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

Abstract

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.

Secretome of obligate intracellularRickettsia

“…4A ). First, Rickettsia ATs typically contain a Sec SS, NT passenger domain, predicted linker (described below), and CT β domain, with AT genes occurring as single transcriptional units or within operons that include genes not associated with type 5 secretion (Sears et al ., 2012) . POTRA domains are unknown from Rickettsia ATs, and there are no homologs to T5SSb TpsB-like proteins encoded in Rickettsia genomes (data not shown).…”

“…Nearly a decade ago, phylogenomics was employed to name 17 distinct Sca families encoded across nine Rickettsia genomes (Blanc et al ., 2005) . Recent analyses based on dozens more genomes suggest that only five families are conserved in most rickettsial genomes: Sca0 (formerly RompA), Sca1, Sca2, Sca4 (formerly PS 120 or protein D) and Sca5 (formerly RompB) (Gillespie et al ., 2012, Sears et al ., 2012) . Interestingly, Sca3 proteins are encoded only within the genomes of typhus group (TG) rickettsiae and R .…”

“…felis ), yet other than their expression and surface localization in R . typhi (Sears et al ., 2012) , nothing is known regarding their function.…”

Detecção de proteínas imunorreativas de Rickettsia sp. cepa Mata Atlântica

“…GroEL é uma proteína de choque térmico que tem demonstrado estar localizado nas superfícies de diversas bactérias rickettsiais (Renesto et al 2005a, Ogawa et al 2007, Pornwiroon et al 2009, Sears et al 2012. Proteínas de choque térmico são componentes essenciais distribuídos por toda a superfície bacteriana e tem como função garantir que as proteínas do genoma codificado sejam expressas como moléculas funcionais (Hartl 1991).…”

Proteomic analysis of Lawsonia intracellularis reveals expression of outer membrane proteins during infection

“…Shotgun proteomic analysis of complex protein mixtures has proved to be a successful approach for investigating the proteomes of obligate intracellular bacteria where technical difficulties in obtaining preparations of organisms free from extraneous host cell material has previously hampered protein identification. To date, such methods have been extensively employed to analyse proteomes and sub-proteomes of a number of obligate intracellular bacteria including Rickettsia typhi, Anaplasma phagocytophilum, Neorickettsia sennetsu, and Ehrlichia chaffeensis, (Lin et al, 2011;Sears et al, 2012;Troese et al, 2011).…”