Analysis of pili heterogeneity and regulation in Enterococcus faecalis

• Main Author: Quek, Keefe Qingyao
• Other Authors: Kimberly Kline
• Format: Final Year Project
• Language: English
• Published: Nanyang Technological University 2022
• Subjects: Science::Biological sciences::Microbiology::Bacteria
• Online Access: https://hdl.handle.net/10356/157136
• Institution: Nanyang Technological University

Enterococcus faecalis undergoes phase variation to regulate the number of cells expressing pili, a characteristic virulence factor, allowing it to evade a host immune response. Understanding phase variation is important for the development of therapeutic strategies against pili. Preliminary RNA sequencing analysis results showed that four genes 11745, 13215, merR and degV were upregulated in the pili-enriched subpopulation, and flow cytometry analysis of transposon mutants for 11745, 13215 and degV showed a decrease in the proportion of pili-expressing cells. This study aims to determine if there is a relationship between these genes and phase variation. Complementation strains for 11745::tn, 13215::tn and degV::tn were generated except merR, where a transposon mutant was unavailable. Flow cytometry, immunofluorescence microscopy and western blot were utilized to quantify pilus phase variation levels in the transposon mutants and compared to the wild-type. We found that 13215 and 11745 transposon mutants do not appear to affect pilus phase variation levels as there was no decrease in pili-expressing cells and restoration to wild-type levels. Interestingly, degV::tn was found to maintain a high proportion of pili-expressing cells, which was higher than the wild-type. We concluded that DegV, a fatty acid-binding protein, may play a role in phase variation indirectly.