Activity-based protein profiling of the vaginal microbiome

Seattle University is receiving $511,763 in Project Grant funding from the National Institute of Allergy and Infectious Diseases (NIAID) under the Allergy and Infectious Diseases Research program (CFDA 93.855) for a three-year project period beginning September 1, 2025 and concluding August 31, 2028. The award supports research to develop and apply activity-based protein profiling (ABPP), a chemical proteomics technique, to analyze molecular function within cervicovaginal fluid samples. The research will investigate the relationship between enzymatic activity—specifically glycogen-degrading enzymes and microbial proteases—and vaginal microbiome community composition in healthy conditions versus bacterial vaginosis (BV).

The primary products of this project include identification and characterization of enzymes associated with healthy vaginal microbiota versus dysbiotic conditions through two focused research aims. The research will generate mechanistic insights into what controls vaginal community composition and may provide a scientific foundation for rational development of probiotic and drug-based therapies to prevent or treat BV and related health conditions, including sexually transmitted infections and preterm birth. The work directly addresses a critical gap in available experimental systems for studying molecular function in human samples and will contribute to the knowledge base supporting improved therapeutic interventions for these prevalent health conditions.

Abstract

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.