coli strains, was negative for the stcE gene. The presence or absence of the stcE gene in all strains was confirmed by Southern blot (data not shown). Analysis of isolated plasmid DNA by Southern blot demonstrated that stcE was encoded on the large plasmid of the four atypical Shigella B13 strains (data not shown). Sequence analysis of the 2.7-kb stcE gene showed only Selleckchem JQ1 three synonymous substitutions shared among
the atypical Shigella B13 strains and a Q727L substitution in strain 3556-77 compared to the EHEC EDL933 allele (data not shown). Six substitutions within 220 nucleotides of the intergenic region upstream of the predicted stcE promoter are present in the plasmids of all four atypical Shigella B13 strains compared to pO157. To determine whether the StcE protein was expressed and secreted by the atypical Shigella B13 strains, TCA-precipitated supernatants of overnight cultures were analyzed by immunoblot. StcE protein was identified in supernatants from strains 3556-77, 3052-94, and 3053-94, but not from 3557-77 or 5216-70 (Table 2). StcE activity in culture supernatants was assayed for C1-INH proteolysis by immunoblots click here and detected with all atypical Shigella B13 strains except 3557-77 and 5216-70 (Fig. 1, Table 2). To determine whether the atypical Shigella B13 plasmid encoding stcE is similar to the large invasion plasmid of Shigella
(pINV), several pINV-encoded virulence factors were sought by PCR amplification (Table 2). None of the pINV-encoded virulence factors could be amplified from the atypical why Shigella B13 strains. PCR analysis using primers specific for pO157-encoded genes resulted in amplification of etpD, but not katP. The gene, traC, which is an F plasmid gene that is also encoded on the large virulence plasmid of E. coli O157:H-, pSFO157, did not PCR amplify from any of the atypical Shigella B13 strains tested. The presence of additional E. coli-specific chromosomally encoded genes was determined by colony PCR (Table 2). The LEE-encoded
regulator (Ler) is a global virulence regulator that has been shown to positively regulate the expression of LEE (Mellies et al., 1999), stcE, and the etp operon in E. coli O157:H7 (Lathem et al., 2002). PCR analysis of the atypical Shigella B13 strains identified the ler gene in the four atypical Shigella B13 strains encoding eae and stcE. An additional LEE-encoded gene, espA, encodes a subunit of the type III secretion system unique to EPEC and EHEC and is encoded by the atypical Shigella B13 strains encoding eae and stcE. PCR analysis of cadA, which encodes lysine decarboxylase and is universally absent in Shigella but present in most E. coli strains (Day et al., 2001), revealed that none of the atypical Shigella B13 strains encoded cadA. The abilities of the atypical Shigella B13 strains to invade HEp-2 cells were determined.