Understanding how and where a particular virulence protein is displayed to the host is critical to understanding the role that protein contributes to pathogenesis. This issue is of central importance for understanding the pathogenesis of any of the myriad serious diseases caused by the Gram- positive pathogen Streptococcus pyogenes (group A streptococcus). What is remarkable is that is that while this bacterium secretes upwards of 100 different polypeptides, it exclusively uses the General Secretory (Sec) pathway for protein export. A key question is how this single pathway can coordinate the secretion and folding of virulence proteins and how these proteins are then trafficked to their final destinations. An important insight into how secretion, folding and trafficking may be coordinated has come from our identification of the ExPortal, a single membrane microdomain that accumulates a high concentration of Sec translocons and accessory biogenesis factors including HtrA and Sortase. Our hypothesis is that the ExPortal functions as an organelle to promote the interaction of nascent unfolded polypeptides secreted by Sec with accessory factors required for folding and biogenesis in order to coordinate these activities with protein trafficking pathways. A more fundamental understanding of secretion and trafficking will require an understanding of the structural basis of ExPortal organization. In this regard, an important advance has been our observation that the ExPortal has characteristics of a membrane microdomain, in that it has an asymmetric lipid content enriched for specific anionic phospholipids. How this asymmetry is maintained, and whether it is necessary for ExPortal function are questions to be addressed in this proposal. Analysis of ExPortal asymmetry will also allow an investigation of how pathways for membrane targeting of presecretory proteins may direct their post-secretory fate, and how the innate immune system, specifically cationic antimicrobial peptides, may directly target the anionic ExPortal to disrupt secretion of surface proteins and toxins. This latter analysis will also serve as the basis for biochemical characterization of the ExPortal and for genetic screens to identify factors required to support ExPortal organization. This analysis may reveal potential drug targets that can be exploited to disrupt the essential processes of protein secretion and trafficking in S. pyogenes and potentially other pathogenic bacteria. )

Public Health Relevance

We have previously shown that the secretion of toxins and virulence-associated surface proteins by the Gram-positive pathogen Streptococcus pyogenes occurs through a single small region of the membrane that we named the ExPortal. In this project, we will investigate how the ExPortal is assembled by looking at the types of negatively charged lipids that are enriched at the ExPortal, to find streptococcal genes that encode factors required to form the ExPortal and how this unique lipid composition may make it the specific target of host defense peptides with positive charge. This information will be valuable for development of drugs that can mimic these peptides to target the ExPortal to disrupt toxin secretion in S. pyogenes and other pathogenic streptococci.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01AI046433-15
Application #
8625265
Study Section
Bacterial Pathogenesis Study Section (BACP)
Program Officer
GU, Xin-Xing
Project Start
Project End
Budget Start
Budget End
Support Year
15
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Washington University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Port, Gary C; Vega, Luis A; Nylander, Andrew B et al. (2014) Streptococcus pyogenes polymyxin B-resistant mutants display enhanced ExPortal integrity. J Bacteriol 196:2563-77
Vega, Luis Alberto; Port, Gary C; Caparon, Michael G (2013) An association between peptidoglycan synthesis and organization of the Streptococcus pyogenes ExPortal. MBio 4:e00485-13
Guiton, Pascale S; Hannan, Thomas J; Ford, Bradley et al. (2013) Enterococcus faecalis overcomes foreign body-mediated inflammation to establish urinary tract infections. Infect Immun 81:329-39
Guiton, Pascale S; Hung, Chia S; Hancock, Lynn E et al. (2010) Enterococcal biofilm formation and virulence in an optimized murine model of foreign body-associated urinary tract infections. Infect Immun 78:4166-75
Guiton, Pascale S; Hung, Chia S; Kline, Kimberly A et al. (2009) Contribution of autolysin and Sortase a during Enterococcus faecalis DNA-dependent biofilm development. Infect Immun 77:3626-38
Weston, Benjamin F; Brenot, Audrey; Caparon, Michael G (2009) The metal homeostasis protein, Lsp, of Streptococcus pyogenes is necessary for acquisition of zinc and virulence. Infect Immun 77:2840-8
Kline, Kimberly A; Kau, Andrew L; Chen, Swaine L et al. (2009) Mechanism for sortase localization and the role of sortase localization in efficient pilus assembly in Enterococcus faecalis. J Bacteriol 191:3237-47
Lin, Ada; Loughman, Jennifer A; Zinselmeyer, Bernd H et al. (2009) Streptolysin S inhibits neutrophil recruitment during the early stages of Streptococcus pyogenes infection. Infect Immun 77:5190-201
Brenot, Audrey; Weston, Benjamin F; Caparon, Michael G (2007) A PerR-regulated metal transporter (PmtA) is an interface between oxidative stress and metal homeostasis in Streptococcus pyogenes. Mol Microbiol 63:1185-96
Loughman, Jennifer A; Caparon, Michael G (2007) Comparative functional analysis of the lac operons in Streptococcus pyogenes. Mol Microbiol 64:269-80

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