The binding of bacteria with platelets is 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. We have recently identified a novel genetic locus of Streptococcus gordonii that encodes GspB, a large, cell wall glycoprotein that binds human platelets. The locus also encodes four proteins mediating the intracellular glycosylation of GspB, and seven proteins comprising a specialized export pathway (the accessory Sec system). This system appears to be dedicated to the export of GspB exclusively. It is unknown how the components of this pathway interact to mediate GspB export. Two members, SecA2 and SecY2, are homologs of SecA and SecY of the canonical Sec system, suggesting they may function similarly. The five other components (accessory Sec proteins Asp1 - Asp5) have no significant homology to proteins of known function, but are essential for GspB export. This project seeks to delineate the interactions of the accessory Sec system that mediate GspB export, and in particular, the roles of Asp1, Asp2, and Asp3. Our previous studies indicate that Asp3 binds Asp1, Asp2, and itself, suggesting that these proteins may form complexes.
Aim 1 explores the role of Asp1-3 binding in GspB export. The size and composition of Asp complexes will be assessed by chromatography and co-immunoprecipitation, respectively. The importance of Asp complex formation in vivo will also be tested, by determining whether variants of Asp3 that do not form multimers can still support export.
Aim 2 examines whether Asp1-3 facilitate the interaction of GspB with SecA2 (the motor protein for export). The ability of Asp1-3 (either individually or as complexes) to bind GspB or SecA2 in vitro will be assessed, as measured by native gel electrophoresis, surface plasmon resonance, and isothermal titration calorimetry. The impact of such binding on SecA2 motor function will also be determined.
Aim 3 looks at whether Asp1-3 associate with SecY2/Asp4/Asp5, the putative channel (translocon) for GspB export. The binding of Asp1-3 to the translocon will be examined, as described above. In addition, the in vitro translocation of GspB into proteolipsomes by SecA2 and the translocon, and whether this process requires Asp1-3, will be evaluated. These experiments should provide considerable mechanistic insights as to how the components of the accessory Sec system interact to form a dedicated pathway for GspB export, and in particular, how Asp1, Asp2, and Asp3 contribute to this process. Since this system is conserved among numerous other Gram-positive pathogens, these studies should be highly applicable to other organisms, and may identify novel targets for vaccine development or new classes of therapeutic agents.
GspB is a surface protein of streptococci that promotes the infection of heart valves by these bacteria. This project explores the inner workings of a novel system that transports GspB to the bacterial surface, where it can then function to attach these microbes to human tissue. By examining how GspB is transported, this research may identify unique targets for new vaccines or new classes of antibiotics.
|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|
|Bensing, Barbara A; Loukachevitch, Lioudmila V; McCulloch, Kathryn M et al. (2016) Structural Basis for Sialoglycan Binding by the Streptococcus sanguinis SrpA Adhesin. J Biol Chem 291:7230-40|
|Loukachevitch, Lioudmila V; Bensing, Barbara A; Yu, Hai et al. (2016) Structures of the Streptococcus sanguinis SrpA Binding Region with Human Sialoglycans Suggest Features of the Physiological Ligand. Biochemistry :|
|Bensing, Barbara A; Khedri, Zahra; Deng, Lingquan et al. (2016) Novel aspects of sialoglycan recognition by the Siglec-like domains of streptococcal SRR glycoproteins. Glycobiology :|
|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|
|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|
|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|
|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|
|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|
Showing the most recent 10 out of 47 publications