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 of redox-regulated processes in the interactions between bacteria and their hosts.

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. Studies of cellular stress responses to HOCl to date have been limited, and have focused largely on lab-adapted E. coli and Bacillus subtilis strains. Our lab takes advantage of genetically tractable model organisms from phyla known to be important members of the microbiota (e.g. symbiotic E. coli strains or the probiotic Lactobacillus reuteri) to define functional roles for genes that may be involved in bacteria-host interactions.

Our long-term goal is to explore the role that HOCl stress response plays in medically important bacteria, particularly focusing on roles in colonization, inflammation, and pathogenesis. The basic science performed in our lab lays the groundwork for a greater understanding 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.

The Architecture of the Bacterial HOCl Response

figure 1Unlike, for example, the bacterial response to the ROS hydrogen peroxide, where a single transcription factor (OxyR in E. coli) controls stress response regulation, we already know of at least 3 HOCl-responsive transcription factors and multiple post-transcriptional regulatory mechanisms leading to HOCl resistance in E. coli (Figure 1). Given that our knowledge of the HOCl stress response is still incomplete, this complexity is surprising. We are using transcriptomic and bioinformatic techniques to fully characterize the bacterial HOCl response and to understand the physiological reason for this complexity, both in E. coli and in other symbiotic bacteria. In particular, we are exploring the global gene responses to HOCl under conditions expected to mimic those found in the gut during chronic inflammatory diseases (such as Crohn’s disease or ulcerative colitis), diseases which are associated with dramatic shifts in the composition of the microbiome.

Molecular Mechanisms of HOCl Defense

figure 2The diversity of the human microbiome is enormous. The gut contains ~1014 bacterial cells from more than 1000 species. While high-throughput sequencing technology has given us access to the more than 5 million unique genes of the microbiome, sequencing alone cannot assign functions to genes with no close matches in reference databases. That assignment depends on genetic and biochemical characterization of novel genes in model organisms.

One example that we are currently working on is the HOCl-specific transcriptional activator RclR and its regulatory target RclA, both of which are required for HOCl resistance in E. coli (Figure 2). The mechanism by which RclR, a regulator found in more than 70 bacterial species, specifically senses HOCl remains to be determined. RclA is a predicted reductase whose physiological substrate is unknown. Intriguingly, using bioinformatic approaches, we have found RclA homologs with similarity scores of e < 10-126 in more than 250 diverse bacterial species, nearly all of which are symbionts or pathogens associated with mammalian host epithelia. We are using genetic and biochemical methods to identify the physiological substrate(s) of RclA in E. coli and explain how RclA protects bacteria against oxidative stress.

Control of Polyphosphate Accumulation

figure 3Perhaps the most striking discovery resulting from recent studies of HOCl response in E. coli is the critical role of inorganic polyphosphate (polyP) as a potent protein-stabilizing chaperone that directly prevents the aggregation of HOCl-damaged proteins (Figure 3). PolyP consists of linear chains of phosphate groups up to 1000 units long. In bacteria, polyP is synthesized by polyphosphate kinase (PPK) and degraded by exopolyphosphatase (PPX), and mutants lacking PPK (∆ppk strains) have severe defects in virulence, stress resistance, persistence, motility, and biofilm formation. However, remarkably little is known about how bacteria regulate polyP accumulation. In E. coli two independent signals (HOCl and the stress alarmone ppGpp) are able to reversibly inhibit PPX activity, allowing polyP to accumulate, but our recent results showthat there are additional regulatory mechanism(s) involved that do not depend on modulation of PPX activity. This mechanism does not depend on transcription of the ppk and ppx genes, requires the stringent response regulator DksA but not the alarmone ppGpp, and probably also involves post-translational activation of PPK by a currently unknown factor or factors. We are taking advantage of the powerful tools available for genetic and physiological analysis of E. coli to identify the regulatory mechanism that leads to stress-dependent  activation of polyP synthesis.

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

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