Unravelling the mechanism of E. faecalis intracellular persistence in keratinocytes

• Main Author: Thong, Chor Ming
• Other Authors: Kevin Pethe
• Format: Thesis-Master by Research
• Language: English
• Published: Nanyang Technological University 2025
• Subjects: Medicine, Health and Life Sciences
• Online Access: https://hdl.handle.net/10356/182801
• Institution: Nanyang Technological University

Enterococcus faecalis, traditionally regarded as an extracellular pathogen, is one of the leading causes of hospital-acquired infections and one of the most frequently isolated pathogens from chronic wounds. However, E. faecalis has long been known to persist within various mammalian cell types, and recent findings from our group demonstrate that it can replicate within both non-professional and professional phagocytes. However, the mechanisms by which E. faecalis replicates and persists within non-professional phagocytes remain largely unknown. To investigate the intracellular lifestyle of E. faecalis, we designed and optimized a transposon screening assay using the strain OG1RF to identify bacterial genes involved in the uptake and survival within keratinocytes. After validating the primary hits from the screen, we identified genes when disrupted impair the intracellular persistence of E. faecalis. Our findings showed that fewer intracellular bacteria were recovered when the genes encoding ireK and components of the fsr locus were disrupted by transposon insertions. Using immunofluorescence assay, we also showed that ireK and fsr could play a role in entrapping E. faecalis within intracellular compartments, to protect the bacteria from the host cell defenses. By performing RNAseq analysis, we also identify potential downstream targets that ireK and/or fsr could be regulating to augment E. faecalis persistence in keratinocytes. We demonstrated that ireK and fsr contribute to E. faecalis persistence in wound infections using a murine mouse infection model. Together, these studies shed insights into how E. faecalis is capable of persisting within keratinocytes and wound infections over a prolonged period. We hope that our findings will facilitate the deepening of knowledge of the intracellular lifestyle of E. faecalis, thereby enhancing our understanding of Enterococcal pathogenesis and leading to the development of improved treatment for recalcitrant infections.