Targeted Deletion of the Cytopathogenic Toxin A Gene in <i>Sneathia vaginalis</i>

Sneathia vaginalis is a common component of the vaginal microbiome and is emerging as a marker for preterm birth. It produces the cytopathogenic toxin A (CptA), which is capable of lysing human red blood cells and permeabilizing epithelial cells. However, the role of CptA and other potential virulence factors in pathogenesis has been difficult to characterize due to the lack of genetic tools for targeted deletion in S. vaginalis. The objective of this study was to create the first isogenic gene deletion mutant in S. vaginalis. We chose the cptA gene as a target for deletion because of its role in virulence. We characterized the restriction-modification profile in S. vaginalis to increase the chances that exogenous DNA would resist restriction digestion, and we identified an antibiotic resistance cassette that is functional in this species. We identified a genetic locus encoding a Dam methylase and a restriction endonuclease with DpnII-like activity in S. vaginalis strain SN35. By convention, this newly described restriction endonuclease would be named SvaSI for S. vaginalis SN35. Using plasmid DNA purified from a Dam+ E. coli strain to evade SvaSI restriction, we successfully replaced cptA with an erythromycin resistance cassette encoding the ermF and ermAM genes, creating the first genetically engineered deletion mutation in this species. Results revealed that CptA is necessary for the hemolytic and cytopathogenic activities of S. vaginalis. This work is a resource that lays the foundation for the development of additional genetic tools for S. vaginalis and facilitates the characterization of additional genes in this emerging pathogen.