Investigating the role of bacterial sialidases in vaginal microbial communities

Abstract

The microbial community that inhabits the human vagina (the vaginal microbiome) is important for sexual and reproductive health. The composition of the vaginal microbiome can differ substantially between individuals1. While positive health outcomes have been associated with Lactobacillus-dominated vaginal microbiomes, more diverse communities containing facultative and strict anaerobic bacteria have been associated with increased risk for preterm birth and bacterial vaginosis (BV)2. Despite these strong connections to health, the specific mechanisms by which vaginal bacteria influence human health are poorly understood. An understanding of the vaginal bacterial functions most strongly linked to adverse health outcomes is needed to enhance our understanding of this microbial community and guide the design of vaginal microbiome-targeted therapeutics. Here we identify and characterize novel sialidase enzymes from vaginal bacteria of the genus Prevotella. Sialidases are mucus degrading enzymes which are correlated to preterm birth and BV but the specific impacts of this enzymatic activity on vaginal health are unknown. Our work highlights Prevotella bacteria as an underappreciated source of sialidase activity with important implications for both our understanding of vaginal health and therapeutic development. Chapter 2 describes our efforts to identify and biochemically characterize four new sialidases from vaginal Prevotella. We identified these new sialidases by sialidase activity screens amongst a diverse panel of vaginal bacteria and bioinformatic searches for sialidase homologs. Sialidase enzymes release sialic acid from host-derived glycans in the vaginal environment, altering their structures and functions. However, biochemical studies of vaginal bacterial sialidases have been limited to one genus, Gardnerella. We biochemically characterized Prevotella species and demonstrate they produce active sialidases that display variable activity toward mucin. Chapter 3 describes our collaborative bioinformatic studies of sialidases in vaginal bacterial whole genome, metagenome, and metatranscriptome datasets. With Fatima Hussain (Ragon Institute) we demonstrate sialidases are highly conserved across clades of Prevotella from different geographies. We also expanded our search sialidases across diverse vaginal bacteria and identified 67 candidate sialidases using a hidden Markov model. This led to us identify and biochemically characterize a novel sialidase from Lactobacillus mulieris, the first sialidase identified among lactobacilli. Our bioinformatic investigation of metagenomic and metatranscriptomic datasets (provided by the Vaginal Microbiome Research Consortium) revealed Prevotella sialidases are more prevalent and abundant in vaginal microbial communities than Gardnerella sialidases. Finally, Chapter 4 describes our studies of small-molecule inhibitor interactions with Prevotella and Gardnerella sialidases and our many collaborative efforts to study sialidase inhibition in complex settings. We demonstrate the broad sialidase inhibitor Neu5Ac2en is effective towards all sialidases characterized here, while Zanamivir is only effective towards P. timonensis sialidases. We describe our ongoing efforts to structurally characterize P. timonensis sialidases with Gabriel Birrane (Beth Israel X-ray Crystallography core) and utilize structural predictions to guide future inhibitor studies. We demonstrated Neu5Ac2en and Zanamivir can inhibit sialidase activity to varying degrees in clinical cervicovaginal lavage samples provided by Dr. Caroline Mitchell (Massachusetts General Hospital). We utilized a novel chemiluminescent sialidase activity probe from Doron Shabat’s lab (Tel-Aviv University) and improved the sensitivity of sialidase activity detection in the lavage samples by 125-fold compared to the standard fluorogenic probe. With collaborators from the Karin Strijbis lab (Utrecht University) we demonstrate sialidase inhibitors can reduce P. timonensis sialidase activity towards vaginal epithelial cells in culture. Finally, with Zohreh Izadifar (Ingber lab) we propose applying sialidase inhibitors to a Cervix-on-a-chip microfluidic system co-cultured with sialidase producing bacteria to investigate the effects of sialidases in this model system. Future studies will continue our efforts to structurally characterize P. timonensis sialidases and use sialidase inhibitors to further characterize the potential effects of sialidase activity on the Cervix-Chip. Our work lays the foundation to use small-molecule sialidase inhibitors as a tool to understand the effects of sialidase activity in more complex systems to build hypothesis on how this enzymatic activity could contribute to BV or preterm birth.