Conference 2021 Pre-Recorded Video
Regulation of the efflux pump FarE in response to antimicrobial fatty acids in Staphylococcus aureus.
Authors and Affiliations
Camryn Bonn1, Katherine Ferguson2, Martin McGavin3
1. Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, London, Canada
2. Western University, London, Canada.
USA100 is the most common cause of HA-MRSA infection and clinical isolates have been recovered that possess amino acid substitutions in the C-terminal ligand-binding domain of FarR: C116Y; P165L; and a GAG duplication at Gly 93. FarR is a TetR family regulator that binds upstream of the farE promoter to induce transcription of the resistance-nodulation-division (RND) efflux pump FarE and alleviate toxicity caused by cytoplasmic buildup of unsaturated free fatty acids (uFFA) on the skin and in the nares. S. aureus is also a common cause of chronic bronchitis in cystic fibrosis (CF) patients. Several USA100 isolates were recovered from CF patients coinfected with Pseudomonas aeruginosa that possess a E160G substitution in FarR. P. aeruginosa produces hydrophobic 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO) that has a similar structure to uFFA and is toxic to S. aureus through increased membrane fluidity. Since the strain FAR7, possessing a H121Y substitution in FarR, provides increased uFFA resistance, we hypothesize that these amino acid substitutions will provide increased uFFA resistance, as well as resistance to P. aeruginosa infection in CF.
USA100 clinical isolates were obtained that possess C116Y, P165L and E160G substitutions, as well as a GAG duplication at Gly 93. Isolates will be subjected to minimum inhibitory concentration (MIC) assays at 1200μM linoleic acid (LA) and growth curves at 50μM LA to determine uFFA resistance. Then, pLIFarR constructs in DH5α will be used in a Western blot with USA300 and FAR7 to determine FarR expression. The same MIC and growth curves will then be repeated with isogenic USA300pLIFarR constructs to confirm the resistance phenotype.
Preliminary evidence suggests the C116Y substitutions and GAG duplications show MIC growth at 1200μM LA, comparable to FAR7. However, only isolates M0330 and M1545 with the C116Y substitution have shown growth at 50μM LA, comparable to FAR7.
MRSA represents a significant burden on global health, representing 74% of global S. aureus cases. The success of S. aureus colonization can be contributed to the diverse array of resistance mechanisms it possesses, including circumvention of toxicity caused by uFFA. In particular, these data support the importance of amino acid substitutions in the ligand-binding domain of FarR in increased uFFA resistance and demonstrate a potential mechanism of adaptive evolution to human hosts in a clinical setting.