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Structural basis for cell surface siganling by a Gram-negative bacteria sigma-regulator

Award Number: R01GM126207
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 09/20/2018
PERIOD OF PERFORMANCE END DATE: 08/31/2023

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Structural basis for cell surface siganling by a Gram-negative bacteria sigma-regulator - PROJECT SUMMARY/ABSTRACT The CDC recently released a report detailing antibiotic resistant threats in the US. Of particular emphasis in the CDC report is the increased prevalence of multidrug-resistant, Gram-negative bacteria (MDR- GNB) and the need to develop the next generation of antibiotics to combat them. All Gram-negative bacteria rely on a set of homologous, yet highly-specific, outer membrane TonB-dependent transporters (TBDTs) to import critical nutrients from their environment, especially metals like iron, which are bound by high-affinity, metal chelating compounds called siderophores. Recent antibiotic developments have shown that siderophore-antibiotic conjugates can be selectively targeted to specific bacteria, and that this delivery mechanism overcomes several key antibiotic resistance mechanisms. A significant limitation of this delivery system is the low expression levels of the TBDTs. However, a subset of these TBDTs controls their own expression through a cell-surface signaling (CSS) process that up-regulates their own expression. The long- term objective of this proposal is to understand the CSS regulatory process and manipulate TBDT expression to enhance siderophore-antibiotic conjugate therapy for treatment of MDR-GNB infections. The results of this proposal will help elucidate the structural basis for CSS by a Gram-negative bacteria sigma-regulator. As a model system the pseudobactin BN7/8 transport system from Psuedomonas putida that consists of the TBDT, PupB, the inner membrane σ-regulator, PupR, and the cytoplasmic σ-factor, PupI, is being used. To accomplish this proposal's objective the following three specific aims will be pursued: 1) establish that PupR anti-σ-factor domain dimerization influences transcriptional activation by PupI, 2) identify the structural determinants and delineate the role of the PupR:PupB periplasmic interactions on the stability of the PupR periplasmic C-terminal CSS domain (CCSSD), and 3) determine changes in the full-length PupB:PupR CCSSD complex in the presence and absence of its cognate siderophore, pseudobactin BN7/8. For the successful completion of these aims a multidisciplinary approach; including X-ray crystallography, small-angle X-ray scattering, molecular biology, cellular assays, and biophysical techniques such as isothermal titration calorimetry and circular dichroism spectroscopy; will be employed. This research will provide critical structural information about a σ-regulator; explain how it interacts with a σ-factor at the inner membrane, and the extent to which periplasmic conformational changes between the TBDT and σ-regulator lead to proteolytic degradation that is important for controlling transcriptional activation.


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: 2025 ( Subtotal = -$90 )
2025	2021	NORTH DAKOTA STATE UNIVERSITY	1340 ADMINISTRATION AVE	FARGO	ND	58105	CASS	USA	Biomedical Research and Research Training	000	4	4/2/2025	NON-COMPETING CONTINUATION	-$53
2025	2021	NORTH DAKOTA STATE UNIVERSITY	1340 ADMINISTRATION AVE	FARGO	ND	58105	CASS	USA	Biomedical Research and Research Training	001	4	4/2/2025	SUPPLEMENT FOR EXPANSION	-$37
														Subtotal = -$90

Issue Date FY: 2023 ( Subtotal = $0 )
2023	2021	NORTH DAKOTA STATE UNIVERSITY	1310 BOLLEY DR	FARGO	ND	58105	CASS	USA	Biomedical Research and Research Training	000	4	9/4/2023	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2021 ( Subtotal = $489,943 )
2021	2021	NORTH DAKOTA STATE UNIVERSITY	1919 UNIVERSITY DR N, STE 102	FARGO	ND	58102	CASS	USA	Biomedical Research and Research Training	000	4	8/26/2021	NON-COMPETING CONTINUATION	$290,000
2021	2021	NORTH DAKOTA STATE UNIVERSITY	1919 UNIVERSITY DR N, STE 102	FARGO	ND	58102	CASS	USA	Biomedical Research and Research Training	001	4	8/30/2021	SUPPLEMENT FOR EXPANSION	$199,943
														Subtotal = $489,943

Issue Date FY: 2020 ( Subtotal = $290,000 )
2020	2020	NORTH DAKOTA STATE UNIVERSITY	1919 UNIVERSITY DR N, STE 102	FARGO	ND	58102	CASS	USA	Biomedical Research and Research Training	000	3	8/17/2020	NON-COMPETING CONTINUATION	$290,000
														Subtotal = $290,000

Issue Date FY: 2019 ( Subtotal = $290,000 )
2019	2019	NORTH DAKOTA STATE UNIVERSITY	1919 UNIVERSITY DR N, STE 102	FARGO	ND	58102	CASS	USA	Biomedical Research and Research Training	000	2	8/29/2019	NON-COMPETING CONTINUATION	$290,000
														Subtotal = $290,000

Issue Date FY: 2018 ( Subtotal = $290,000 )
2018	2018	NORTH DAKOTA STATE UNIVERSITY	1919 UNIVERSITY DR N, STE 102	FARGO	ND	58102	CASS	USA	Biomedical Research and Research Training	000	1	9/20/2018	NEW	$290,000
														Subtotal = $290,000

Grand Total All Awards = $1,359,853

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Structural basis for cell surface siganling by a Gram-negative bacteria sigma-regulator

Award Number: R01GM126207

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

PERIOD OF PERFORMANCE START DATE: 09/20/2018

PERIOD OF PERFORMANCE END DATE: 08/31/2023

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