Pseudomonas aeruginosa (PA) is an opportunistic pathogen that is notorious for causing persistent infections in people suffering from chronic lung disorders such as cystic fibrosis. A major factor that contributes to antibiotic resistance in these persistent PA infections is the complex structure of the PA cell envelope, which is composed of an inner membrane, a thin layer of peptidoglycan, and a lipopolysaccharide-containing outer membrane. These membranes present different physiochemical barriers to antibiotics that make delivery of drugs into PA cells difficult.

In collaboration with Prof. Dao Nguyen in the Department of Medicine, we have recently been awarded a grant from the Canadian Institutes of Health Research (CIHR) to investigate the role of cyclopropane fatty acid (CFA) synthase in PA membrane biogenesis (Figure 5). CFA synthase is an interesting enzyme that installs a methylene group derived from the ubiquitous methyl donor S-adenosyl-methionine (SAM) across the olefin moiety of unsaturated fatty acyl chains to make cyclopropane fatty acids (CFAs) in inner membrane phospholipids.

Figure 5.  Investigating the biophysical properties of CFA synthase (CFAS) from Pseudomonas aeruginosa (PA) and its role in antimicrobial resistance. A) The reaction catalyzes by CFAS.  B) Genetic deletion of the CFAS gene from PA sensitizes the bacterium to ofloxacin in a mouse model of infection. C and D) Structural modeling of the CFAS enzyme, highlighting the functional domains (C) and structural homology with the E. coli enzyme (D).  E) Expression and purification of the PA-CFAS enzyme.  F) In vitro enzymatic activity of the PA-CFAS.  G) Native MS characterization of PA-CFAS, showing the presence of different conformations.  H) Preliminary HDX-MS characterization of PA-CFAS, showing good coverage of the protein.  The lipid binding domain and interface between the lipid binding domain and catalytic domain are more dynamic.

CFA synthase is expressed as cells enter stationary phase and CFAs are enriched in PA biofilms. Preliminary evidence suggests that the CFAs play an important role in making the PA membranes less permeable to several antibiotics (see Figure 5B). Our work on CFA synthase involves understanding the changes in the PA proteome and lipidome that occur upon disruption of CFA synthesis. To achieve this, we are developing liquid chromatography mass spectrometry (LC-MS) based workflows to profile changes in protein expression and lipid content at the systems level.  We are also preforming biochemical and biophysical studies to develop a better understanding of the mechanism by which CFA synthase associates with phospholipid membranes and gains access to its unsaturated fatty acid substrate.