Mechanisms DecreasingIn VitroSusceptibility to the LpxC Inhibitor CHIR-090 in the Gram-Negative Pathogen Pseudomonas aeruginosa

Abstract

ABSTRACTTestingP. aeruginosaefflux pump mutants showed that the LpxC inhibitor CHIR-090 is a substrate for MexAB-OprM, MexCD-OprJ, and MexEF-OprN. UtilizingP. aeruginosaPAO1 with a chromosomalmexC::luxCDABEfusion, luminescent mutants arose on medium containing 4 μg/ml CHIR-090, indicating upregulation of MexCD-OprJ. These mutants were less susceptible to CHIR-090 (MIC, 4 μg/ml) and had mutations in themexCD-oprJrepressor genenfxB. Nonluminescent mutants (MIC, 4 μg/ml) that had mutations in themexAB-oprMregulator genemexRwere also observed. Plating the clinical isolate K2153 on 4 μg/ml CHIR-090 selected mutants with alterations inmexS(immediately upstream ofmexT), which upregulates MexEF-OprN. A mutant altered in the putative1ribosomal binding site (RBS) upstream oflpxCand overexpressing LpxC was selected on a related LpxC inhibitor and exhibited reduced susceptibility to CHIR-090. 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 amutS(hypermutator) strain, a mutant with an alteredlpxCtarget gene (LpxC L18V) was also selected. Purified LpxC L18V had activity similar to that of wild-type LpxC in anin vitroassay but had reduced inhibition by CHIR-090. Finally, an additional class of mutant, typified by an extreme growth defect, was identified. These mutants had mutations infabG, indicating that alteration in fatty acid synthesis conferred resistance to LpxC inhibitors. Passaging experiments showed progressive decreases in susceptibility to CHIR-090. Therefore,P. aeruginosacan employ several strategies to reduce susceptibility to CHIR-090in vitro.