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At least 19 recordsLinked to original sources
PubMed1995

Evaluation of pig lungs following an experimental challenge with Actinobacillus pleuropneumoniae serotype 1 and 5 in pigs inoculated with either hemolysin protein and/or outer membrane proteins.

Histopathological changes were compared in pigs challenged with Actinobacillus pleuropneumoniae serotype 1 and serotype 5 after inoculation with subunit vaccines. The vaccines consisted of outer membrane protein and/or hemolysin protein isolated from Actinobacillus pleuropneumoniae serotype 1 or both subunits combined. Twenty-seven cross-bred pigs were separated into six groups: Groups I and IV were vaccinated and boostered with 1500 micrograms outer membrane protein; Groups II and V were vaccinated and boostered with 250 micrograms hemolysin protein; Groups III and VI were vaccinated and boostered with a combination of 1500 micrograms outer membrane protein and 250 micrograms hemolysin protein. Groups I, II and III were challenged with A. pleuropneumoniae serotype 1; and Groups IV, V and VI were challenged with A. pleuropneumoniae serotype 5. Groups III and VI demonstrated the least severe lung tissue damage, with significantly lower (P < 0.05) lung involvement as compared to the other groups. Lesions were noted in all six groups. These results showed that complete protection against A. pleuropneumoniae infection was not feasible using a subunit vaccine consisting of just outer membrane protein and hemolysin protein, and that some cross-protection did occur.

Actinobacillus Infections↗
PubMed1991

Pore-forming bacterial protein hemolysins (cytolysins).

Protein toxins forming pores in biological membranes occur frequently in Gram-positive and Gram-negative bacteria. They kill either bacteria or eukaryotic cells (at most, a few seem to act on both groups of organisms). Most of the toxins affecting eukaryotes have clearly been shown to be related to the pathogenicity of the producing organisms. Toxin formation frequently involves a number of genes which encode the toxin polypeptide as well as proteins for toxin activation and secretion. Regulation of toxin production is usually coupled with that of the synthesis of a number of other virulence factors. Iron is the only known environmental factor that regulates transcription of a number of toxin genes by a Fur repressor-type mechanism, as has been originally described in Escherichia coli. Interestingly, the thiol-activated hemolysins (cytolysins) of Gram-positive bacteria contain a single cysteine which can be replaced by alanine without affecting the cytolytic activity. The Gram-negative hemolysins (cytolysins) are usually synthesized as precursor proteins, then covalently modified to yield an active hemolysin and secreted via specific export systems, which differ for various types of hemolysins.

Amino Acid Sequence↗
PubMed1989

Isolation and analysis of the C-terminal signal directing export of Escherichia coli hemolysin protein across both bacterial membranes.

We have studied the C-terminal signal which directs the complete export of the 1024-amino-acid hemolysin protein (HlyA) of Escherichia coli across both bacterial membranes into the surrounding medium. Isolation and sequencing of homologous hlyA genes from the related bacteria Proteus vulgaris and Morganella morganii revealed high primary sequence divergence in the three HlyA C-termini and highlighted within the extreme terminal 53 amino acids the conservation of three contiguous sequences, a potential 18-amino-acid amphiphilic alpha-helix, a cluster of charged residues, and a weakly hydrophobic terminal sequence rich in hydroxylated residues. Fusion of the C-terminal 53 amino acid sequence to non-exported truncated Hly A directed wild-type export but export was radically reduced following independent disruption or progressive truncation of the three C-terminal features by in-frame deletion and the introduction of translation stop codons within the 3' hlyA sequence. The data indicate that the HlyA C-terminal export signal comprises multiple components and suggest possible analogies with the mitochondrial import signal. Hemolysis assays and immunoblotting confirmed the intracellular accumulation of non-exported HlyA proteins and supported the view that export proceeds without a periplasmic intermediate. Comparison of cytoplasmic and extracellular forms of an independently exported extreme C-terminal 194 residue peptide showed that the signal was not removed during export.

Amino Acid Sequence↗
PubMed1997

Temperature-sensitive mutants of Actinobacillus pleuropneumoniae induce protection in mice.

Temperature-sensitive mutants of Actinobacillus pleuropneumoniae 4074, serotype 1, were isolated after treatment with nitrosoguanidine and enrichment with penicillin and D-cycloserine. Of the four temperature-sensitive mutants evaluated in mice, one (A-1) had a tight phenotype (i.e., it ceased replication immediately after transfer to the nonpermissive temperature [37 degrees C]) and three (1-2, 4-1, and 12-1) were coasters that continued replication for up to three generations after transfer to 37 degrees C. The reversion frequencies ranged from 10(-6) to 10(-9), and cutoff temperatures ranged from 33 to 35 degrees C. No major changes were detected in the biochemical profiles; agglutination reactions; electrophoretic profiles of the lipopolysaccharides, outer membrane proteins, and hemolysin proteins; hemolytic titers; or CAMP factor reactions of the mutants and the wild-type bacteria. Groups of 3- to 5-week-old, female ICR mice were immunized intranasally with three doses of 3.5 x 10(6) CFU of the mutants over 3 weeks and subsequently challenged intranasally with 5 50% lethal doses of the parental wild-type. Protection was induced by both the tight and the coaster mutants, with the 4-1 and 12-1 coasters eliciting greater protection (67 and 82%, respectively) than that induced by the A-1 tight mutant (57%). Intranasal immunization with both phenotypes induced serum antibody responses against the surface antigens and the hemolysin protein.

Actinobacillus pleuropneumoniae↗
PubMed2006

Purification and characterization of a hemolysin-like protein, Sll1951, a nontoxic member of the RTX protein family from the Cyanobacterium Synechocystis sp. strain PCC 6803.

The hemolysin-like protein (HLP) Sll1951, characterized by the GGXGXDXUX nonapeptide motif implicated in Ca(2+) binding, was purified from the glucose-tolerant strain (GT) of Synechocystis sp. strain PCC 6803. HLP was eluted at 560 kDa after gel filtration chromatography. Atomic absorption spectroscopy indicated that the protein bound calcium. The bound Ca(2+) was not chelated with EGTA; however, it was released after being heated at 100 degrees C for 1 min, and it rebound to the Ca(2+)-depleted protein at room temperature. The apparent HLP molecular mass increased to 1,000 kDa and reverted to 560 kDa during the release and rebinding of Ca(2+), respectively. The monomers of the respective forms appeared at 90 and 200 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. HLP showed no apparent hemolytic activity against sheep erythrocytes; however, a slight hemolytic activity was detected during the conformational change caused by the rebinding of Ca(2+). Immunoelectron microscopy using polyclonal antibodies against the 200-kDa monomer revealed that HLP is located in the cell surface layer. The localization and Ca(2+)-induced reversible conformational change suggest that HLP is a member of the repeat in toxin (RTX) protein family despite its latent and low toxicity. In some other cyanobacteria, RTX proteins are reported to be necessary for cell motility. However, the GT was immotile. Moreover, the motile wild-type strain did not express any HLP, suggesting that HLP is one of the factors involved in the elimination of motility in the GT. We concluded that the involvement of RTX protein in cyanobacterial cell motility is not a general feature.

Antibodies↗
PubMed1996

The hemolysin B protein, expressed in Saccharomyces cerevisiae, accumulates in binding-protein (BiP)-containing structures.

The hemolysin B (Hly B) protein of Escherichia coli, a member of the ABC-transporter family, was expressed in Saccharomyces cerevisiae and tested for its ability to complement a defect in the a-factor transporter Ste6. We found that HlyB was not able to restore mating ability to a STE6 deletion strain. The HlyB protein did not co-fractionate with Ste6 on sucrose gradients, indicating that improper localization of the HlyB protein could contribute to the lack of complementation. Immunofluorescence experiments suggest that HlyB is localized to structures derived from the endoplasmic reticulum (ER). The HlyB-expressing cells revealed a perinuclear staining typical of ER-localized proteins and intensely staining ring-like structures (HlyB-bodies). Double-label immunofluorescence experiments show that the HlyB structures also contain the ER binding protein (BiP), the product of the KAR2 gene. The HlyB protein, however, did not co-fractionate with another ER marker protein, the NADPH cytochrome c reductase. The HlyB bodies could be derivatives of a novel compartment of the early secretory pathway which contains BiP but not other resident ER proteins. In this case, HlyB could serve as a tool for the biochemical characterization of this compartment.

ATP-Binding Cassette Transporters↗
PubMed2006

Self-energy-limited ion transport in subnanometer channels.

The current-voltage characteristics of the alpha-Hemolysin protein pore during the passage of single-stranded DNA under varying ionic strength C are studied experimentally. We observe strong blockage of the current, weak superlinear growth of the current as a function of voltage, and a minimum of the current as a function of C. These observations are interpreted as the result of the ion electrostatic self-energy barrier originating from the large difference in the dielectric constants of water and the lipid bilayer. The dependence of DNA capture rate on C also agrees with our model.

Anions↗
PubMed2002

Temperature- and H-NS-dependent regulation of a plasmid-encoded virulence operon expressing Escherichia coli hemolysin.

Proteins H-NS and Hha form a nucleoprotein complex that modulates expression of the thermoregulated hly operon of Escherichia coli. We have been able to identify two H-NS binding sites in the hly regulatory region. One of them partially overlaps the promoter region (site II), and the other is located about 2 kbp upstream (site I). In contrast, Hha protein did not show any preference for specific sequences. In vitro, temperature influences the affinity of H-NS for a DNA fragment containing both binding sites and H-NS-mediated repression of hly operon transcription. Deletion analysis of the hly regulatory region confirms the relevance of site I for thermoregulation of this operon. We present a model to explain the temperature-modulated repression of the hly operon, based on the experiments reported here and other, preexisting data.

Bacterial Proteins↗
PubMed2002

Pleurotus and Agrocybe hemolysins, new proteins hypothetically involved in fungal fruiting.

Novel hemolytic proteins, ostreolysin and aegerolysin, were purified from the fruiting bodies of the edible mushrooms Pleurotus ostreatus and Agrocybe aegerita. Both ostreolysin and aegerolysin have a molecular weight of about 16 kDa, have low isoelectric points of 5.0 and 4.85, are thermolabile, and hemolytic to bovine erythrocytes at nanomolar concentrations. Their activity is impaired by micromolar Hg(2+) but not by membrane lipids and serum low-density lipoproteins (LDL). The sequence of respectively 50 and 10 N-terminal amino acid residues of ostreolysin and aegerolysin has been determined and found to be highly identical with a cDNA-derived amino acid sequence of putative Aa-Pri1 protein from the mushroom A. aegerita, Asp-hemolysin from Aspergillus fumigatus, and two bacterial hemolysin-like proteins expressed during sporulation. We found that ostreolysin is expressed during formation of primordia and fruiting bodies, which is in accord with previous finding that the Aa-Pri1 gene is specifically expressed during fruiting initiation. It is suggestive that the isolated hemolysins play an important role in initial phase of fungal fruiting.

Agaricales↗
PubMed1994

Identification and characterization of two functional domains of the hemolysin translocator protein HlyD.

Secretion of Escherichia coli hemolysin is mediated by a sec-independent pathway which requires the products of at least three genes, hlyB, hlyD and tolC. Two regions of HlyD were studied. The first region (region A), consisting of the 33-amino acid, C-terminal part of the HlyD protein, is predicted to form a potential helix-loop-helix structure. This sequence is conserved among HlyD analogues of similar transport systems of other bacterial species. Using site-directed mutagenesis, we showed that the amino acids Leu475, Glu477 and Arg478 of this region are essential for HlyD function. The last amino acid of HlyD, Arg478, is possibly involved in the release of the HlyA protein, since cells bearing a hlyD gene mutant at this position produce similar amounts of HlyA to the wild-type strain, but most of the protein remains cell-associated. Competition experiments between wild-type and mutant HlyD proteins indicate that region A interacts directly with a component of the secretion apparatus. The second region of HlyD (region B), located between amino acids Leu127 and Leu170, is highly homologous to the otherwise unrelated outer membrane protein TolC. Deletion of this region abolishes secretion of hemolysin. This sequence of HlyD also seems to interact with a component of the hemolysin secretion machinery since a hybrid HlyD protein carrying the corresponding TolC sequence, although inactive in the transport of HlyA, is able to displace wild-type HlyD from the secretion apparatus.

Amino Acid Sequence↗
PubMed1985

Studies on toxin of Aspergillus fumigatus. XXII. Fashion of binding of Asp-hemolysin to human erythrocytes and Asp-hemolysin-binding proteins of erythrocyte membranes.

The fashion of binding of Asp-hemolysin to human erythrocytes and the isolation of Asp-hemolysin-binding proteins from erythrocyte membranes were investigated by the immunocytochemical technique and affinity chromatography. Asp-hemolysin bound best at a pH range from 5 to 7. The erythrocytes treated with Asp-hemolysin showed diffuse, ring-like or cap-like staining by the peroxidase-labeled antibody method under the light microscope. The distribution of Asp-hemolysin on the erythrocyte surface was clearly observed as patches or caps in the scanning electron microscope. The erythrocyte ghosts were extracted with 1% sodium deoxycholate-0.1 M Tris-HC1 buffer (pH 7.5) containing 0.2 M NaCl and 1 mM EDTA, and the extract was chromatographed on an affinity column consisting of Asp-hemolysin attached to activated thiol-Sepharose 4B. Four proteins present in the membrane extract were retained by activated thiol-Sepharose 4B and eluted with 50 mM cysteine as toxin-membrane components. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that the polypeptides correspond to band 2.1, one protein of the 2 region, band 3 and band 7 in the Steck nomenclature system.

Aspergillus fumigatus↗
PubMed1998

Reversible denaturation, self-aggregation, and membrane activity of Escherichia coli alpha-hemolysin, a protein stable in 6 M urea.

Escherichia coli alpha-hemolysin (HlyA) is an extracellular protein toxin (107 kDa) whose cell lytic activity may be preserved for months at -20 degreesC in the presence of 6 M urea, although it decays rapidly in urea-free buffers. This paper describes experiments addressed to unravel the role of urea in HlyA stabilization. Urea up to 8 M inhibits the Ca2+-binding and hemolytic activities of the protein, alters its secondary and tertiary structures, and reduces its tendency to self-aggregation. All these changes are largely reversed upon urea removal by dilution or dialysis, suggesting that they are interrelated. Furthermore, the extent of recovery of the native activities and structural features of alpha-hemolysin that follows urea removal increases with the concentration of urea during the previous phase. Thus, it seems that urea elicits the reversible transition of HlyA to a less active but more stable state whose structure differs significantly from that of the native protein. Moreover dialysis equilibration of the protein with buffers containing 3 M urea induces the formation of a molecular form of HlyA 5-10 times more active than the native protein in the absence of urea. This hyperactive intermediate appears to keep the native secondary structure of HlyA, but with a less compact tertiary structure, that increases the number of exchangeable Ca2+ ions under these conditions. Changes in the intrinsic fluorescence of HlyA also support the notion of a conformational change in the high-activity intermediate. The intermediate is only detected when assayed in the presence of Ca2+ and 3 M urea and can bind a large number of calcium ions (approximately 12 vs approximately 3 for the native protein); it shows a large tendency to self-aggregation and presumably, in the presence of membranes, a similar tendency to irreversible insertion, which may be the reason for its high lytic activity.

Amino Acid Sequence↗
PubMed1998

Construction of chromosomally encoded secreted hemolysin fusion proteins by use of mini-TnhlyAs transposon.

We used the minitransposon TnhlyAs [Gentschev, I., Maier, G., Kranig, A. and Goebel, W. (1996) Mol. Gen. Genet. 252, 266-274] for random insertion of the secretion signal (HlyAs) of Escherichia coli hemolysin (HlyA) into chromosomal genes. Four mini-TnhlyAs derivatives bearing the gltA (citrate synthase), deoC (2 deoxyribose-5 phosphate aldolase), tig (trigger factor) genes and an unknown ORF fused to hlyAs were identified and characterized. Our data suggest that TnhlyAs-generated hemolysin fusion proteins are secreted efficiently by the HlyB/HlyD/TolC hemolysin secretion machinery and that this can be useful for studies of gene expression or function.

Base Sequence↗
PubMedDate not supplied

Aeromonas and plesiomonas as possible causes of diarrhoea.

The number of reports on the isolation of Aeromonas from patients with diarrhoeal disease is now large and suggests an etiological role of the bacterium. It is well established that strains of Aeromonas produce an enterotoxin. This enterotoxin is cytotonic, i.e. it does not damage the membrane, and it does not cross-react immunologically with cholera toxin and Escherichia coli heat-labile (LT) toxin. Most enterotoxigenic strains also produce a cytotoxic protein (hemolysin), the role of which is probably limited in diarrhoea but potentially toxic in humans in other kinds of infections. Strains of Plesiomonas shigelloides also seem to be able to cause diarrhoea in some cases. The pathogenesis of Plesiomonas-induced diarrhoea remains to be elucidated, but a heat-stable enterotoxin may be involved.

Aeromonas↗
PubMed2005

Protection against Leptospira interrogans sensu lato challenge by DNA immunization with the gene encoding hemolysin-associated protein 1.

The use of DNA constructs encoding leptospiral proteins is a promising new approach for vaccination against leptospirosis. In previous work we determined that immunization with hemolysis-associated protein 1 (Hap1) (LipL32) expressed by adenovirus induced significant protection against a virulent Leptospira challenge in gerbils. To avoid the use of the adenovirus vector, we checked for clinical protection against lethal challenge by DNA vaccination. A DNA vaccine expressing Hap1 was designed to enhance the direct gene transfer of this protein into gerbils. A challenge was performed 3 weeks after the last immunization with a virulent strain of serovar canicola. Our results show that the cross-protective effect with pathogenic strains of Leptospira, shared by Hap1, could be mediated by the DNA plasmid vector. This finding should facilitate the design and development of a new generation of vaccines against bacteria, particularly Leptospira interrogans sensu lato.

Animals↗
PubMed1996

Association of RTX toxins with erythrocytes.

A critical step in the target cell attack by RTX cytotoxins is their association with target cells. A binding assay was used to study the association of the Escherichia coli hemolysin protein (HlyA) with erythrocytes. Several parameters required for lysis by HlyA were tested for their effects on its initial association with erythrocytes. The results demonstrate that HlyA binding to target cells is independent of several structural components of the active toxin, including the N-terminal hydrophobic region, the glycine-rich repeat region, and the HlyC-dependent acylation of HlyA. Further, the association with erythrocytes was independent of Ca2+ concentration or temperature, while the lytic event is both Ca2+ dependent and temperature dependent. The association of two other RTX toxin proteins, the Pasteurella haemolytica leukotoxin (LktA) and the enterohemorrhagic E. coli toxin (EhxA), were also examined; these toxins bound to erythrocytes much less efficiently than did HlyA. The association of HlyA with erythrocytes occurred rapidly, within 12 s of incubation, and demonstrated no measurable dissociation. HlyA bound to erythrocytes with a maximum of approximately 2,000 molecules per cell. Competition between active HlyA and unacylated HlyA demonstrated no inhibition of binding by unacylated HlyA; rather, active HlyA appeared to displace unacylated HlyA on the cell surface. These data demonstrate that binding and lysis by HlyA are separable events and challenge the concept of nonspecific binding to the cell surface by RTX toxins.

Acylation↗
PubMed1988

Immune response to Escherichia coli alpha-hemolysin in patients.

The serum antibody response of patients infected with alpha-hemolysin (AH)-producing Escherichia coli was measured by three immunoassays: tube neutralization, microneutralization, and enzyme-linked immunosorbent assay. All three assay results showed good correlation with each other. The mean anti-AH titer in patients with E. coli infection was higher than the mean titer in noninfected patients. The hemolysin-neutralizing activity was immunoglobulin G. The amount of lipopolysaccharide (LPS) antibody did not correlate with the amount of AH antibody. LPS antibody measured by enzyme-linked immunosorbent assay was predominantly of the immunoglobulin G class. Adsorption of LPS antibody by E. coli H79 LPS did not affect anti-AH titers, indicating that LPS and AH have different antigenic determinants. AHs prepared from several different E. coli strains had identical or similar antigenic determinants at the active site. Hemolysin proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were stained identically by human sera with AH antibody and by a mouse monoclonal AH antibody.

Aged↗