Nonhemolytic Strains Research Articles

Influence of calcium on secretion and activity of the cytolysins of Actinobacillus pleuropneumoniae

In vitro production of a secreted hemolytic cytolysin of Actinobacillus pleuropneumoniae has been reported to be dependent on the presence of calcium in culture media. This is not the case with Escherichia coli hemolysins, however, where calcium has been shown to be required only for activation and binding to target cells. Because the cytolysins of A. pleuropneumoniae have structural and functional similarities to those of hemolytic E. coli, we sought to reexamine the role that calcium plays in the secretion and activity of A. pleuropneumoniae cytolysins. A. pleuropneumoniae hemolytic strain S4074 secreted two major proteins into culture supernatants independent of the presence of calcium in growth medium. These proteins were identified with murine monoclonal antibodies as the 105-kDa cytolysin I and the 103-kDa cytolysin II. It was found that both cytolysins required calcium for binding to erythrocyte membranes. Culture fluids from bacteria grown with calcium lysed porcine erythrocytes even after free calcium in the fluid was removed prior to the hemolytic assay. When bacteria were grown in medium depleted of calcium, no lysis of erythrocytes was detected unless calcium was added to assay buffers. Culture supernatants from A. pleuropneumoniae nonhemolytic strain 1421 grown with or without calcium contained two predominant proteins, which were identified with mouse monoclonal antibodies as the 103-kDa cytolysin II and the 120-kDa cytolysin III. Binding to erythrocytes (without hemolysis) by cytolysin II was dependent on calcium. Cytolysin III did not bind to erythrocytes. These results indicate that the ability of strain S4074 to lyse swine erythrocytes (and the inability of strain 1421 to do so) was directly correlated with the presence of cytolysin I. Cytolysins I, II, and III bound to swine neutrophils and purified neutrophil membranes only in the presence of calcium. When calcium was depleted, cytolysin I of strain S4074 had a reduced binding and cytolysis II and III of strain 1421 did not bind at all. The data suggest that regardless of the target cell involved, calcium plays an integral role in the function but not the production of A. pleuropneumoniae cytolysins.

Factors influencing variable oxidative hemolysis of inbred mouse erythrocytes

The hemolysis of erythrocytes from certain inbred mouse strains (e.g., BALB/c) in response to hydrogen peroxide stress has been shown to be correlated with the type of hemoglobin β chain (Kruckeberg, W.C., et al. (1987) Blood 70, 909–914). The characteristic hemolytic response of BALB/c red cells to oxidative stress resembles that of human red cells in that carbon monoxide and iron chelators inhibit hemolysis of both. Gross hemoglobin oxidation rates were similar in hemolytic (BALB/c) and nonhemolytic (C57BL/6) strains. The rate and degree of in vitro catalase inhibition by sodium azide was also the same for the two strains. Even in the presence of this catalase inhibitor the assayable hydrogen peroxide disappeared within seconds of its addition, yet hemolysis was not observed for about 15 min. The mechanism underlying this delay between hydrogen peroxide addition and disappearance and subsequent hemolysis is under investigation.

A nonhemolytic phospholipase C produced byPseudomonas cepacia

Pseudomonas cepacia Pc224c, a nonhemolytic strain originally isolated from the sputum of a cystic fibrosis patient, produced an extracellular, heat-labile phospholipase C activity, which was measured quantitatively on the synthetic substratep-nitrophenylphophorylcholine. Cell-free supernatants from cultures grown to late log phase in MOPS-minimal salts-Tryptose medium contained specific activity at least 38 times greater than that from cultures grown in Tryptose minimal medium, Tryptic Soy broth, or peptone medium. Production was inhibited by the presence of inorganic phosphate in the medium and enhanced by aeration of the culture. The PLC ofP. cepacia is nonhemolytic and does not exhibit lecithinase activity on egg-yolk agar.

Characterization of thermostable direct hemolysins encoded by four representative tdh genes of Vibrio parahaemolyticus

Four tdh genes encoding thermostable direct hemolysin (TDH) cloned from two representative strains of V. parahaemolyticus ( tdh1 and tdh2 from a hemolytic strain, tdh3 and tdh4 from a non-hemolytic strain) have different nucleotide sequences (the maximum divergence: 3.3%). In this study, each tdh gene product was purified from the lysate of Escherichia coli cells carrying the cloned gene and their properties were compared to investigate the influence of the amino acid substitutions caused by these base changes. The four tdh gene products showed different electrophoretic mobilities under non-denaturing conditions. All the gene products had hemolytic activities for various animal erythrocytes, stimulated vascular permeability in the rabbit skin, and were lethal to mice, although their potencies were slightly different. Antigenicities of the four gene products were indistinguishable. These results indicate that the four tdh genes have evolved to maintain a fundamental molecular structure and biological activities of the gene products and minor structural and/or charge differences of the molecules are perhaps responsible for the slight divergence of their biological activities.

Signal transduction in human platelets and inflammatory mediator release induced by genetically cloned hemolysin-positive and -negative Escherichia coli strains

Incubation of human platelets with the hemolysin-producing Escherichia coli strain K-12 (pANN5211) induced the activation of protein kinase C, aggregation of platelets, calcium influx, low amounts of 12-hydroxyeicosatetraenoic acid (12-HETE), and release of serotonin from dense granules. Nonhemolytic isogenic strains of E. coli 536/21 which differed only in their types of adhesins (MSH+ MS-Fim+; S-MRH+ S-Fim+; P-MRH+ P-Fim+) released neither serotonin nor 12-HETE from human platelets nor induced platelet aggregation. All hemolysin-negative bacteria except E. coli 536/21, without any adhesins, were able to activate protein kinase C reversibly but did not induce calcium influx. Activation of platelets with fluoride, an activator of the GTP-binding protein, was associated with protein kinase C activation, calcium influx, platelet aggregation, serotonin release, and 12-HETE formation. The simultaneous stimulation of platelets with NaF and the nonhemolytic E. coli strains suppressed several of the NaF-induced platelet responses. Membrane preparations isolated from stimulated platelets with hemolysin-negative and hemolysin-positive E. coli showed increased binding of guanylylimidodiphosphate, a nonhydrolyzable GTP analog, and enhanced GTPase activity.

Pyelonephritogenic Escherichia coli and killing of cultured human renal proximal tubular epithelial cells: role of hemolysin in some strains

Acute pyelonephritis, a complication of Escherichia coli bacteriuria, must represent a bacterial invasion through the kidney epithelium. To study this process, we overlaid bacterial suspensions onto monolayers of cultured human kidney proximal tubular epithelial cells and measured cytotoxicity by release of lactate dehydrogenase (LDH). Thirty-four isolates cultured from patients with acute pyelonephritis were screened for the ability to cause pyelonephritis in CBA mice by transurethral challenge. The eight most virulent strains (greater than or equal to 70% of mice challenged developed greater than or equal to 10(3) CFU/g of kidney after 48 h) were selected for study. Each strain displayed mannose-resistant hemagglutination of human O erythrocytes; three strains were phenotypically and genotypically hemolytic. Pyelonephritogenic strains were significantly more cytotoxic (30.1 +/- 9.5% LDH release after 18 h) than eight fecal control strains (13.5 +/- 11.5% LDH release; P = 0.0068). We selected the most cytotoxic strain, CFT073, for further study. Sterile filtrate from this hemolytic strain was significantly more cytotoxic than was the filtrate of the fecal control strain, FN414. Transposon mutagenesis of CFT073 with TnphoA abolished hemolytic activity and cytotoxicity by both whole cells and sterile filtrate. Southern blot analysis revealed that the Tnphoa insertion mapped to the E. coli chromosomal hly determinant within a 12-kilobase SalI restriction fragment. Transformation of a nonhemolytic strain, CPZ005 with plasmid pSF4000, which carries a cloned hemolysin determinant, resulted in highly elevated cytotoxicity. Light micrographs of proximal tubular epithelial cell cultures demonstrated cell damage by pyelonephritogenic strains that was not induced by a fecal strain or the hemolysin-deficient mutant. Results indicate that pyelonephritogenic E. coli strains are more frequently cytotoxic for a putative target, that is, human renal tubular epithelium, than are fecal isolates. Hemolysin, in some strains, is apparently responsible for this cytotoxicity.

Comparative characterization of the leukocidic and hemolytic activity of Moraxella bovis

SUMMARY The cytotoxic effect of Moraxella bovis 118F on bovine neutrophils was evaluated and characterized by use of a 51Cr release assay. Neutrophils harvested from healthy adult cattle were labeled with 51Cr. The leukocidic activity produced by M bovis 118F, a hemolytic strain of M bovis, was heat-labile. A live culture of strain 118F, at a ratio of 100 bacteria/neutrophil, released 97.7% of the 51Cr from labeled neutrophils. Neither a heat-killed preparation of M bovis 118F nor a live or heat-killed preparation of M bovis IBH63 (a nonhemolytic and nonpathogenic strain) induced significant (P > 0.05) release of 51Cr. Moraxella bovis 118F broth culture filtrates prepared for evaluation of leukocidic activity also were evaluated for hemolytic activity. These 2 toxic activities had several characteristics in common. Both were filterable, heat-labile, produced by a hemolytic strain, and were released during early logarithmic phase growth from broth cultures. Leukocidic and hemolytic activities were protected from degradation by phenylmethyl sulfonyl fluoride, a serine protease inhibitor. Leukocidic and hemolytic activities were dependent on calcium ions. Filtrate resulted in 54.1% 51Cr release from labeled neutrophils and contained 646.7 hemolytic U/ml, respectively, when saline (0.85% NaCl) + 10 mM CaCl2 solution was used as diluent. Neither saline solution nor saline + 10 mM MgCl2 solution supported leukocidic or hemolytic activity. Serum, obtained from several calves 10 to 38 days after M bovis inoculation, substantially neutralized leukocidic and hemolytic activities, compared with paired preinoculation serum samples. In addition, no significant difference (P > 0.05) was detected when the ability of each calf's postinfection serum to neutralize leukocidic activity was compared with the ability of the serum to neutralize hemolytic activity.

Role of α-Hemolysin for the in vitro Phagocytosis and Intracellular Killing of Escherichia coli

The role of α-hemolysin for the elimination of Escherichia coli by phagocytes in vitro was investigated using sets of isogenic strains which included wild-type α-hemolytic strains, derived strains with a reduced production of α-hemolysin and derived nonhemolytic strains. Phagocytosis and intracellular killing of the bacteria by human blood granulocytes or monocytes were measured using growth inhibition techniques. α-hemolytic strains were phagocytosed and killed to a lesser extent than isogenic strains with a reduced production of α-hemolysin and isogenic nonhemolytic strains. The results obtained with granulocytes were similar to those obtained with monocytes although the elimination of bacteria by monocytes was less than that by granulocytes. These results strongly suggest that production of α-hemolysin is a means by which E. coli counteracts the activity of phagocytes by injuring these cells with the toxin.

Chemotactic response of equine polymorphonuclear leucocytes to Streptococcus equi

Streptococcus equi infection in horses is characterised by intense infiltration of lymph nodes by polymorphonuclear leucocytes (PMNs) suggesting a potent chemotactic response to the organism or its products. Equine PMNs were separated using Ficoll-Hypaque medium and used in an assay of chemotaxis under agarose to study the components of S equi involved in this response. Results showed that complement-derived chemotactic factors generated by activation of the alternative complement pathway were important in chemotactic responses to S equi. Both whole bacteria and peptidoglycan preparations were potent complement activators, whereas purified M protein was less active. In contrast, S equi culture supernatant protein did not activate complement; instead it directly inhibited migration of PMNs. Moreover, PMNs, when incubated with culture supernatant of a non-haemolytic strain, showed signs of cellular degeneration suggesting the presence of a cytotoxin distinct from haemolysin.

Extracellular proteins of Vibrio cholerae: nucleotide sequence of the structural gene (hlyA) for the haemolysin of the haemolytic El Tor strain 017 and characterization of the hlyA mutation in the non-haemolytic classical strain 569B.

The EI T or haemolysin, product of hlyA, is exported from Vibrio cholerae as a Mr 80,000 protein which can be subsequently cleaved to give two proteins of Mr 65,000 and 15,000. Nucleotide sequence analysis has demonstrated that hlyA encodes a protein of Mr 82,250 with a potential 18-amino-acid signal sequence. The non-haemolytic classical strain 569B has been shown to have a structural gene defect rather than a defect in secretion. By non-reciprocal recombination it was possible to transfer this defect onto a plasmid and show that a truncated hlyA product of Mr 27,000 is made in Escherichia coli K-12 minicells. Nucleotide sequence analysis demonstrates an 11-base-pair deletion which would result in a Mr 26,940 protein probably loosely associated with the membrane.

Nucleotide sequences and comparison of the hemolysin determinants of Vibrio cholerae El Tor RV79(Hly+) and RV79(Hly-) and classical 569B(Hly-)

We determined the nucleotide sequences of the hemolysin structural gene, hlyA, of Vibrio cholerae El Tor biotype strains RV79(Hly+) and RV79(Hly-) and the hly determinant of the nonhemolytic classical biotype strain 569B(Hly-). The sequences of the hlyA gene from El Tor strains RV79(Hly+) and RV79(Hly-) have an identical 2,223-base open reading frame which is predicted to encode an 81,977-dalton precursor form of hemolysin. This value is in excellent agreement with the 84,000-Mr hemolysin described in the earlier report of Goldberg and Murphy (S. L. Goldberg and J. R. Murphy, J. Bacteriol. 162:35-41, 1985). In contrast, the sequence of the hly determinant of the classical 569B(Hly-) strain has an 11-base-pair deletion within the hlyA structural gene. In this instance the hly determinant is predicted to encode a 26,765-dalton precursor form of a truncated hemolysin. In each case, the regulatory region encoding the putative hlyA promoter and the predicted 25-amino-acid signal sequence are identical.

Positive reaction in mouse ligated intestinal loop assay with nonenterotoxigenic and nonhemolytic strains of Staphylococcus aureus.

Nonenterotoxigenic and nonhemolytic strains of Staphylococcus aureus exhibited a positive reaction in the mouse intestinal loop assay except for a strain negative for the egg yolk reaction. Edema and swelling in all positive loops, increased bacterial cell numbers within intestinal loops, and extremely close adhesion to or inclusion of numerous bacterial cells in HeLa cells after inoculation of the strains were observed. These results suggest a possible invasive ability of the strains.

Hydrolytic enzymes of "Streptococcus milleri".

Seventy-two isolates classified as "Streptococcus milleri" were examined for the presence of various hydrolytic enzymes. While no protein or lipid-degrading activities were demonstrated, some isolates showed DNase and mucopolysaccharide-degrading activities. Beta-hemolytic isolates were more likely to produce these enzymes than were nonhemolytic strains. Isolates of one "S. milleri" biotype (mannitol fermentation positive) were uniformly devoid of all enzyme activities tested.

High incidence of hemolysin production by Enterococcus (Streptococcus) faecalis strains associated with human parenteral infections

Hemolysin production, clumping (pheromone) response, transferability of the hemolytic trait, and drug resistance were examined in 97 clinical isolates of Enterococcus (Streptococcus) faecalis. The isolates were derived from various sources (i.e., urine, pus, vagina, sputum, bile, and blood), and approximately 60% were found to be hemolytic. About 85% of the hemolytic strains exhibited a clumping response, compared with about 49% of the nonhemolytic strains. Over 50% of the hemolytic strains carried transferable hemolysin determinants, and in no case were drug resistance genes linked. The hemolytic strains exhibited multiple drug resistance more frequently than did the nonhemolytic strains. In contrast to the high frequency of hemolysin producers among parenteral isolates, strains derived from fecal specimens of healthy individuals exhibited a low (17%) incidence of hemolysin production.

Cytotoxic necrotizing factor production by hemolytic strains of Escherichia coli causing extraintestinal infections.

Two hundred and nineteen strains of Escherichia coli from extraintestinal infections and feces of healthy subjects were examined for hemolysin (Hly) and cytotoxic necrotizing factor (CNF) production and for mannose-resistant hemagglutination. Of 105 strains from extraintestinal infections, 42 (40.0%) were positive for production of both Hly and CNF, and 21 (20.0%) were positive for Hly alone; on the contrary, only 1 Hly- and CNF-positive strain and 2 Hly-positive strains were found among 114 strains from normal stools. CNF production was not found to occur among the nonhemolytic strains, confirming the close association existing between these toxic factors. Hemolytic strains positive for CNF showed mannose-resistant hemagglutination less frequently than did Hly-positive, CNF-negative strains (25.6 versus 82.6%), suggesting the existence of two distinct classes among hemolytic strains of E. coli.

Microplate technique to determine hemolytic activity for routine typing of Listeria strains.

Because the hemolysis produced by Listeria monocytogenes and Listeria seeligeri on blood agar is frequently difficult to interpret, we developed a microplate technique for the routine determination of hemolytic activity with erythrocyte suspensions. This microtechnique is a simple and reliable test for distinguishing clearly between hemolytic and nonhemolytic strains and could be used instead of the CAMP (Christie-Atkins-Munch-Petersen) test with Staphylococcus aureus in the routine typing of Listeria strains. Furthermore, our results suggest that the quantitation of the hemolytic activity of the Listeria strains, along with the D-xylose, L-rhamnose, and alpha-methyl-D-mannoside acidification tests, allows the differentiation of L. monocytogenes, L. seeligeri, and Listeria ivanovii. We also observed that the treatment of erythrocytes with crude exosubstances of rhodococcus equi, Pseudomonas fluorescens, Acinetobacter calcoaceticus, and S. aureus enhanced the hemolytic activity of all Listeria strains with this characteristic.

Cloning and characterization of the hemolysin determinants from Vibrio cholerae RV79(Hly+), RV79(Hly-), and 569B.

The Hly region from the chromosome of Vibrio cholerae El Tor strain RV79(Hly-) and the nonhemolytic classical strain 569B were cloned into plasmid vector pBR322. Escherichia coli K-12 transformants possessing these recombinant plasmids were nonhemolytic and were detected with a 32P-labeled hly-specific DNA probe. Restriction endonuclease Sau3AI digestions of the cloned hly loci of two independently obtained RV79(Hly+) convertants, when compared with the digests of cloned RV79(Hly-) loci, revealed that an apparent alteration (10 to 15 base pairs) had occurred. In contrast, an apparent 20-base-pair deletion was present in the cloned hly locus of the classical biotype V. cholerae strain 569B. Maxicell analysis and immunoprecipitation of labeled proteins of E. coli which are encoded by the cloned hly loci of RV79(Hly+) and from nuclease BAL 31-deleted plasmids, as well as immunoprecipitation of [35S]methionine-labeled V. cholerae proteins, suggest that the hemolysin is an 84,000-dalton polypeptide.

A role for hemolysin in Escherichia coli-induced inflammation in granulocytopenic rabbits.

Inflammation induced in the skin of granulocytopenic rabbits by Escherichia coli was examined. Protein exudation and platelet deposition in lesions were measured with 125I-labeled albumin and 111In-labeled platelets. In granulocytopenic rabbits 10(4)-10(7) live serum-resistant E. coli induced protein exudation and platelet deposition beginning at 3 hr and then progressing over the next 24 hr to much higher levels than in normal rabbits. These responses were associated with interstitial edema and progressive venous thrombosis in the absence of leukocytes; no such reactions were observed in normal rabbits. No reactions were induced in granulocytopenic rabbits by killed E. coli. Of six E. coli strains tested, all three hemolytic strains induced lesions with four to five times more thrombosis (platelet deposition) than did nonhemolytic strains. Two hemolysin-negative mutants lost most of their thrombogenic activity. All three hemolytic strains had cell-associated hemolysin, but only one of these elaborated appreciable free, filterable hemolysin as well.

In vitro cytotoxic effect of alpha-hemolytic Escherichia coli on human blood granulocytes.

The cytotoxic effect of Escherichia coli bacteria on human blood granulocytes was measured by recording numbers of nonlysed cells and percentages of viable cells after in vitro incubation with bacteria in the presence of plasma. A total of 179 strains from various sources of infection were tested. Of 117 alpha-hemolytic strains, 59 were cytotoxic. Five nonhemolytic mutant strains, derived from alpha-hemolytic cytotoxic strains, were nontoxic. None of the 62 nonhemolytic strains were toxic. Four spontaneously occurring alpha-hemolytic, nontoxic mutant strains were isolated from cytotoxic ones. Cytotoxicity of bacteria reached a maximum after log-phase growth at 30 to 37 degrees C for 2.5 h, and the toxic capacity was equal after growth in various media, including human urine and plasma. The cytotoxic effect increased with the length of exposure of granulocytes to bacteria and with increasing numbers of bacteria per granulocyte. Cytotoxic strains showed different degrees of toxicity, highly cytotoxic strains lysing about 90% of the granulocytes and killing about one-half of nonlysed cells in 1 h. Bacteria killed by heat, formaldehyde, or UV light were nontoxic. Alpha-hemolytic strains of O groups 2, 4, 6, 25, and 75 originating from various infections in humans were more frequently cytotoxic than alpha-hemolytic strains of other O groups derived from human infections. Culture supernatants containing free alpha-hemolysin were highly cytotoxic to human blood granulocytes, monocytes, and lymphocytes in vitro, whether supernatants originated from cytotoxic or noncytotoxic bacteria. Cytotoxicity to phagocytes, which is mediated by or closely linked genetically to alpha-hemolysin, may be a mechanism by which alpha-hemolytic strains of E. coli strengthen their ability to establish and maintain infections.

Characterization of Escherichia coli hemolysins conferring quantitative differences in virulence.

Recombinant plasmids encoding hemolysins (hly) isolated from four different Escherichia coli strains were found to be very similar by restriction endonuclease fragment analysis within the hemolysin region. Each of the four recombinant plasmids were used to transform a nonhemolytic fecal strain of E. coli. The comparative virulence of the transformants was tested in a rat model of peritonitis. Despite the physical similarity among the hemolysin recombinant plasmids, each conferred different normally avirulent fecal E. coli strain. Reciprocal exchange of similar restriction endonuclease fragments enabled the construction of hybrid hemolysin determinants with two hemolysins of disparate extracellular hemolysin production and relative virulence levels. The hybrid plasmids were introduced into the standard avirulent fecal strain and tested in the rat peritonitis model. The region conferring quantitative differences in extracellular hemolysin production and virulence was found to be within a region of less than 1 kilobase where the hly c cistron is encoded as well as the probable transcription initiation ares for the entire hemolysin operon. A 750-base pair (bp) AvaI fragment from this region was isolated from a virulent hemolysin recombinant and inserted into a common AvaI site present in an avirulent hemolysin plasmid. This insertion resulted in an increase in the amount of extracellular hemolysin activity and the associated virulence when in the fecal E. coli background.