Functional and genetic characterization of an in vivo derived single-nucleotide polymorphism mutant of Enterococcus faecalis OG1RF with increased colonization ability in the gut

The human oral isolate E. faecalis OG1RF is a well characterized strain of E. faecalis that induces slowly progressive predominantly rectal colitis in mouse experimental colitis models that mimic the progression of inflammatory bowel diseases (IBDs). Recent in vivo studies in our lab isolated a small colony morphology variant of OG1RF (OG1RF-SV) from E. faecalis OG1RF mono-associated mice that spontaneously arises and outcompetes the wild-type strain (OG1RF-WT) in vivo despite having an in vitro growth deficiency. Whole genome sequencing demonstrated that this variant has a point mutation at the 177065 bp in the DNA dependent RNA polymerase (RpoA) gene, encoding a G>A substitution that causes a missense glutamic acid > lysine mutation. This small variant reverts to large colony phenotype at a low frequency in vivo and in vitro. This study aims to characterize the in vitro growth properties and genetics of E. faecalis OG1RF-SV to understand its increased persistence in vivo in the gut. Using a growth selection screen, we identified OG1RF-SV clones that revert to a large colony phenotype (referred to as OG1RF-Suppressor) yet maintained the G>A substitution in the RpoA gene, indicating that they arose due to a secondary mutation independent of the RpoA gene. In vitro growth assays confirmed the growth deficiency of OG1RF-SV, while demonstrating that OG1RF Suppressor partially rescues this deficiency to achieve near WT growth kinetics. Whole genome sequencing of OG1RF Suppressor strains revealed multiple independent mutations in the GrvRS operon, a two-component system that allows E. faecalis to respond to stressors, such as bile acids. We postulate that this system is constitutively active in OG1RF-SV, and that disruption of this system allows OG1RF-Suppressor to regain WT growth kinetics. Furthermore, in vitro bile acid assays show that OG1RF-SV is more tolerant to bile acid stressors. These results are significant because they demonstrate that E. faecalis OG1RF is readily able to adapt to varying selective pressures and environmental conditions in vivo and in vitro, increasing its colonization abilities.