Genetic transformation of Gardnerella species and characterization of vaginolysin and sialidase mutants

Bacterial vaginosis (BV) is the most prevalent vaginal disorder in women of childbearing age and causes pregnancy complications, including preterm birth, amnionitis, and postpartum endometritis. BV also interferes with sexual health and increases stress. BV is a vaginal dysbiosis that occurs when Lactobacillus species are displaced by facultative and anaerobic bacterial species, including Gardnerella, Prevotella, Fannyhessea, Sneathia, Megasphaera, Mycoplasma , and others. Species of Gardnerella increase just prior to the onset of symptoms and are considered to play major roles in the development and transmission of BV. However, Gardnerella species have remained genetically intractable, limiting investigations of their virulence mechanisms. Here, we describe methods for genetic manipulation of Gardnerella . Through trial and error, we optimized methods for electrotransformation of Gardnerella and created methods for making mutations and complements. We mutated the gene for the toxin vaginolysin ( vly ) in G. vaginalis and the gene for sialidase nanH3 in G. pickettii . A vly point mutant was tested in human cervix tissue and found to lack lytic activity. The nanH3 mutant lost sialidase and mucus degradation activity. Overall, this genetic toolkit opens a door for molecular characterization of Gardnerella and its mechanisms in BV.