Project 1 Mechanistic Project

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 translational development and prospective validation of rationally optimized combination dosing strategies with new and available BL, BLI, and non-β-lactam-PBP-binders, as well as other partner antibiotics in Projects #2 & #3.