Engineering Lactococcus lactis for nisin-inducible keratinocyte growth factor delivery: in vitro evaluation of a candidate strategy for vaginal epithelial repair

Introduction Vaginal atrophy associated with the physiological decline in estrogen levels leads to mucosal thinning, impaired epithelial cell proliferation and differentiation, compromised barrier integrity, and increased susceptibility to infection. Hormonal therapies may cause adverse effects and pose risks for patients with cancer. Keratinocyte growth factor (KGF) has been proposed as a local alternative, but rapid release necessitates daily administration. Genetically engineered lactic acid bacteria offer a potential tool for sustained delivery of therapeutic proteins to mucosal surfaces. Methods We engineered a L. lactis strain for inducible KGF production using the nisin-controlled expression (NICE) system. Following generation of a Usp45–KGF fusion construct, induction conditions were optimized to maximize KGF production and secretion. The biological activity of cell-free supernatant from KGF-producing Lactococcus lactis (CFS-KGF) was evaluated in skin keratinocytes and a vaginal epithelial cell line by assessing proliferation, differentiation, migration, and epithelial barrier-associated markers. Results Induction with 10 ng/mL nisin for 3 h yielded the highest KGF levels in the supernatant. CFS-KGF increased ERK1/2 and Akt phosphorylation and total p38 levels and promoted cell cycle entry by upregulating cyclins D1 and B1. The proportion of Ki67-positive cells increased from 25.3% to 53.4% at 24 h, alongside increased metabolic activity. CFS-KGF also promoted cytoskeletal reorganization and cell migration, reducing the residual open area in scratch assays performed on mitomycin C-pretreated cells from 52.6% to 25.7% at 48 h. The initial proliferative response was followed by increased expression of early (K1 and KRT10 ) and late ( IVL , FLG , and LORICRIN ) differentiation markers at 48 h. Furthermore, 1.4- to 5.3-fold increases at 24 and 48 h in MUC1 and intercellular junction-associated transcripts ( OCLN , TJP1 , CDH1 , and DSG1 ) suggested a potential beneficial effect on the vaginal epithelial barrier. Discussion These findings support engineered L. lactis as a source of biologically active recombinant KGF with potential therapeutic effects on vaginal epithelial cells. Further studies in physiologically representative models and in vivo are needed to evaluate its therapeutic relevance as a non-hormonal approach to vaginal atrophy.