickel-nitrilotriacetic acid (Ni-NTA) His-Bind Resin method showed phospholipase activity on an egg yolk emulsion plate and hemolytic activity against flounder erythrocytes with a specific activity of 18 hemolytic units microg(-1). The addition of divalent cations at different concentrations decreased hemolytic activity of the purified TLH, but monavalent cations did not affect hemolytic activity. The hemolytic activity of TLH was also markedly inhibited by protein modification reagents, i.e. beta-mercaptoethanol, phenylmethylsulfonyl fluoride, and 5,5'-dithio-bis(2-nitrobenzoic acid). Moreover, TLH was toxic to zebrafish when injected intraperitoneally, with a median lethal dose (LD50) of 0.8 microg protein g(-1) fish. This work shows that TLH could potentially be developed as a vaccine and used as a diagnostic tool for vibriosis.
Topics: Animals; Fish Diseases; Gene Expression Regulation, Bacterial; Hemolysin Proteins; Vibrio alginolyticus; Zebrafish
PubMed: 20662368
DOI: 10.3354/dao02225 -
Communications Biology Apr 2020Protein post-translational translocation is found at the plasma membrane of prokaryotes and protein import into organellae. Translocon structures are becoming available,...
Protein post-translational translocation is found at the plasma membrane of prokaryotes and protein import into organellae. Translocon structures are becoming available, however the dynamics of proteins during membrane translocation remain largely obscure. Here we study, at the single-molecule level, the folding landscape of a model protein while forced to translocate a transmembrane pore. We use a DNA tag to drive the protein into the α-hemolysin pore under a quantifiable force produced by an applied electric potential. Using a voltage-quench approach we find that the protein fluctuates between the native state and an intermediate in the translocation process at estimated forces as low as 1.9 pN. The fluctuation kinetics provide the free energy landscape as a function of force. We show that our stable, ≈15 kT, substrate can be unfolded and translocated with physiological membrane potentials and that selective divalent cation binding may have a profound effect on the translocation kinetics.
Topics: Bacterial Toxins; Cell Membrane; Escherichia coli Proteins; Hemolysin Proteins; Kinetics; Membrane Potentials; Mutation; Protein Folding; Protein Transport; Protein Unfolding; Single Molecule Imaging; Structure-Activity Relationship; Thioredoxins
PubMed: 32246057
DOI: 10.1038/s42003-020-0841-4 -
Microbiology and Molecular Biology... Jun 1998The pore-forming hemolysin (HlyA) of Escherichia coli represents a unique class of bacterial toxins that require a posttranslational modification for activity. The... (Review)
Review
The pore-forming hemolysin (HlyA) of Escherichia coli represents a unique class of bacterial toxins that require a posttranslational modification for activity. The inactive protoxin pro-HlyA is activated intracellularly by amide linkage of fatty acids to two internal lysine residues 126 amino acids apart, directed by the cosynthesized HlyC protein with acyl carrier protein as the fatty acid donor. This action distinguishes HlyC from all bacterial acyltransferases such as the lipid A, lux-specific, and nodulation acyltransferases, and from eukaryotic transferases such as N-myristoyl transferases, prenyltransferases, and thioester palmitoyltransferases. Most lipids directly attached to proteins may be classed as N-terminal amide-linked and internal ester-linked acyl groups and C-terminal ether-linked isoprenoid groups. The acylation of HlyA and related toxins does not equate to these but does appear related to a small number of eukaryotic proteins that include inflammatory cytokines and mitogenic and cholinergic receptors. While the location and structure of lipid moieties on proteins vary, there are common effects on membrane affinity and/or protein-protein interactions. Despite being acylated at two residues, HlyA does not possess a "double-anchor" motif and does not have an electrostatic switch, although its dependence on calcium binding for activity suggests that the calcium-myristoyl switch may have relevance. The acyl chains on HlyA may provide anchorage points onto the surface of the host cell lipid bilayer. These could then enhance protein-protein interactions either between HlyA and components of a host signal transduction pathway to influence cytokine production or between HlyA monomers to bring about oligomerization during pore formation.
Topics: Acyl Carrier Protein; Acylation; Amino Acid Sequence; Bacterial Proteins; Bacterial Toxins; Escherichia coli; Escherichia coli Proteins; Fatty Acids; Hemolysin Proteins; Humans; Membrane Lipids; Molecular Sequence Data; Sequence Alignment; Signal Transduction
PubMed: 9618444
DOI: 10.1128/MMBR.62.2.309-333.1998