High-Throughput Assays for Direct Comparison of Recognition and Entry via Natural and Engineered Receptor Binding Proteins

PROJECT 1: HIGH-THROUGHPUT ASSAYS FOR DIRECT COMPARISON OF RECOGNITION AND ENT RY VIA NATURAL AND ENGINEERED RECEPTOR BINDING PROTEINS SUMMARY/ABSTRACT This project will develop a suite of high-throughput, sequencing-based assays that quantify distinct steps of the phage infection cycle-recognition, entry, replication, packaging, and lysis-to transform phage therapy development for ESKAPE pathogens. Instead of relying on conventional clearance assays that conflate all infection steps and produce binary outcomes, our new approaches yield graded, quantitative readouts that enable precise rate constant estimation for each discrete event. In Aim 1, we will construct a multiplexed nanopore-based assay to measure phage adsorption to Klebsiella pneumoniae both in bacteria-only cultures and in human airway organoid co-cultures to uncover off-target interactions. In Aim 2, we will uncouple recognition and entry from downstream events by packaging reporter cosmids into pseudotyped phages, measuring genome delivery directly in target cells without replication or lysis. We will further leverage barcoded "recombitrons" that attach phage- and strain-specific barcodes for massively multiplexed testing of many phages against many strains in a single experiment. In Aim 3, we will deliver purified phage genomes via electroporation to quantify replication, packaging, and lysis independently of recognition and entry, enabling calculation of distinct rate constants for each phase of infection. By merging these assays with advanced computational pipelines that predict optimal receptor-binding proteins and by validating them in relevant 2D and 3D human tissue models, this work will yield a powerful platform to identify and engineer the most effective phages and phage components for clinical use against antibiotic-resistant K. pneumoniae, while providing a broad framework for all ESKAPE pathogens.