Gram-positive adhesive pili are assembled at the cell surface by a series of sortase enzymes which covalently link pilin subunits and then anchor resulting polymers to the cell wall. Prior to interacting with sortase, nascent pilins are translocated out of the cytoplasm, tethered to the exoplasmic face of the membrane, and folded. Although, the mechanism for sortase-mediated pilus assembly has been well described, the manner in which pilin subunits fold within the exoplasm is less clear. The goal of this proposal is to describe an extracellular folding pathway for these virulence factors. Recently structural studies of FimA and SpaA, the pilus shaft proteins expressed by the Actinobacteria Actinomyces oris and Corynebacterium diphtheriae, respectively, revealed insight into this biological problem. The crystal structures for both proteins revealed disulfide bonds with the C-termini. When cysteine residues forming these were mutated, FimA and SpaA were no longer polymerized, but secreted into culture media as monomers and degradation products. Deletion of genes encoding extracellular proteins with predicted thiol-oxidoreductase-like domains produced similar phenotypes suggesting that the pilins require oxidative protein folding. A pathway for oxidative protein folding within Actinobacteria has yet to be elucidated. We hypothesize that disulfide bond forming machinery is required to properly fold pilus proteins and other virulence factors within the exoplasm. Using Actinobacteria A. oris and C. diphtheriae as experimental models, we aim to (1) investigate disulfide bond formation within pilus proteins and their role in pilus assembly, (2) elucidate the mechanism of disulfide bond formation, and (3) determine whether disulfide bond formation is a general folding mechanism to fold both pilus and non-pilus virulence factors. The study of disulfide bond formation within these models will provide new targets to develop antimicrobial drugs.

Public Health Relevance

To infect hosts pathogenic bacteria secrete an arsenal of virulence factors including adhesive pili, which facilitate bacterial adherence to host tissues, cultivate biofilm formation, and modulate host immunity. This study aims to elucidate the pathways that Gram-positive bacteria maintain proper folding of these virulence factors, thus providing targets for the development of new antimicrobial therapeutics which can serve to prevent disease.

National Institute of Health (NIH)
National Institute of Dental & Craniofacial Research (NIDCR)
Predoctoral Individual National Research Service Award (F31)
Project #
Application #
Study Section
NIDR Special Grants Review Committee (DSR)
Program Officer
Frieden, Leslie A
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Texas Health Science Center Houston
Schools of Medicine
United States
Zip Code
Siegel, Sara D; Reardon, Melissa E; Ton-That, Hung (2017) Anchoring of LPXTG-Like Proteins to the Gram-Positive Cell Wall Envelope. Curr Top Microbiol Immunol 404:159-175
Wu, Chenggang; Reardon-Robinson, Melissa Elizabeth; Ton-That, Hung (2016) Genetics and Cell Morphology Analyses of the Actinomyces oris srtA Mutant. Methods Mol Biol 1440:109-22
Reardon-Robinson, Melissa E; Ton-That, Hung (2015) Disulfide-Bond-Forming Pathways in Gram-Positive Bacteria. J Bacteriol 198:746-54
Reardon-Robinson, Melissa E; Osipiuk, Jerzy; Jooya, Neda et al. (2015) A thiol-disulfide oxidoreductase of the Gram-positive pathogen Corynebacterium diphtheriae is essential for viability, pilus assembly, toxin production and virulence. Mol Microbiol 98:1037-50
Reardon-Robinson, Melissa E; Osipiuk, Jerzy; Chang, Chungyu et al. (2015) A Disulfide Bond-forming Machine Is Linked to the Sortase-mediated Pilus Assembly Pathway in the Gram-positive Bacterium Actinomyces oris. J Biol Chem 290:21393-405
Wu, Chenggang; Huang, I-Hsiu; Chang, Chungyu et al. (2014) Lethality of sortase depletion in Actinomyces oris caused by excessive membrane accumulation of a surface glycoprotein. Mol Microbiol 94:1227-41
Reardon-Robinson, Melissa E; Wu, Chenggang; Mishra, Arunima et al. (2014) Pilus hijacking by a bacterial coaggregation factor critical for oral biofilm development. Proc Natl Acad Sci U S A 111:3835-40