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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI041513-14
Application #
8416992
Study Section
Bacterial Pathogenesis Study Section (BACP)
Program Officer
GU, Xin-Xing
Project Start
1997-07-01
Project End
2014-11-30
Budget Start
2012-12-01
Budget End
2014-11-30
Support Year
14
Fiscal Year
2013
Total Cost
$357,002
Indirect Cost
$126,678
Name
Northern California Institute Research & Education
Department
Type
DUNS #
613338789
City
San Francisco
State
CA
Country
United States
Zip Code
94121
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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
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
Seo, Ho Seong; Sullam, Paul M (2011) Characterization of the fibrinogen binding domain of bacteriophage lysin from Streptococcus mitis. Infect Immun 79:3518-26
Nallapareddy, Sreedhar R; Sillanpaa, Jouko; Mitchell, Jennifer et al. (2011) Conservation of Ebp-type pilus genes among Enterococci and demonstration of their role in adherence of Enterococcus faecalis to human platelets. Infect Immun 79:2911-20
Pyburn, Tasia M; Bensing, Barbara A; Xiong, Yan Q et al. (2011) A structural model for binding of the serine-rich repeat adhesin GspB to host carbohydrate receptors. PLoS Pathog 7:e1002112
Yen, Yihfen T; Seepersaud, Ravin; Bensing, Barbara A et al. (2011) Asp2 and Asp3 interact directly with GspB, the export substrate of the Streptococcus gordonii accessory Sec System. J Bacteriol 193:3165-74
Baum, Paul D; Sullam, Paul M; Stoddart, Cheryl A et al. (2011) Abacavir increases platelet reactivity via competitive inhibition of soluble guanylyl cyclase. AIDS 25:2243-8
Pyburn, Tasia M; Yankovskaya, Victoria; Bensing, Barbara A et al. (2010) Purification, crystallization and preliminary X-ray diffraction analysis of the carbohydrate-binding region of the Streptococcus gordonii adhesin GspB. Acta Crystallogr Sect F Struct Biol Cryst Commun 66:1503-7

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