# High-throughput characterization of antimicrobial peptide-PhoPQ interactions

> **NIH NIH R01** · RICE UNIVERSITY · 2022 · $377,111

## Abstract

Project Summary/Abstract
 The human immune system produces at least 140 different antimicrobial peptides (AMPs) to kill invading
bacteria. However, pathogenic bacteria use specialized pathways called two-component systems (TCSs) to
detect these AMPs and activate the expression of AMP-resistance and virulence genes. This response enables
pathogens to survive immune attacks and mount deadly infections. Therefore, elucidating the mechanisms by
which peptides interact with TCSs is critical to understanding how infections progress. This knowledge could
also lead to the design of new antimicrobial drugs that interfere with TCS-mediated AMP sensing. Gram-negative
Enterobacteriaceae, such as the common pathogen Salmonella Typhimurium, cause 200,000 infections and
10,000 deaths in the United States each year. The most important AMP-sensing TCS in Gram-negative
Enterobacteriaceae is named PhoPQ. Here, the membrane bound histidine kinase PhoQ senses AMPs and
responds by phosphorylating the cytoplasmic response regulator PhoP, which activates a gene expression
response. Though its interactions with a small number of model AMPs have been characterized, little is known
about the broader peptide binding and sensing capabilities of PhoQ. The major limitations have been the cost
and time required to chemically synthesize peptides and characterize their effects on TCSs using traditional
microbiological or biochemical methods. In preliminary work, we have developed a new technology named
SLAY-TCS that combines bacterial peptide display, fluorescence-activated cell sorting, and next-generation
DNA sequencing to measure how S. Typhimurium PhoQ responds to millions of peptides in a single experiment.
Using SLAY-TCS, we have already revealed that PhoQ senses a far wider range of peptides than previously
known. Here, we propose to use SLAY-TCS to characterize how S. Typhimurium PhoQ responds to nearly every
AMP produced by the human immune system, and thousands of mutants thereof, in order to reveal the identities,
sequence motifs, and biophysical properties of PhoQ-activating peptides (Aim 1). We will also combine this
approach with PhoQ mutational analyses to reveal how PhoQ sensing specificity has evolved across diverse
pathogens, which may have enabled them to adapt to different biogeographical locations in vivo (Aim 2). Finally,
we will use SLAY-TCS to perform the first large-scale characterization of peptide inhibitors of PhoQ, and explore
the efficacy of the strongest inhibitors we identify in preventing S. Typhimurium virulence in primary mouse
macrophages (Aim 3). The work in Aim 3 will reveal mechanisms by which exogenously-delivered peptides can
inhibit PhoQ, and could lead to the design of novel antimicrobial therapeutics based on modified peptides in the
future. Taken together, this proposal will substantially enhance our understanding of how a dangerous family of
bacteria causes infections in humans and accelerate the design of sorely-needed antimicrobial ...

## Key facts

- **NIH application ID:** 10378042
- **Project number:** 5R01AI155586-02
- **Recipient organization:** RICE UNIVERSITY
- **Principal Investigator:** Jeffrey Jay Tabor
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $377,111
- **Award type:** 5
- **Project period:** 2021-03-25 → 2026-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10378042

## Citation

> US National Institutes of Health, RePORTER application 10378042, High-throughput characterization of antimicrobial peptide-PhoPQ interactions (5R01AI155586-02). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10378042. Licensed CC0.

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