Activity-based protein profiling of the vaginal microbiome - PROJECT SUMMARY Microorganisms present in the vaginal tract have a profound effect on human health. High abundance of certain bacteria, predominantly Lactobacillus species, is associated with positive health outcomes. In contrast, high abundance of non-Lactobacillus taxa is often associated with increased rates of bacterial vaginosis, increased susceptibility to sexually transmitted infections, and increased rates of preterm birth. Developing probiotic or drug-based therapies that shift the community composition of the vaginal microbiome could be one way to prevent or treat these health conditions. Rational development of such therapies requires a mechanistic understanding of what controls community composition in the vaginal tract. Achieving such an understanding has faced major barriers related to the lack of suitable experimental systems. As such, techniques capable of studying molecular function in human samples of cervicovaginal fluid (CVF) are needed. Activity-based protein profiling (ABPP) is a chemical proteomics technique is uniquely suited to such scenarios. By using small molecule probes, it is possible to covalently label any active enzymes within complex biological matrices. The labeled enzymes can then be fluorescently tagged for SDS-PAGE or biotinylated to allow enrichment and identification using mass- spectrometry based proteomics. This proposal seeks to use ABPP to test the hypothesis that two classes of enzymes, glycogen degrading enzymes (GDEs) and microbial proteases, are associated with vaginal community composition and BV. We will achieve this goal in two Aims. The first will test the hypothesis that distinct GDEs are more active under healthy conditions relative to BV and identify GDEs that are preferentially active during BV. The second will test the hypothesis that microbial proteases are more prevalent and active in BV relative to healthy conditions and determine the molecular identity of these proteases. By applying ABPP, we will be able to identify the specific enzymes responsible for each activity, providing clear resolution about which enzymes control each process. This understanding will allow the development of probiotic strains or novel inhibitors to promote vaginal health and provide a much-needed treatment option for BV.
