Mechanisms decreasing in vitro susceptibility to the LpxC inhibitor CHIR-090 in the gram-negative pathogen Pseudomonas aeruginosa.
Testing P. aeruginosa efflux pump 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 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 the mexCD-oprJ repressor gene nfxB. Nonluminescent mutants (MIC, 4 μg/ml) that had mutations in the mexAB-oprM regulator gene mexR were also observed. Plating the clinical isolate K2153 on 4 μg/ml CHIR-090 selected mutants with alterations in mexS (immediately upstream of mexT), which upregulates MexEF-OprN. A mutant altered in the putative1ribosomal binding site (RBS) upstream of lpxC 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 a mutS (hypermutator) strain, a mutant with an altered lpxC target gene (LpxC L18V) was also selected. Purified LpxC L18V had activity similar to that of wild-type LpxC in an in 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 in fabG, 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-090 in vitro.
Infectious Diseases, Novartis Institutes for Biomedical Research, Emeryville, California 94608, USA.
Ruth E
RE
Caughlan
Adriana K
AK
Jones
Angela M
AM
Delucia
Angela L
AL
Woods
Lili
L
Xie
Bing
B
Ma
S Whitney
SW
Barnes
John R
JR
Walker
Elizabeth R
ER
Sprague
Xia
X
Yang
Charles R
CR
Dean
D000581:Amidohydrolases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D024901:Drug Resistance, Multiple, Bacterial / Q000187:drug effects*; D005227:Fatty Acids / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D017930:Genes, Reporter; D006877:Hydroxamic Acids / Q000494:pharmacology*; D008163:Luminescent Measurements; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010957:Plasmids; D011552:Pseudomonas Infections / Q000188:drug therapy / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013912:Threonine / Q000031:analogs & derivatives* / Q000494:pharmacology; D014169:Transformation, Bacterial
D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; C525369:CHIR 090; D005227:Fatty Acids; D006877:Hydroxamic Acids; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D013912:Threonine; D000581:Amidohydrolases; C514484:LpxC deacetylase, Pseudomonas