Pathogenic bacteria display polymeric virulence factors to establish and maintain infections. We will investigate the mechanisms through which these polymers are produced and search for small molecule polymer assembly inhibitors. Two types of surface polymers in Gram-positive bacteria will be studied, (i) pili, proteinaceous fibers that project from the cell surface to mediate adhesion to host tissues, and (ii) Wall Teichoic Acids (WTAs), highly abundant glycopolymers that play a fundamental role in maintaining the integrity of the cell wall. Both pili and WTA polymers are important virulence factors, but how they are synthesized is poorly understood.
In aims #1 - 2, we will study how the archetypal SpaA-pilus from Corynebacterium diphtheria is assembled by sortase polymerases. These enzymes catalyze a unique transpeptidation reaction that covalently links adjacent protein pilus subunits together via a lysine isopeptide bond, thereby conferring enormous tensile strength that enables bacterial adherence. By synergistically employing structural, biochemical, cellular and chemical tools, we will learn how sortase polymerases build the pilus shaft and define the structure of the fundamental building block from which all Gram-positive pili are constructed. We will also determine the molecular basis through which pilus biogenesis is terminated via a novel handoff mechanism in which the pilus is transferred between tandemly arranged sortases on the cell surface. This work will have a broad impact, as a wide range of pathogenic microbes use a similar mechanism to assemble their pili.
In aim #3, we will study how Gram-positive bacteria produce WTA using the TarA enzyme, a novel glycosyltransferase that catalyzes the first committed step in polymer biosynthesis. TarA is a promising drug target, as clinically relevant methicillin-resistant Staphylococcus aureus (MRSA) is defective in host colonization and re-sensitized to Beta-lactam drugs when WTA biosynthesis is disrupted. Crystal structures of TarA in its apo- and substrate-bound forms will be determined, facilitating the rational exploration of its catalytic mechanism. High-throughput screening using a novel, cell-based bacterial cytological profiling assay will also be performed to discover small molecule TarA inhibitors that could have useful therapeutic properties. Combined, studies of pilus and WTA biogenesis will provide fundamental insight into the chemistry underlying polymer assembly in Gram-positive bacteria and could lead to novel antibiotics to treat infections caused by MRSA and other multi-drug resistant bacteria.

Public Health Relevance

Bacterial pathogens display protein- and glyco-polymers that play essential roles in microbial cell structure and pathogenicity. We will determine how these polymers are assembled and perform a cell-based screen to identify small molecule polymer assembly inhibitors that could be useful in treating infections caused by methicillin- resistant strains of Staphylococcus aureus.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Macromolecular Structure and Function C Study Section (MSFC)
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Huntley, Clayton C
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University of California Los Angeles
Schools of Arts and Sciences
Los Angeles
United States
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Chang, Chungyu; Amer, Brendan R; Osipiuk, Jerzy et al. (2018) In vitro reconstitution of sortase-catalyzed pilus polymerization reveals structural elements involved in pilin cross-linking. Proc Natl Acad Sci U S A 115:E5477-E5486
Macdonald, Ramsay; Cascio, Duilio; Collazo, Michael J et al. (2018) The Streptococcus pyogenes Shr protein captures human hemoglobin using two structurally unique binding domains. J Biol Chem 293:18365-18377
McConnell, Scott A; Amer, Brendan R; Muroski, John et al. (2018) Protein Labeling via a Specific Lysine-Isopeptide Bond Using the Pilin Polymerizing Sortase from Corynebacterium diphtheriae. J Am Chem Soc 140:8420-8423
Huang, Grace L; Gosschalk, Jason E; Kim, Ye Seong et al. (2018) Stabilizing displayed proteins on vegetative Bacillus subtilis cells. Appl Microbiol Biotechnol 102:6547-6565
Sjodt, Megan; Macdonald, Ramsay; Marshall, Joanna D et al. (2018) Energetics underlying hemin extraction from human hemoglobin by Staphylococcus aureus. J Biol Chem 293:6942-6957
Jacobitz, Alex W; Kattke, Michele D; Wereszczynski, Jeff et al. (2017) Sortase Transpeptidases: Structural Biology and Catalytic Mechanism. Adv Protein Chem Struct Biol 109:223-264
Chan, Albert H; Yi, Sung Wook; Weiner, Ethan M et al. (2017) NMR structure-based optimization of Staphylococcus aureus sortase A pyridazinone inhibitors. Chem Biol Drug Des 90:327-344
Sjodt, Megan; Clubb, Robert T (2017) Nitroxide Labeling of Proteins and the Determination of Paramagnetic Relaxation Derived Distance Restraints for NMR Studies. Bio Protoc 7:
Amer, Brendan R; Macdonald, Ramsay; Jacobitz, Alex W et al. (2016) Rapid addition of unlabeled silent solubility tags to proteins using a new substrate-fused sortase reagent. J Biomol NMR 64:197-205
Sjodt, Megan; Macdonald, Ramsay; Spirig, Thomas et al. (2016) The PRE-Derived NMR Model of the 38.8-kDa Tri-Domain IsdH Protein from Staphylococcus aureus Suggests That It Adaptively Recognizes Human Hemoglobin. J Mol Biol 428:1107-1129

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