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

<div><p>Surface proteins of the obligate intracellular bacterium <em>Rickettsia typhi</em>, 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 <em>R. typhi</em> 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 <em>Rickettsia</em> has a different complement of <em>sca</em> genes in various states; <em>R. typhi</em>, has genes <em>sca1</em> thru <em>sca5</em>. <em>In silico</em> analyses indicate divergence of the Sca paralogs across the four <em>Rickettsia</em> groups and concur with previous evidence of positive selection. Transcripts for each <em>sca</em> 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 <em>R. typhi</em> Sca protein is expressed during <em>in vitro</em> infections and selected Sca proteins were expressed during <em>in vivo</em> infections. Using biotin-affinity pull down assays, negative staining electron microscopy, and flow cytometry, we demonstrate that the Sca proteins in <em>R. typhi</em> 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 (<em>Ctenocephalides felis</em>). 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 <em>R. typhi</em> and the lack of host restricted expression may indicate a strategy of preparedness for infection of a new host.</p>