Mutant Alleles of lptD Increase the Permeability of Pseudomonas aeruginosa and Define Determinants of Intrinsic Resistance to Antibiotics

Abstract

Gram-negative bacteria provide a particular challenge to antibacterial drug discovery due to their cell envelope structure. Compound entry is impeded by the lipopolysaccharide (LPS) of the outer membrane (OM), and those molecules that overcome this barrier are often expelled by multidrug efflux pumps. Understanding how efflux and permeability affect the ability of a compound to reach its target is paramount to translating in vitro biochemical potency to cellular bioactivity. Herein, a suite of Pseudomonas aeruginosa strains were constructed in either a wild-type or efflux-null background in which mutations were engineered in LptD, the final protein involved in LPS transport to the OM. These mutants were demonstrated to be defective in LPS transport, resulting in compromised barrier function. Using isogenic strain sets harboring these newly created alleles, we were able to define the contributions of permeability and efflux to the intrinsic resistance of P. aeruginosa to a variety of antibiotics. These strains will be useful in the design and optimization of future antibiotics against Gram-negative pathogens.

FIG 1

Alignment of LptE, LpxC, and LptD protein sequences from E. coli (Ec) and P. aeruginosa (Pa). Permeability-conferring alleles are listed with the resulting amino acid changes indicated in parentheses, and mutations are illustrated above the sequence with asterisks. Identical residues are highlighted in gray.

FIG 2

Mutations in P. aeruginosa lptD cause cells to produce LPS palmitoylated by PagP. LPS was extracted from stationary-phase cultures of MB5890 as well as ΔpagP, lptD208, or lptD4213 derivative strains that were grown in CAMHB and radiolabeled with ³²P. Where indicated, cultures were treated with 25 mM EDTA for 15 min before sample collection. Samples were developed by TLC and visualized using a phosphor screen. The two major lipid A species produced in wild-type cells are denoted as “wt.” The two major PagP-dependent lipid A species are marked with asterisks.