MOLECULAR ANALYSIS OF THE PLASMID-ENCODED HEMOLYSIN OF ESCHERICHIA-COLI-0157-H7 STRAIN EDL-933

In this study, we determined the nucleotide sequence of the 5.4-kb Sal I restriction fragment of the recombinant plasmid pEO40-1, cloned from the large plasmid of enterohemorrhagic Escherichia coli (EHEC) 0157:H 7 strain EDL 933. This revealed two open reading frames which shared a pproximately 60% homology to the hlyC and hlyA genes of the E. coli al pha-hemolysin (alpha-hly) operon. We termed these genes EHEC-hlyA and EHEC-hlyC to distinguish them from the alpha-hly genes. Preliminary se quence analysis indicated that another open reading frame homolog to t he hlyB gene is located close to the 3' end of EHEC-hlyA. The predicte d molecular masses of the EHEC-hlyA and EHEC-hlyC gene products were 1 07 and 19.9 kDa, respectively. The EHEC hemolysin protein (EHEC-Hly) w as not secreted into the culture supernatant by the strain EDL 933. Ho wever, hemolytic activity was found in the broth culture supernatant a fter transforming EDL 933 with the recombinant plasmid pRSC6 carrying the hlyB and hlyD genes from the E. coli alpha-hemolysin operon. The E HEC hemolysin was precipitated and used as an antigen for immunoblot a nalysis. This demonstrated that 19 of 20 reconvalescent-phase serum sa mples from patients with hemolytic uremic syndrome reacted specificall y with the antigen; conversely, only 1 of 20 control serum samples dem onstrated reactivity. To investigate the prevalence of EHEC hemolysin genes in diarrheagenic E. coli, a PCR was developed to specifically de tect EHEC-hlyA. All Shiga-like toxin-producing 0157 strains and 12 of 25 Shiga-like toxin-producing non-O157 strains were PCR positive; stra ins of other categories of diarrheagenic E. coli were PCR negative. Al l PCR-positive strains hybridized with the CVD 419 probe. We found the CVD 419 probe to be identical to the 3.4-kb HindIII fragment of plasm id pEO40 carrying most of the EHEC-hlyA gene and a part of the putativ e EHEC-hlyB gene, In this study, the newly discovered EHEC hemolysin w as shown to be responsible for the enterohemolytic phenotype and demon strated to be related but not identical to alpha-hemolysin. The EHEC h emolysin appears to have clinical importance because it occurs in all 0157 strains tested and is reactive to sera of patients with hemolytic uremic syndrome.