Molecular determinants of Burkholderia pseudomallei BpeEF-OprC efflux pump expression

Abstract

<p class="first" id="d2642416e309"> <i>Burkholderia pseudomallei</i>, the cause of melioidosis, is intrinsically resistant to many antibiotics. Acquired multidrug resistance, including resistance to doxycycline and co-trimoxazole used for melioidosis eradication phase therapy, is mainly attributed to constitutive expression of the BpeEF-OprC efflux pump. Constitutive expression of this pump is caused by mutations affecting two highly similar LysR-type transcriptional regulators (LTTR), BpeT and BpeS, but their interaction with the regulatory region governing BpeEF-OprC expression has not yet been studied. The <i>bpeE-bpeF-opr</i>C genes are distally located in the <i>llpE-bpeE-bpeF-oprC</i> operon. The <i>llpE</i> gene encodes a putative lipase/esterase of unknown function. We show that in a <i>bpeT</i> mutant <i>llpE</i> is constitutively co-transcribed with <i>bpeE-bpeF-oprC</i>. As expected from previous studies with <i>B. cenocepacia</i>, deletion of <i>llpE</i> does not affect antibiotic efflux. Using transcriptional <i>bpeE′-lacZ</i> fusions, we demonstrate that the 188 bp <i>bpeT-llpE</i> intergenic region located between <i>bpeT</i> and the <i>llpE-bpeE-bpeF-oprC</i> operon contains regulatory elements needed for control of <i>bpeT</i> and <i>llpE-bpeE-bpeF-oprC</i> operon expression. By native polyacrylamide gel electrophoresis and electrophoretic mobility shift assays with purified recombinant BpeT and BpeS proteins, we show BpeT and BpeS form oligomers that share a 14 bp binding site overlapping the essential region required for <i>llpE-bpeE-bpeF-oprC</i> expression. The binding site contains the conserved T-N ₁₁-A LTTR box motif involved in binding of LysR proteins, which in concert with two other possible LTTR boxes may mediate BpeT and BpeS regulation of BpeEF-OprC expression. These studies form the basis for further investigation of BpeEF-OprC expression and regulation at the molecular level by yet unknown external stimuli. </p>