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Mechanism and activity of beta-lactam resistant enzymes in E. faecium and E. faecalis

Award Number: R01AI141522
ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 05/09/2019
PERIOD OF PERFORMANCE END DATE: 04/30/2025

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Mechanism and activity of beta-lactam resistant enzymes in E. faecium and E. faecalis - Enterococci (e.g. E. faecalis and E. faecium) cause severe and often fatal nosocomial and community-acquired infections. Therapy of enterococcal infections is frequently compromised by their decreased susceptibility (increased resistance) to many classes of antibiotics, including β-lactams. This resistance is overwhelmingly attributable to the expression of low-affinity penicillin-binding proteins PBP4 (E. faecalis) and PBP5 (E. faecium), both of which are members of a family of low-affinity PBPs that also includes PBP2a from methicillin-resistant S. aureus. In the clinical setting, E. faecium strains show widespread high-level penicillin resistance due to amino acid substitutions, while similar highly-resistant E. faecalis strains are rare. Building on our extensive structural and functional preliminary data, we will leverage the unique synergy of scientific expertise of the investigators to answer the following key fundamental questions: how do low affinity PBPs bind and catalyze transpeptidation, how do sequence changes in these PBPs further reduce their affinity for β-lactam antibiotics while retaining their ability to synthesize peptidoglycan, and what cellular factors beyond low affinity PBP substitutions augment levels of resistance expressed by clinical strains? To answer these questions, we will pursue four specific aims that integrate structural biology, chemical synthesis, biochemistry and microbiology. Aim 1 will use structural biology, especially biomolecular NMR spectroscopy, to determine why PBP5 is an inferior target of β-lactam antibiotics. Our extensive preliminary data shows that this tour-de-force effort (at ~75 kDa, PBP5 is the second largest single-chain protein studied using NMR spectroscopy) is not only feasible but, combined with our extensive crystallographic data, will reveal why β-lactams only poorly inhibit PBP5 and, by extension, the entire family of low affinity PBPs. Aims 2 and 3 will use newly developed chemical synthesis schemes coupled with structure and dynamics (NMR spectroscopy) to determine how, at a molecular level, these PBPs catalyze transpeptidation. We have achieved high-yield syntheses of PBP5-specific pentapeptide precursors and variants of lipid II, enabling us to use NMR spectroscopy and transpeptidase assays to determine how substrates bind and ultimately become cross-linked by PBP5. The impact of resistance-causing mutations in PBP5 on transpeptidase activity will also be determined. Aim 4 will identify the orthogonal factors that contribute to resistance in E. faecalis. Our preliminary data suggest that E. faecalis PBP2 likely contributes to β-lactam resistance in the highly resistant LS4828 E. faecalis strain. We will quantify the contribution of PBP2 to LS4828 β-lactam resistance. In parallel, we will use BioID (proximity labeling) to identify PBP4 and PBP2 interacting proteins (our recently published crystallographic data revealed that the PBP4 N-terminal domains are dynamic and are likely involved in protein interactions). Together, these studies will reveal the structural and functional details of enterococcal low-affinity PBP function, providing critical data upon which to base future strategies for inhibiting these important enzymes.


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: 2026 ( Subtotal = $0 )
2026	2023	RHODE ISLAND HOSPITAL	593 EDDY ST	PROVIDENCE	RI	02903	PROVIDENCE	USA	Allergy and Infectious Diseases Research	000	5	1/26/2026	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2025 ( Subtotal = $751,387 )
2025	2025	RHODE ISLAND HOSPITAL	593 EDDY ST	PROVIDENCE	RI	02903	PROVIDENCE	USA	Allergy and Infectious Diseases Research	000	6	8/11/2025	COMPETING CONTINUATION	$751,387
														Subtotal = $751,387

Issue Date FY: 2023 ( Subtotal = $701,209 )
2023	2023	RHODE ISLAND HOSPITAL	593 EDDY ST	PROVIDENCE	RI	02903	PROVIDENCE	USA	Allergy and Infectious Diseases Research	000	5	4/24/2023	NON-COMPETING CONTINUATION	$701,209
														Subtotal = $701,209

Issue Date FY: 2022 ( Subtotal = $719,203 )
2022	2022	RHODE ISLAND HOSPITAL	593 EDDY ST	PROVIDENCE	RI	02903	PROVIDENCE	USA	Allergy and Infectious Diseases Research	000	4	5/18/2022	NON-COMPETING CONTINUATION	$719,203
														Subtotal = $719,203

Issue Date FY: 2021 ( Subtotal = $724,573 )
2021	2021	RHODE ISLAND HOSPITAL	593 EDDY ST	PROVIDENCE	RI	02903	PROVIDENCE	USA	Allergy and Infectious Diseases Research	001	3	4/9/2021	NON-COMPETING CONTINUATION	$724,573
2021	2020	RHODE ISLAND HOSPITAL	593 EDDY ST	PROVIDENCE	RI	02903	PROVIDENCE	USA	Allergy and Infectious Diseases Research	000	2	3/2/2021	NON-COMPETING CONTINUATION	$0
														Subtotal = $724,573

Issue Date FY: 2020 ( Subtotal = $728,363 )
2020	2020	RHODE ISLAND HOSPITAL	593 EDDY ST	PROVIDENCE	RI	02903	PROVIDENCE	USA	Allergy and Infectious Diseases Research	001	2	9/29/2020	NON-COMPETING CONTINUATION	$0
2020	2020	RHODE ISLAND HOSPITAL	593 EDDY ST	PROVIDENCE	RI	02903	PROVIDENCE	USA	Allergy and Infectious Diseases Research	000	2	4/3/2020	NON-COMPETING CONTINUATION	$728,363
														Subtotal = $728,363

Issue Date FY: 2019 ( Subtotal = $777,570 )
2019	2019	RHODE ISLAND HOSPITAL	593 EDDY ST	PROVIDENCE	RI	02903	PROVIDENCE	USA	Allergy and Infectious Diseases Research	000	1	5/8/2019	NEW	$777,570
														Subtotal = $777,570

Grand Total All Awards = $4,402,305

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Mechanism and activity of beta-lactam resistant enzymes in E. faecium and E. faecalis

Award Number: R01AI141522

ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

PERIOD OF PERFORMANCE START DATE: 05/09/2019

PERIOD OF PERFORMANCE END DATE: 04/30/2025

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