Molecular Mechanisms of Bacterial Stress Response Relevant to Host-Microbe Interactions

PROJECT SUMMARY/ABSTRACT The interface between bacteria and the immune system is absolutely fundamental to human health. The broad, long-term objective of this research project is to understand this interface. The innate immune system controls bacterial colonization of epithelial surfaces with a variety of heme peroxidase enzymes that generate powerful antimicrobial oxidants, including hypochlorous acid (HOCI; the active ingredient in household bleach), hypobromous acid, and hypothiocyanite. Different kinds of bacteria are able to mount a variety of defenses against these toxic chemicals, which determines their ability to survive on host epithelial surfaces. For pathogenic bacteria, these defensive pathways are important for their ability to cause disease, while for health-promoting probiotics and commensal members of the microbiome, they are determinants of how well those species are able to colonize their hosts and survive during periods of inflammation. In response to HOCI, Escherichia coli (a proinflammatory member of the gut microbiota) and other bacteria synthesize large amounts of inorganic polyphosphate (polyP), a universally conserved biopolymer of phosphate up to 1000 units long. PolyP contributes to bacterial survival by, among other functions, stabilizing damaged proteins and regulating protease activity and chromosome replication. However, very little is known about how the production of this protective compound is regulated in response either to HOCI or to other stress conditions. The most highly upregulated genes in HOCl-exposed E. coli are those belonging to the re/ABC operon, controlled by the HOCl-sensing transcription factor RclR. RclA has copper (11) reductase activity, protects E. coli against the combination of HOCI and intracellular copper toxicity, and is conserved among a very wide range of hostcolonizing bacteria, including both health- and disease-associated species. The precise mechanism by which RclA protects bacteria against these stresses remains unclear, however, as do the functions of the RclB and RclC proteins, which are conserved only in proinflammatory enterobacteria. Therefore, the key gaps in understanding addressed in this research proposal are the following: 1. How do bacteria regulate the synthesis of inorganic polyphosphate? 2. How do the RclABCR proteins protect host-associated bacteria against oxidative stress? 3. How do these pathways affect host-microbe interactions? We will address these questions using a validated, collaborative research strategy that uses genetic and transcriptomic analysis of specific stress-sensing regulators to identify relevant genes and pathways in host-associated bacteria, followed by combined genetic, bioinformatic, and biochemical approaches to establish a mechanistic understanding of the relevant microbial physiology.