Expression and Purification of the Crystalline Surface Layer Protein of <i>Rickettsia typhi</i>

Hahn, Myong‐Joon; Chang, Woo-Hyun

doi:10.1111/j.1348-0421.1996.tb03339.x

Cited by 4 publications

(4 citation statements)

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“…Discrepancies between the number of predicted outer membrane or extracellular proteins and the number actually identified may be due to low abundance of some peptides and or inaccessibility to the biotinylation reagent. It is predicted that Sca5 comprises the S-layer of rickettsiae and constitutes 15% of the total protein mass [56], [57] and might block many potential interactions. The properties of the labeling reagent must also be taken into account.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2012

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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.

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Section: Discussionmentioning

confidence: 99%

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

et al. 2012

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“…conorii mediates both adhesion and invasion of cultured mammalian cells (Cardwell & Martinez, 2009) . Sca5 is the predominant protein of the rickettsial S-layer, comprising 15% of the total protein mass (Ching et al ., 1990, Hahn & Chang, 1996) . Sca5 of R .…”

Section: Sec-dependent Secretory Pathwaysmentioning

confidence: 99%

“…Specifically, it is critical to determine if the β domains of processed Scas can be used to translocate the passenger domains of other Scas lacking β domains (e.g., Sca4, and other less conserved Scas, such as Sca9), or even serve as OM channels for secretion of other proteins. Such stable β-barrel proteins are likely prevalent in the rickettsial OM given the abundance of Scas at the bacterial surface (Ching et al ., 1990, Hahn & Chang, 1996) , and probably would only turn over during cell growth and division (Grijpstra et al , 2013). Indeed, families of proteins comprising solitary AT-like β domains have been identified in some proteobacterial genomes (Prakash et al ., 2011) .…”

Section: Sec-dependent Secretory Pathwaysmentioning

confidence: 99%

Secretome of obligate intracellularRickettsia

et al. 2014

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The genus Rickettsia (Alphaproteobacteria; Rickettsiales; Rickettsiaceae) is comprised of obligate intracellular parasites, with virulent species of interest both as causes of emerging infectious diseases and for their potential deployment as bioterrorism agents. Currently there are no effective commercially available vaccines, with treatment limited primarily to tetracycline antibiotics, though others (e.g., josamycin, ciprofloxacin, chloramphenicol and azithromycin) are also effective. Much of the recent research geared towards understanding mechanisms underlying rickettsial pathogenicity has centered on characterization of secreted proteins that directly engage eukaryotic cells. Herein, we review all aspects of the Rickettsia secretome, including six secretion systems, 19 characterized secretory proteins, and potential moonlighting proteins identified on surfaces of multiple Rickettsia species. Employing bioinformatics and phylogenomics, we present novel structural and functional insight on each secretion system. Unexpectedly, our investigation revealed that the majority of characterized secretory proteins have not been assigned to their cognate secretion pathways. Furthermore, for most secretion pathways, the requisite signal sequences mediating translocation are poorly understood. As a blueprint for all known routes of protein translocation into host cells, this resource will assist research aimed at uniting characterized secreted proteins with their apposite secretion pathways. Furthermore, our work will help in the identification of novel secreted proteins involved in rickettsial “life on the inside”.

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“…Two homologous autotransporter proteins, rOmpA and rOmpB, have been identified among the Rickettsiales (25,29,52,54,55). The rOmpB proteins appear to form an S layer on the surface of the bacteria (20,62,63), whereas a surface structure for rOmpA has not been described. No function has been clearly assigned to these proteins.…”

Section: Rickettsial Autotransportersmentioning

confidence: 99%