How do mutations in pssA lead to antiseptic/antibiotic resistance in Pseudomonas aeruginosa?

It is increasingly clear that Gram-negative bacteria can overcome antiseptic challenges by remodelling both the lipopolysaccharide rich outer membrane and the inner, plasma membrane. Since membrane active biocides are widely used in the clinic, bacterial adaptions that lead to loss of sensitivity to these antiseptics are becoming common. Compounding this issue, these resulting adaptations can lead to cross-resistance to key components of the human immune system including membrane-active defensin antimicrobial peptides as well as even last-resort antibiotics.

This project focuses on pssA, a gene annotated as coding for phosphatidylserine synthase, in Pseudomonas aeruginosa. We have recently identified that two mutations in pssA confer resistance in several clinical isolates of this multidrug resistant organism to octenidine and cross-resistance to chlorhexidine, other antiseptics and at least one antimicrobial peptide. It is now essential to understand the role of these mutations in (cross)resistance to a wider range of antiseptics to inform diagnostic and surveillance programmes e.g. in clinical or agricultural settings. Specifically, this project seeks to understand the effects of two known mutations in pssA, the conditions required for these mutations to arise and the mechanisms by which these mutations lead to varying resistance phenotypes in P. aeruginosa isolates of varying clinical origin. If time allows, elements of the project will be replicated in Acinetobacter baumanii, the pssA protein of which is predicted to have a similar single domain trans-membrane structure to that of P. aeruginosa, which is unlike that of most other Gram-negative pathogens.

The project relies on a combination of expertise, skills and techniques available only through extensive collaboration between KCL and Public Health England (PHE). The project will incorporate classical molecular microbiology techniques, next-generation sequencing, NMR metabolomics and membrane biophysical approaches.

Year 1: At PHE - Create site-directed mutants of pssA in a variety of P. aeruginosa lineages (both with/without suspected potentiating mutations in smvR, an efflux pump found to be involved in cationic compound resistance.), evaluate (cross)resistance phenotypes and determine whether mutations in pssA are themselves potentiating mutations for further adaptation to antiseptics. Use reporter fusions and/or tagged PssA to understand the molecular basis for alteration in PssA activity associated with point mutations. Techniques: bacterial culture and susceptibility testing, recombineering, experimental evolution, qPCR (of pssA and smvR expression), whole genome sequencing and MALDI-Biotyper.

Years 2-3: At KCL - Determine the effect of each pssA mutation on bacterial metabolism and membrane composition in each P. aeruginosa isolate background, understand resistance/susceptibility to a range of antiseptics and membrane active host defence peptides (hBD2 and hBD3) and investigate the possible effect of pssA mutations on infection models using a Transwell lung epithelial cell co-culture system. Techniques: HR-MAS NMR metabolomics, HPLC-MS lipidomics (ion-trap MS and evaporative light scattering detection), RNAseq, biophysical experiments (primarily fluorescence spectroscopy and imaging and computational modelling of interaction of different antiseptics with bacterial membranes supported where appropriate by liquid and solid-state NMR and circular dichroism spectroscopy), mammalian cell culture and confocal microscopy.

Year 4: At PHE and KCL - Depending on project outcomes 1) use results to inform preliminary study of A. baumanii adaptation to octenidine or 2) translate results to qPCR and/or MALDI-TOF based diagnostic assays that can be deployed to screen for markers of pssA mutation induced resistance in P. aeruginosa from clinical or agricultural environments where octenidine and related antiseptics are applied routinely.