The serine-rich repeat (SRR) glycoproteins are a unique family of adhesins that are found in numerous species of Gram-positive bacteria. These cell wall-anchored surface proteins bind a wide range of host ligands, and are associated with increased virulence in a diversity of infections, including endocarditis, meningitis, and pneumonia. The biogenesis of SRR glycoproteins requires both the intracellular glycosylation of the adhesins, and their export to the bacterial cell surface by a specialized transporter, the accessory Sec (aSec) system. This export pathway is dedicated exclusively to the transport of SRR adhesins, and is comprised of SecA2 (the motor protein for transport), SecY2 (the transmembrane channel), and at least three accessory Sec proteins (Asps). The goal of this project is to better define the mechanisms for SRR glycoprotein export by the aSec system and to determine how transport is coordinated with glycosylation. We hypothesize that the biogenesis of SRR glycoproteins requires the precise interplay of glycosylation and export, and that Asps1-3 are bifunctional proteins that serve as a nexus for these two processes. By accurately coordinating glycosylation and transport, the Asps assure that that the binding properties of the SRR adhesins are optimized. To address these hypotheses, we will use GspB (an SRR protein of Streptococcus gordonii) as a model for SRR biogenesis. GspB mediates streptococcal binding to human platelets and enhances virulence in the setting of infective endocarditis.
Aim 1 will examine the mechanisms by which Asps1-3 control the interaction of SecA2 with GspB, as measured by changes in SecA2 conformation and ATPase activity. The binding sites on SecA2 for the Asps will also be identified.
Aim 2 will explore whether Asp1 modulates Asp3-GspB binding, such that GspB is engaged and then transported by SecA2.
Aim 3 will investigate the roles of Asps1-3 in glycosylation, and whether the correct (WT) glycosylation of GspB only occurs during aSec transport. We will generate point mutations in Asps1-3 that have no impact on transport, but result in altered glycoforms of GspB. Analysis of the glycan composition and structure of these variants will reveal the precise roles of each Asp in GspB glycosylation.
Aim 4 will assess the importance of accurate glycosylation on virulence, as measured by GspB- mediated binding to human platelets in vitro, and a well-established animal model of infective endocarditis. These studies will provide fundamental insights into the biogenesis of this important family of adhesins, and the roles of the Asps in glycosylation and transport. In turn, this research may lead to innovative antimicrobial therapies that specifically target the glycosylation or transport of SRR adhesins.

Public Health Relevance

A key step in many infections is the attachment of microbes onto host tissues. This research examines how bacteria such as streptococci produce a family of specialized proteins on their surface (the SRR glycoproteins) that mediate binding to human cells. By examining the pathways for SRR glycoprotein production, this project will provide important insights into a central mechanism for infection, and may lead to novel antimicrobial therapies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI041513-17
Application #
9172643
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
GU, Xin-Xing
Project Start
1997-07-01
Project End
2019-11-30
Budget Start
2016-12-01
Budget End
2017-11-30
Support Year
17
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Northern California Institute Research & Education
Department
Type
DUNS #
613338789
City
San Francisco
State
CA
Country
United States
Zip Code
94121
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

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