Cheng Catherine, Tennant Sharon M, Azzopardi Kristy I, Bennett-Wood Vicki, Hartland Elizabeth L, Robins-Browne Roy M, Tauschek Marija
Department of Microbiology and Immunology, The University of Melbourne, Victoria 3010, Australia.
Infect Immun. 2009 May;77(5):1936-44. doi: 10.1128/IAI.01246-08. Epub 2009 Mar 2.
Strains of enteropathogenic Escherichia coli (EPEC) generally employ the adhesins bundle-forming pili (Bfp) and intimin to colonize the intestine. Atypical EPEC strains possess intimin but are negative for Bfp and, yet, are able to cause disease. To identify alternative adhesins to Bfp in atypical EPEC, we constructed a transposon mutant library of atypical EPEC strain E128012 (serotype O114:H2) using TnphoA. Six mutants that had lost the ability to adhere to HEp-2 cells were identified, and in all six mutants TnphoA had inserted into the pstSCAB-phoU (Pst) operon. To determine if the Pst operon is required for adherence, we used site-directed mutagenesis to construct a pstCA mutant of E128012. The resultant mutant showed a reduced ability to adhere to HEp-2 cells and T84 intestinal epithelial cells, which was restored by trans-complementation with intact pstCA. To determine if pst contributes to bacterial colonization in vivo, a pstCA mutation was made in the EPEC-like murine pathogen, Citrobacter rodentium. C57BL/6 mice infected perorally with the pstCA mutant of C. rodentium excreted significantly lower numbers of C. rodentium than those given the wild-type strain. Moreover, colonic hyperplasia and diarrhea, which are features of infections with C. rodentium, were not observed in mice infected with the pstCA mutant but did occur in mice given the trans-complemented mutant. As mutations in pst genes generally lead to constitutive expression of the Pho regulon, our findings suggested that the Pho regulon may contribute to the reduced virulence of the pstCA mutants. To investigate this, we inactivated phoB in the pstCA mutants of EPEC E128012 and C. rodentium and found that the phoB mutation restored the adherent phenotype of both mutant strains. These results demonstrate that Pst contributes to the virulence of atypical EPEC and C. rodentium, probably by causing increased expression of an unidentified, Pho-regulated adhesin.
肠道致病性大肠杆菌（EPEC）菌株通常利用束状菌毛（Bfp）和紧密黏附素在肠道中定植。非典型EPEC菌株拥有紧密黏附素，但Bfp呈阴性，然而，它们仍能够引发疾病。为了在非典型EPEC中鉴定出替代Bfp的黏附素，我们使用TnphoA构建了非典型EPEC菌株E128012（血清型O114:H2）的转座子突变文库。鉴定出6个失去黏附HEp-2细胞能力的突变体，并且在所有6个突变体中，TnphoA都插入到了pstSCAB-phoU（Pst）操纵子中。为了确定Pst操纵子对于黏附是否必需，我们使用定点诱变构建了E128012的pstCA突变体。所得突变体显示出黏附HEp-2细胞和T84肠上皮细胞的能力降低，通过用完整的pstCA进行反式互补，这种能力得以恢复。为了确定Pst是否有助于体内细菌定植，我们在类EPEC鼠病原体鼠柠檬酸杆菌中制造了pstCA突变。经口感染鼠柠檬酸杆菌pstCA突变体的C57BL/6小鼠排出的鼠柠檬酸杆菌数量明显低于给予野生型菌株的小鼠。此外，在感染pstCA突变体的小鼠中未观察到鼠柠檬酸杆菌感染所特有的结肠增生和腹泻，但在给予反式互补突变体的小鼠中确实出现了这些症状。由于pst基因的突变通常会导致Pho调节子的组成型表达，我们的研究结果表明Pho调节子可能导致了pstCA突变体毒力的降低。为了对此进行研究，我们使EPEC E128012和鼠柠檬酸杆菌的pstCA突变体中的phoB失活，发现phoB突变恢复了两种突变菌株的黏附表型。这些结果表明，Pst可能通过导致一种未鉴定的、受Pho调节的黏附素表达增加，从而有助于非典型EPEC和鼠柠檬酸杆菌的毒力。
