KtrB-mediated alkaline adaptation drives Enterococcus faecalis persistence in the gastrointestinal tract of Helicoverpa zea

<i>Enterococcus faecalis</i> is a commensal enteric bacterium capable of surviving in extreme and diverse environments. Here, we characterized the role of the gene <i>ntpJ</i>, which encodes the KtrB subunit of the KtrAB Na⁺/K⁺ symporter, during the adaptation of <i>E. faecalis</i> to alkaline stress and persistence in <i>Helicoverpa zea</i> (corn earworm). We assessed growth kinetics, biofilm formation, surface adhesion, and gastrointestinal persistence <i>in vivo</i> using an <i>E. faecalis</i> OG1RF mariner transposon mutant (<i>ntpJ</i>-Tn). The <i>ntpJ</i>-Tn mutant showed delayed entry into mid-log phase growth and biofilm formation under standard and alkaline-adjusted conditions relative to the wild-type strain, while adherence to a low-density substrate was not affected, indicating KtrB-mediated transport was important for early-stage planktonic growth but unnecessary for surface attachment. Interestingly, elevated K⁺ and Na⁺ ions differentially influenced biofilm morphology and the distribution of adherent cells, highlighting an ion-specific response to alkalinity. The <i>ntpJ</i>-Tn mutant was undetectable 48 hours following ingestion in the novel non-destructive <i>H. zea</i> model, suggesting the loss of KtrB resulted in a persistence defect. These findings reinforce the significance of KtrB-mediated transport in sustaining optimal ionic homeostasis during microbial survival of alkaline stress and demonstrate the efficacy of lepidopteran models for interrogating host-microbe interactions.