Gray Lab

Research Overview

Bacteria are critical determinants of human health. The ability of pathogens to cause acute infectious disease has been clear since the late 1800’s, but there is a growing consensus that the vast numbers of symbiotic and commensal microbes inhabiting our bodies also have a profound effect on our health and well-being. Our laboratory uses a combination of bacterial physiology, genetics, and biochemistry to explore the role and regulation of pathways involved in the interactions between bacteria and their hosts, with a particular focus on understanding how bacteria sense and respond to environmental stressors produced by the innate immune system.

The basic science performed in our lab lays the groundwork for a greater understanding of how bacteria respond to their environments and of the factors controlling the dynamics and composition of the human microbiome. Ultimately, we hope this will lead to new therapeutic strategies that could either sensitize pathogenic bacteria to killing by the immune system or make health-promoting bacteria more robust.

Control of Polyphosphate Accumulation

One long-known but poorly understood element of bacterial stress response is the production of inorganic polyphosphate (polyP), a linear biopolymer of phosphate up to 1,000 units in length that is synthesized by the widely conserved bacterial enzyme polyP kinase (PPK). In response to a variety of stressful changes in conditions, including nutrient limitation, heat, salt, and hypochlorous acid (HOCl) treatment, E. coli transiently accumulates large amounts of polyP. The physiological functions of polyP are not fully understood, but it is known to promote survival under different stress conditions by stabilizing unfolded proteins, chelating toxic metals, acting as a phosphate store, increasing translation fidelity, and as a second messenger that regulates the activity of a variety of proteins. Importantly, in many bacterial pathogens, deleting the gene encoding PPK (ppk) results in the loss of the ability to cause disease, indicating that polyP metabolism may be a potential therapeutic target for use against bacterial infections.

polyP regulationWe know surprisingly little about the mechanism by which polyP synthesis is regulated. Several different regulators have been implicated in modulating polyP production in E. coli under different stress conditions, including RpoS, the PhoB and PhoU regulators of phosphate transport, and the ppGpp synthases RelA and SpoT , but no convincing mechanistic model has been developed that explains how any of these systems controls polyP accumulation or why different regulators appear to be required under different conditions. Transcription of the operon containing ppk and ppx, which encodes the exopolyphosphatase PPX, does not increase upon stress treatment in E. coli, and while ppGpp potently inhibits degradation of polyP by PPX, neither PPX nor ppGpp is necessary for the induction of polyP by nutrient limitation stress. However, we have recently reported that the stringent response regulator DksA, which normally works in concert with ppGpp, is required for polyP synthesis under those conditions, and this phenotype can be reverted by deletion of the RNA polymerase-binding elongation factor GreA, indicating that there is an important role for transcription in polyP control.

Current work in the lab is focused on understanding the control of polyP both at a transcriptional level, where the alternative sigma factors RpoN and RpoE appear to play key roles, and the post-transcriptional level, by which the activity of the PPK enzyme itself appears to be regulated by a currently unknown mechanism.

Molecular Mechanisms of HOCl Defense

Cellular responses to reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), superoxide, (O2-), and hydroxyl radicals (OH) are well known as critical factors in virulence, development, and physiology. Hypochlorous acid (HOCl), the powerful oxidant that is the active component of household bleach, is one of the most commonly used disinfectants in the world, responsible for preventing as many as 70,000 deaths per day from water-borne disease. It is less well known that HOCl is naturally generated during the antimicrobial oxidative burst of neutrophils and has recently been shown to play a role in controlling bacterial populations in the animal gut. HOCl is more reactive and more toxic than ROS because it reacts with nearly all biomolecules and causes extensive protein unfolding and aggregation. Oxidative damage caused by excessive HOCl production is also implicated in many human diseases, including chronic inflammation, atherosclerosis, Crohn’s disease, and cancer. Despite its important disinfection functions both in drinking water and when produced within our own bodies, little is known about how bacteria sense or respond to HOCl stress, especially when compared to the extensive literature on bacterial responses to ROS.

Rcl protein treeOne example that we are currently working on is the HOCl-specific transcriptional activator RclR and its regulatory targets RclA, RclB, and RclC, all of which are required for HOCl resistance in E. coli, and are widely conserved among bacteria that live on animal epithelia. The mechanism by which RclR, a regulator found in more than 70 bacterial species, specifically senses HOCl remains to be determined. RclB and RclC are periplasmic and inner membrane proteins of currently unknown function.

However, the best-conserved of the Rcl proteins is RclA, which we have recently found to be specifically required for E. coli to resist the combination of HOCl and intracellular copper, the first indication that these two antimicrobial compounds deployed by the innate immune system interact in a physiologically significant way. We are currently working to understand this interaction and to clarify the molecular mechanism by which the RclABC proteins protect bacteria against stress.

copper phenotype

This project is being carried out in collaboration with Dr. Frederick Stull (Western Michigan University) and Dr. Nichole Broderick (Johns Hopkins University).

Understanding and Preventing Neonatal Sepsis

sepsisLate-onset sepsis (LOS) is a leading cause of morbidity and mortality in premature infants. It is thought to be triggered by perturbations of the developing microbiome (dysbiosis) that leads to systemic spread of commensal microbes. Dysbiosis is far more common in preterm infants than in full-term infants, and is thought to underlie their heightened susceptibility to LOS. Various lactic acid bacteria (LAB) are used clinically as probiotics to prevent dysbiosis in the preterm microbiome, but efficacy has proven quite variable and there is limited evidence to guide dose or rational choice of bacterial species and strain. The mechanism(s) by which LAB prevent neonatal dysbiosis is poorly understood.

Using a newly-developed mouse model of LOS, we are currently working to identify the molecular mechanisms by which a specific strain of Ligilactobacillus murinus prevents dysbiosis, with the ultimate goal of identifying or engineering probiotic LAB able to prevent LOS in human infants.

This work is being done in collaboration with Dr. Casey Weaver (UAB Department of Pathology).

© 2021-01-23,All Rights Reserved.
Gray Laboratory, University of Alabama at Birmingham Feedback/Questions

Design by Big Rose Web Design

UAB Department of Microbiology
654 Bevill Biomedical Research Building
1720 2nd Avenue South
Birmingham, AL 35294

Contact Info