Genetic methods for studying Gardnerella virulence

PROJECT SUMMARY Bacterial vaginosis (BV) occurs in nearly one third of women of reproductive age, causing significant problems for sexual and reproductive health. Women with BV are predisposed to pregnancy complications including amnionitis and preterm delivery. Gardnerella spp. are major players in the BV dysbiosis, displacing Lactobacillus spp. and forming a robust biofilm supporting the growth of multiple anaerobes. The biofilm also serves as the infectious form for transmitting bacterial vaginosis between people. Gardnerella make a number of putative virulence factors including multiple pili, lipases, sialidases, and the pore-forming toxin vaginolysin. It has been difficult to study Gardnerella virulence factors due to the inability to make targeted mutations or introduce genetic constructs. We discovered a way to make targeted insertion mutations using suicide plasmids in Gardnerella spp., and we propose to further develop these methods to produce a full set of tools for Gardnerella genetic manipulation. First, we will improve methods for making insertional mutants by determining factors affecting transformation frequency and insert stability, and by developing use of a counter- selectable marker to allow for creation of selectable unmarked mutations such as deletions and point mutations. Furthermore, we will test different methods of insert replacement to optimize creation of unmarked mutations. Second, we will develop methods and constructs for gene expression from the Gardnerella chromosome in order to produce constitutive or regulated complement gene expression, transcriptional fusions to Gardnerella ‘lacZ, or translational fusions to the FLAG epitope. The usefulness of the methods will be tested with mutations in the vaginolysin gene.