A Two-Component Regulatory System Interconnects Resistance to Polymyxins, Aminoglycosides, Fluoroquinolones, and β-Lactams inPseudomonas aeruginosa

Abstract

Constitutive overexpression of the active efflux system MexXY/OprM is a major cause of resistance to aminoglycosides, fluoroquinolones, and cefepime in clinical strains of Pseudomonas aeruginosa. Upregulation of this pump often results from mutations occurring in mexZ, the local repressor gene of the mexXY operon. In this study, analysis of MexXY-overproducing mutants selected in vitro from reference strain PAO1Bes on amikacin (at a concentration 1.5-fold higher than the MIC) led to identification of a new class of mutants harboring an intact mexZ gene and exhibiting increased resistance to colistin and imipenem in addition to aminoglycosides, fluoroquinolones, and cefepime. Reverse transcription-quantitative PCR (RT-qPCR) experiments on a selected clone named PAOW2 demonstrated that mexXY overexpression was independent of mexZ and the PA5471 gene, which is required for drug-dependent induction of mexXY. Furthermore, the transcript levels of the oprD gene, which encodes the carbapenem-selective porin OprD, were found to be reduced drastically in PAOW2. Wholegenome sequencing revealed a single mutation resulting in an M59I substitution in the ParR protein, the response regulator of the ParRS two-component regulatory system (with ParS being the sensor kinase), which is required for adaptive resistance of P. aeruginosa to polycationic peptides such as colistin. The multidrug resistance phenotype was suppressed in PAOW2 by deletion of the parS and parRS genes and conferred to PAO1Bes by chromosomal insertion of the mutated parRS locus from PAOW2. As shown by transcriptomic analysis, only a very small number of genes were expressed differentially between PAOW2 and PAO1Bes, including the lipopolysaccharide (LPS) modification operon arnBCADTEF-ugd, responsible for resistance to polycationic agents. Exposure of wild-type PAO1Bes to different polycationic peptides, including colistin, was shown to result in increased mexY and repressed oprD expression via ParRS, independent of PA5471. In agreement with these results, colistin antagonized activity of the MexXY/OprM substrates in PAO1Bes but not in a ⌬parRS derivative. Finally, screening of clinical strains exhibiting the PAOW2 resistance phenotype allowed the identification of additional alterations in ParRS. Collectively, our data indicate that ParRS may promote either induced or constitutive multidrug resistance to four different classes of antibiotics through the activation of three distinct mechanisms (efflux, porin loss, and LPS modification).

Adaptive and Mutational Resistance: Role of Porins and Efflux Pumps in Drug Resistance

“…However, two-component regulators can also exert an influence on the transcription of efflux pumps encoded by genes located in separate regions of the chromosome as part of a more general response. Recently, the ParRS system of P. aeruginosa was identified as an important regulator of resistance to several antibiotics in response to the presence of certain antimicrobial peptides, including the clinically relevant polymyxins (61,169). The transcriptional responses mediated by ParRS include the upregulation of the arn operon, involved in peptide resistance, as well as the enhanced expression of the MexXY pump and the downregulation of the OprD porin, which determine susceptibility to aminoglycosides and imipenem, respectively.…”

“…Potentially, mutations in any of these loci could lead to changes in porin expression and, consequently, changes in antibiotic resistance. Another example is the lower level of expression of the porin OprD of P. aeruginosa following the occurrence of mutations that constitutively activate the ParRS two-component system, which is necessary for the downregulation of this porin in the presence of certain antimicrobial peptides (169), while a variety of other regulatory mutants exist, including nfxC mutations that upregulated the efflux pump MexEF-OprN while downregulating OprD (110,186,187).…”

Primary mechanisms mediating aminoglycoside resistance in the multidrug-resistant Pseudomonas aeruginosa clinical isolate PA7

“…mexX expression in PA7 was much higher (~50-fold) than in DSM 1128 (0.017±0.0104 as a ratio to PA7), indicating that P. aeruginosa PA7 is a mexXY-oprA-upregulated strain. To date, three kinds of mutants (agrZ, agrW1 and agrW2) have been recognized as MexXY-overproducers as a result of genetic mechanisms: mutants with alterations in mexZ, the cognate repressor gene located upstream of, but transcribed divergently from, mexXY (type agrZ); mutants with impaired protein synthesis (type agrW1); and mutants with alterations in parRS, a recently discovered two-component regulatory system (type agrW2) (Llanes et al, 2004;Muller et al, 2011; de Bentzmann & Plésiat, 2011). Although Roy et al (2010) reported that the regulatory gene mexZ (PSPA7_3268) of mexXY-oprA in P. aeruginosa PA7 was intact, we found an obvious inconsistency in the N-terminal portion in comparison with the MexZs of P. aeruginosa PAO1 and PA14, while there was significantly high similarity in the C-terminal portion.…”

Alternativas terapêuticas para o tratamento de infecções por Pseudomonas aeruginosa multirresistentes endêmicas no Brasil.

“…Dentre os diversos mecanismos de resistência ressaltam-se: inativação ou degradação enzimática dos antimicrobianos por enzimas, super expressão de bombas de efluxo, perda de porina, alterações no sítio alvo (Kanj, Kanafani, 2011;Muller et al, 2011;Zavascki et al, 2010).…”

“…Dentre os múltiplos mecanismos de resistência ressaltam-se: enzimas modificadoras de aminoglicosídeos, super expressão de bombas de efluxo, perda de porina, alterações no sítio alvo (Kanj, Kanafani, 2011;Muller et al, 2011;Poole, 2011;Strateva, Yordanov, 2009;Zavascki et al, 2010).…”