The microenvironment: how it impacts enterococcus faecalis wound pathogenesis

• Main Author: Tan, Casandra Ai Zhu
• Other Authors: Kevin Pethe
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2023
• Subjects: Science::Biological sciences
• Online Access: https://hdl.handle.net/10356/165582
• Institution: Nanyang Technological University

Enterococcus faecalis is commonly isolated from single- and mixed-species biofilm-associated wound infections. As a defense strategy, the host innately restricts iron availability at infection sites. Despite E. faecalis prevalence in wounds, the mechanism of E. faecalis wound pathogenesis during single- and mixed-species wound infection are poorly understood. The overall aim of this dissertation is to understand the mixed-species interactions of E. faecalis with commonly co-isolated Pseudomonas aeruginosa in biofilms under iron-restricted conditions, as well as to determine and explore the genetic determinants that contributes to E. faecalis wound pathogenesis. To achieve the first aim, the mixed-species interactions were explored in biofilm conditions, and I found that E. faecalis inhibits P. aeruginosa growth within biofilms when iron is restricted. E. faecalis lactate dehydrogenase (encoded by ldh1) gives rise to L-lactate during fermentative growth and I found that an E. faecalis ldh1 mutant fails to inhibit P. aeruginosa growth. Additionally, ldh1 expression was induced under iron-restricted conditions, resulting in increased lactic acid exported and consequently, a reduction in the local environmental pH which contributes to P. aeruginosa growth inhibition. To achieve the second aim, in vivo E. faecalis transposon and RNA sequencing were performed to identify genetic determinants that are crucial for acute replication and persistence of E. faecalis during wound infection. I found that E. faecalis purine biosynthesis genes were important for bacterial replication during the early stages of wound infection, a time when purine metabolites are also low within wounds as quantified by liquid chromatography-mass spectrometry. I also identified the E. faecalis MptABCD phosphotransferase system, involved in the import of galactose and mannose, is crucial for E. faecalis persistence within wounds where carbohydrate availability also changes during the course of infection. During in vitro growth with mannose as the sole carbohydrate source, shikimate and purine biosynthesis genes were downregulated in the E. faecalis OG1RF ∆mptD mutant compared to the isogenic wild-type strain, indicating that mannose transport, shikimate and purine biosynthesis are linked. Overall, these findings emphasize the importance of the wound microenvironment during single- and mixed-species wound infection, and how manipulation of the microenvironment can affect the pathogenesis of an infection.