Proper protein folding is critical to cellular function. Disulfide bond-forming machines that facilitate proper protein folding are well recognized in eukaryotes and Gram-negative bacteria. Disulfide bond formation contributes to the overall protein folding process, stabilizing structures and protecting against degradation. In Gram-negative bacteria, this process occurs in the oxidizing periplasmic space and is required a pair of oxidoreductase enzymes DsbA and DsbB. In contrast, little is known about oxidative protein folding in single- membrane Gram-positive bacteria, which are not considered to have periplasms. Specifically, how protein precursors translocated across the cytoplasmic membrane by the general secretion Sec translocon in an unfolded state manage to fold correctly is poorly understood. Recent findings of oxidoreductase-encoding genes in the genome of actinobacteria and Vitamin K epoxide reductase in Mycobacterium tuberculosis, considered as a functional homolog of Escherichia coli DsbB, offer some clue to an oxidative folding mechanism in these organisms. Therefore, our laboratory recently began to investigate this fundamental problem using an experimental model in Actinomyces oris, an actinobacterium known to play an important role in the formation of oral biofilms or dental plaque. By structural analysis, we identified disulfide bonds in FimA of A. oris. FimA is the fimbrial shaft required for biofilm formation and interspecies interactions. We demonstrated that the C-terminal disulfide bond of FimA is essential for fimbrial assembly and biofilm formation. More recently, we revealed that disruption of a disulfide bond in coaggregation factor CafA eliminates A. oris coaggregation with Streptococcus oralis. To find additional factors that affect interspecies interactions, we performed a large-scale screen with a Tn5 transposon mutant library in A. oris and identified coaggregation- defective mutants mapped to genes potentially encoding various components of an oxidative protein folding pathway. By using a multidisciplinary approach that combines genetics, biophysics, biochemistry, crystallography, mass spectrometry, cell-based assays, and models of dental caries and bacterial infection, we aim to elucidate the mechanism of oxidative protein folding in A. oris, to determine the conservation of this pathway in other actinobacteria, and to explore preventive strategies for dental caries and bacterial infections.

Public Health Relevance

This study examines a pathway of how Gram-positive pathogens maintain proper folding of virulence factors during their assembly and explores strategies to block this pathway. A clear understanding of these aspects will provide new targets for anti-infective therapies and prevention of oral biofilm-associated diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE025015-06
Application #
9647439
Study Section
Oral, Dental and Craniofacial Sciences Study Section (ODCS)
Program Officer
Lunsford, Dwayne
Project Start
2018-09-01
Project End
2020-02-29
Budget Start
2019-03-01
Budget End
2020-02-29
Support Year
6
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Dentistry
Type
Schools of Dentistry/Oral Hygn
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
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
Luong, Truc Thanh; Tirgar, Reyhaneh; Reardon-Robinson, Melissa E et al. (2018) Structural Basis of a Thiol-Disulfide Oxidoreductase in the Hedgehog-Forming Actinobacterium Corynebacterium matruchotii. J Bacteriol 200:
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
Wittchen, Manuel; Busche, Tobias; Gaspar, Andrew H et al. (2018) Transcriptome sequencing of the human pathogen Corynebacterium diphtheriae NCTC 13129 provides detailed insights into its transcriptional landscape and into DtxR-mediated transcriptional regulation. BMC Genomics 19:82
Sanchez, Belkys C; Chang, Chungyu; Wu, Chenggang et al. (2017) Electron Transport Chain Is Biochemically Linked to Pilus Assembly Required for Polymicrobial Interactions and Biofilm Formation in the Gram-Positive Actinobacterium Actinomyces oris. MBio 8:
Luong, Truc Thanh; Reardon-Robinson, Melissa E; Siegel, Sara D et al. (2017) Reoxidation of the Thiol-Disulfide Oxidoreductase MdbA by a Bacterial Vitamin K Epoxide Reductase in the Biofilm-Forming Actinobacterium Actinomyces oris. J Bacteriol 199:
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
Siegel, Sara D; Liu, Jun; Ton-That, Hung (2016) Biogenesis of the Gram-positive bacterial cell envelope. Curr Opin Microbiol 34:31-37
Echelman, Daniel J; Alegre-Cebollada, Jorge; Badilla, Carmen L et al. (2016) CnaA domains in bacterial pili are efficient dissipaters of large mechanical shocks. Proc Natl Acad Sci U S A 113:2490-5

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