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Post-translocational protein folding in Gram-positive bacteria

Award Number: R01DE025015
ORGANIZATION: NATIONAL INSTITUTE OF DENTAL AND CRANIOFACIAL RESEARCH
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 03/01/2015
PERIOD OF PERFORMANCE END DATE: 08/31/2025

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Post-translocational protein folding in Gram-positive bacteria - 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. .


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2024 ( Subtotal = $565,238 )
2024	2024	UNIVERSITY OF CALIFORNIA, LOS ANGELES	10889 WILSHIRE BLVD STE 700	LOS ANGELES	CA	90024	LOS ANGELES	USA	Oral Diseases and Disorders Research	002	11	8/1/2024	SUPPLEMENT FOR EXPANSION	$89,503
2024	2024	UNIVERSITY OF CALIFORNIA, LOS ANGELES	10889 WILSHIRE BLVD STE 700	LOS ANGELES	CA	90024	LOS ANGELES	USA	Oral Diseases and Disorders Research	001	11	7/31/2024	NON-COMPETING CONTINUATION	$353,206
2024	2024	UNIVERSITY OF CALIFORNIA, LOS ANGELES	10889 WILSHIRE BLVD STE 700	LOS ANGELES	CA	90024	LOS ANGELES	USA	Oral Diseases and Disorders Research	000	10	6/18/2024	SUPPLEMENT FOR EXPANSION	$122,529
														Subtotal = $565,238

Issue Date FY: 2023 ( Subtotal = $360,195 )
2023	2023	UNIVERSITY OF CALIFORNIA, LOS ANGELES	10889 WILSHIRE BLVD STE 700	LOS ANGELES	CA	90024	LOS ANGELES	USA	Oral Diseases and Disorders Research	000	10	7/31/2023	NON-COMPETING CONTINUATION	$360,195
														Subtotal = $360,195

Issue Date FY: 2022 ( Subtotal = $356,379 )
2022	2022	UNIVERSITY OF CALIFORNIA, LOS ANGELES	10889 WILSHIRE BLVD STE 700	LOS ANGELES	CA	90024	LOS ANGELES	USA	Oral Diseases and Disorders Research	000	9	8/25/2022	NON-COMPETING CONTINUATION	$356,379
														Subtotal = $356,379

Issue Date FY: 2021 ( Subtotal = $359,770 )
2021	2021	UNIVERSITY OF CALIFORNIA, LOS ANGELES	10889 WILSHIRE BLVD STE 700	LOS ANGELES	CA	90024	LOS ANGELES	USA	Oral Diseases and Disorders Research	002	8	8/3/2021	NON-COMPETING CONTINUATION	$359,770
2021	2020	UNIVERSITY OF CALIFORNIA, LOS ANGELES	10889 WILSHIRE BLVD STE 700	LOS ANGELES	CA	90024	LOS ANGELES	USA	Oral Diseases and Disorders Research	001	7	2/28/2021	COMPETING CONTINUATION	$239,141
2021	2019	UNIVERSITY OF CALIFORNIA, LOS ANGELES	10889 WILSHIRE BLVD STE 700	LOS ANGELES	CA	90024	LOS ANGELES	USA	Oral Diseases and Disorders Research	000	6	2/28/2021	NON-COMPETING CONTINUATION	-$239,141
														Subtotal = $359,770

Issue Date FY: 2020 ( Subtotal = $372,924 )
2020	2020	UNIVERSITY OF CALIFORNIA, LOS ANGELES	10889 WILSHIRE BLVD STE 700	LOS ANGELES	CA	90095	LOS ANGELES	USA	Oral Diseases and Disorders Research	001	7	9/22/2020	COMPETING CONTINUATION	$372,924
2020	2019	UNIVERSITY OF CALIFORNIA, LOS ANGELES	10889 WILSHIRE BLVD STE 700	LOS ANGELES	CA	90095	LOS ANGELES	USA	Oral Diseases and Disorders Research	000	6	9/21/2020	NON-COMPETING CONTINUATION	$0
														Subtotal = $372,924

Issue Date FY: 2019 ( Subtotal = $358,106 )
2019	2019	UNIVERSITY OF CALIFORNIA, LOS ANGELES	10889 WILSHIRE BLVD STE 700	LOS ANGELES	CA	90095	LOS ANGELES	USA	Oral Diseases and Disorders Research	004	6	2/14/2019	NON-COMPETING CONTINUATION	$355,740
2019	2018	UNIVERSITY OF TEXAS HEALTH SCIENCE CENTER AT HOUSTON, THE	7000 FANNIN ST	HOUSTON	TX	77030	HARRIS	USA	Oral Diseases and Disorders Research	002	4	2/3/2019	NON-COMPETING CONTINUATION	-$39,145
2019	2018	UNIVERSITY OF TEXAS HEALTH SCIENCE CENTER AT HOUSTON, THE	7000 FANNIN ST	HOUSTON	TX	77030	HARRIS	USA	Oral Diseases and Disorders Research	000	4	1/16/2019	NON-COMPETING CONTINUATION	-$275,561
2019	2018	UNIVERSITY OF CALIFORNIA, LOS ANGELES	10889 WILSHIRE BLVD STE 700	LOS ANGELES	CA	90095	LOS ANGELES	USA	Oral Diseases and Disorders Research	003	5	2/3/2019	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$39,145
2019	2018	UNIVERSITY OF CALIFORNIA, LOS ANGELES	10889 WILSHIRE BLVD STE 700	LOS ANGELES	CA	90095	LOS ANGELES	USA	Oral Diseases and Disorders Research	001	5	1/17/2019	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$277,927
														Subtotal = $358,106

Issue Date FY: 2018 ( Subtotal = $391,094 )
2018	2018	UNIVERSITY OF TEXAS HEALTH SCIENCE CENTER AT HOUSTON, THE	7000 FANNIN ST	HOUSTON	TX	77030	HARRIS	USA	Oral Diseases and Disorders Research	001	4	4/26/2018	NON-COMPETING CONTINUATION	$39,110
2018	2018	UNIVERSITY OF TEXAS HEALTH SCIENCE CENTER AT HOUSTON, THE	7000 FANNIN ST	HOUSTON	TX	77030	HARRIS	USA	Oral Diseases and Disorders Research	000	4	2/13/2018	NON-COMPETING CONTINUATION	$351,984
														Subtotal = $391,094

Issue Date FY: 2017 ( Subtotal = $390,071 )
2017	2017	UNIVERSITY OF TEXAS HEALTH SCIENCE CENTER AT HOUSTON	7000 FANNIN ST STE 1006	HOUSTON	TX	77030	HARRIS	USA	Oral Diseases and Disorders Research	000	3	2/7/2017	NON-COMPETING CONTINUATION	$351,063
2017	2017	UNIVERSITY OF TEXAS HEALTH SCIENCE CENTER AT HOUSTON	7000 FANNIN ST STE 1006	HOUSTON	TX	77030	HARRIS	USA	Oral Diseases and Disorders Research	001	3	5/26/2017	NON-COMPETING CONTINUATION	$39,008
														Subtotal = $390,071

Issue Date FY: 2016 ( Subtotal = $389,270 )
2016	2016	UNIVERSITY OF TEXAS, GRADUATE SCHOOL BIOMEDICAL SCIENCE	POST OFFICE BOX 20367	HOUSTON	TX	77225	HARRIS	USA	Oral Diseases and Disorders Research	000	2	2/12/2016	NON-COMPETING CONTINUATION	$389,270
														Subtotal = $389,270

Issue Date FY: 2015 ( Subtotal = $402,647 )
2015	2015	UNIVERSITY OF TEXAS, GRADUATE SCHOOL BIOMEDICAL SCIENCE	POST OFFICE BOX 20367	HOUSTON	TX	77225	HARRIS	USA	Oral Diseases and Disorders Research	000	1	2/4/2015	NEW	$397,547
2015	2015	UNIVERSITY OF TEXAS, GRADUATE SCHOOL BIOMEDICAL SCIENCE	POST OFFICE BOX 20367	HOUSTON	TX	77225	HARRIS	USA	Oral Diseases and Disorders Research	001	1	2/5/2015	NEW	$5,100
														Subtotal = $402,647

Grand Total All Awards = $3,945,694

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Post-translocational protein folding in Gram-positive bacteria

Award Number: R01DE025015

ORGANIZATION: NATIONAL INSTITUTE OF DENTAL AND CRANIOFACIAL RESEARCH

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 03/01/2015

PERIOD OF PERFORMANCE END DATE: 08/31/2025

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