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Regulation of polymyxin B and cationic antimicrobial peptide resistance in pseudomonas aeruginosa McPhee, Joseph B.

Abstract

Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that is noted for its environmental ubiquity, its metabolic potential and its intrinsic resistance to a wide variety of antimicrobials, detergents, dyes, and biocides. These properties are consequences of a large (6.3 Mb) genome containing ~5500 genes of which 9.4% encode regulatory proteins. One of the largest classes of regulators in the P. aeruginosa genome is the two-component regulators. This work describes the contribution of two two-component regulatory systems, PhoP-PhoQ and PmrA-PmrB to Mg²⁺-limitation induced polymyxin B and cationic antimicrobial peptide resistance. Both of these systems respond to limiting Mg²⁺ and cause increased transcription of an eight-gene operon, pmrHFIJKLM-ugd, that is responsible for the addition of aminoarabinose to 1 and 4' phosphates on Lipid A. In addition, the PmrA-PmrB system regulates a three gene operon, PA4773-PA4775 that also contributes to polymyxin B and cationic antimicrobial peptide resistance. In addition to regulating polymyxin B and cationic antimicrobial peptide resistance, the PhoP-PhoQ system also directly regulates several small ORFs, one of which PA0921 contributes to swimming motility via an unknown mechanism. Similarly, PmrA-PmrB regulate other phenotypes, including the growth of P. aeruginosa in the presence of Fe³⁺. This growth phenotype occurs through gene products encoded by the feoAB operon. Interestingly, all genes identified in this study that are PmrA-PmrB regulated are also regulated by the presence of sub-inhibitory concentrations of cationic antimicrobial peptides. The regulation of PA4773-PA4775 and pmrHFIJKLM-ugd via cationic peptides is mostly independent of the PmrA-PmrB and PhoP-PhoQ systems. This observation explains why adaptive resistance to cationic antimicrobial peptides occurs and suggests that another, as yet unidentified, regulator is responsible for the detection of cationic antimicrobial peptides. A third regulatory system, PxrRS, is also identified. Mutants in this system show increased susceptibility to cationic antimicrobial peptides and polymyxin B. This susceptibility was not due to loss of regulation of the PA4773-PA4775 or pmrHFIJKLM-ugd. Microarray analysis demonstrated downregulation of a number of heat-shock proteins, as well as two operons potentially involved in efflux. The combined downregulation of heat-shock proteins involved in response to cellular stress and efflux systems suggests that intrinsic cationic peptide resistance is altered in these mutants.

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