Gong Wenping, Xiong Xiaolu, Qi Yong, Jiao Jun, Duan Changsong, Wen Bohai
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.
PLoS One. 2014 Jun 20;9(6):e100253. doi: 10.1371/journal.pone.0100253. eCollection 2014.
Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is the most pathogenic member among Rickettsia spp. Surface-exposed proteins (SEPs) of R. rickettsii may play important roles in its pathogenesis or immunity. In this study, R. rickettsii organisms were surface-labeled with sulfo-NHS-SS-biotin and the labeled proteins were affinity-purified with streptavidin. The isolated proteins were separated by two-dimensional electrophoresis, and 10 proteins were identified among 23 protein spots by electrospray ionization tandem mass spectrometry. Five (OmpA, OmpB, GroEL, GroES, and a DNA-binding protein) of the 10 proteins were previously characterized as surface proteins of R. rickettsii. Another 5 proteins (Adr1, Adr2, OmpW, Porin_4, and TolC) were first recognized as SEPs of R. rickettsii herein. The genes encoding the 5 novel SEPs were expressed in Escherichia coli cells, resulting in 5 recombinant SEPs (rSEPs), which were used to immunize mice. After challenge with viable R. rickettsii cells, the rickettsial load in the spleen, liver, or lung of mice immunized with rAdr2 and in the lungs of mice immunized with other rSEPs excluding rTolC was significantly lower than in mice that were mock-immunized with PBS. The in vitro neutralization test revealed that sera from mice immunized with rAdr1, rAdr2, or rOmpW reduced R. rickettsii adherence to and invasion of vascular endothelial cells. The immuno-electron microscopic assay clearly showed that the novel SEPs were located in the outer and/or inner membrane of R. rickettsii. Altogether, the 5 novel SEPs identified herein might be involved in the interaction of R. rickettsii with vascular endothelial cells, and all of them except TolC were protective antigens.
落基山斑疹热的病原体立氏立克次体是立克次体属中致病性最强的成员。立氏立克次体的表面暴露蛋白（SEP）可能在其发病机制或免疫过程中发挥重要作用。在本研究中，用磺基-NHS-SS-生物素对立氏立克次体进行表面标记，并用链霉亲和素对标记蛋白进行亲和纯化。分离得到的蛋白通过二维电泳进行分离，通过电喷雾电离串联质谱在23个蛋白点中鉴定出10种蛋白。这10种蛋白中的5种（外膜蛋白A、外膜蛋白B、60kDa热休克蛋白、10kDa热休克蛋白和一种DNA结合蛋白）先前已被鉴定为立氏立克次体的表面蛋白。另外5种蛋白（Adr1、Adr2、外膜孔蛋白W、孔蛋白_4和外膜转运蛋白C）在此首次被确认为立氏立克次体的SEP。编码这5种新型SEP的基因在大肠杆菌细胞中表达，产生了5种重组SEP（rSEP），用于免疫小鼠。用活的立氏立克次体细胞攻击后，用rAdr2免疫的小鼠脾脏、肝脏或肺中的立克次体载量以及用除rTolC之外的其他rSEP免疫的小鼠肺中的立克次体载量显著低于用PBS进行假免疫的小鼠。体外中和试验表明，用rAdr1、rAdr2或rOmpW免疫的小鼠血清可减少立氏立克次体对血管内皮细胞的黏附和侵袭。免疫电子显微镜检测清楚地表明，这些新型SEP位于立氏立克次体的外膜和/或内膜中。总之，本文鉴定出的5种新型SEP可能参与立氏立克次体与血管内皮细胞的相互作用，除TolC外，它们均为保护性抗原。
