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 #
5R01AI041513-19
Application #
9603694
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Lu, Kristina
Project Start
1997-07-01
Project End
2019-11-30
Budget Start
2018-12-01
Budget End
2019-11-30
Support Year
19
Fiscal Year
2019
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
Bensing, Barbara A; Li, Qiongyu; Park, Dayoung et al. (2018) Streptococcal Siglec-like adhesins recognize different subsets of human plasma glycoproteins: implications for infective endocarditis. Glycobiology 28:601-611
Yakovenko, Olga; Nunez, Jamie; Bensing, Barbara et al. (2018) Serine-Rich Repeat Adhesins Mediate Shear-Enhanced Streptococcal Binding to Platelets. Infect Immun 86:
Lin, Shun-Mei; Jang, A-Yeung; Zhi, Yong et al. (2017) Vaccination With a Latch Peptide Provides Serotype-Independent Protection Against Group B Streptococcus Infection in Mice. J Infect Dis 217:93-102
Garcia-de-la-Maria, C; Xiong, Y Q; Pericas, J M et al. (2017) Impact of High-Level Daptomycin Resistance in the Streptococcus mitis Group on Virulence and Survivability during Daptomycin Treatment in Experimental Infective Endocarditis. Antimicrob Agents Chemother 61:
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:
Seepersaud, Ravin; Sychantha, David; Bensing, Barbara A et al. (2017) O-acetylation of the serine-rich repeat glycoprotein GspB is coordinated with accessory Sec transport. PLoS Pathog 13:e1006558
Yim, Juwon; Smith, Jordan R; Singh, Nivedita B et al. (2017) Evaluation of daptomycin combinations with cephalosporins or gentamicin against Streptococcus mitis group strains in an in vitro model of simulated endocardial vegetations (SEVs). J Antimicrob Chemother 72:2290-2296
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
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 26:1222-1234
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 :

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