Open AccessMechanisms Decreasing In Vitro Susceptibility to the LpxC Inhibitor CHIR-090 in the Gram-Negative Pathogen Pseudomonas aeruginosa
Author(s) -
Ruth E. Caughlan,
Adriana K. Jones,
Angela M. DeLucia,
Angela Woods,
Lili Xie,
Bing Ma,
S. Whitney Barnes,
John R. Walker,
Elizabeth R. Sprague,
Xia Yang,
Charles R. Dean
Publication year - 2012
Publication title -
antimicrobial agents and chemotherapy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.07
H-Index - 259
eISSN - 1070-6283
pISSN - 0066-4804
DOI - 10.1128/aac.05417-11
Subject(s) - mutant , pseudomonas aeruginosa , biology , efflux , microbiology and biotechnology , bacterial outer membrane , wild type , gene , escherichia coli , biochemistry , bacteria , genetics
TestingP. aeruginosa efflux pump mutants showed that the LpxC inhibitor CHIR-090 is a substrate for MexAB-OprM, MexCD-OprJ, and MexEF-OprN. UtilizingP. aeruginosa PAO1 with a chromosomalmexC ::luxCDABE fusion, 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-oprJ repressor genenfxB . Nonluminescent mutants (MIC, 4 μg/ml) that had mutations in themexAB-oprM regulator genemexR were 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 oflpxC and 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 alteredlpxC target gene (LpxC L18V) was also selected. Purified LpxC L18V had activity similar to that of wild-type LpxC in anin vitro assay 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. aeruginosa can employ several strategies to reduce susceptibility to CHIR-090in vitro .