Identification of a Locus Involved in Systemic Dissemination of Yersinia enterocolitica

A putative LysR-type transcriptional activator, Hre20, was identified previously in an in vivo expression technology screen designed to identify factors which are expressed early during infection by Yersinia enterocolitica (G. M. Young and V. L. Miller, Mol. Microbiol. 25:319–328, 1997). An insertion in hre20, now designated rscR, resulted in increased splenic dissemination of bacteria during infection in a BALB/c mouse model. A nonpolar mutation was generated in rscR, and examination of this strain in the BALB/c mouse model demonstrated that the mutation in rscR was responsible for the increased dissemination to the spleen that was seen in the original experiments. RscR is homologous to the LysR family of transcriptional regulators; thus, a screen was undertaken to identify genes regulated by RscR. A strain containing an insertion in the chromosomal rscR gene and carrying rscR on a plasmid under the control of the inducible araBAD promoter was mutagenized with an mTn5Km-2 transposon containing a promoterless lacZY. Eighteen insertions were identified which appeared to respond to levels of RscR, and these were classified into four allelic groups based on Southern blot hybridization analysis. Representative members were sequenced from three allelic groups. Sequencing revealed insertions in an ORF with no known homologues, a homologue of OmpF of Serratia marcescens, and a locus (designated rscBAC) with similarity to the hmwABC locus of Haemophilus influenzae. The hmwABC locus promotes adherence of H. influenzae to host cells (S. J. Barenkamp and J. W. St. Geme III, Infect. Immun. 62:3320–3328, 1994; J. W. St. Geme III, S. Falkow, and S. J. Barenkamp, Proc. Natl. Acad. Sci. USA 90:2875–2879, 1993). A strain containing a deletion mutant of rscA, the hmwA homologue, exhibits increased splenic dissemination of bacteria during infection in a BALB/c mouse model, similar to the rscR mutant. This suggests that the phenotype of an rscR mutant is due to the loss of RscA.

此前在一项旨在鉴定小肠结肠炎耶尔森氏菌（Yersinia enterocolitica）感染早期表达因子的体内表达技术筛选中，研究人员鉴定出一种推定的LysR型转录激活因子Hre20（G. M. Young与V. L. Miller, Mol. Microbiol. 25:319–328, 1997）。hre20基因的一处插入突变现被命名为rscR，该突变可使菌株在BALB/c小鼠感染模型中的脾脏播散能力增强。本研究构建了rscR的非极性突变株，并在BALB/c小鼠模型中对该菌株进行验证，结果证实rscR突变正是导致原始实验中脾脏播散能力增强的原因。RscR与LysR家族转录调控因子同源，因此本研究开展筛选以鉴定RscR调控的基因。以携带染色体rscR基因插入突变、且携带有由可诱导araBAD启动子调控的质粒表达rscR的菌株为材料，使用携带无启动子lacZY的mTn5Km-2转座子进行诱变。最终鉴定得到18个响应RscR表达水平的插入突变株，通过Southern印迹杂交分析将其划分为4个等位基因组。对3个等位基因组的代表性菌株进行测序，结果显示插入位点分别位于：一个无已知同源物的开放阅读框（ORF）、粘质沙雷氏菌（Serratia marcescens）OmpF的同源基因，以及一个与流感嗜血杆菌（Haemophilus influenzae）hmwABC基因座同源的位点（命名为rscBAC）。hmwABC基因座可促进流感嗜血杆菌黏附宿主细胞（S. J. Barenkamp与J. W. St. Geme III, Infect. Immun. 62:3320–3328, 1994；J. W. St. Geme III、S. Falkow与S. J. Barenkamp, Proc. Natl. Acad. Sci. USA 90:2875–2879, 1993）。作为hmwA同源基因的rscA缺失突变株，在BALB/c小鼠感染模型中同样表现出细菌脾脏播散能力增强的表型，与rscR突变株一致。这表明rscR突变株的表型是由于RscA的表达缺失所导致的。