Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA.
J Bacteriol. 2012 Sep;194(18):4920-32. doi: 10.1128/JB.00793-12. Epub 2012 Jul 6.
Rickettsia typhi, the causative agent of murine (endemic) typhus, is an obligate intracellular pathogen with a life cycle involving both vertebrate and invertebrate hosts. In this study, we characterized a gene (RT0218) encoding a C-terminal ankyrin repeat domain-containing protein, named Rickettsia ankyrin repeat protein 1 (RARP-1), and identified it as a secreted effector protein of R. typhi. RT0218 showed differential transcript abundance at various phases of R. typhi intracellular growth. RARP-1 was secreted by R. typhi into the host cytoplasm during in vitro infection of mammalian cells. Transcriptional analysis revealed that RT0218 was cotranscribed with adjacent genes RT0217 (hypothetical protein) and RT0216 (TolC) as a single polycistronic mRNA. Given one of its functions as a facilitator of extracellular protein secretion in some Gram-negative bacterial pathogens, we tested the possible role of TolC in the secretion of RARP-1. Using Escherichia coli C600 and an isogenic tolC insertion mutant as surrogate hosts, our data demonstrate that RARP-1 is secreted in a TolC-dependent manner. Deletion of either the N-terminal signal peptide or the C-terminal ankyrin repeats abolished RARP-1 secretion by wild-type E. coli. Importantly, expression of R. typhi tolC in the E. coli tolC mutant restored the secretion of RARP-1, suggesting that TolC has a role in RARP-1 translocation across the outer membrane. This work implies that the TolC component of the putative type 1 secretion system of R. typhi is involved in the secretion process of RARP-1.
鼠型（地方性）斑疹伤寒立克次体（Rickettsia typhi）是一种专性细胞内病原体，其生命周期涉及脊椎动物和无脊椎动物宿主。在这项研究中，我们鉴定了一个编码 C 端锚蛋白重复结构域的基因（RT0218），命名为立克次体锚蛋白重复蛋白 1（Rickettsia ankyrin repeat protein 1，RARP-1），并鉴定其为 R. typhi 的一种分泌效应蛋白。RT0218 在 R. typhi 细胞内生长的不同阶段表现出差异转录丰度。RARP-1 在体外感染哺乳动物细胞时由 R. typhi 分泌到宿主细胞质中。转录分析显示，RT0218 与相邻基因 RT0217（假定蛋白）和 RT0216（TolC）一起作为单个多顺反子转录。鉴于其在某些革兰氏阴性细菌病原体中作为细胞外蛋白分泌促进因子的一个功能，我们测试了 TolC 在 RARP-1 分泌中的可能作用。使用大肠杆菌 C600 和一个同源 tolC 插入突变体作为替代宿主，我们的数据表明 RARP-1 是通过 TolC 依赖的方式分泌的。野生型大肠杆菌中缺失 N 端信号肽或 C 端锚蛋白重复序列均会导致 RARP-1 分泌的缺失。重要的是，在大肠杆菌 tolC 突变体中表达 R. typhi tolC 恢复了 RARP-1 的分泌，表明 TolC 在 RARP-1 跨外膜转运中起作用。这项工作表明，R. typhi 假定的 I 型分泌系统的 TolC 成分参与了 RARP-1 的分泌过程。
