Targeting the Nonmevalonate Pathway in Burkholderia cenocepacia Increases Susceptibility to Certain β-Lactam Antibiotics

Abstract

The nonmevalonate pathway is the sole pathway for isoprenoid biosynthesis in Burkholderia cenocepacia and is possibly a novel target for the development of antibacterial chemotherapy. The goals of the present study were to evaluate the essentiality of dxr, the second gene of the nonmevalonate pathway, in B. cenocepacia and to determine whether interfering with the nonmevalonate pathway increases susceptibility toward antibiotics. To this end, a rhamnose-inducible conditional dxr knockdown mutant of B. cenocepacia strain K56-2 (B. cenocepacia K56-2dxr) was constructed, using a plasmid which enables the delivery of a rhamnose-inducible promoter in the chromosome. Expression of dxr is essential for bacterial growth; the growth defect observed in the dxr mutant could be complemented by expressing dxr in trans under the control of a constitutive promoter, but not by providing 2-C-methyl-d-erythritol-4-phosphate, the reaction product of DXR (1-deoxy-d-xylulose 5-phosphate reductoisomerase). B. cenocepacia K56-2dxr showed markedly increased susceptibility to the β-lactam antibiotics aztreonam, ceftazidime, and cefotaxime, while susceptibility to other antibiotics was not (or was much less) affected; this increased susceptibility could also be complemented by in trans expression of dxr A similarly increased susceptibility was observed when antibiotics were combined with FR900098, a known DXR inhibitor. Our data confirm that the nonmevalonate pathway is essential in B. cenocepacia and suggest that combining potent DXR inhibitors with selected β-lactam antibiotics is a useful strategy to combat B. cenocepacia infections.

Keywords: Burkholderia cenocepacia; Burkholderia cepacia complex; DXR; beta-lactam antibiotics; nonmevalonate pathway.

FIG 1

Relative mRNA expression of dxr measured by qPCR. Expression levels shown are relative to that of the expression observed in B. cenocepacia K56-2. Error bars represent range; n = 2.

FIG 2

Growth of B. cenocepacia K56-2 wild type and the K562dxr conditional mutant in permissive and nonpermissive conditions. Top panels: planktonic growth. Solid squares, K56-2dxr; open circles, K56-2 wild type; orange, BSM-O with 0.5% glucose; blue, BSM-O with 0.5% rhamnose. Absence of rhamnose inhibits growth of K56-2dxr, but growth resumes after a delay (left). Addition of rhamnose enables growth of the conditional mutant to near wild-type levels. Growth inhibition in the presence of glucose is less pronounced if precultures are grown in the presence of rhamnose (right). Growth curves are representative of three biological replicates, performed in 96-well round bottom microtiter plates. Bottom panel: colony formation on agar plates. A, K56-2 + pSCrhaB2; B, K56-2dxr. Cultures grown in BSM-O with 200 mg/liter trimethoprim without addition of sugars were normalized to an optical density at 590 nm (OD₅₉₀) of 1.0, and then a 1:10 dilution series was performed. Aliquots of 15 μl of the diluted cultures were spotted onto BSM-O (with 200 mg/liter trimethoprim) with 0.5% rhamnose (left) or 0.5% glucose (right).

FIG 3

Expression of dxr in trans complements the growth defect observed in B. cenocepacia K56-2dxr. Growth medium, LBB with 200 mg/liter trimethoprim. Orange, 0.5% glucose; blue, 0.5% rhamnose. pDA-dxr expresses dxr constitutively. Growth curves represent the average of 4 biological replicates, performed in 96-well round bottom microtiter plates.

FIG 4

Growth of B. cenocepacia K56-2dxr in the presence of 2-C-methyl-d-erythritol 4-phosphate (MEP). Growth medium, BSM-O. Solid squares, K56-2dxr; open circles, K56-2 wild type; orange, 0.5% glucose; blue, 0.5% rhamnose; yellow, 5 mM MEP.