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PubMed1999

Hemolysin of Aeromonas sobria stimulates production of cyclic AMP by cultured cells.

Hemolysin of Aeromonas sobria possesses both cytotoxic activity against mammalian cells and enterotoxic activity. Histopathological examination revealed that hemolysin causes diarrhea without damaging the intestinal epithelial cells. And the fluid accumulated in the mouse intestinal loop by the action of the hemolysin is watery. These observations indicated that the enterotoxic activity of hemolysin is not dependent on its cytotoxic activity. To clarify the mechanism of the enterotoxic action of hemolysin, we examined cyclic nucleotide levels in cultured cells exposed to this toxin. These results showed that hemolysin stimulates the production of cyclic AMP in cultured cells and the cyclic AMPs thus produced emerge in the milieu of cells.

Aeromonas↗
PubMed1965

Hemolysin of Mycoplasma pneumoniae: tentative identification as a peroxide.

Mycoplasma pneumoniae produces a soluble hemolysin active against guinea pig erythrocytes. This hemolysin appears to be a peroxide, since catalase or peroxidase inhibits its activity. The action of catalase and peroxidase is specific, since heating the enzymes abolishes their effect on the hemolysin. In addition, 3-amino- 1,2,4-triazole, a potent inhibitor of catalase, reverses the inhibitory effect of the enzyme. The hemolysin of M. laidlawii is also a peroxide. The hemolysins of M. pneumoniae and M. laidlawii seem unique for microbial organisms since the bacterial hemolysins which have been described have been protein or lipid in nature.

Animals↗
PubMed1974

Demonstration of a temperature-dependent inactivating factor of the thermostable direct hemolysin in Vibrio parahaemolyticus.

A factor was found in Vibrio parahaemolyticus which inactivated the hemolytic activity of a purified, thermostable direct hemolysin. The inactivating factor was associated with the hemolysin but could be separated from it by diethylaminoethyl-cellulose column chromatography. The inactivating factor was activated by heating at 50 to 60 C, but was itself thermolabile and lost activity on heating to 70 to 100 C. The mechanism of the Arrhenius effect, observed with crude hemolysin of V. parahaemolyticus, is as follows. At 50 to 60 C, the temperature-dependent inactivating factor associated with hemolysin inactivates the hemolysin, whereas at 80 to 100 C the crude hemolysin retains activity because at this temperature the factor is inactivated.

Ammonium Sulfate↗
PubMed1975

Characterization of the temperature-dependent inactivating factor of the thermostable direct hemolysin in Vibrio parahaemolyticus.

The termostable direct hemolysin of Vibrio parahaemolyticus is inactivated by heating at around 55 C with an inactivating factor isolated from culture filtrates of V. parahaemolyticus. The characteristics of the temperature-dependent inactivating factor were studied by using polyacrylamide gel disc electrophoresis. It was found that when heated with the hemolysin, the inactivating factor destroyed the hemolysin and thus inactivated the hemolytic activity, suggesting that the inactivating factor has proteolytic activity. It was also demonstrated that the inactivating factor itself is heat labile, losing its activity on heating with the hemolysin at 95 C for 15 min. The inactivating factor was stimulated by the presence of NaCl or MgCl2 and showed maximal activity at around pH 8.0. The results support our previous hypothesis that the Arrhenius effect observed with crude hemolysin of V. parahaemolyticus is due to the presence of a temperature-dependent inactivating factor. The fact that the factor is activated on heating at 50 to 60 C but is inactivated on heating at 70 to 100 C explains the Arrheius effect of crude hemolysin of V. parahaemolyticus.

Cell-Free System↗
PubMed1983

In vivo function of hemolysin in the nephropathogenicity of Escherichia coli.

The role of hemolysin in the nephropathogenicity of Escherichia coli was studied in a hematogenous pyelonephritis model in mice. The nephropathogenicity of a nonhemolytic, avirulent E. coli strain was increased by simultaneous injection with its hemolytic, nephropathogenic parent. This helper mechanism could be attributed to hemolysin, since the simultaneous injection of partially purified hemolysin gave a similar enhancement of nephropathogenicity. Intraperitoneal injection of hemoglobin or iron sulfate before intravenous challenge with this avirulent strain also led to increased virulence. The nephropathogenicity-enhancing effect of hemolysin is therefore supposed to depend on increasing the level of available iron in the host. Under conditions of plentiful iron, hemolysin production was repressed, as shown by in vitro growth experiments in the presence of exogenous iron. These results suggest that the production of hemolysin is regulated by feedback inhibition.

Animals↗
PubMed1990

Intracellular hemolysin-producing Listeria monocytogenes strains inhibit macrophage-mediated antigen processing.

We found that virulent hemolysin-producing (Hly+) Listeria monocytogenes strains inhibit antigen processing and presentation when added to macrophages in vitro. A virulent Hly- bacteria caused little or no inhibition. Live Hly+ bacteria inhibited presentation of both heat-killed L. monocytogenes and ovalbumin. Several observations indicate that hemolysin produced by intracellular bacteria was responsible for the inhibition. First, inhibition was observed even when extracellular bacteria were removed after a brief 10-min bacterial uptake period. Second, inhibition was not prevented by the addition of cholesterol, a substance which inactivates soluble hemolysin. Third, only very high concentrations of soluble hemolysin were inhibitory. Under conditions which inhibit antigen presentation (10(5) per well), macrophages retained normal levels of Ia, maintained normal morphology, and were not permeable when assayed by chromium release. The uptake and catabolism of 35S-labeled live bacteria by macrophages were similar for both Hyl+ and Hly- bacteria. Only a small decrease in uptake and catabolism of surface-iodinated heat-killed L. monocytogenes by macrophages pretreated with inhibitory numbers of live Hly+ bacteria was observed. Additionally, macrophages pretreated with live Hly+ bacteria and fixed 1.5 h later were able to effectively present an ovalbumin peptide (amino acids 323 to 339) to the T-cell hybridoma DO11.10. Hemolysin-producing bacteria inhibited the presentation of antigens that need processing better than they did of antigens that do not require a processing event. Thus, we have demonstrated inhibition of an intracellular antigen processing pathway by hemolysin-producing L. monocytogenes, which may contribute to the virulence of this pathogen.

Animals↗
PubMed1990

Effects of Escherichia coli hemolysin on endothelial cell function.

Escherichia coli hemolysin is considered an important virulence factor in extraintestinal E. coli infections. The present study demonstrates that cultured pulmonary artery endothelial cells are susceptible to attack by low concentrations of E. coli hemolysin (greater than or equal to 0.05 hemolytic units/ml; greater than or equal to 5 ng/ml). Sublytic amounts of hemolysin increased the permeability of endothelial cell monolayers in a time- and dose-dependent manner. The hydraulic conductivity increased approximately 30-fold and the reflection coefficient for large molecules dropped from 0.71 to less than 0.05, indicating a toxin-induced loss of endothelial barrier function. The alterations of endothelial monolayer permeability were accompanied by cell retraction and interendothelial gap formation. In addition, E. coli hemolysin stimulated prostacyclin synthesis in endothelial cells. This effect was strictly dependent on the presence of extracellular Ca2+ but not of Mg2+. An enhanced passive influx of 45Ca2+ and 3H-sucrose but not of tritiated inulin and dextran was noted in toxin-treated cells, indicating that small transmembrane pores comparable to those detected in rabbit erythrocytes had been generated in endothelial cell membranes. These pores may act as nonphysiologic Ca2+ gates, thereby initiating different Ca2+-dependent cellular processes. We conclude that endothelial cells are highly susceptible to E. coli hemolysin and that two major endothelial cell functions are altered by very low concentrations of hemolysin.

Animals↗
PubMed1990

Humoral antibody response and protective immunity in swine following immunization with the 104-kilodalton hemolysin of Actinobacillus pleuropneumoniae.

Five cesarean-derived, colostrum-deprived pigs were given three adjuvant-supplemented subcutaneous and one intravenous injection of the purified 104-kDa hemolysin from serotype 1 Actinobacillus pleuropneumoniae CM-5. Six control animals received phosphate-buffered saline only. Five of six control pigs died within 24 h after challenge. The sixth control pig was moribund and euthanized after 48 h. All six pigs had pleuropneumonia, and A. pleuropneumoniae was isolated from all six lungs. None of the vaccinated pigs died as a result of challenge. After being euthanized, two pigs in this group had no lung lesions but three had chronic pleuropneumonia involving 10, 20, and 40% of the lung tissue. A. pleuropneumoniae was isolated from lung lesions of these three animals but not from the two pigs without lesions. The prechallenge hemolysin-neutralizing antibody titers in the vaccinated pigs were 1:10,900, 1:10,600, 1:4,800, 1:3,900, and 1:3,000, in order of increasing lung involvement. None of the control pigs had neutralizing antibodies. Enzyme-linked immunosorbent assay (ELISA) antibodies to capsule, lipopolysaccharide, and hemolysin were not detected in serum samples collected from the control pigs. In the vaccinated group, prechallenge sera did not contain ELISA antibodies to capsule or lipopolysaccharide. ELISA antibodies to the hemolysin were detected only in the prechallenge and postchallenge serum samples. These results indicate that pigs immunized with the 104-kDa hemolysin of serotype 1 A. pleuropneumoniae are protected against challenge with virulent bacteria. The association between neutralizing antibodies and protection indicates indirectly that the hemolysin is an important virulence factor.

Actinobacillus↗
PubMed1990

The HpmA hemolysin is more common than HlyA among Proteus isolates.

Two different hemolysins, HpmA and HlyA, have been reported in Proteus spp. To study the distribution of these hemolysins among Proteus strains, isolates from various infections and normal feces were screened for hemolysin production. All 63 Proteus mirabilis strains and 23 of the 24 Proteus vulgaris strains produced a calcium-independent hemolytic activity detectable in cell-free supernatants. The calcium-independent activity was due to HpmA; this activity correlated with the presence of hpmA sequences and the production of an extracellular 166-kilodalton (kDa) protein that reacted with anti-HpmA antiserum. HpmA- mutants, constructed by deletion of the central portion of the hpmA gene, did not produce the 166-kDa protein and were no longer hemolytic when compared with their respective parent strains. Among the 87 P. mirabilis and P. vulgaris isolates examined, calcium-dependent hemolytic activity was produced by only two P. vulgaris strains. These strains produced a 110-kDa protein which comigrated with the Escherichia coli hemolysin (HlyA) antibodies in immunoblots. These studies show that Proteus spp. produce two distinct extracellular hemolysins, with nearly all strains producing the calcium-independent hemolysin, HpmA, but only an occasional P. vulgaris isolate producing HlyA.

Bacterial Proteins↗
PubMed1991

Cytotoxicity of the HpmA hemolysin and urease of Proteus mirabilis and Proteus vulgaris against cultured human renal proximal tubular epithelial cells.

Proteus mirabilis, a common agent of nosocomially acquired and catheter-associated bacteriuria, can cause acute pyelonephritis. In ascending infections, bacteria colonize the bladder and ascend the ureters to the proximal tubules of the kidney. We postulate that Proteus species uses the HpmA hemolysin and urease to elicit tissue damage that allows entry of these bacteria into the kidney. To study this interaction, strains of Proteus mirabilis and P. vulgaris and their isogenic hemolysin-negative (hpmA) or isogenic urease-negative (ureC) constructs were overlaid onto cultures of human renal proximal tubular epithelial cells (HRPTEC) isolated from kidneys obtained by immediate autopsy. Cytotoxicity was measured by release of soluble lactate dehydrogenase (LDH). Two strains of P. mirabilis inoculated at 10(6) CFU caused a release of 80% of total LDH after 6 h, whereas pyelonephritogenic hemolytic Escherichia coli CFT073 released only 25% at 6 h (P less than 0.012). Ten P. mirabilis isolates and five P. vulgaris isolates were all hemolytic and cytotoxic and produced urease which was induced by urea. The HpmA hemolysin is apparently responsible for the majority of cytotoxicity in vitro since the hemolysin-negative (hpmA) mutants of P. mirabilis and P. vulgaris were significantly less cytotoxic than wild-type strains. P. mirabilis WPM111 (hemolysin negative) was used to test the effect of urease-catalyzed urea hydrolysis on HRPTEC viability. In the presence of 50 mM urea, WPM111 caused the release of 42% of LDH versus 1% at 6 h in the absence of substrate (P = 0.003). We conclude that the HpmA hemolysin of Proteus species acts as a potent cytotoxin against HRPTEC. In addition, urease apparently contributes to this process when substrate urea is available.

Bacterial Proteins↗
PubMed1992

Cloning and expression of a Serpula (Treponema) hyodysenteriae hemolysin gene.

Serpula (Treponema) hyodysenteriae, the etiologic agent of swine dysentery, produces a hemolysin which is thought to be an important factor in the pathogenesis of the disease. We report the cloning, sequencing, and expression of a hemolysin gene (tly) from S. hyodysenteriae B204. A pUC19 gene bank of strain B204 was constructed in the Escherichia coli K-12 strain DH5 alpha, and hemolytic recombinants were identified by plating the library on blood agar plates. From the hemolytic recombinants, a 1.5-kb DNA fragment could be isolated that contained information necessary for the production of a hemolysin/cytotoxin in E. coli. Nucleotide sequence determination of this 1.5-kb fragment showed that it contained an open reading frame capable of encoding a 26.9-kDa protein. The recombinant hemolysin was easily released from E. coli by osmotic shock. As with the native hemolysin, the recombinant hemolysin is EDTA insensitive, thermolabile, and cytotoxic for several eukaryotic cell lines. Southern blot hybridization showed that the cloned S. hyodysenteriae hemolysin gene tly is present in all pathogenic strains of S. hyodysenteriae tested and absent in the nonpathogenic, weakly hemolytic spirochete S. innocens.

Amino Acid Sequence↗
PubMed1992

Effects of Actinobacillus pleuropneumoniae hemolysin on porcine neutrophil function.

In an attempt to gain insight into the events that take place during Actinobacillus pleuropneumoniae infection, the present study was designed to ascertain the effects of bacterial toxicity on porcine neutrophil functions and viability. Incubation of phagocytes (2 x 10(6)) with opsonized A. pleuropneumoniae 4074 (2 x 10(7) CFU) resulted in phagocytic uptake of less than or equal to 4%. At the same bacterium-to-phagocyte ratio, levels of lactate dehydrogenase activity of 74 and 81% were detected in the extracellular medium after 1.5 and 3 h of incubation, respectively. Furthermore, the ingested bacteria were not killed by the phagocytes. These effects were ascribed to hemolysin produced by the bacteria, because the presence of hemolysin-neutralizing antibody prevented overt cellular damage, significantly increased phagocytic uptake (P less than 0.001), and resulted in an approximately 10-fold decrease in the number of CFU of the ingested bacteria. Cytolytic doses of isolated hemolysin caused dose-related loss of cell viability, diminished bactericidal activity of toxin-treated phagocytes for Escherichia coli, and decreased the ability of the phagocytes to undergo a respiratory burst upon stimulation with phorbol myristic acetate. In contrast, sublytic doses of the hemolysin activated the phagocytes and caused them to respond to phorbol myristic acetate with increased generation of superoxide anion. Because heated (100 degrees C, 5 min) hemolysin preparations did not produce similar effects, we contend that the observed effects were not due to contaminating endotoxin. The data presented herein indicate that A. pleuropneumoniae hemolysin is a potent antiphagocytic virulence factor by virtue of its leukocidal activity. Sublytic doses of the toxin may have important effects on the oxidative metabolism of phagocytic cells.

Actinobacillus pleuropneumoniae↗
PubMed1992

Identification of a second hemolysin (HlyII) in Actinobacillus pleuropneumoniae serotype 1 and expression of the gene in Escherichia coli.

Hemolysin genes of the reference strains of Actinobacillus pleuropneumoniae serotypes 1 and 2 were identified, cloned, and expressed in Escherichia coli by using polymerase chain reaction amplification with oligonucleotides derived from the DNA sequence of the corresponding appA gene from A. pleuropneumoniae serotype 5. The three genes from serotypes 1, 2, and 5 have identical restriction maps and appear to encode a hemolysin which was previously identified in serotype 2 and designated HlyII. Gene appA is different from hlyIA encoding the major hemolysin type I (HlyI) which was identified earlier in serotype 1. Polymerase chain reaction amplification with oligonucleotides derived from the DNA sequence of hlyIA of serotype 1 showed that the gene encoding HlyI is present in serotype 1 but not in serotype 2, in contrast to the gene encoding HlyII that was present in both serotypes. This was confirmed by Western blot (immunoblot) experiments using monoclonal antibodies specific for either recombinant HlyI or recombinant HlyII, which showed that A. pleuropneumoniae serotype 1 strain 4074 produces both HlyI and HlyII, whereas serotype 2 strain S1536 produces only HlyII. The expression of both hemolysins was investigated in all serotypes by the use of monoclonal antibodies. HlyI was shown to be expressed by the reference strains of serotypes 1, 5a, 5b, 9, 10, and 11, whereas HlyII was shown to be expressed by the reference strains of all 12 serotypes tested except serotype 10. A. pleuropneumoniae serotype 1 strain 4074 is the first bacterium which has been shown to contain two different actively expressed RTX toxin genes. Comparison of our data with those from other groups shows that the originally described strongly hemolytic hemolysin type I (HlyI) corresponds to cytolysin I (ClyI) which was recently described by others, while the weakly hemolytic hemolysin type II (HlyII) seems to be identical to ClyII and AppA.

Actinobacillus pleuropneumoniae↗
PubMed1996

Group B streptococcal beta-hemolysin expression is associated with injury of lung epithelial cells.

Group B streptococci (GBS) are the leading cause of serious bacterial infection in newborns. Early-onset disease is heralded by pneumonia and lung injury, and the lung may serve as a portal of entry for GBS into the bloodstream. To examine a potential role for GBS beta-hemolysin in lung epithelial injury, five wild-type strains varying in beta-hemolysin expression were chosen, along with five nonhemolytic (NH) and five hyperhemolytic (HH) variants of these strains derived by chemical or transposon mutagenesis. Monolayers of A549 alveolar epithelial cells were exposed to log-phase GBS or stabilized hemolysin extracts of GBS cultures, and cellular injury was assessed by lactate dehydrogenase (LDH) release and trypan blue nuclear staining. Whereas NH strains produced no detectable injury beyond baseline (medium alone), hemolysin-producing strains induced LDH release from A549 cells in direct correlation to their ability to lyse sheep erythrocytes. HH strains were also associated with marked increases in trypan blue nuclear staining of A549 monolayers. The extent of LDH release produced by HH strains was significantly reduced in the presence of dipalmitoyl phosphatidylcholine, a known inhibitor of hemolysin and the major phospholipid component of human surfactant. Electron microscopic studies of A549 cell monolayers exposed to HH GBS mutants revealed global loss of microvillus architecture, disruption of cytoplasmic and nuclear membranes, and marked swelling of the cytoplasm and organelles. We conclude that GBS hemolysin expression correlates with lung epithelial cell injury and may be important in the initial pathogenesis of early-onset disease, particularly when pulmonary surfactant is deficient.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1998

Evolution of enterohemorrhagic Escherichia coli hemolysin plasmids and the locus for enterocyte effacement in shiga toxin-producing E. coli.

This study assessed the diversity of the enterohemorrhagic Escherichia coli (EHEC) hemolysin gene (ehxA) in a variety of Shiga toxin-producing E. coli (STEC) serotypes and the relationship between ehxA types and virulence markers on the locus for enterocyte effacement (LEE). Restriction fragment length polymorphism of the ehxA gene and flanking sequences and of the E. coli attaching and effacing (eae) gene was determined for 79 EHEC hemolysin-positive STEC isolates of 37 serotypes. Two main groups of EHEC hemolysin sequences and associated plasmids, which corresponded to the eae-positive and the eae-negative isolates, were delineated. Comparisons of the ehxA gene sequences of representative isolates of each group showed that this gene and the rest of the EHEC hemolysin operon are highly conserved. Digestion of an ehxA PCR product with the restriction endonuclease TaqI showed a unique restriction pattern for eae-negative isolates and another one for isolates of serotypes O157:H7 and O157:NM. A conserved fragment of 5.6 kb with four potential open reading frames was identified on the EHEC hemolysin plasmid of eae-positive STEC. Phylogenetic analysis of a subset of 27 STEC isolates, one enteropathogenic E. coli isolate, and a K-12 reference isolate showed that eae-positive STEC isolates all belong to a single evolutionary lineage and that the EHEC hemolysin plasmid and the ehxA gene evolved within this lineage without recent horizontal transfer. However, the eae gene and the LEE appear to have been transferred horizontally within this STEC lineage on several occasions. The reasons for the lack of transfer or maintenance of the LEE in other STEC lineages are not clear and require further study.

Adhesins, Bacterial↗
PubMed1998

Hemolytically active (acylated) alpha-hemolysin elicits interleukin-1beta (IL-1beta) but augments the lethality of Escherichia coli by an IL-1- and tumor necrosis factor-independent mechanism.

Many pathogenic Escherichia coli produce the toxin alpha-hemolysin (Hly), and lipopolysaccharide (LPS), interleukin-1 (IL-1), and tumor necrosis factor (TNF) have all been recognized as important effector molecules during infections by gram-negative organisms. Despite the characterization of many in vitro effects of hemolysin, no direct relationship has been established between hemolysin, LPS, proinflammatory cytokine production, and E. coli-induced mortality. Previously, we have shown in vivo that hemolysin elicits a distinct IL-1alpha spike by 4 h into a lethal hemolytic E. coli infection. Using three transformed E. coli strains, WAF108, WAF270, and WAH540 (which produce no Hly [Hlynull], acylated Hly [Hlyactive], or nonacylated Hly [Hlyinactive], respectively), we sought to determine the specific roles of hemolysin acylation, LPS, IL-1, and TNF in mediating the lethality of E. coli infection in mice. WAF270 was 100% lethal in BALB/c, C3H/HeJ, and C57BL/6 mice; in mice pretreated with antibody to the type 1 IL-1 receptor; in type 1 IL-1 receptor-deficient mice; and in dual (type 1 IL-1 receptor-type 1 TNF receptor)-deficient mice at doses which were nonlethal (0%) with both WAF108 and WAH540. At lethal doses, WAF270 killed by 6 +/- 2.3 h while WAF108 and WAH540 killed at 36 +/- 9.4 and 36 +/- 13.8 h, respectively. These differences in mortality were not due to IL-1 or TNF release, and the enhanced expression of LPS, which corresponded to Hly expression, was not likely the primary factor causing mortality. We demonstrate that bacterial fatty acid acylation of hemolysin is required in order for it to elicit IL-1 release by monocytes and to confer its virulence on E. coli.

Acylation↗
PubMed1974

Existence of two distinct hemolysins in Vibrio parahaemolyticus.

Two distinct hemolysins were demonstrated in Vibrio parahaemolyticus. A thermostable direct hemolysin purified from V. parahemolyticus WP-1, a Kanagawa phenomenon (KP)-positive strain, is antigenically different from a thermolabile hemolysin produced by V. parahaemolyticus T-3454, a KP-negative strain. The thermostable direct hemolysin was found in KP-positive strains but not in KP-negative strains. On the other hand, the thermolabile hemolysins were found in both KP-positive and -negative strains, although some KP-positive strains did not produce this hemolysin.

Animals↗
PubMed1967

Identification of staphylococcal hemolysins by an electrophoretic localization technique.

A technique for identifying and characterizing staphylococcal hemolysins by first separating them electrophoretically in barbital-buffered agar gel (pH 8.4) at 5 ma/cm for 2 hr and then determining their hemolytic activities by exposing them to human, horse, rabbit, and sheep erythrocytes is described. The alpha-hemolysin produced by a White variant of the Wood 46 strain of Staphylococcus aureus migrated 18 mm towards the cathode, and it lysed horse, rabbit, and sheep erythrocytes, whereas a Clear variant of the Wood 46 strain of S. aureus produced a lysin which migrated similarly to the alpha-hemolysin but lysed only rabbit cells. This latter lysin was tentatively named alpha(1)-lysin. This strain of S. aureus also produced beta-hemolysin which migrated 36 mm towards the cathode and lysed sheep cells. beta-Hemolysin produced by some strains of S. aureus showed considerable tailing during electrophoresis, whereas beta-hemolysin produced by other strains of S. aureus migrated as a well-defined peak. A lysin migrating 11 mm towards the anode was probably delta-lysin. It was, however, not produced in sufficient concentration when the cultures were grown in semisolid medium.

Animals↗