Imbalance of envelope biosynthesis pathways promotes drug resistance in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen particularly resistant to antimicrobials owing to its set of multidrug efflux pumps and its impermeable outer membrane (OM). Although cell envelope biosynthesis is well studied in Escherichia coli, far less is known in P. aeruginosa. LptD is an essential component of the Lpt complex involved in lipopolysaccharide (LPS) transport from the inner membrane to the OM. An hypomorphic lptD mutant has a greater OM permeability and shows increased sensitivity to antibiotics normally inactive against Gram-negative bacteria, including vancomycin (VNC) that targets cell wall synthesis. This mutant in P. aeruginosa was also unable to grow in presence of NaCl, a phenotype not described in E. coli. To dissect the cell envelope homeostasis of P. aeruginosa, we isolated suppressors of salt and VNC sensitivity. A salt suppressor mutant in bamD validates the role of the Bam complex, which drives the insertion of LptD in the OM. A second mutation in a LPS O-antigen synthesis gene improved VNC resistance. This mutation also conferred cross-resistance to antibiotics targeting the peptidoglycan (PG) synthesis but did not re-establish the OM permeability barrier. Further analyses prompt us to hypothesize that specific mutations in O-antigen synthesis leads to a buildup of undecaprenyl phosphate, an essential lipid carrier implicated in O-antigen and PG synthesis, which in turn leads to the increase of lipid II, the target of VNC. Our work highlights how the complex balance between LPS and PG synthesis underlying cell envelope homeostasis can affect drug resistance in P. aeruginosa.