Identification of Novel Surface-Exposed Proteins of <i>Rickettsia rickettsii</i> by Affinity Purification and Proteomics

<div><p><i>Rickettsia rickettsii</i>, the causative agent of Rocky Mountain spotted fever, is the most pathogenic member among <i>Rickettsia</i> spp. Surface-exposed proteins (SEPs) of <i>R. rickettsii</i> may play important roles in its pathogenesis or immunity. In this study, <i>R. rickettsii</i> 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 <i>R. rickettsii</i>. Another 5 proteins (Adr1, Adr2, OmpW, Porin_4, and TolC) were first recognized as SEPs of <i>R. rickettsii</i> herein. The genes encoding the 5 novel SEPs were expressed in <i>Escherichia coli</i> cells, resulting in 5 recombinant SEPs (rSEPs), which were used to immunize mice. After challenge with viable <i>R. rickettsii</i> 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 <i>in vitro</i> neutralization test revealed that sera from mice immunized with rAdr1, rAdr2, or rOmpW reduced <i>R. rickettsii</i> 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 <i>R. rickettsii</i>. Altogether, the 5 novel SEPs identified herein might be involved in the interaction of <i>R. rickettsii</i> with vascular endothelial cells, and all of them except TolC were protective antigens.</p>