Subhemolytic doses of Escherichia coli hemolysin evoke large quantities of lipoxygenase products in human neutrophils

Polymorphonuclear leukocytes (PMN) have been identified as preferred target cells for Escherichia coli hemolysin in human blood (Bhakdi, S., Greulich, S., Muhly, M., Ebersp?cher, B., Becker, H., Thiele, A., and Hugo, F. (1989) J. Exp. Med. 169, 737-754). Leukotriene and 5-hydroxyeicosatetraenoic acid generation was investigated in human PMN challenged with E. coli hemolysin in the absence or presence of free arachidonic acid or eicosapentaenoic acid (EPA). In the absence of exogenous free fatty acids, E. coli hemolysin (0.01-10 hemolytic units/ml) induced moderate generation of leukotriene B4 (LTB4) and its omega-oxidation products. The presence of free arachidonic acid (10 microM) during E. coli hemolysin (0.1 hemolytic unit/ml) challenge evoked the generation of large quantities of these products (greater than 100 pmol/1.5 x 10(7) PMN). In parallel, large amounts of 5-hydroxyeicosatetraenoic acid and nonenzymatic LTA4 hydrolysis products appeared. Product release peaked or plateaued 5-10 min after E. coli hemolysin challenge. The presence of exogenous EPA upon E. coli hemolysin challenge resulted in the exclusive generation of LTB5 and metabolites, LTA5 decay products and 5-hydroxyeicosapentaenoic acid. Dose and time dependences corresponded to those with arachidonic acid provision, and the total of EPA-derived products surpassed that of arachidonic acid metabolites in corresponding experiments approximately 2-fold. Increasing the time between free fatty acid provision and E. coli hemolysin challenge resulted in a rapid decline in the generation of arachidonic acid or EPA metabolites. Thus, subhemolytic doses of E. coli hemolysin evoke marked PMN eicosanoid generation that is dependent on exogenous free fatty acid supply, with total amounts approximating those found in calcium ionophore-stimulated neutrophils.     

Purification and characterization of hemolysin from Porphyromonas gingivalis A7436

Porphyromonas gingivalis, a periodontal pathogen, has the ability to lyse erythrocytes. The hemolytic activity of P. gingivalis A7436 was purified as a 45-kDa protein from the culture supernatant of a 3-days old culture using nickel-nitrilotriacetic acid chromatography. Erythrocytes treated with purified P. gingivalis hemolysin showed the presence of pores and extracellular debris by scanning electron microscopy. Active immunization of mice with 15 micrograms hemolysin induced neutralizing antibodies to hemolysin. Heating at 60 degrees C and treatment with trypsin and dithiothreitol abolished hemolytic activity, while incubation with the protease inhibitor Na-p-tosyl-L-lysine chloromethyl ketone caused no effect. We report here for the first time purification of a hemolysin from P. gingivalis A7436. The amino acid sequence of an internal peptide of hemolysin showed sequence similarity with fimbrillin from P. gingivalis HG564. However, the amino acid composition of purified hemolysin was different from that of P. gingivalis fimbrillin. Also, the ability to lyse but not agglutinate erythrocytes and to bind to nickel-nitrilotriacetic acid differentiates P. gingivalis hemolysin from fimbrillin.     

Effect of sodium chloride and citric acid on growth and toxin production by A. caviae and A. sobria at moderate and low temperatures

The effect of sodium chloride and citric acid on hemolysin and caseinase production by Aeromonas caviae and Aeromonas sobria at 32 degrees C and 5 degrees C was investigated. At 32 degrees C, although both strains were tolerant to 3% NaCl in TSB, the production of caseinase was decreased in the presence of 1-3% NaCl, and the production of hemolysin was abolished by 2-3% NaCl. Citric acid (0.03%) was less effective than NaCl in reducing hemolysin and caseinase production by both strains at 32 degrees C. A combination of low temperature (5 degrees C) and citric acid treatment reduced hemolysin and caseinase production by both strains. A combination of low temperature (5 degrees C) and NaCl (3%) treatment was the most effective procedure in reducing growth and hemolysin and caseinase production by the tested strains.     

Amino acid sequence of thermostable direct hemolysin produced by Vibrio parahaemolyticus

The complete amino acid sequence of the subunit of thermostable direct hemolysin, a dimeric protein composed of identical subunits isolated from Vibrio parahaemolyticus, was determined by sequencing BrCN-peptides, their tryptic peptides, and overlaps obtained by Achromobacter protease I digestion. The subunit consists of 165 amino acid residues with the sole disulfide bond between Cys 151 and Cys 161. It is deduced that the biologically active hemolysin is formed by noncovalent association of subunits which are not linked together by disulfide bonds. The primary structure of hemolysin elucidated in the present study is essentially the same as that deduced from the nucleotide sequence of a gene encoding the protein but differs in 9 amino acid residues, suggesting the possibility of the presence of multiple genes for the thermostable direct hemolysin in Vibrio parahaemolyticus.     

Effect of carrier molecules on production and properties of extracellular hemolysin produced byStreptococcus agalactiae

Streptococcus agalactiae type la strain 090 produced a cell-associated hemolysin during exponential growth in medium lacking proteins. Growth of the organism in medium containing proteins or medium supplemented with Tween 40 resulted in the appearance of extracellular hemolytic activity that was filterable. Maximum extracellular hemolytic activity was obtained in the late exponential phase of growth corresponding to the maximum number of cells. Extracellular hemolysin released in medium containing proteins could be precipitated by ammonium sulfate. Cell-associated hemolysin could be extracted in the cold by purified lipoteichoic acid from the organism. Purification and characterization of the extracellular hemolysin by column chromatography showed that the hemolysin was associated with molecules eliciting its release. Hemolysin associated with lipoteichoic acid or Tween 40 had an apparent molecular weight of 1,800,000 or 60,000 daltons, respectively.     

Effects of penicillin on the synthesis of membrane proteins of Chlamydia trachomatis LGV2 serotype

Abstract Actinobacillus (Hemophilus) pleuropneumoniae type strain 4074, serotype 1, secretes a potent hemolysin. This hemolysin is thermolabile and inactivated by proteinase K. We have purified the hemolysin to homogeneity and characterized it as a protein of 105 kDa by SDS-Polyacrylamide gel electrophoresis. Using a calibrated gel filtration column, the active hemolysin was identified as a monomer of the 105 kDa polypeptide. This hemolysin is an acid protein with an isoelectric point at p I 4.3.     

An aberrant hemolysin of Vibrio cholerae non-O1.

An aberrant hemolysin produced by a Vibrio cholerae non-O1 strain N037 (N037-hly) was purified and characterized. N037-Hly was antigenically very similar to El Tor hemolysin but differed in molecular weight (48,000 vs. 60,000), interaction with glucose, and hemolytic activity. Of 100 V. cholerae non-O1 strains other than the N037 strain examined, none produced this aberrant hemolysin. The N-terminal amino acid sequence of N037-hly was highly homologous to that of El Tor hemolysin.     

Cloning and characterization of a gene encoding a new thermostable hemolysin from Vibrio parahaemolyticus

A new thermostable hemolysin (delta-VPH) gene was cloned from a Kanagawa-negative Vibrio parahaemolyticus strain into vector pBR322 in Escherichia coli K12. The nucleotide and amino acid sequences had no homology with those of the thermostable direct hemolysin (TDH) which causes the Kanagawa phenomenon, and of the thermolabile hemolysin (TLH) of V. parahaemolyticus. The gene was present in all V. parahaemolyticus strains tested and also in one strain of V. damsela.     

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

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.     

Analysis of a gene (vch) encoding hemolysin isolated and sequenced from Vibrio campbellii

This study describes the amplification, localization, and sequence analysis of a hemolysin gene from type strain V. campbellii NBRC 15631--the first report of a full-length hemolysin gene for the species. An amplicon ( approximately 600 bp) of polymerase chain reaction performed using V. campbellii DNA template and primers previously designed to target a fragment of V. harveyi hemolysin gene (vhh) was shotgun-cloned and sequenced, generating 576 bp nucleotide sequences of the V. campbellii hemolysin gene. PCR primers designed based on these initial sequences were used to amplify a 551-bp V. campbellii hemolysin gene fragment that was used as probe in Southern hybridization, which localized the complete hemolysin gene within a 3.5-kb HindIII restriction fragment of the V. campbellii genomic DNA. To obtain the remaining DNA sequences upstream and downstream of the 576-bp hemolysin gene sequences, inverse PCR was performed using a self-ligated (circularized) V. campbellii HindIII restriction fragment as the template and PCR primers designed to amplify flanking regions of the 576-bp gene fragment. Nucleotide sequences from the terminal regions of the 3.1-kb product of inverse PCR provided the flanking sequences, resulting in the complete sequence for the V. campbellii hemolysin gene. A VCH PCR primer set was designed to amplify a 1.3-kb region containing the entire hemolysin gene even from other V. campbellii strains, which was sequenced to confirm the V. campbellii hemolysin gene sequence. An open reading frame (ORF) of 1,254 bp (designated as vch) was identified, sharing 79% sequence identity with V. harveyi hemolysin gene vhh, representing 262 base substitutions between V. campbellii and V. harveyi. The deduced amino acid sequence of V. campbellii hemolysin (VCH) shows homologies to the V. harveyi hemolysin (VHH), thermolabile hemolysin of V. parahaemolyticus, proteins such as phospholipase of V. vulnificus and lecithinases of V. mimicus and V. cholerae. The VCH primer set did not produce any amplicon in PCR using V. harveyi DNA, and may therefore be used to distinguish environmental strains of V. campbellii from V. harveyi.     

A low molecular weight enterotoxic hemolysin from clinical Enterobacter cloacae

Seven of 50 Enterobacter cloacae strains from clinical isolates produced small turbid zones of hemolysis in horse and sheep blood agar plates, and the culture supernatants were also positive for hemolytic activity. The hemolysin was partially purified from the culture supernatant of E. cloacae by ultrafiltration (PM-10 membrane) and extraction with acetone. Semipurified hemolysin was stable to heating (100 degrees C, 30 min) and was soluble in organic solvents (acetone, ethanol, and methanol). The toxin showed no loss of biological activity after treatment with trypsin and was stable to acid treatment at pH 2.0 but not at a pH greater than 7.0. In the rat intestinal loop assay, the hemolysin caused hemorrhagic fluid accumulation and severe histological alterations. These findings indicate that this hemolysin may be a putative virulence factor in E. cloacae infections.     

The erythrocyte receptor for Fusobacterium necrophorum hemolysin: phosphatidylcholine as a possible candidate

An attempt was made to determine the receptor for the hemolysin of Fusobacterium necrophorum using horse erythrocyte or its membranes as target. The spectrum of erythrocyte sensitivity has indicated that horse, dog and mouse erythrocytes are highly sensitive whereas cattle, sheep, goat and chicken red blood cells are insensitive to this hemolysin. A high correlation between sensitivity and phosphatidylcholine content of the erythrocyte membranes was noted. Binding of hemolysin to horse erythrocyte membranes was reduced significantly by prior treatment of membranes with phospholipase A₂ but not with phospholipase C. Pretreatment of erythrocyte membranes with pronase, proteinase K, trypsin or neuraminidase did not alter binding of hemolysin to the membranes, suggesting that protein or sialyl residues are not involved as receptors. Gas liquid chromatography analysis showed that the fatty acid profile from hydrolysis of bovine liver phosphatidylcholine by hemolysin and phospholipase A₂ were similar. In conclusion, this report presents evidence that phosphatidylcholine may be acting as a possible receptor for the hemolysin of F. necrophorum.     

A novel extracellular protease of Vibrio mimicus that mediates maturation of an endogenous hemolysin

Vibrio mimicus, a human pathogen that causes gastroenteritis, produces an enterotoxic hemolysin as a virulence factor. The hemolysin is secreted extracellularly as an inactive protoxin and converted to a mature toxin through removal of the N‐terminal propeptide, which comprises 151 amino acid residues. In this study, a novel protease having the trypsin‐like substrate specificity was purified from the bacterial culture supernatant. The N‐terminal amino acid sequence of the purified protein was identical with putative trypsin VMD27150 of V. mimicus strain VM573. The purified protease was found to cause maturation of the protoxin by cleavage of the Arg¹⁵¹? Ser¹⁵² bond. Deletion of the protease gene resulted in increased amounts of the protoxin in the culture supernatant. In addition, expression of the hemolysin and protease genes was detected during the logarithmic growth phase. These findings indicate that the protease purified may mediate maturation of the hemolysin.

     

Hybridization analysis of the gene encoding a hemolysin (suilysin) of Streptococcus suis type 2: evidence for the absence of the gene in some isolates

A hemolysin gene was cloned from a virulent strain of Streptococcus suis type 2 strain 1933. Analysis of the gene and its product revealed that it is identical to a previously reported hemolysin (suilysin) of S. suis type 2. Southern hybridization analysis of the digested total genomic DNA from S. suis with the cloned hemolysin DNA sequences as probe indicated that the hemolysin gene is present as a single copy on the genome. Genomic DNA of 63 isolates of S. suis encompassing all known serotypes were examined by DNA hybridization and polymerase chain reaction (PCR) studies for the presence of the hemolysin gene homolog. The results of both techniques were identical and demonstrated the absence of the hemolysin gene in some isolates. In DNA hybridization studies, three DNA probes derived from the hemolysin encoding gene were used. Results showed that sequences encoding the C-terminal 257 amino acid residues (Probe 1) were the most conserved and hybridized to a 1.2 kb fragment in 32 (51%) strains and a 4.0 kb fragment in 23 (36%) strains respectively. Thus, Probe 2 hybridized to the DNA of 55 (87%) of the isolates tested. The first probe (Probe 1) comprising almost the entire hemolysin gene and the third probe (Probe 3) which consisted of the N-terminal sequences hybridized only to a 4.0 kb fragment in 23 (36%) of the strains tested. Eight (13%) of the strains tested were hybridization and PCR negative. The hybridization of the C-terminal end sequences (Probe 2) to the 1.2 kb fragment in 32 (51%) of the strains and the lack of hybridization of the probes to eight (13%) strains may suggest the presence of different types of hemolysin molecule in S. suis strains.     

Secretion of hemolysin of Aeromonas sobria as protoxin and contribution of the propeptide region removed from the protoxin to the proteolytic stability of the toxin

The sequence at the amino terminus region of the hemolysin ofAeromonas sobria is homologous with that of aerolysin of A. hydrophila. However, there is no homology between the two toxins in the sequence at the carboxy terminal region. It has been shown that aerolysin is secreted into culture supernatant as a protoxin. This proaerolysin is activated by the proteolytic removal of a carboxy terminal peptide. However, the role of the carboxy terminal region, which is removed in the activation process, has not been elucidated. In this study, we showed that hemolysin is also secreted as a protoxin into culture supernatant and that prohemolysin is cleaved by the protease of A. sobria between Ser-446 and Ala-447, resulting in the removal of a 42 amino acid peptide. The removal of the peptide converts the prohemolysin into active hemolysin. Subsequently, we mutated the hemolysin gene to delete the last several amino acid residues and expressed the genes in Escherichia coli, in order to examine the role of the carboxy terminal region of prohemolysin. The amounts of these mutant hemolysins accumulated in the periplasmic space of E. coli were very low compared with that of the wild-type. This observation indicated that the carboxy terminal region of prohemolysin contributes to the proteolytic stability of the toxin.     

Characterization of inhibitor to leptospiral hemolysin present in bovine serum

Hemolysis by leptospiral hemolysin was strongly inhibited by bovine serum. The inhibitory activity was observed in the chloroform-methanol-soluble fraction of bovine serum. The inhibitor was eluted in a complex lipid fraction and was separated into two fractions (Fr. I and II) by silicic acid column chromatography. Fractions I and II inhibited approximately 75% and 95%, respectively, of hemolysis by leptospiral hemolysin. Fraction I was identified as phosphatidylethanolamine (PdE) by silica gel thin-layer chromatography (TLC). Two kinds of phospholipids (PLs) were detected in Fr. II by TLC. One was resistant to alkaline treatment and was identified as sphingomyelin (Spm), and the other was sensitive to such treatment and was identified as phosphatidylcholine (PdC). PLs, such as Spm, PdC, phosphatidylglycerol, PdE, phosphatidylserine and cardiolipin, inhibited hemolysis by leptospiral hemolysin, but phosphatidylinositol did not show any inhibitory activity. PLs lacking the amino group in the polar backbone of the molecules were more effective. From experiments using erythrocytes of various kinds of animals, it was revealed that the hemolytic sensitivity of mammalian erythrocytes to leptospiral hemolysin depended on the Spm content in the erythrocyte membrane. On the other hand, phospholipase C (PLase C) activity with Spm and PdC as substrates was detected in the culture supernatant of Leptospira. Therefore, leptospiral hemolysin was presumed to be PLase C, perhaps sphingomyelinase. The inhibitors of leptospiral hemolysin present in bovine serum were identified as PLs. PLs in bovine serum were suggested to function as inhibitors of the interaction between leptospiral hemolysin and the surface of the erythrocyte membrane.     

Identification of a new hemolysin from diarrheal isolate SSU of Aeromonas hydrophila

A clinical strain SSU of Aeromonas hydrophila produces a potent cytotoxic enterotoxin (Act) with cytotoxic, enterotoxic, and hemolytic activities. A new gene, which encoded a hemolysin of 439-amino acid residues with a molecular mass of 49 kDa, was identified. This hemolysin (HlyA) was detected based on the observation that the act gene minus mutant of A. hydrophila SSU still had residual hemolytic activity. The new hemolysin gene (hlyA) was cloned, sequenced, and overexpressed in Escherichia coli. The hlyA gene exhibited 96% identity with its homolog found in a recently annotated genome sequence of an environmental isolate, namely the type strain ATCC 7966 of A. hydrophila subspecies hydrophila. The hlyA gene did not exhibit any homology with other known hemolysins and aerolysin genes detected in Aeromonas isolates. However, this hemolysin exhibited significant homology with hemolysin of Vibrio vulnificus as well as with the cystathionine beta synthase domain protein of Shewanella oneidensis. The HlyA protein was activated only after treatment with trypsin and the resulting hemolytic activity was not neutralizable with antibodies to Act. The presence of the hlyA gene in clinical and water Aeromonas isolates was investigated and DNA fingerprint analysis was performed to demonstrate its possible role in Aeromonas virulence.     

Superlytic hemolysin mutants of Serratia marcescens.

Hemolysis by Serratia marcescens is caused by two proteins, ShlA and ShlB. ShlA is the hemolysin proper, and ShlB transports ShlA through the outer membrane, whereby ShlA is converted into a hemolysin. Superhemolytic ShlA derivatives that displayed 7- to 20-fold higher activities than wild-type ShlA were isolated. ShlA80 carried the single amino acid replacement of G to D at position 326 (G326D), ShlA87 carried S386N, and ShlA80III carried G326D and N236D. Superhemolysis was attributed to the greater stability of the mutant ShlA derivatives because they aggregated less than the wild-type hemolysin, which lost activity within 3 min at 20 degrees C. In contrast to the highly hemolytic wild-type ShlA at 0 degrees C, the hyperlytic hemolysins were nonhemolytic at 0 degrees C, suggesting that the hyperlytic derivatives differed from wild-type ShlA in adsorption to and insertion into the erythrocyte membrane. However, the size of the pores formed at 20 degrees C by superhemolytic hemolysins could not be distinguished from that of wild-type ShlA. In addition to the N-terminal sequence up to residue 238, previously identified to be important for activation and secretion, sites 326 and 386 contribute to hemolysin activity since they are contained in regions that participate in hemolysin inactivation through aggregation.     

Comparison of Vibrio parahaemolyticus hemolysin (Vp-TRH) produced by environmental and clinical isolates

TDH-related hemolysin (Vp-TRH) produced by Kanagawa-phenomenon-negative (KP-) Vibrio parahaemolyticus has been demonstrated to be a possible virulence determinant. Though almost half of KP- isolates examined from diarrhoeal patients produced Vp-TRH, few reports mentioned the ability of environmental isolates to produce Vp-TRH. Considering the route of infection with V. parahaemolyticus, this toxin must be produced by the organisms in the sea or in sea food. To confirm that Vp-TRH produced by V. parahaemolyticus could be involved in sea-food-borne diarrhoeas, Vp-TRH-producing strains were isolated from the environment, identified and hemolysin purified from these strains was compared to hemolysin (Vp-TRH) isolated from diarrhoeal patients. The results showed that the hemolytic activity, antigenicity, reactivity in the rabbit ileal loop test and N-terminal amino acid sequence of Vp-TRH from environmental strains was indistinguishable from the toxin of clinical origin.     

Comparative amino acid sequence analysis of hemolysins produced by Vibrio hollisae and Vibrio parahaemolyticus.

Vibrio hollisae produces a hemolysin (Vh-rTDH) that is related to the thermostable direct hemolysin of Vibrio parahaemolyticus (Vp-TDH). Although both hemolysins are essentially similar biologically and immunologically, they differ markedly in heat stability; Vp-TDH is heat stable, whereas Vh-rTDH is heat labile. To elucidate the relationships between their characteristics and molecular structures, we analyzed the amino acid sequence of Vh-rTDH and compared it with that of Vp-TDH. Vh-rTDH consisted of 165 residues, of which 23 residues, spread over the peptide chain, differed from those of Vp-TDH.