Chemical Strategies to Map Bacterial Response to Environmental Change

The Carlson Group applies the diverse tools of chemical biology to understand how bacteria harness their limited genome to inhabit nearly every ecological niche on our planet. These “simple” single-celled organisms are remarkable in their ability to respond to and survive in the presence of diverse environmental stressors. This

is largely accomplished through related but specialized proteins that are activated under disparate conditions. With NIH support, the PI has been investigating two protein families that are crucial to a bacteria’s ability to respond to its environment and maintain cell wall integrity, histidine kinases (HKs) and penicillin-binding proteins

(PBPs). The PBPs are required for cell wall construction and are the targets of b-lactams, the most used class of antibiotics in the world. The HKs sense changes in the periplasmic space and transmit this information inside of the cell, allowing bacteria to respond to fluctuating environments. The HKs are also important in infection and

antibiotic resistance. Clearly, a deeper understanding of when, where, and how the PBPs and HKs are functionally differentiated and used to enable cell survival will be crucial in combating the rapidly approaching

“post-antibiotic era.” Chemical probes are an essential tool for the characterization of these differentiating factors. We have pioneered the development of activity-based probes (ABPs) for the PBP and HK families that specifically label catalytically active enzymes, yielding a read-out of their functional state. These molecules are

required to investigate how functionally related proteins differ from one another in their catalytic activation, substrate or ligand recognition, localization, and biomolecular interactions. The overall vision for this MIRA project is to build on the PI’s foundation of results and leadership in this area and apply a chemical approach to

expand the understanding of these two enzyme families. We will achieve this vision by expanding our library of PBP-selective ABPs and applying these chemical probes to investigation of the roles of these proteins in cell growth and division, as well as adaptation to environmental perturbations. We will also optimize the chemical

probes and internalization agents needed to deliver ABPs into the bacterial cytoplasm, enabling global mapping of HK activation upon exposure to stimuli. Finally, we will validate the HKs as viable targets for the development of anti-virulence agents and adjuvants and optimize the potency of our lead compounds.

Our combined expertise in chemical synthesis, ABPs, mass spectrometry-based -omics, biochemistry, and microbiology make us uniquely qualified to untangle the web of apparent functional redundancy within these enzyme families. We also have long-standing collaborators with expertise in the HKs and PBPs, as well as

methods critical to this project. The overall impact of this work will be a drastically increased and rigorously tested understanding of cell wall biosynthesis and signal transduction in bacteria and a suite of freely accessible research tools. Ultimately, the amassed knowledge and tools will enable us to understand and predict how a

signal is propagated into bacterial action and to hijack the involved proteins for disease treatment.