Surface Proteome Analysis and Characterization of Surface Cell Antigen (Sca) or Autotransporter Family of Rickettsia typhi

Surface proteins of the obligate intracellular bacterium Rickettsia typhi, the agent of murine or endemic typhus fever, comprise an important interface for host-pathogen interactions including adherence, invasion and survival in the host cytoplasm. In this report, we present analyses of the surface exposed proteins of R. typhi based on a suite of predictive algorithms complemented by experimental surface-labeling with thiol-cleavable sulfo-NHS-SS-biotin and identification of labeled peptides by LC MS/MS. Further, we focus on proteins belonging to the surface cell antigen (Sca) autotransporter (AT) family which are known to be involved in rickettsial infection of mammalian cells. Each species of Rickettsia has a different complement of sca genes in various states; R. typhi, has genes sca1 thru sca5. In silico analyses indicate divergence of the Sca paralogs across the four Rickettsia groups and concur with previous evidence of positive selection. Transcripts for each sca were detected during infection of L929 cells and four of the five Sca proteins were detected in the surface proteome analysis. We observed that each R. typhi Sca protein is expressed during in vitro infections and selected Sca proteins were expressed during in vivo infections. Using biotin-affinity pull down assays, negative staining electron microscopy, and flow cytometry, we demonstrate that the Sca proteins in R. typhi are localized to the surface of the bacteria. All Scas were detected during infection of L929 cells by immunogold electron microscopy. Immunofluorescence assays demonstrate that Scas 1–3 and 5 are expressed in the spleens of infected Sprague-Dawley rats and Scas 3, 4 and 5 are expressed in cat fleas (Ctenocephalides felis). Sca proteins may be crucial in the recognition and invasion of different host cell types. In short, continuous expression of all Scas may ensure that rickettsiae are primed i) to infect mammalian cells should the flea bite a host, ii) to remain infectious when extracellular and iii) to infect the flea midgut when ingested with a blood meal. Each Sca protein may be important for survival of R. typhi and the lack of host restricted expression may indicate a strategy of preparedness for infection of a new host.专性胞内菌莫氏立克次体（Rickettsia typhi）是鼠型（地方性）斑疹热的致病菌，其表面蛋白是宿主-病原体互作的重要界面，介导黏附、入侵以及在宿主细胞质内的存活过程。本研究基于一系列预测算法，结合巯基可裂解磺基-NHS-SS-生物素的实验性表面标记技术与液相色谱-串联质谱（LC MS/MS）对标记肽段的鉴定结果，对莫氏立克次体的表面暴露蛋白进行了系统分析。本研究重点关注了表面细胞抗原（Sca）自转运蛋白（AT）家族成员，这类蛋白已被证实参与立克次体对哺乳动物细胞的感染过程。不同立克次体物种所携带的sca基因组成存在差异，且基因状态各不相同；莫氏立克次体携带sca1至sca5共5个基因。计算机辅助分析显示，四类立克次体的Sca旁系同源蛋白存在序列分化，这与此前关于正选择的研究证据相符。研究人员在感染L929细胞（L929 cells）的样本中检测到了所有sca基因的转录本，且在表面蛋白质组分析中鉴定出5个Sca蛋白中的4个。实验证实，莫氏立克次体的所有Sca蛋白均在体外感染过程中表达，部分Sca蛋白可在体内感染模型中检测到。通过生物素亲和下拉实验、负染电子显微镜技术与流式细胞术，本研究证明莫氏立克次体的Sca蛋白定位于细菌表面。免疫金电子显微镜技术结果显示，在感染L929细胞的过程中可检测到所有Sca蛋白。免疫荧光实验证实，在感染斯普拉格-道利大鼠（Sprague-Dawley rats）的脾脏组织中可检测到Sca1、Sca2、Sca3与Sca5的表达，而在猫蚤（Ctenocephalides felis）体内可检测到Sca3、Sca4与Sca5的表达。Sca蛋白可能在识别并入侵不同宿主细胞类型的过程中发挥关键作用。综上，所有Sca蛋白的持续表达可能确保立克次体具备三重感染预备策略：一是当蚤类叮咬宿主时，可快速感染哺乳动物细胞；二是在胞外环境中维持感染性；三是当随血液餐被猫蚤摄入后，可感染蚤类中肠。每一种Sca蛋白均可能对莫氏立克次体的存活至关重要，而无宿主限制性的表达模式可能预示着其具备感染新宿主的预备策略。