Characterization of a conserved streptococcal membrane protein affecting manganese utilization - PROJECT SUMMARY Manganese uptake has been shown to be essential for oxygen tolerance, oxidative stress resistance, and virulence in streptococci. Through our work with the oral commensal Streptococcus sanguinis and the work of others, the role of manganese in oxidative stress resistance is partially understood. Yet, many fundamental questions remain. We have previously shown that S. sanguinis strains containing mutations in the high-affinity manganese ABC transport system lose the ability to grow in aerobic serum. This growth defect can be corrected by the addition of manganese. We have identified suppressor mutants that have regained the ability to grow under these conditions, and yet are indistinguishable from the parent transporter mutants with regard to cellular levels of manganese. Genome sequencing of these suppressor strains identified mutations in a gene encoding a highly conserved integral membrane protein of unknown function. Complete deletion of this gene produced the same phenotype. Until recently, no structural or functional studies had been performed with the suppressor protein encoded by this gene or any other member of the large family to which this protein belongs. Recently, an integral membrane protein of unknown function in humans was characterized as an acyltransferase and was shown to have predicted structural homology to the S. sanguinis suppressor protein family, although the structure of the human protein was not determined experimentally. Based on that data and our new preliminary data, we hypothesize that the suppressor protein is an acyltransferase that incorporates polyunsaturated fatty acids into the S. sanguinis membrane, and that oxidative stress results in lipid peroxidation leading to further toxicity. We will test this hypothesis by determining the crystal structure of the suppressor protein and characterizing its function. This multidisciplinary application proposes to determine the first crystal structure of any protein belonging to this large superfamily and the first functional characterization of a bacterial member of this family. This project promises to provide fundamental new insights into fatty acid incorporation and oxidative stress resistance in streptococci and other bacteria of great importance to human health.
