Bacterial anti-inflammatory lipid mediators

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Project Summary The rapid response of the innate immune system to infections relies on the detection of pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharides, that activate a robust inflammatory response to invading organisms. My research program focuses on an unappreciated countermeasure deployed by commensal bacteria and pathogens that attenuates the immune response to PAMPs. Oleate hydratase (OhyA) is an enzyme that is expressed in commensal bacteria and the important human pathogen Staphylococcus aureus. OhyA stereospecifically hydrates the 9-cis double bond of unsaturated fatty acids to produce 10- hydroxy-fatty acids (hFAs); however, bacteria expressing OhyA do not synthesize unsaturated fatty acids. OhyA substrates are only encountered at the interface of host tissues, where unsaturated fatty acids are abundant. I hypothesize that hFAs produced by commensal or pathogenic organisms serve as signaling molecules that blunt the innate immune response to PAMPs. Indeed, S. aureus ohyA knockouts fail to establish infections in mice, illustrating the importance of OhyA and hFAs to S. aureus virulence. Thus, hFAs, which were first discovered in the gut microbiome, may provide an important mechanism for commensal bacteria to attenuate gastrointestinal inflammation and create tolerant, symbiotic environments. My pilot experiments and some previous reports provide preliminary evidence for the anti-inflammatory action of hFAs, but the mechanism(s) of immune suppression by hFAs are unknown. To address this shortcoming, I will receive formal immunology instruction through coursework and hands-on training in the laboratory of Dr. Paul Thomas, an expert immunologist, during the K99 phase. We will determine whether hFAs directly antagonize extracellular PAMP binding to immune cell receptors, bind to CD1 lipid-presenting molecules for T-cell receptor recognition to antagonize T cell activation, or intercept signaling downstream of PAMP-receptor activation by stimulating G-protein coupled receptors and/or PAPR. The results of these experiments will uncover the mechanistic basis for hFA modification of immune cell responses. OhyA has no mammalian homolog, and validating the importance of OhyA for virulence coupled with the X-ray structures of OhyA will provide the key information required to determine whether OhyA is a candidate target for antimicrobial drug discovery. During the R00 phase, I will test the hypothesis that OhyA is packaged into extracellular vesicles released by S. aureus to interact with surrounding host tissues to form a halo of hFA mediators around the infection site. My findings will provide an atomistic mechanistic understanding of how OhyA binds to and influences membrane structure to promote vesicle formation. The K99/R00 research plan is designed to leverage my expertise in biochemistry and structural biology, while receiving new training in immunology, to advance the understanding of immune regulation by bacteria and launch a productive and successful career in which I will lead a research program to elucidate the role bacteria lipid products play in controlling host responses.