# Project 1 Mechanistic Project

> **NIH NIH P01** · UNIVERSITY OF FLORIDA · 2024 · $852,109

## Abstract

Project Summary/Abstract Project #1
Acinetobacter baumannii (AB) and Klebsiella pneumoniae (KP) are bacterial “superbugs” listed in the highest
threat category (‘Urgent’) by the United States Centers for Disease Control and Prevention. Globally, more than
100,000 deaths per year are attributable to resistance in carbapenem-resistant isolates (i.e. CRAB and CRKP).
New antibiotics, such as β-lactam (BL) / β-lactamase inhibitor (BLI) combinations, have become available to
combat CRKP. The combination of sulbactam with durlobactam to combat CRAB is currently under FDA review.
However, no clinically available BLI covers metallo-β-lactamases (MBL), which are often produced together with
OXA-enzymes in CRAB as well as with KPC and ESBL in CRKP. For serious infections, such as ventilator-
associated bacterial pneumonia (VABP), the clinical outcomes remain suboptimal, even with this latest
generation of BL/BLI, especially for patients with a high bacterial burden. The BL have been used to successfully
treat infections by susceptible isolates of AB and KP for decades which clearly proves that the penicillin-binding
proteins (PBP; i.e. the high affinity targets of all BL) are highly valuable antibiotic targets. While it is known that
each BL inactivates one or multiple PBPs, which have different biochemical functions, our preliminary data
present the first comprehensive dataset on PBP binding of ≥45 BL and BLI in lysed cells of KP and AB. In our
Gram-negative toolbox R01 (AI136803), we created a series of target site penetration assays for PBP-binders
and other antibiotics, as well as intact-cell PBP binding assays that leverage ‘clickable’ probe BL. We further
developed a highly efficient approach to identify BL-induced bacterial morphology changes using automated
confocal microscopy and flow cytometry that can thereby reverse-engineer PBP occupancy patterns directly in
intact cells of CRAB and CRKP. We found that the expression of some PBPs changes extensively over time (i.e.
with growth phase). This may be important for serious infections with a high bacterial burden. Likewise, certain
non-essential PBPs are highly expressed and may serve as decoy targets that prevent BL and other PBP-binders
from inactivating the most important PBPs. Studies in Aim 1 of this Project (#1) will identify the optimal sets of
PBPs that need to be simultaneously inactivated to maximize killing of bacteria at high densities, including rapidly
and slowly replicating bacteria, as well as non-replicating persisters (NRP), which are hard to kill. In Aim 2, this
Project will further optimize strategies for maximizing PBP binding by partner antibiotics. We will use an
innovative combination of biochemical, molecular, chemical biology, and mathematical modeling approaches, in
close integration with the Mechanistic Assay Core #2 and the Mathematical Modeling Core #3. This will be greatly
facilitated by the Administrative Core #1. These novel mechanistic insights will underpin the tran...

## Key facts

- **NIH application ID:** 10763470
- **Project number:** 1P01AI179409-01
- **Recipient organization:** UNIVERSITY OF FLORIDA
- **Principal Investigator:** Jurgen Bernd Bulitta
- **Activity code:** P01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $852,109
- **Award type:** 1
- **Project period:** 2024-08-08 → 2029-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10763470

## Citation

> US National Institutes of Health, RePORTER application 10763470, Project 1 Mechanistic Project (1P01AI179409-01). Retrieved via AI Analytics 2026-06-12 from https://api.ai-analytics.org/grant/nih/10763470. Licensed CC0.

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