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PubMed1985

Escherichia coli hemolysin is released extracellularly without cleavage of a signal peptide.

A 110-kilodalton polypeptide isolated from cell-free culture supernatants of hemolytic Escherichia coli was shown to be associated with hemolytic activity. The relative amount of the extracellular 110-kilodalton species detected directly reflects the extracellular hemolysin activity associated with Escherichia coli strains harboring different hemolysin recombinant plasmids. The predicted molecular mass of the hemolysin structural gene (hlyA) based on DNA sequence analysis was 109,858 daltons. Amino-terminal amino acid sequence analysis of the 110-kilodalton polypeptide provided direct evidence that it was encoded by hlyA. Based on this information, it was also demonstrated that the HlyA polypeptide was released extracellularly without signal peptidase-like cleavage. An examination of hemolysin-specific polypeptides detected by use of recombinant plasmids in a minicell-producing strain of Escherichia coli was performed. These studies demonstrated how hemolysin-associated 110- and 58-kilodalton polypeptides detected in the minicell background could be misinterpreted as a precursor-product relationship.

Amino Acid Sequence↗
PubMed1988

Transcriptional organization of the Escherichia coli hemolysin genes.

The transcriptional organization of the Escherichia coli hemolysin genes (hlyCABD) encoded by pSF4000 was examined. The use of different hemolysin gene-specific radiolabeled probes in blots containing isolated in vivo RNA revealed 4.0-kilobase hlyCA and 8.0-kilobase hlyCABD transcripts. The treatment of cells with rifampin just before RNA isolation showed the half-lives of these mRNAs to be 10.2 and 4.4 min, respectively. The 5' ends of the hly transcripts were 462 and 464 nucleotides from the putative initiation codon of hlyC based on a primer extension method of RNA mapping. Deletion analysis of pSF4000 combined with quantification of the hemolysin structural protein HlyA by immunoblotting confirmed that major control of HlyA expression occurs within a 168-base-pair PstI fragment located 433 base pairs upstream of the start of hlyC. A second recombinant plasmid, pANN202-312, encoding an E. coli hemolysin of different origin expressed 6-fold less total HlyA and 50-fold less extracellular HlyA than pSF4000 in identical cell backgrounds. The pANN202-312 recombinant had a different hly promoter, with the hly mRNA beginning 264 nucleotides upstream from the start of hlyC. We showed by RNA blotting that cells harboring pANN202-312 compared with pSF4000 have similar steady-state levels of the hlyCA transcript but they lack a consistently detectable hlyCABD transcript. We propose that one reason for the disparate levels of extracellular hemolysin produced by hemolytic E. coli is dissimilar levels of mRNA encoding in part the transport genes hlyB and hlyD.

Base Sequence↗
PubMed1989

Synthesis, inactivation, and localization of extracellular and intracellular Escherichia coli hemolysins.

Extra- and intracellular Escherichia coli hemolysin expressed by two cloned hly determinants, both under the control of the activator element hlyR, were analyzed. One determinant carried all four hly genes (hlyC, hlyA, hlyB, and hlyD), whereas the other carried only the two genes (hlyC and hlyA) required for synthesis of active hemolysin but not those essential for its secretion. It was shown that the total amounts of HlyA protein and of hemolytic activity are similar in both cases in logarithmically growing cultures. The E. coli strain carrying the complete hly determinant released most hemolysin into the media and accumulated very little HlyA intracellularly. The active extracellular hemolysin (HlyA*) was inactivated in the stationary phase without degradation of the HlyA protein. In contrast, the hemolysin which accumulated intracellularly in the E. coli strain carrying hlyA and hlyC only was proteolytically degraded at the end of the logarithmic growth phase. Immunogold labeling indicates that active intracellular HlyA bound preferentially to the inner membrane, whereas that part of the extracellular HlyA which remained cell-bound was located exclusively at the cell surface. It was shown by fluorescence-activated cell sorter analysis that active extra- and intracellular HlyA* bound with similar efficiency to erythrocytes, whereas hemolytically inactive HlyA protein did not bind to these target cells.

Biological Transport↗
PubMed2001

Isolation and characterization of Escherichia coli tolC mutants defective in secreting enzymatically active alpha-hemolysin.

This study describes the isolation and characterization of a unique class of TolC mutants that, under steady-state growth conditions, secreted normal levels of largely inactive alpha-hemolysin. Unlike the reduced activity in the culture supernatants, the cell-associated hemolytic activity in these mutants was identical to that in the parental strain, thus reflecting a normal intracellular toxin activation event. Treatment of the secreted toxin with guanidine hydrochloride significantly restored cytolytic activity, suggesting that the diminished activity may have been due to the aggregation or misfolding of the toxin molecules. Consistent with this notion, sedimentation and filtration analyses showed that alpha-hemolysin secreted from the mutant strain has a mass greater than that secreted from the parental strain. Experiments designed to monitor the time course of alpha-hemolysin release showed delayed appearance of toxin in the culture supernatant of the mutant strain, thus indicating a possible defect in alpha-hemolysin translocation or release. Eight different TolC substitutions displaying this toxin secretion defect were scattered throughout the protein, of which six localized in the periplasmically exposed alpha-helical domain, while the remaining two mapped within the outer membrane-embedded beta-barrel domain of TolC. A plausible model for the secretion of inactive alpha-hemolysin in these TolC mutants is discussed in the context of the recently determined three-dimensional structure of TolC.

Bacterial Outer Membrane Proteins↗
PubMed1966

Action of streptolysin S, the group D hemolysin, and phospholipase C on whole cells and spheroplasts.

Davie, Joseph M. (Indiana University, Bloomington), and Thomas D. Brock. Action of streptolysin S, the group D hemolysin, and phospholipase C on whole cells and spheroplasts. J. Bacteriol. 91:595-600. 1966.-The effect of streptolysin S, the group D hemolysin, and phospholipase C (the alpha toxin of Clostridium perfringens) on whole cells and spheroplasts or protoplasts of three strains of streptococci and Micrococcus lysodeikticus was tested. Viability, C(14)-glycine uptake, and lysis were measured. The group D hemolysin and phospholipase C were active against whole bacteria; streptolysin S was not. All three substances were active on spheroplasts. A partially resistant mutant derived from a strain sensitive to the group D hemolysin was also partially resistant to streptolysin S and phospholipase C. Antimycin A protected spheroplasts from streptolysin S but not from the group D hemolysin.

Antimycin A↗
PubMed1968

Transformation of type polysaccharide antigen synthesis and hemolysin synthesis in streptococci.

Transformation of the ability to synthesize type polysaccharide antigen and beta-hemolysin has been obtained in group F streptococci. Colonies possessing cells transformed to antigen synthesis were detected on the agar surface with fluorescein-labeled anti-type serum. This selection method, in contrast to those with antibiotics, allowed both transformed and nontransformed cells to grow, resulting in sectored colonies. These colonies could be subcultured to further establish the synthesis of antigen. Group F, group A, and group-like z deoxyribonucleic acid (DNA) labeled with type II antigen and hemolysin, and streptomycin resistance transferred each marker to a group F strain lacking a type antigen. DNA from group F and z3 strains labeled with type III antigen, and streptomycin resistance transferred both markers to group F and z3 strains lacking type antigen. A second F strain without type antigen was not transformed with any of these markers. A group H strain was transformed to streptomycin resistance only by the same types of DNA. Transformation to type II antigen synthesis always resulted in the formation of beta-hemolysin. All strains isolated from natural sources contained both markers. A mutant, obtained by nitrosoguanidine treatment of an FII(sr) strain, did not synthesize either the hemolysin or the antigen. This mutant still possessed the group antigen and streptomycin resistance. A close linkage of type II antigen and beta-hemolysin is indicated. The fluorescent-antibody staining of cells containing both group and type antigens showed a more intense ultraviolet adsorption for type than group antigen. A surface location (microcapsular) for the type antigen appeared likely. These results are of interest for studies on antigen biosynthesis, genetics, and classification of the streptococci.

Drug Resistance, Microbial↗
PubMed1990

Production of hemolysin and other extracellular enzymes by clinical isolates of Pseudomonas pseudomallei.

One hundred clinical isolates of Pseudomonas pseudomallei from humans were tested for their ability to produce extracellular, biologically active substances which are thought to contribute to the virulence of Pseudomonas species. All isolates produced at least on extracellular enzyme; 91 strains were positive for lecithinase, lipase, and protease; but none was positive for elastase. Ninety-three strains produced a hemolysin which was detectable around the heavy growth on saline-washed sheep erythrocyte brain heart infusion agar but not demonstrable around individual colonies or in broth culture filtrate. In contrast, a hemolysin which was cytolytic around individual colonies of P. pseudomallei on the assay plate and in broth culture filtrate was exhibited by four strains. By using one of these four isolates as the test strain, the latter hemolysin was characterized further. It was heat labile, most active in an acid environment (pH 5.5), and cytolytic in broth culture filtrate for a variety of animal and human erythrocytes. Sterols, particularly cholesterol and 7-dehydrocholesterol, inhibited its hemolytic activity, but the activity was not enhanced by reducing agents or suppressed by reagents which modify sulfhydryl-activated hemolysins. A nonhemolytic mutant of the test strain of P. pseudomallei retained the extracellular enzymes of its parent, indicating that the hemolysin was not a lecithinase, lipase, or protease.

Adolescent↗
PubMed1978

Production and characterization of two hemolysins of Bacillus cereus.

Bacillus cereus strain B-48 produced two hemolysins with molecular weights of 52,000 (H-I) and 31,000 (H-II). A mutant was isolated that produced only H-II but was identical with the wild type in all other respects. We exploited this mutant to produce H-II for study that was free of contamination by H-I. By manipulation of media composition, we produced H-I in the absence of H-II. The hemolysins were precipitated differently by ammonium sulfate, and both exhibited the Arrhenius effect when heated. Both hemolysins attached rapidly to erythrocytes; however, lysis by H-I was immediate, while lysis by H-II followed after a lag. Hemolysis by H-I and H-II increased in rate with increasing temperature and was absent at 0 degrees C. Only H-I was inhibited by cholesterol. The hemolysins of B. cereus appeared similar to the hemolysins of B. thuringiensis. H-I probably is identical with cereolysin.

Animals↗
PubMed1985

A thiol-activated hemolysin in gram-negative bacteria.

An investigation of Klebsiella pneumoniae hemolytic activity was carried out. Strains isolated from different infected specimens were hemolytic in tryptic soy agar with rabbit blood; incubation at 4 degrees C enhanced the hemolysis. There was no evident red blood cell lysis in plates with human, sheep, mouse, and chicken erythrocytes. The culture in tryptic soy broth, its supernatant and bacterial lysate did produce evident hemolysis of rabbit red blood cells when they were preincubated with 2-mercaptoethanol. Klebsiella pneumoniae hemolysin showed the Arrhenius effect, while temperatures over 60 degrees C for 10 min reduced the activity of crude hemolysin to zero; purified hemolysin, however, was heat stable. Two hemolysins active on rabbit red blood cells were purified and both shared several properties. This work represents the initial description of a thiol-activated hemolysin in Gram-negative bacteria.

Animals↗
PubMed1999

Group B streptococcal beta-hemolysin promotes injury of lung microvascular endothelial cells.

Group B streptococci (GBS) are the leading cause of pneumonia and sepsis in human newborns. Exudative pulmonary edema and alveolar hemorrhage seen in GBS pneumonia indicate vascular damage, and we reported that GBS injure lung microvascular endothelial cells (LMvEC) both in vivo and in vitro. The specific GBS factors causing LMvEC injury are uncertain, but GBS beta-hemolysin activity is associated with lung epithelial cell injury. We hypothesized that GBS beta-hemolysin contributes to LMvEC injury and exudative pulmonary edema. To test this hypothesis we used isogenic nonhemolytic and hyperhemolytic GBS mutants derived by transposon insertional mutagenesis from three different wild-type strains. Hemolytic titers for each strain were calculated using live GBS and Tween 80/starch-stabilized extracts of log-phase GBS. All nonhemolytic mutants lacked detectable hemolytic activity, whereas hyperhemolytic mutants produced 4-16 times the hemolytic activity of their parent strains. LMvEC injury was assayed by light microscopy, the release of lactate dehydrogenase, trypan blue nuclear staining and Evans blue-albumin flux. Compared with the parent strains, all nonhemolytic mutants caused significantly reduced, and all hyperhemolytic mutants caused significantly greater lactate dehydrogenase release from and trypan blue nuclear staining of LMvEC. Moreover, a nonhemolytic mutant caused reduced and a hyperhemolytic mutant caused increased Evans-blue albumin flux across polar LMvEC monolayers. These findings were corroborated by light microscopic evidence of hemolysin-associated damage to the LMvEC monolayers. We conclude that GBS beta-hemolysin promotes LMvEC injury and increases permeability in vitro, and speculate that GBS beta-hemolysin contributes to the pathogenesis of alveolar edema and hemorrhage in early onset GBS pneumonia.

Animals↗
PubMed1982

Isolation and some properties of the hemolysins from the crude venom extract of scorpion telson, Heterometrus gravimanus.

THe hemolytic and enzymic investigations of the crude venom from Heterometrus gravimanus scorpion have revealed the presence of both direct and indirect (phospholipase A) hemolysins. The direct hemolysins were isolated from the lyophilized crude venom extract by Sephadex G-100 gel filtration, SP-Sephadex C-25 column chromatography, gel filtration of Sepharose 4B and Sephacryl S-300. The two direct hemolysins having high molecular weights, P (A) (M.W.: nearly 200 X 10(4)) and P (B) (M.W.: 22 X 10(4)), were obtained with the activity recoveries of 8% and 39.4%, respectively. P (B) was further separated into three fractions by Sephadex G-200 gel filtration, namely F (A) (M.W.: 17 X 10(4), F (B) (M.W.: 4.5 X 10(4) and F (C) (M.W.: below 1 x 10(4), with the significant losses of direct hemolytic activities. However, F (B) had phospholipase A activity and this activity was partially inhibited by p-bromophenacyl bromide. F (B) and F (C) were soluble, even after the treatment of dialysis and lyophilization, in the isotonic saline and distilled water, and were homogeneous in polyacrylamide gels after going through disc and vertical plate electrophoreses. Furthermore, the schlieren pattern of F (B) also showed homogeneity of the preparation. The protein parts of hemolysins were found to be acidic ones. Although ultraviolet spectra of them were somewhat varied in accordance with their protein contents in the samples, the spectra of exitation and emission were almost identical. The direct hemolysins are presumably glycoproteins, since they are positive in the sugar reactions, and the difference between P (A) and P (B) is supposed to consist in the sugar part rather than that of proteins.

Amino Acids↗
PubMed2001

[Effects of low density lipoprotein and oxidized low density lipoprotein on the cytotoxic activity of Asp-hemolysin to murine macrophages].

We examined the effects of human low density lipoprotein (LDL) and oxidized LDL (Ox-LDL) on the cytotoxic activity of Asp-hemolysin from Aspergillus fumigatus Fresenius-Muramatsu strain to mouse peritoneal macrophages (M phi). The inhibitory effects of LDL and Ox-LDL on the cytotoxic activity of Asp-hemolysin to M phi increased in a dose-dependent manner, and the effect of Ox-LDL was greater than the inhibitory effect of LDL. Furthermore, the binding of Asp-hemolysin to LDL or Ox-LDL was observed by western blot analysis of the culture medium. These results suggest that the inhibition by LDL or Ox-LDL on the cytotoxic activity of Asp-hemolysin to M phi was due to the binding of LDL or Ox-LDL to Asp-hemolysin in the culture medium.

Animals↗
PubMed1995

Purification and some properties of S-Hemolysin produced by Streptomyces sp. strain no. A-6288.

A new cytolytic toxin, designated as S-Hemolysin, was found in the culture filtrate of Streptomyces sp. strain No. A-6288, isolated from a soil sample. The molecular weight of S-Hemolysin was estimated to be 10,000 by SDS-polyacrylamide gel electrophoresis and to be 20,000 by Sephadex G-100. S-Hemolysin is a glycoprotein that is composed of 102 amino acid residues with 11.6% glucose, and the isoelectric point is around pH 5.8. The phospholipase C activity of S-Hemolysin was specific for the following substrates in this order: sphingomyelin > lysophosphatidylethanolamine > lysophosphatidylcholine > phosphatidylethanolamine > phosphatidylcholine. S-Hemolysin had hemolytic activity against rabbit, human, and sheep erythrocytes, but did not cause aggregation of human platelets. These activities were accelerated with Mg2+, Mn2+, and Co2+ ions and inhibited by the addition of Ca2+, Cu2+, and Zn2+ ions. This enzyme was shown to be different from the known bacterial phospholipase C.

Amino Acids↗
PubMed1990

[Antibacterial and anti-hemolysin activities of tea catechins and their structural relatives].

Among catechins tested, (-)epigallocatechin (EGC), (-)epicatechin gallate (ECg), (-) epigallocatechin gallate (EGCg) inhibited the growth of Staphylococcus aureus, Vibrio cholerae O1 classical Inaba 569B and El Tor Inaba V86. S. aureus was more sensitive than V. cholerae O1 to these compounds. EGCg showed also a bactericidal activity against V. cholerae O1 569B. Pyrogallol showed a stronger antibacterial activity against S. aureus and V. cholerae O1 than tannic and gallic acid. Rutin or caffein had no effect on them. ECg and EGCg showed the most potent anti-hemolysin activity against S. aureus alpha-toxin, Vibrio parahaemolyticus thermostable direct hemolysin (Vp-TDH) and cholera hemolysin. Among catechin relatives, only tannic acid had a potent anti-hemolysin activity against alpha-toxin. These results suggest that the catechol and pyrogallol groups are responsible for the antibacterial and bactericidal activities, while the conformation of catechins might play an important role in the anti-hemolysin activity.

Antitoxins↗
PubMed1992

Virulence factors in Serratia marcescens: cell-bound hemolysin and aerobactin.

1. A total of 60 nosocomial isolates of Serratia marcescens were screened for the presence of markers related to virulence, i.e., cell-bound hemolysin and production of siderophore aerobactin. 2. No aerobactin-producing strains were found, and the incidence of cell-bound hemolysin was 97%. 3. Hemolysin-positive (58 strains) and hemolysin-negative (2 strains) Serratia marcescens showed the same LD50 (3 x 10(7) bacteria) in a test of virulence for mice. 4. These results indicate that cell-bound hemolysin is not a main factor of virulence for mice in Serratia marcescens.

Animals↗
PubMed1992

A hemolysin-encoding plasmid contributes to bacterial virulence in experimental Enterococcus faecalis endophthalmitis.

Beta-hemolysin production is a variable trait of the Lancefield group D streptococcus, Enterococcus faecalis. The E. faecalis hemolysin is encoded by large transmissible plasmids. The variable nature of this putative virulence factor provided an ideal system for testing its contribution in experimental endophthalmitis. In this study, isogenic E. faecalis strains were compared to determine whether the presence of the hemolysin-encoding plasmid affected the severity of disease in a rabbit endophthalmitis model. Experimental infections (n = 6) with 10(1)-10(4) E. faecalis organisms harboring the hemolysin-encoding plasmid resulted in a 98% loss of retinal function (by electroretinography [ERG]) and white reflex by postoperative day 3. By contrast, infections of similar numbers of plasmid-free E. faecalis organisms (n = 5) resulted in retention of some retinal function (23% per ERG) with a red reflex demonstrated on postoperative day 3. Results of light microscopy, slit-lamp examination, ERG, and indirect ophthalmoscopy indicated that infections with hemolysin-encoding plasmid-containing E. faecalis resulted in a more aggressive endophthalmitis compared with the endophthalmitis caused by plasmid-free E. faecalis. This is the first endophthalmitis model to the authors' knowledge that specifically evaluates bacterial virulence using isogenic strains.

Animals↗
PubMed1989

Identification of the heat-labile hemolysin of Actinobacillus pleuropneumoniae serotype 1.

The heat-labile hemolysin of Actinobacillus pleuropneumoniae serotype 1 strain CM-5 was partially purified using ammonium sulfate precipitation and gel permeation chromatography. This partially purified material was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nylon filters. The filters were treated with convalescent pig serum and subsequently with CM-5 culture supernatant containing active hemolysin. A 104 kd peptide was identified as the hemolysin because it bound antibodies in convalescent pig serum which cross-linked active hemolysin. The same 104 kd protein when injected into a rabbit produced neutralizing antibodies to the CM-5 hemolysin in culture supernatant.

Actinobacillus↗
PubMed1986

[Effects of subinhibitory concentrations of pefloxacine on hemolysin production and adherence of urinary Escherichia coli].

The in vitro effect of subinhibitory concentrations of pefloxacin on E. coli's production of hemolysin and adherence to eucaryotic cells was studied. Six uropathogenic E. coli strains, with mannose-resistant and/or mannose-sensitive adhesins were tested. All strains produced an alpha-hemolysin and were susceptible to pefloxacin (0.125 less than or equal to MIC less than or equal to 0.250 mg/l). The effect on hemolysin production was studied using microtitration plates to determine the concentration inhibiting 50% of E. coli's hemolytic activity. The inhibition of adhesion was tested following adhesion of E. coli to uroepithelial cells. Sub-MICs of pefloxacin strongly inhibited the production of alpha-hemolysin and adhesion of E. coli regardless of adhesin type. A correlation between the inhibition of hemolysin production and the inhibition of adhesion to uroepithelial cells was found for four E. coli strains.

Bacterial Adhesion↗