Core 2 Mechanistic Assay Core

Project Summary/Abstract: Mechanistic Assay Core #2 Carbapenem-resistant Acinetobacter baumannii (CRAB) and Klebsiella pneumoniae (CRKP) were categorized in the highest threat category (‘Urgent’) according to the U.S. CDC in 2019. While β-lactam antibiotics (BL) have been used for decades to successfully treat infections by susceptible isolates of AB and KP, resistant isolates

often produce several different types of β-lactamases that can inactivate virtually every BL in monotherapy. Moreover, efflux pumps and loss of outer membrane porin channels present additional clinically relevant resistance mechanisms. In 2017, NIAID set forth the Gram-negative toolbox development program (RFA-AI-16-

081) to create novel approaches that generate mechanistic insights to combat these bacterial ‘superbugs’. Our established team collectively developed a novel mechanistic assay platform as our Gram-negative toolbox (under R01 AI136803). These assays underpin the present P01. The Mechanistic Assay Core (#2) will provide

the mechanistic foundation to rationally optimize clinically relevant combination dosage regimens of available and new antibiotics. This core will mutually integrate all Projects and all Cores, and will provide the latest assays for outer membrane permeability of BL and β-lactamase in inhibitors (BLI). Moreover, it will provide the latest

assays for studying the intracellular drug accumulation and washout kinetics of non-PBP-binding partner antibiotics and for quantifying intracellular energy metabolite concentrations (e.g. ATP). The latter will be particularly relevant for mechanistic studies to characterize non-replicating persisters (NRP), along with

automated confocal microscopy and flow cytometry (with sorting) of bacterial strains labelled with fluorescent protein(s) to determine the rate of protein synthesis. Flow cytometry (with and without sorting) and confocal microscopy will be used to efficiently identify PBP occupancy patterns that yield extensive morphology changes

for combinations of BL, BLI, and non-β-lactam-PBP-binders in Project #1. This approach has proven highly beneficial in R01 AI136803. Moreover, the latest proteomic assays will provide important insights into the expression of proteins involved in antibiotic action (e.g. PBP) and resistance (e.g. β-lactamases, efflux pumps

and outer membrane porin channels). These resistance mechanism studies will be further enhanced by whole- genome sequencing and other molecular techniques, which are particularly relevant for evaluating the ability of optimized dosage regimens to suppress resistance in Projects #2 and #3. For the latter projects, this Core will

provide extracellular drug concentration determinations for the in vitro hollow fiber and mouse studies. Taken together, the Mechanistic Assay Core will provide novel mechanistic insights to support rational optimization of combination dosage regimens in Project #1, and to prospectively validate the synergistic bacterial killing and

resistance suppression of these dosage regimens in Projects #2 & #3. Facilitated by the Administrative Core #1, these mechanistic data will be analyzed by the Mathematical Modeling Core #3 to optimally benefit all Projects.