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Mechanisms decreasing in vitro susceptibility to the LpxC inhibitor CHIR-090 in the Gram negative pathogen Pseudomonas aeruginosa.

Caughlan, Ruth and Jones, Adriana and Delucia, Angela and Woods, Angela and Xie, Lili and Ma, Bing and Barnes, Whitney and Walker, John and Sprague, Elizabeth and Yang, Xia and Dean, Charles (2011) Mechanisms decreasing in vitro susceptibility to the LpxC inhibitor CHIR-090 in the Gram negative pathogen Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy. ISSN 0066-4804

Abstract

Testing defined P. aeruginosa efflux mutants showed that the LpxC inhibitor CHIR-090 is a substrate for MexAB-OprM, MexCD-OprJ and MexEF-OprN. Utilizing P. aeruginosa PAO1 with a chromosomal mexC::luxCDABE fusion, luminescent mutants arose when on medium containing 4 g/ml CHIR-090, indicating constitutive expression of the MexCD-OprJ efflux pump. These mutants were less susceptible to CHIR-090 (MIC 4 g/ml), and had mutations in the mexCD-oprJ repressor gene nfxB. Non-luminescent mutants (MIC 4 g/ml) were also observed that had mutations in the mexAB-oprM regulator mexR. Plating the clinical isolate K2153 on 4 g/ml CHIR-090 selected mutants with alterations in mexS (immediately upstream of mexT), the alteration of which upregulates MexEF-OprN. Beyond efflux, a mutant altered in the putative RBS upstream of lpxC and overexpressing LpxC was selected on a related LpxC inhibitor and exhibited reduced susceptibility to CHIR-090. Consistent with this, overexpression of LpxC from a plasmid reduced susceptibility to CHIR-090, and introduction of the altered RBS in this construct further increased expression of LpxC and decreased susceptibility to CHIR-090. Using a mutS (hypermutator) strain in selection experiments, a mutant with an altered lpxC target gene, (encoding LpxC L18V) was isolated. Introduction of this allele into PAO1 and PA14 confirmed this mutation decreased susceptibility. Purified LpxC L18V had similar activity to wild type LpxC in an in vitro assay, but was less sensitive to inhibition by CHIR-090. Finally, an additional class of mutant, typified by an extreme growth defect, was identified. These had mutations in fabG indicating that alteration in fatty acid synthesis could confer resistance to LpxC inhibitors. Passaging experiments showed progressive decrease in susceptibility to CHIR-090. Therefore, P. aeruginosa can employ several strategies to reduce susceptibility to CHIR-090 in vitro.

Item Type: Article
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Keywords: efflux LpxC CHIR-090 Pseudomonas susceptibility
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Date Deposited: 13 Oct 2015 13:15
Last Modified: 13 Oct 2015 13:15
URI: https://oak.novartis.com/id/eprint/4591

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