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

et al. (2012) Surface Proteome Analysis and Characterization of Surface Cell Antigen (Sca) or Autotransporter Family of Rickettsia typhi. PLoS Pathog 8(8): e1002856. doi:10.1371/journal.ppat.1002856 Surface Proteome Analysis and Characterization of Surface Cell Antigen (Sca) or Autotransporter Family of Rickettsia typhi Khandra T. Sears 0 Shane M. Ceraul 0 Joseph J. Gillespie 0 Edwin D. Allen Jr. 0 Vsevolod L. Popov 0 Nicole C. Ammerman 0 M. Sayeedur Rahman 0 Abdu F. Azad 0 Craig R. Roy, Yale University School of Medicine, United States of America 0 1 Department of Microbiology and Immunology, School of Medicine, University of Maryland Baltimore , Baltimore , Maryland, United States of America, 2 Virginia Bioinformatics Institute at Virginia Tech, Blacksburg, Virginia, United States of America, 3 Department of Biological Sciences, Towson University , Towson, Maryland , United States of America, 4 Center for Biodefense and Emerging Infectious Diseases, WHO Collaborating Center for Tropical Diseases, The University of Texas Medical Branch , Galveston, Texas , United States of America, 5 Center for Tuberculosis Research, Johns Hopkins University , Baltimore, Maryland , United States of America 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. - Funding: The research presented in this article was supported by funds from the National Institutes of Health/National Institute of Allergy and Infectious Diseases (AI017828 to AFA). JJG is grateful for support from the National Institute of Allergy and Infectious Diseases (NIAID Contract No. HHSN272200900040C awarded to Bruno Sobral, Virginia Bioinformatics Institute at Virginia Tech). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Rickettsia (Rickettsiales: Rickettsiaceae) are Gram-negative, obligate intracellular bacteria that are maintained in enzootic cycles involving both hematophagous arthropod vectors and vertebrate hosts [1]. Rickettsiae are the causative agents of significant human diseases such as Rocky Mountain spotted fever (R. rickettsii) and epidemic typhus (R. prowazekii). Classically, the members of the genus Rickettsia have been divided into two groups: the ticktransmitted spotted fever group (SFG) and the insect-transmitted typhus group (TG) based on their antigenic and molecular profiles. However, these groups share some antigenic proteins such as outer membrane protein B (OmpB) and 17 kDa lipoprotein [2]. The tick-transmitted SFG currently includes over 16 species, several of which are known human pathogens (R. rickettsii, R. conorii, and R. sibirica). The louse and flea transmitted TG rickettsia contain the pathogenic species R. prowazekii and R. typhi (causative agent of murine typhus). Extensive phylogenetic and comparative genomic analyses have resulted in the proposal of the ancestral group (AG) and transitional group (TRG) rickettsia and these include species with mild or unknown pathogenicity as well as broad arthropod host ranges [3,4,5]. Despite the recent advances made in rickettsial molecular biology and genomics, their determinants of pathogenicity still remain undefined. Because of the involvement of rickettsial Omps in cell surface recognition, initial binding of bacteria to host cells, invasion processes [6,7] as well as their immunogenicity and utility as vaccine candidates, this group of proteins has been a target of Rickettsia typhi, a member of the typhus group (TG) rickettsia, is the agent of murine or endemic typhus fever a disease exhibiting mild to severe flu-like symptoms resulting in significant morbidity. It is maintained in a flearodent transmission cycle in urban and suburban environments. The obligate intracellular lifestyle of rickettsiae makes genetic manipulation difficult and impedes progress towards identification of virulence factors. All five Scas were detected on the surface of R. typhi using a combination of a biotin-labeled affinity assay, negative stain electron microscopy and flow cytometry. Sca proteins are members of the autotransporter (AT) family or type V secretion system (TVSS). We employed detailed bioinformatic analyses and evaluated their transcript abundance in an in vitro infection model where sca transcripts are detected at varying levels over the course of a 5 day in vitro infection. We also observe expression of selected Sca proteins during infection of fleas and rats. Our study provides a proteomic analysis of the bacterial surface and an initial characterization of the Sca family as it exists in R. typhi. interest [8,9,10,11]. Bacterial surface-exposed proteins are involved in an array of processes including sensing the environm (...truncated)