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

> **NIH NIH R01** · RHODE ISLAND HOSPITAL · 2020 · $728,363

## Abstract

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 inv...

## Key facts

- **NIH application ID:** 9927573
- **Project number:** 5R01AI141522-02
- **Recipient organization:** RHODE ISLAND HOSPITAL
- **Principal Investigator:** Wolfgang Peti
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $728,363
- **Award type:** 5
- **Project period:** 2019-05-09 → 2024-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9927573

## Citation

> US National Institutes of Health, RePORTER application 9927573, Mechanism and activity of beta-lactam resistant enzymes in E. faecium and E. faecalis (5R01AI141522-02). Retrieved via AI Analytics 2026-06-11 from https://api.ai-analytics.org/grant/nih/9927573. Licensed CC0.

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