Enterococcus faecalis membrane micro-domain formation and antimicrobial targeting

• Main Author: Nair, Zeus Jaren
• Other Authors: Kimberly Kline
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2021
• Subjects: Science::Biological sciences::Microbiology::Bacteria
• Online Access: https://hdl.handle.net/10356/146760
• Institution: Nanyang Technological University

Enterococcus faecalis is an opportunistic pathogen commonly isolated with nosocomial infections where its ability to acquire resistance to last-line antimicrobials poses a public health threat. Previous literature suggests the existence of septal membrane microdomains and the tendency of cationic antimicrobials to target them. Given the importance of microdomains and antimicrobial targeting as a potential drug target for this often-resistant organism, this thesis takes a multi-faceted approach to investigate the dynamics behind microdomain targeting, microdomain assembly, and membrane and genetic factors involved in targeting and resistance. We first investigated a phosphatidyl glycerol (PG) modifying enzyme, the multiple peptide resistance factor (MprF), since it is implicated in focal targeting and resistance of cationic antimicrobial peptides (CAMPs). Apart from its catalytic activity in lysinylating PG to lysyl-PG, MprF was discovered to affect the global lipidome, de novo fatty acid biosynthesis regulation and dependence on exogenous fatty acids under nutrient limitation. Loss of mprF also leads to functional outcomes in terms of secretion defects, increased membrane rigidity and susceptibility to cationic antimicrobial peptides. Interestingly, ΔmprF also provides a permissive environment for loss of function of an otherwise essential metalloprotease, FtsH, which results in enhanced rate of acquisition of daptomycin resistance through increased basal mutation rates. Given their possible roles in coordinating antimicrobial targeting of the membrane, microdomains were also characterised, with results suggesting that flotillins, and sortase A associated microdomains are likely part of separate domains. Flotillin homologs in E. faecalis demonstrate atypical functions in growth and membrane fluidity that are only apparent under nutrient limitation, and they are not enriched within detergent resistant membrane fractions. We also provided proof of concept in the use of styrene-maleic acid to generate lipid particles as an alternative method to sample microdomains. Together this work provides a better understanding of E. faecalis membrane physiology and homeostasis, and how metabolic networks, membrane lipid composition, microdomains and functional outcomes are all closely intertwined.