Occurrence of hlyA and sheA Genes in Extraintestinal Escherichia coli Strains
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微生物临床杂志 2005年第6期
Departments of Medical Microbiology and Immunology
Anesthesiology and Intensive Therapy, Medical School, University of Pecs, Hungary
School of Biological Sciences, Division of Microbiology and Genomics, University of Liverpool, Liverpool, United Kingdom
Department of Medical Microbiology, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al-Ain 17666, United Arab Emirates
ABSTRACT
The association of a hemolytic phenotype with the carriage of the -hemolysin gene (hlyA) and/or the silent hemolysin gene (sheA or clyA) among 540 extraintestinal clinical isolates of Escherichia coli and 110 fecal isolates from healthy individuals was investigated. Though HlyA is an important virulence factor in extraintestinal E. coli infection, the role of SheA is not completely clarified. Two hemolytic sheA+ E. coli strains that lacked hlyA and possessed no other hemolysin genes were identified. No hlyA+ sheA+ strains were identified, suggesting that there is possible incompatibility between hlyA and sheA in the chromosome of E. coli.
TEXT
Escherichia coli strains that cause extraintestinal infections possess numerous virulence factors, including hemolysin production. E. coli -hemolysin (HlyA) produces large, clear zones of hemolysis around colonies on blood agar. The hemolysin is present in cell-free filtrates and is the best characterized member of the RTX (repeat in toxin) toxin family (25). HlyA lyses cells by the creation of pores in the target cell membrane and affects erythrocytes, leukocytes (4, 5), and renal tubular cells (12). Its activity on polymorphonuclear granulocytes liberates leukotrienes, histamine, and ATP (13) and is neutralized by specific antiserum.
Cytolysin A or "silent hemolysin" (SheA) causes hemolysis when its gene, sheA (also known as clyA or hlyE), is present on high-copy-number plasmids; when certain regulator genes, mprA and slyA, are overexpressed (8); or when the transcription of the chromosomal sheA is derepressed (i.e., in hns mutant E. coli strains) (26). The activities of these two E. coli hemolysins differ (Table 1) (1). It has been proven that SheA is cytotoxic to human and mouse macrophages and is capable of inducing apoptosis, and so SheA has been implicated as a factor contributing to the pathogenicity of some E. coli strains (14).
The sheA gene is present in certain nonpathogenic E. coli strains (K-12), in the pathogenic Shiga toxin-producing E. coli strain O157:H7 (9), and in other enteropathogenic (enteroinvasive, enteroaggregative, and enterotoxigenic) E. coli strains (16), but its presence in E. coli strains associated with extraintestinal infections has not been well studied. We examined the distribution of sheA and hlyA genes among 540 E. coli strains isolated from human extraintestinal infections (isolated in the diagnostic laboratory at the Department of Medical Microbiology, University of Pecs, from urinary, genital, and respiratory tract infections and at the University Teaching Hospitals from bloodstream and abdominal wound infections during 2001-2002); 110 strains isolated from the feces of different healthy subjects, aged 20 to 55 years, who had received no antibiotics in the previous 6 months or suffered urinary tract infections (including asymptomatic bacteriuria) or diarrheagenic disease within the previous 6 months; the 72-member ECOR collection (18); and 5 laboratory K-12 strains, namely, HB101 (22), MC4100 (24), J53 (7), DH5- (27), and XL1-Blue (6).
The presence or absence of a hemolytic phenotype was determined for all strains following growth on Columbia blood agar plates containing 5% (wt/vol) defibrinated ox erythrocytes following incubation for 24 h at 37°C. Using the primer sets described in Table 2, hemolysin genes (sheA, hlyA, ehx, and ehly) were detected by PCR. Briefly, a single bacterial colony was suspended in 100 μl sterile ultrapure water, and 1 μl of this suspension was added to a 10-μl reaction mixture (REDTaq [3 U Taq DNA polymerase, 1.5 mM MgCl2, 200 μM dNTP] and 40 pmol of each primer]; Sigma). The template was denatured for 2 min at 94°C followed by 30 cycles of denaturation at 94°C for 60 s, annealing at 52°C (sheA and ehx), 58°C (hlyA), or 61°C (ehly) for 60 s, and extension at 72°C for 60 s. All products were analyzed by 1% agarose gel electrophoresis. The strains DH5- (K-12) carrying pCFP60 (9), J96 (O4) (10), and EDL933 (O157:H7) (21) served as positive controls for the presence of sheA, hlyA, and phage-associated enterohemolysin (ehly) and plasmid-borne enterohemolysin (ehx) genes, respectively. The results are given in Table 3. No E. coli K-12 strains tested carried the hlyA gene. Nine members of the ECOR collection carried the hlyA gene, supporting results of a previous study (15), while 198 extraintestinal clinical isolates and only 8 normal fecal isolates carried hlyA. The sheA gene was identified in all tested K-12 strains, 55 of the ECOR strains, 241 of the extraintestinal clinical isolates, and 94 of the fecal isolates from healthy individuals (Table 3). Of the hlyA+ strains from the ECOR group (nine strains) and the fecal isolates (eight strains), two strains from each group did not possess hemolytic activity. Of the 198 hlyA+ strains from extraintestinal infections, 26 strains were nonhemolytic, as were 2 members of the ECOR group and 2 of the fecal isolates. We identified two strains from the extraintestinal infection group that lacked hlyA, ehx (enterohemolysin associated with some virulent E. coli strains) (3), and ehly but possessed sheA and were hemolytic.
The most common extraintestinal infections caused by E. coli are urinary tract infections. The -hemolysin is present in about 25 to 56% of isolated strains from urinary tract infections (17, 19), similar to our rate of 35%. The presence of the sheA gene in normal fecal strains is higher (85.4%; P < 0.001) than in the isolates from urinary tract infection (47.1%). In our study, the occurrence of hlyA gene in normal fecal isolates was 7.3%, similar to that previously reported (11). Though it is accepted that the -hemolysin is an important virulence factor to the pathogenic profile of E. coli, the role played by the silent hemolysin SheA in disease is unknown.
This study focused on correlating the hemolytic phenotype of extraintestinal E. coli isolates with the presence of hlyA and sheA genes. The absence of a hemolytic phenotype in the presence of hlyA has been well characterized and can be due to defects in the hlyBCD genes or to defects in the transcriptional activator rfaH (2). We detected 174 hemolytic E. coli strains out of our pool of 540 extraintestinal clinical isolates. Two of these isolates did not possess the hlyA gene and also lacked the other common E. coli hemolysin, enterohemolysin (ehx). However, these two strains did possess the sheA gene (Table 3). Oscarsson et al. reported that the bacteriophage-associated Ehly determinant does not encode enterohemolysins but causes the release of SheA or ClyA cytolysin (20). Although this ehly was not detected, it may be that sheA is responsible for the hemolytic phenotype for these two strains. The observed hemolytic phenotype was identical to the HlyA-mediated phenotype. Further studies are required to determine if this hemolytic phenotype is due to SheA and the role SheA plays in the pathogenic profile of these strains. The sheA gene was deleted in the 14 uropathogenic E. coli (UPEC) strains examined by Ludwig et al. in a recent study (16). Their 14 UPEC strains, including the well-known hlyA+ J96 and 536 strains, were assayed for the presence of an hlyA fragment by Southern blot hybridization, but it was identified in only 10 of the 14 strains (28, 29). In our study, we found that 47.1% of the 486 UPEC strains contained the sheA gene (Table 3). The smaller number of UPEC strains in the first two studies may explain the differences in sheA gene carriage. The surprising finding of this study was that out of the 727 E. coli strains tested, 610 possessed either the hlyA or the sheA gene and none carried both of them. The complete absence of hlyA+ sheA+ individuals within a sample of 540 individuals is sufficiently compelling to make additional statistical confirmation redundant (however, if we look at the subset of individuals with one of these two genes and calculate the expected proportion of hlyA+, sheA+, and hlyA+ sheA+ individuals that would exist by random assortment, the probability that the actual frequencies match this null hypothesis is vanishingly small [P < 0.0001; chi square = 439]). The sheA gene in Shiga toxin-producing E. coli (enterohemorrhagic E. coli) can be found in ehx gene-positive isolates (9, 16), even though the plasmid-encoded ehx gene exhibits 60 to 65% sequence homology to hlyA (23). The reasons for the mutual exclusivity between sheA and hlyA in the chromosome found in this study are not understood. It might be that the complex mechanisms regulating expression of hlyA and sheA interfere with each other, posing a selective pressure against their coexistence in the same cell. Further studies will shed light on these questions.
ACKNOWLEDGMENTS
We thank Gyula Mestyán and Anita Novák for their help in collecting the clinical isolates, Gabor Nagy for the hlyA primers, and Mike Speed (University of Liverpool) for his help with statistical analyses.
This work was supported by grant OTKA T037833 from the Hungarian Science Foundation and a Bolyai scholarship from the Hungarian Academy of Sciences.
REFERENCES
Atkins, A., N. R. Wyborn, A. J. Wallace, T. J. Stillman, L. K. Black, A. B. Fielding, M. Hisakado, P. J. Artymiuk, and J. Green. 2000. Structure-function relationships of a novel bacterial toxin, hemolysin E. The role of alpha G. J. Biol. Chem. 275:41150-41155.
Bailey, M. J., V. Koronakis, T. Schmoll, and C. Hughes. 1992. Escherichia coli HlyT protein, a transcriptional activator of haemolysin synthesis and secretion, is encoded by the rfaH (sfrB) locus required for expression of sex factor and lipopolysaccharide genes. Mol. Microbiol. 6:1003-1012.
Beutin, L., J. Prada, S. Zimmermann, R. Stephan, I. Orskov, and F. Orskov. 1988. Enterohemolysin, a new type of hemolysin produced by some strains of enteropathogenic E. coli (EPEC). Zentbl. Bakteriol. Mikrobiol. Hyg. A 267:576-588.
Bhakdi, S., S. Greulich, M. Muhly, B. Eberspacher, H. Becker, A. Thiele, and F. Hugo. 1989. Potent leukocidal action of Escherichia coli hemolysin mediated by permeabilization of target cell membranes. J. Exp. Med. 169:737-754.
Bhakdi, S., M. Muhly, S. Korom, and G. Schmidt. 1990. Effects of Escherichia coli hemolysin on human monocytes. Cytocidal action and stimulation of interleukin 1 release. J. Clin. Investig. 85:1746-1753.
Bullock, W. O., J. M. Fernandez, and J. M. Short. 1987. Xl1-Blue: a high efficiency plasmid transforming recA Escherichia coli strain with beta-galactosidase selection. BioTechniques 5:376-378.
Clowes, R., and D. Rowley. 1954. Some observations on linkage effects in genetic recombination in Escherichia coli K-12. J. Gen. Microbiol. 11:250-260.
del Castillo, F. J., S. C. Leal, F. Moreno, and I. del Castillo. 1997. The Escherichia coli K-12 sheA gene encodes a 34-kDa secreted haemolysin. Mol. Microbiol. 25:107-115.
del Castillo, F. J., F. Moreno, and I. del Castillo. 2000. Characterization of the genes encoding the SheA haemolysin in Escherichia coli O157:H7 and Shigella flexneri 2a. Res. Microbiol. 151:229-230.
Hull, R. A., R. E. Gill, P. Hsu, B. H. Minshew, and S. Falkow. 1981. Construction and expression of recombinant plasmids encoding type 1 or D-mannose-resistant pili from a urinary tract infection Escherichia coli isolate. Infect. Immun. 33:933-938.
Johnson, J. R. 1991. Virulence factor in Escherichia coli urinary tract infection. Clin. Microbiol. Rev. 4:80-128.
Keane, W. F., R. Welch, G. Gekker, and P. K. Peterson. 1987. Mechanism of Escherichia coli alpha-hemolysin-induced injury to isolated renal tubular cells. Am. J. Pathol. 126:350-357.
Konig, B., A. Ludwig, W. Goebel, and W. Konig. 1994. Pore formation by the Escherichia coli alpha-hemolysin: role for mediator release from human inflammatory cells. Infect. Immun. 62:4611-4617.
Lai, X. H., I. Arencibia, A. Johansson, S. N. Wai, J. Oscarsson, S. Kalfas, K. G. Sundqvist, Y. Mizunoe, A. Sjostedt, and B. E. Uhlin. 2000. Cytocidal and apoptotic effects of the ClyA protein from Escherichia coli on primary and cultured monocytes and macrophages. Infect. Immun. 68:4363-4367.
Lai, X. H., S. Y. Wang, and B. E. Uhlin. 1999. Expression of cytotoxicity by potential pathogens in the standard Escherichia coli collection of reference (ECOR) strains. Microbiology 145:3295-3303.
Ludwig, A., C. von Rhein, S. Bauer, C. Hüttinger, and W. Goebel. 2004. Molecular analyses of cytolysin A (ClyA) in pathogenic Escherichia coli strains. J. Bacteriol. 186:5311-5320.
Minshew, B. H., J. Jorgensen, M. Swanstrum, G. A. Grootes-Reuvecamp, and S. Falkow. 1978. Some characteristics of Escherichia coli strains isolated from extraintestinal infections of humans. J. Infect. Dis. 137:648-654.
Ochman, H., and R. K. Selander. 1984. Standard reference strains of Escherichia coli from natural populations. J. Bacteriol. 157:690-693.
Opal, S. M., A. S. Cross, P. Gemski, and L. W. Lyhte. 1990. Aerobactin and alpha-hemolysin as virulence determinants in Escherichia coli isolated from human blood, urine, and stool. J. Infect. Dis. 161:794-796.
Oscarsson, J., M. Westermark, L. Beutin, and B. E. Uhlin. 2002. The bacteriophage-associated Ehly1 and Ehly2 determinants from Escherichia coli O26:H– strains do not encode enterohemolysins per se but cause release of the ClyA cytolysin. Int. J. Med. Microbiol. 291:625-631.
Perna, N. T., G. Plunkett III, V. Burland, B. Mau, J. D. Glasner, D. J. Rose, G. F. Mayhew, P. S. Evans, J. Gregor, H. A. Kirkpatrick, G. Posfai, J. Hackett, S. Klink, A. Boutin, Y. Shao, L. Miller, E. J. Grotbeck, N. W. Davis, A. Lim, E. T. Dimalanta, K. D. Potamousis, J. Apodaca, T. S. Anantharaman, J. Lin, G. Yen, D. C. Schwartz, R. A. Welch, and F. R. Blattner. 2001. Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature 409:529-533.
Sambrook, J. E., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Schmidt, H., L. Beutin, and H. Karch. 1995. Molecular analyses of the plasmid-encoded hemolysin of Escherichia coli O157:H7 strain EDL 933. Infect. Immun. 63:1055-1061.
Silhavy, T. J., M. L. Berman, L. W. Enquist, and Cold Spring Harbor Laboratory. 1984. Experiments with gene fusions. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Welch, R. A., C. Forestier, A. Lobo, S. Pellett, W. Thomas, Jr., and G. Rowe. 1992. The synthesis and function of the Escherichia coli hemolysin and related RTX exotoxins. FEMS Microbiol. Immunol 5:29-36.
Westermark, M., J. Oscarsson, Y. Mizunoe, J. Urbonaviciene, and B. E. Uhlin. 2000. Silencing and activation of ClyA cytotoxin expression in Escherichia coli. J. Bacteriol. 182:6347-6357.
Woodcock, D. M., P. J. Crowther, J. Doherty, S. Jefferson, E. DeCruz, M. Noyer-Weidner, S. S. Smith, M. Z. Michael, and M. W. Graham. 1989. Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants. Nucleic Acids Res. 17:3469-3478.
Zingler, G., M. Ott., G. Blum, U. Falkenhagen, G. Naumann, W. Sokolowska-Khler, and J. Hacker. 1992. Clonal analysis of Escherichia coli serotype O6 from urinary tract infection. Microb. Pathog. 12:299-311.
Zingler, G., G. Blum, U. Falkenhagen, I. Orskov, F. Orskov, J. Hacker, and M. Ott. 1993. Clonal differentiation of uropathogenic Escherichia coli isolates of serotype O6:K5 by fimbrial antigen typing and long-range mapping techniques. Med. Microbiol. Immunol. 182:13-24.(Monika Kerenyi, Heather E)
Anesthesiology and Intensive Therapy, Medical School, University of Pecs, Hungary
School of Biological Sciences, Division of Microbiology and Genomics, University of Liverpool, Liverpool, United Kingdom
Department of Medical Microbiology, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al-Ain 17666, United Arab Emirates
ABSTRACT
The association of a hemolytic phenotype with the carriage of the -hemolysin gene (hlyA) and/or the silent hemolysin gene (sheA or clyA) among 540 extraintestinal clinical isolates of Escherichia coli and 110 fecal isolates from healthy individuals was investigated. Though HlyA is an important virulence factor in extraintestinal E. coli infection, the role of SheA is not completely clarified. Two hemolytic sheA+ E. coli strains that lacked hlyA and possessed no other hemolysin genes were identified. No hlyA+ sheA+ strains were identified, suggesting that there is possible incompatibility between hlyA and sheA in the chromosome of E. coli.
TEXT
Escherichia coli strains that cause extraintestinal infections possess numerous virulence factors, including hemolysin production. E. coli -hemolysin (HlyA) produces large, clear zones of hemolysis around colonies on blood agar. The hemolysin is present in cell-free filtrates and is the best characterized member of the RTX (repeat in toxin) toxin family (25). HlyA lyses cells by the creation of pores in the target cell membrane and affects erythrocytes, leukocytes (4, 5), and renal tubular cells (12). Its activity on polymorphonuclear granulocytes liberates leukotrienes, histamine, and ATP (13) and is neutralized by specific antiserum.
Cytolysin A or "silent hemolysin" (SheA) causes hemolysis when its gene, sheA (also known as clyA or hlyE), is present on high-copy-number plasmids; when certain regulator genes, mprA and slyA, are overexpressed (8); or when the transcription of the chromosomal sheA is derepressed (i.e., in hns mutant E. coli strains) (26). The activities of these two E. coli hemolysins differ (Table 1) (1). It has been proven that SheA is cytotoxic to human and mouse macrophages and is capable of inducing apoptosis, and so SheA has been implicated as a factor contributing to the pathogenicity of some E. coli strains (14).
The sheA gene is present in certain nonpathogenic E. coli strains (K-12), in the pathogenic Shiga toxin-producing E. coli strain O157:H7 (9), and in other enteropathogenic (enteroinvasive, enteroaggregative, and enterotoxigenic) E. coli strains (16), but its presence in E. coli strains associated with extraintestinal infections has not been well studied. We examined the distribution of sheA and hlyA genes among 540 E. coli strains isolated from human extraintestinal infections (isolated in the diagnostic laboratory at the Department of Medical Microbiology, University of Pecs, from urinary, genital, and respiratory tract infections and at the University Teaching Hospitals from bloodstream and abdominal wound infections during 2001-2002); 110 strains isolated from the feces of different healthy subjects, aged 20 to 55 years, who had received no antibiotics in the previous 6 months or suffered urinary tract infections (including asymptomatic bacteriuria) or diarrheagenic disease within the previous 6 months; the 72-member ECOR collection (18); and 5 laboratory K-12 strains, namely, HB101 (22), MC4100 (24), J53 (7), DH5- (27), and XL1-Blue (6).
The presence or absence of a hemolytic phenotype was determined for all strains following growth on Columbia blood agar plates containing 5% (wt/vol) defibrinated ox erythrocytes following incubation for 24 h at 37°C. Using the primer sets described in Table 2, hemolysin genes (sheA, hlyA, ehx, and ehly) were detected by PCR. Briefly, a single bacterial colony was suspended in 100 μl sterile ultrapure water, and 1 μl of this suspension was added to a 10-μl reaction mixture (REDTaq [3 U Taq DNA polymerase, 1.5 mM MgCl2, 200 μM dNTP] and 40 pmol of each primer]; Sigma). The template was denatured for 2 min at 94°C followed by 30 cycles of denaturation at 94°C for 60 s, annealing at 52°C (sheA and ehx), 58°C (hlyA), or 61°C (ehly) for 60 s, and extension at 72°C for 60 s. All products were analyzed by 1% agarose gel electrophoresis. The strains DH5- (K-12) carrying pCFP60 (9), J96 (O4) (10), and EDL933 (O157:H7) (21) served as positive controls for the presence of sheA, hlyA, and phage-associated enterohemolysin (ehly) and plasmid-borne enterohemolysin (ehx) genes, respectively. The results are given in Table 3. No E. coli K-12 strains tested carried the hlyA gene. Nine members of the ECOR collection carried the hlyA gene, supporting results of a previous study (15), while 198 extraintestinal clinical isolates and only 8 normal fecal isolates carried hlyA. The sheA gene was identified in all tested K-12 strains, 55 of the ECOR strains, 241 of the extraintestinal clinical isolates, and 94 of the fecal isolates from healthy individuals (Table 3). Of the hlyA+ strains from the ECOR group (nine strains) and the fecal isolates (eight strains), two strains from each group did not possess hemolytic activity. Of the 198 hlyA+ strains from extraintestinal infections, 26 strains were nonhemolytic, as were 2 members of the ECOR group and 2 of the fecal isolates. We identified two strains from the extraintestinal infection group that lacked hlyA, ehx (enterohemolysin associated with some virulent E. coli strains) (3), and ehly but possessed sheA and were hemolytic.
The most common extraintestinal infections caused by E. coli are urinary tract infections. The -hemolysin is present in about 25 to 56% of isolated strains from urinary tract infections (17, 19), similar to our rate of 35%. The presence of the sheA gene in normal fecal strains is higher (85.4%; P < 0.001) than in the isolates from urinary tract infection (47.1%). In our study, the occurrence of hlyA gene in normal fecal isolates was 7.3%, similar to that previously reported (11). Though it is accepted that the -hemolysin is an important virulence factor to the pathogenic profile of E. coli, the role played by the silent hemolysin SheA in disease is unknown.
This study focused on correlating the hemolytic phenotype of extraintestinal E. coli isolates with the presence of hlyA and sheA genes. The absence of a hemolytic phenotype in the presence of hlyA has been well characterized and can be due to defects in the hlyBCD genes or to defects in the transcriptional activator rfaH (2). We detected 174 hemolytic E. coli strains out of our pool of 540 extraintestinal clinical isolates. Two of these isolates did not possess the hlyA gene and also lacked the other common E. coli hemolysin, enterohemolysin (ehx). However, these two strains did possess the sheA gene (Table 3). Oscarsson et al. reported that the bacteriophage-associated Ehly determinant does not encode enterohemolysins but causes the release of SheA or ClyA cytolysin (20). Although this ehly was not detected, it may be that sheA is responsible for the hemolytic phenotype for these two strains. The observed hemolytic phenotype was identical to the HlyA-mediated phenotype. Further studies are required to determine if this hemolytic phenotype is due to SheA and the role SheA plays in the pathogenic profile of these strains. The sheA gene was deleted in the 14 uropathogenic E. coli (UPEC) strains examined by Ludwig et al. in a recent study (16). Their 14 UPEC strains, including the well-known hlyA+ J96 and 536 strains, were assayed for the presence of an hlyA fragment by Southern blot hybridization, but it was identified in only 10 of the 14 strains (28, 29). In our study, we found that 47.1% of the 486 UPEC strains contained the sheA gene (Table 3). The smaller number of UPEC strains in the first two studies may explain the differences in sheA gene carriage. The surprising finding of this study was that out of the 727 E. coli strains tested, 610 possessed either the hlyA or the sheA gene and none carried both of them. The complete absence of hlyA+ sheA+ individuals within a sample of 540 individuals is sufficiently compelling to make additional statistical confirmation redundant (however, if we look at the subset of individuals with one of these two genes and calculate the expected proportion of hlyA+, sheA+, and hlyA+ sheA+ individuals that would exist by random assortment, the probability that the actual frequencies match this null hypothesis is vanishingly small [P < 0.0001; chi square = 439]). The sheA gene in Shiga toxin-producing E. coli (enterohemorrhagic E. coli) can be found in ehx gene-positive isolates (9, 16), even though the plasmid-encoded ehx gene exhibits 60 to 65% sequence homology to hlyA (23). The reasons for the mutual exclusivity between sheA and hlyA in the chromosome found in this study are not understood. It might be that the complex mechanisms regulating expression of hlyA and sheA interfere with each other, posing a selective pressure against their coexistence in the same cell. Further studies will shed light on these questions.
ACKNOWLEDGMENTS
We thank Gyula Mestyán and Anita Novák for their help in collecting the clinical isolates, Gabor Nagy for the hlyA primers, and Mike Speed (University of Liverpool) for his help with statistical analyses.
This work was supported by grant OTKA T037833 from the Hungarian Science Foundation and a Bolyai scholarship from the Hungarian Academy of Sciences.
REFERENCES
Atkins, A., N. R. Wyborn, A. J. Wallace, T. J. Stillman, L. K. Black, A. B. Fielding, M. Hisakado, P. J. Artymiuk, and J. Green. 2000. Structure-function relationships of a novel bacterial toxin, hemolysin E. The role of alpha G. J. Biol. Chem. 275:41150-41155.
Bailey, M. J., V. Koronakis, T. Schmoll, and C. Hughes. 1992. Escherichia coli HlyT protein, a transcriptional activator of haemolysin synthesis and secretion, is encoded by the rfaH (sfrB) locus required for expression of sex factor and lipopolysaccharide genes. Mol. Microbiol. 6:1003-1012.
Beutin, L., J. Prada, S. Zimmermann, R. Stephan, I. Orskov, and F. Orskov. 1988. Enterohemolysin, a new type of hemolysin produced by some strains of enteropathogenic E. coli (EPEC). Zentbl. Bakteriol. Mikrobiol. Hyg. A 267:576-588.
Bhakdi, S., S. Greulich, M. Muhly, B. Eberspacher, H. Becker, A. Thiele, and F. Hugo. 1989. Potent leukocidal action of Escherichia coli hemolysin mediated by permeabilization of target cell membranes. J. Exp. Med. 169:737-754.
Bhakdi, S., M. Muhly, S. Korom, and G. Schmidt. 1990. Effects of Escherichia coli hemolysin on human monocytes. Cytocidal action and stimulation of interleukin 1 release. J. Clin. Investig. 85:1746-1753.
Bullock, W. O., J. M. Fernandez, and J. M. Short. 1987. Xl1-Blue: a high efficiency plasmid transforming recA Escherichia coli strain with beta-galactosidase selection. BioTechniques 5:376-378.
Clowes, R., and D. Rowley. 1954. Some observations on linkage effects in genetic recombination in Escherichia coli K-12. J. Gen. Microbiol. 11:250-260.
del Castillo, F. J., S. C. Leal, F. Moreno, and I. del Castillo. 1997. The Escherichia coli K-12 sheA gene encodes a 34-kDa secreted haemolysin. Mol. Microbiol. 25:107-115.
del Castillo, F. J., F. Moreno, and I. del Castillo. 2000. Characterization of the genes encoding the SheA haemolysin in Escherichia coli O157:H7 and Shigella flexneri 2a. Res. Microbiol. 151:229-230.
Hull, R. A., R. E. Gill, P. Hsu, B. H. Minshew, and S. Falkow. 1981. Construction and expression of recombinant plasmids encoding type 1 or D-mannose-resistant pili from a urinary tract infection Escherichia coli isolate. Infect. Immun. 33:933-938.
Johnson, J. R. 1991. Virulence factor in Escherichia coli urinary tract infection. Clin. Microbiol. Rev. 4:80-128.
Keane, W. F., R. Welch, G. Gekker, and P. K. Peterson. 1987. Mechanism of Escherichia coli alpha-hemolysin-induced injury to isolated renal tubular cells. Am. J. Pathol. 126:350-357.
Konig, B., A. Ludwig, W. Goebel, and W. Konig. 1994. Pore formation by the Escherichia coli alpha-hemolysin: role for mediator release from human inflammatory cells. Infect. Immun. 62:4611-4617.
Lai, X. H., I. Arencibia, A. Johansson, S. N. Wai, J. Oscarsson, S. Kalfas, K. G. Sundqvist, Y. Mizunoe, A. Sjostedt, and B. E. Uhlin. 2000. Cytocidal and apoptotic effects of the ClyA protein from Escherichia coli on primary and cultured monocytes and macrophages. Infect. Immun. 68:4363-4367.
Lai, X. H., S. Y. Wang, and B. E. Uhlin. 1999. Expression of cytotoxicity by potential pathogens in the standard Escherichia coli collection of reference (ECOR) strains. Microbiology 145:3295-3303.
Ludwig, A., C. von Rhein, S. Bauer, C. Hüttinger, and W. Goebel. 2004. Molecular analyses of cytolysin A (ClyA) in pathogenic Escherichia coli strains. J. Bacteriol. 186:5311-5320.
Minshew, B. H., J. Jorgensen, M. Swanstrum, G. A. Grootes-Reuvecamp, and S. Falkow. 1978. Some characteristics of Escherichia coli strains isolated from extraintestinal infections of humans. J. Infect. Dis. 137:648-654.
Ochman, H., and R. K. Selander. 1984. Standard reference strains of Escherichia coli from natural populations. J. Bacteriol. 157:690-693.
Opal, S. M., A. S. Cross, P. Gemski, and L. W. Lyhte. 1990. Aerobactin and alpha-hemolysin as virulence determinants in Escherichia coli isolated from human blood, urine, and stool. J. Infect. Dis. 161:794-796.
Oscarsson, J., M. Westermark, L. Beutin, and B. E. Uhlin. 2002. The bacteriophage-associated Ehly1 and Ehly2 determinants from Escherichia coli O26:H– strains do not encode enterohemolysins per se but cause release of the ClyA cytolysin. Int. J. Med. Microbiol. 291:625-631.
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