The role of iron in Enterococcus faecalis biofilm formation
Lam, Ling Ning
Date of Issue2018-12-31
School of Biological Sciences
Singapore Centre for Environmental Life Sciences and Engineering
Enterococcal biofilm-associated infections are a major challenge in hospitals due to their tolerance to host defenses and their intrinsic and acquired antimicrobial resistance. Enterococcus faecalis is a member of the gut microbiota and is a clinically important opportunistic pathogen associated with endocarditis, and several other nosocomial infections such as wound and urinary tract infections. Owing to its malleable genome and metabolic flexibility, E. faecalis is well adapted to colonize a variety of niches in the host. Furthermore, E. faecalis possesses several cell surface virulence factors including endocarditis and biofilm associated pili (Ebp) which are important for host colonization. Many pathogenic organisms require iron (Fe) for essential metabolic processes, and the host has multiple mechanisms to restrict iron availability to limit bacterial growth. Even though E. faecalis is highly tolerant to both iron restriction and high iron concentrations, its homeostatic response to iron availability has been minimally studied and the role of iron in enterococcal biofilm physiology and infection has been largely overlooked. In this thesis, we show that iron augments E. faecalis biofilm formation and alters biofilm structure. Using a transposon library screen, we identified genes associated with respiration and metal efflux that contribute to iron-augmented biofilm growth. Using chronocoulometry assay, we also uncovered that E. faecalis uses iron for extracellular electron transfer (EET), and absence of ldh1 attenuate electron transfer. Presence of Ldh1 contributes to generate growth-promoting energy. In addition to Ldh1, further characterization of other genes identified through the library screen uncovered additionally mechanisms driving iron-augmented biofilm growth. We demonstrate that pili, which are important for surface adherence and host colonization, contribute to both iron mediated biofilm growth and EET. We also show that MenB and Ndh3 which are essential for demethylmenaquinone (DMK) biosynthesis and flavin-mediated electron transfer respectively are involved in iron-mediated EET. Global transcriptional analysis of E. faecalis biofilm grown in iron supplemented media revealed upregulation of feoB iron transporter which is important for intracellular iron uptake. Furthermore, we demonstrate that iron co-purifies with Ebp on cell surface, and presence of Ebp and FeoB are essential for iron uptake in E. faecalis biofilm cells. We also show that a high iron diet promoted E. faecalis gastrointestinal tract (GI) colonization in the mouse colon, in an ebp-dependent manner. Together, these findings demonstrate that iron-pili interaction facilitate electron transfer, biofilm augmentation and intracellular iron accumulation in Enterococcus faecalis biofilm. In addition to iron uptake, we also found that iron and manganese export have a profound effect on biofilm growth. Among the genes identified in the transposon library screen, we uncovered that E. faecalis has a dual specificity MntE-like manganese exporter (OG1RF_10589) which contributes to iron-augmented biofilm growth. We show that MntE functions as a manganese (Mn) and iron (Fe) efflux pump in E. faecalis. Deletion of mntE results in accumulation of intracellular Mn and Fe which can be reverted either with mntE complementation or magnesium (Mg) supplementation. However, metal-induced growth inhibition is only observed in response to Mn, which suggest that MntE contributes to a larger extent, in Mn homeostasis. In support of this, we also observed upregulation of mntE in response to Mn, but not in Fe. Global transcriptional analysis of mntE mutant grown in high iron-supplemented media resulted in upregulation of three glycerol catabolic genes (glpF2, glpK, glpO), which leads to enhanced iron-mediated biofilm growth. Furthermore, we also demonstrate that deletion of mntE resulted in attenuated colonization in mouse gastrointestinal tract (GI) model. These findings represent the first mechanistic description of extracellular electron transfer (EET) by Enterococcus faecalis, a gastrointestinal tract commensal and opportunistic pathogen, and support a previously unappreciated, additional role for E. faecalis endocarditis and biofilm associated pili in this process, beyond their adhesive function. Moreover, this is also the first study describing the importance of manganese tolerance by the manganese exporter MntE (OG1RF_10589) in E. faecalis. Through these findings, we better understand E. faecalis biofilm physiology and the role for transition metals in E. faecalis colonization, biofilm formation, and persistence in the host.