PROJECT SUMMARY/ABSTRACT: One-third of women in the U.S. have bacterial vaginosis (BV), a condition associated with greater risks of reproductive tract infections and preterm birth. BV is characterized by loss of lactobacilli and polymicrobial overgrowth of diverse potential pathogens, including Gardnerella vaginalis. Vaginal sialidase activity is found in nearly all women with BV and is independently associated with adverse outcomes. However, little is known regarding how sialidase supports dysbiosis and disease. This proposal examines how the sialidase produced by certain subtypes of G. vaginalis participates in the molecular pathogenesis of vaginal and intrauterine infection and encourages overgrowth of diverse BV bacteria. We have identified the genetic basis of G.v. sialidase activity and found that 80% of BV cases have G.v. encoding the sialidase genes nanH2 or nanH3, and 80% of women with nanH2-positive or H3-positive vaginal microbiota have BV. Our data suggests that G.v. sialidase fundamentally transforms the cellular glycan landscape in BV and shows that glycan degradation encourages pathogen colonization. In three specific aims, this proposal will use human vaginal samples, diverse G.v. isolates, a mouse model that replicates relevant features of BV, new wild-type and paired nanH3 mutant G.v. strains, and novel ex vivo models employing native human vaginal communities to test several hypotheses about the molecular mechanism of G. vaginalis sialidase in the causes and complications of BV. In Aim 1, we will examine the relationship between G.v. clades and biochemical and cellular phenotypes in human clinical samples and compare sialidase positive and negative G.v. clades and a G.v. sialidase mutant in our mouse vaginal colonization model. In Aim 2, we will use our repository of live native vaginal communities in vitro to test how the availability of free sialic acid and exposed glycans, both consequences of sialidase activity, influence BV community composition and also examine the relationship between nanH2/H3-positive G.v. and bacterial burden and diversity in clinical specimens. Finally, Aim 3 we will evaluate samples from a longitudinal cohort of pregnant women to determine whether women who went on to experience preterm birth have higher levels of vaginal nanH2/H3-positive G.v., larger numbers of exfoliated epithelial cells, degraded mucus and epithelial glycans, and more diverse microbiomes with higher vaginal bacterial burden compared to term controls. The successful completion of these aims will have an important positive impact on this field by identifying G.v. subtypes that drive pathophysiology, defining mechanisms by which G.v. sialidase supports the development of dysbiosis and disease, and identifying biomarkers that might be used in clinical surveillance efforts to prevent preterm birth.