# Post-translocational protein folding in Gram-positive bacteria

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA LOS ANGELES · 2023 · $360,195

## Abstract

PROJECT SUMMARY
From eukaryotes to prokaryotes, proper protein folding is essential to cellular function. Disulfide bond formation
contributes to the overall protein folding process, stabilizing structures and protecting against degradation.
Disulfide bond-forming machines that facilitate proper protein folding are well recognized in eukaryotes and
Gram-negative bacteria. In contrast, a major disulfide bond-forming pathway has only recently been identified in
the Gram-positive Actinobacteria Actinomyces oris, Corynebacterium diphtheriae, and Corynebacterium
matruchotii. In these organisms, a membrane-bound thiol-disulfide oxidoreductase named MdbA catalyzes post-
translocational folding of exported proteins. Importantly, genetic disruption of mdbA abrogates assembly of
adhesive pili and biofilm formation, alters cell morphology, and attenuates bacterial virulence. Nonetheless, how
actinobacterial cells cope with stress and protein misfolding is not well understood. To address this fundamental
question, we began to analyze the proteomes of Actinobacteria and found that most PBPs harbor 2 or more
cysteines; intriguingly, deletion of pbp1A or pbp1B in C. diphtheriae resulted in a cell morphology defect that
mirrors that of mdbA mutations. With a genetic approach, we then screened for viable suppressor mutants when
C. diphtheriae mdbA mutant cells grown at non-permissive temperatures. Serendipitously, we discovered
another thiol-disulfide oxidoreductase, which we named TsdA (tsd for temperature-sensitive dsb-forming).
Preliminary studies reveal that TsdA contains a thioredoxin-like fold found in MdbA, suggesting that TsdA may
serve as a specialized disulfide bond-forming machine to encounter cell stress. Finally, we identified a potential
protein disulfide bond isomerase that may serve as a safeguarding system to rescue misfolded proteins. As we
continue employing A. oris and C. diphtheriae as experimental models in this renewal application, by using a
multidisciplinary approach that combines genetics, biochemical and biofilm assays, and crystallography, we aim
to examine the molecular coupling between oxidative protein folding and cell wall biosynthesis in Actinobacteria,
to elucidate the mechanism of oxidative protein folding mediated by a compensatory thiol-oxidoreductase
machine in response to stress, and to elucidate a pathway for protein disulfide bond isomerization in
Actinobacteria.
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## Key facts

- **NIH application ID:** 10691229
- **Project number:** 5R01DE025015-10
- **Recipient organization:** UNIVERSITY OF CALIFORNIA LOS ANGELES
- **Principal Investigator:** Hung Ton-That
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $360,195
- **Award type:** 5
- **Project period:** 2015-03-01 → 2025-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10691229

## Citation

> US National Institutes of Health, RePORTER application 10691229, Post-translocational protein folding in Gram-positive bacteria (5R01DE025015-10). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/10691229. Licensed CC0.

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