The binding of bacteria with platelets appears to be a central event in the pathogenesis of infective endocarditis. This interaction may be important both for the initial attachment of blood borne organisms to the endocardium, and for the subsequent formation of macroscopic vegetations on the cardiac valve surface. Our research has identified a novel genetic locus of Streptococcus gordonii that encodes a cell wall-anchored, serine-rich glycoprotein (GspB) that mediates the binding of streptococci to human platelets, and enhances virulence, as measured by animal models of endocardial infection. The locus also encodes four proteins that glycosylate GspB intracellularly, and seven proteins comprising a specialized system (the accessory Sec system) that are essential for GspB export. Two components (SecA2 and SecY2) are homologs of SecA and SecY of the canonical Sec system, and appear to have analogous functions. The other five members of this specialized system (Asp1-5) are also required for GspB export, but have no homologs of known function. The goal of this project is to further define the mechanisms for GspB trafficking and export to the bacterial surface. We will specifically examine the features of GspB and Asp1, Asp2, and Asp3 that contribute to the selective transport of the substrate by the accessory Sec system. Although both the extended N region of the GspB signal peptide and the novel AST domain (amino acids 91 - 117) of GspB are essential for export, it is unknown how these regions facilitate this process. To address these issues, Aim 1 uses in vivo site-specific photo cross-linking and mass spectroscopy to identify proteins that bind to the N and AST domains during transport. These studies should identify not only which components of the accessory Sec system interact directly with GspB, but will also provide insights into the sequence of these events. In addition, this work could identify additiona novel co-factors needed for transport.
Aim 2 addresses the roles Asps 1, 2, and 3 in the targeting of GspB to the transmembrane channel complex (SecA2, SecY2, Asp4/5). The subcellular localization of GspB and the Asps will be examined by cell fractionation and fluorescence microscopy. By examining the location of GspB and the Asps, and how their distribution is affected by selected deletions and mutations, these studies will provide further information as to the roles of Asp1-3 in export.
Aim 3 explores the bi-functional properties of Asp2, i.e., the role of this protein in mediating both GspB export and glycosylation. The impact of Asp2 binding on GspB export will be addressed through a series of targeted mutations. In addition, we will assess how mutagenesis of Asp2 alters the glycosylation of GspB, as measured by monosaccharide compositional analysis and mass spectroscopy. These experiments should provide considerable insights into the selective trafficking and biogenesis of GspB. Since the accessory Sec system is conserved among numerous Gram- positive pathogens, these studies should be highly applicable to a broad range of clinically- important organisms and infections, and may also yield targets for the development of novel therapeutic agents.
Endocarditis is a life-threatening infection of the valves of the heart. A key step in the development of this disease is the attachment of microbes onto the heart valves. For many bacteria, this binding is mediated by a family of proteins on the surface of the organisms. The goal of the proposed research is to examine how these proteins are transported from inside the bacterium to the microbial surface by a specialized pathway (the accessory Sec system). This research will examine how the seven components of this system work as a machine to transport one specific protein (called GspB) made by the bacterium Streptococcus gordonii. By examining the inner workings of the accessory Sec system, this research will provide important insights into a central process for the production of endocarditis and related infections. In addition, it may provide a basis for developing novel therapies for these diseases.
|Mishra, Nagendra N; Tran, Truc T; Seepersaud, Ravin et al. (2017) Perturbations of Phosphatidate Cytidylyltransferase (CdsA) Mediate Daptomycin Resistance in Streptococcus mitis/oralis by a Novel Mechanism. Antimicrob Agents Chemother 61:|
|Chen, Yu; Seepersaud, Ravin; Bensing, Barbara A et al. (2016) Mechanism of a cytosolic O-glycosyltransferase essential for the synthesis of a bacterial adhesion protein. Proc Natl Acad Sci U S A 113:E1190-9|
|Deng, Lingquan; Bensing, Barbara A; Thamadilok, Supaporn et al. (2014) Oral streptococci utilize a Siglec-like domain of serine-rich repeat adhesins to preferentially target platelet sialoglycans in human blood. PLoS Pathog 10:e1004540|
|Bensing, Barbara A; Seepersaud, Ravin; Yen, Yihfen T et al. (2014) Selective transport by SecA2: an expanding family of customized motor proteins. Biochim Biophys Acta 1843:1674-86|
|Wang, Nai-Yu; Patras, Kathryn A; Seo, Ho Seong et al. (2014) Group B streptococcal serine-rich repeat proteins promote interaction with fibrinogen and vaginal colonization. J Infect Dis 210:982-91|
|Seo, Ho Seong; Minasov, George; Seepersaud, Ravin et al. (2013) Characterization of fibrinogen binding by glycoproteins Srr1 and Srr2 of Streptococcus agalactiae. J Biol Chem 288:35982-96|
|Yen, Yihfen T; Cameron, Todd A; Bensing, Barbara A et al. (2013) Differential localization of the streptococcal accessory sec components and implications for substrate export. J Bacteriol 195:682-95|
|Seo, Ho Seong; Xiong, Yan Q; Sullam, Paul M (2013) Role of the serine-rich surface glycoprotein Srr1 of Streptococcus agalactiae in the pathogenesis of infective endocarditis. PLoS One 8:e64204|
|Bensing, Barbara A; Yen, Yihfen T; Seepersaud, Ravin et al. (2012) A Specific interaction between SecA2 and a region of the preprotein adjacent to the signal peptide occurs during transport via the accessory Sec system. J Biol Chem 287:24438-47|
|Seepersaud, Ravin; Bensing, Barbara A; Yen, Yihfen T et al. (2012) The accessory Sec protein Asp2 modulates GlcNAc deposition onto the serine-rich repeat glycoprotein GspB. J Bacteriol 194:5564-75|
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