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. Species ofGardnerellaincrease just prior to the onset of symptoms and are considered to play major roles in the development and transmission of BV. However,Gardnerellaspecies have remained genetically intractable, limiting investigations of their virulence mechanisms. Here we describe methods for genetic manipulation ofGardnerella. Through trial and error we optimized methods for electrotransformation ofGardnerellaand created methods for making mutations and complements. We mutated the gene for the toxin vaginolysin (vly) inG. vaginalisand the gene for sialidasenanH3inG. pickettii. Avlypoint mutant was tested in human cervix tissue and found to lack lytic activity. ThenanH3mutant lost sialidase and mucus degradation activity. Overall, this genetic toolkit opens a door for molecular characterization ofGardnerellaand its mechanisms in BV.