UBC Theses and Dissertations
Structural and functional studies on the role of the outer membrane of Pseudomonas aeruginosa in resistance and permeability to antibiotics Angus, Barbara Lee
The intrinsic antibiotic resistance of aeruginosa was examined using an antibiotic hypersusceptible mutant strain. This multiple mutant (M1) was characterised with respect to antibiotic susceptibility, outer membrane permeability and outer membrane structure. It was found to be hypersusceptible to all antibiotics tested. Two spontaneous revertants of the mutant strain were isolated (P1-4 and P1-6), using carbenicillin or gentamicin as the selective agent, and characterised, revealing that these had recovered partial resistance to all antibiotics. Two other strains, for which two genetic loci (apparently responsible for antibiotic hypersusceptibility in the mutant M1) had been separately conjugated into a strain with a wild type genetic background, were also characterised. One of these transconjugants (P2-6) was found to be partially hypersusceptible to a wide range of antibiotics, whereas the other (P2-8) was partially hypersusceptible to B-lactam and aminoglycoside antibiotics but not to the hydrophobic agent, trimethoprim. Outer membrane permeability of the above strains was characterised by three different methods. Using an assay involving periplasmic beta-lactamase, the mutant strain M1 was found to be five-fold more permeable to the chromogenic cephalosporin nitrocefin than its wild type parent WT1. In addition, mutant M1 was much more permeable to the hydrophobic fluorescent compound 1-N-phenyl-N-naphthylamine than was wild type strain WT1. In contrast, mutant strain M1 was less susceptible to permeabilisation of the outer membrane to lysozyme by aminoglycoside antibiotics or ethylenediaminetetraacetate. Partially resistant mutants were used to confirm and clarify these results. It was concluded from these data that the mutations responsible for antibiotic susceptibility in the mutant were acting upon one or more of the outer membrane constituents, protein and lipopolysaccharide. Characterisation of outer membrane proteins by SDS-polyacrylamide gel electrophoresis showed no observable differences between outer membrane proteins of the wild type and mutant strains. Further examination showed that the lipopolysaccharide in the mutant strain M1 was altered with respect to its composition, mobility on SDS-polyacrylamide gels and in its Mg++-binding properties. From these data, it appeared that at least three separate mutations existed in the mutant which contributed to antibiotic susceptibility. Two of these mutations were responsible for separate alterations to lipopolysaccharide, while the third mutation could not be characterised. It was proposed that the chief alteration leading to antibiotic susceptibility in strain M1 involved partial loss of an outer membrane-stabilising, Mg++-binding site on lipopolysaccharide. In another portion of the study, native oligomers of three P. aeruginosa outer membrane proteins and one E. coli porin were demonstrated using a chemical crosslinking technique. P. aeruginosa protein F, the major constitutive outer membrane porin, was crosslinked to dimers in outer membranes and whole cell crosslinking experiments. Purified preparations of P. aeruginosa proteins F, P (phosphate starvation-induced) and coli protein PhoE (Ic) were also crosslinked to reveal dimers and trimers upon two-dimensional SDS-polyacrylamide gel electrophoretic analysis. Crosslinking of protein F was abolished by pretreatment of the protein with SDS, indicating that the crosslinked products were due to native associations in the outer membrane.
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