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Volume 164, Issue 9

Molecular determinants of Burkholderia pseudomallei BpeEF-OprC efflux pump expression Free

Abstract

Burkholderia pseudomallei, 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 bpeE-bpeF-oprC genes are distally located in the llpE-bpeE-bpeF-oprC operon. The llpE gene encodes a putative lipase/esterase of unknown function. We show that in a bpeT mutant llpE is constitutively co-transcribed with bpeE-bpeF-oprC. As expected from previous studies with B. cenocepacia, deletion of llpE does not affect antibiotic efflux. Using transcriptional bpeE′-lacZ fusions, we demonstrate that the 188 bp bpeT-llpE intergenic region located between bpeT and the llpE-bpeE-bpeF-oprC operon contains regulatory elements needed for control of bpeT and llpE-bpeE-bpeF-oprC 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 llpE-bpeE-bpeF-oprC expression. The binding site contains the conserved T-N11-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.

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Keyword(s): antibiotic resistance , Burkholderia pseudomallei , efflux , melioidosis and regulation
© 2018 The Authors
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2018-07-19
2024-04-18
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References

  1. Cheng AC, Currie BJ, Dance DA, Funnell SG, Limmathurotsakul D et al. Clinical definitions of melioidosis. Am J Trop Med Hyg 2013; 88:411–413 [View Article][PubMed]
     [Google Scholar]
  2. Wiersinga WJ, Virk HS, Torres AG, Currie BJ, Peacock SJ et al. Melioidosis. Nat Rev Dis Primers 2018; 4:17107 [View Article][PubMed]
     [Google Scholar]
  3. Currie BJ, Kaestli M. Epidemiology: a global picture of melioidosis. Nature 2016; 529:290–291 [View Article][PubMed]
     [Google Scholar]
  4. Limmathurotsakul D, Golding N, Dance DA, Messina JP, Pigott DM et al. Predicted global distribution of Burkholderia pseudomallei and burden of melioidosis. Nat Microbiol 2016; 1:15008 [View Article][PubMed]
     [Google Scholar]
  5. Dance D. Treatment and prophylaxis of melioidosis. Int J Antimicrob Agents 2014; 43:310–318 [View Article][PubMed]
     [Google Scholar]
  6. Lipsitz R, Garges S, Aurigemma R, Baccam P, Blaney DD et al. Workshop on treatment of and postexposure prophylaxis for Burkholderia pseudomallei and B. mallei Infection, 2010. Emerg Infect Dis 2012; 18:e2 [View Article][PubMed]
     [Google Scholar]
  7. Schweizer HP. Mechanisms of antibiotic resistance in Burkholderia pseudomallei: implications for treatment of melioidosis. Future Microbiol 2012; 7:1389–1399 [View Article][PubMed]
     [Google Scholar]
  8. Sarovich DS, Price EP, von Schulze AT, Cook JM, Mayo M et al. Characterization of ceftazidime resistance mechanisms in clinical isolates of Burkholderia pseudomallei from Australia. PLoS One 2012; 7:e30789 [View Article][PubMed]
     [Google Scholar]
  9. Viberg LT, Sarovich DS, Kidd TJ, Geake JB, Bell SC et al. Within-host evolution of Burkholderia pseudomallei during chronic infection of seven Australasian cystic fibrosis patients. MBio 2017; 8:e00356-17 [View Article][PubMed]
     [Google Scholar]
  10. Chantratita N, Rholl DA, Sim B, Wuthiekanun V, Limmathurotsakul D et al. Antimicrobial resistance to ceftazidime involving loss of penicillin-binding protein 3 in Burkholderia pseudomallei. Proc Natl Acad Sci USA 2011; 108:17165–17170 [View Article][PubMed]
     [Google Scholar]
  11. Rholl DA, Papp-Wallace KM, Tomaras AP, Vasil ML, Bonomo RA et al. Molecular investigations of PenA-mediated β-lactam resistance in Burkholderia pseudomallei. Front Microbiol 2011; 2:139 [View Article][PubMed]
     [Google Scholar]
  12. Sarovich DS, Price EP, Limmathurotsakul D, Cook JM, von Schulze AT et al. Development of ceftazidime resistance in an acute Burkholderia pseudomallei infection. Infect Drug Resist 2012; 5:129–132 [View Article][PubMed]
     [Google Scholar]
  13. Podnecky NL, Rhodes KA, Schweizer HP. Efflux pump-mediated drug resistance in Burkholderia. Front Microbiol 2015; 6:305 [View Article][PubMed]
     [Google Scholar]
  14. Moore RA, Deshazer D, Reckseidler S, Weissman A, Woods DE. Efflux-mediated aminoglycoside and macrolide resistance in Burkholderia pseudomallei. Antimicrob Agents Chemother 1999; 43:465–470[PubMed]
     [Google Scholar]
  15. Webb JR, Price EP, Currie BJ, Sarovich DS. Loss of methyltransferase function and increased efflux activity leads to doxycycline resistance in Burkholderia pseudomallei. Antimicrob Agents Chemother 2017; 61:e00268-17 [View Article][PubMed]
     [Google Scholar]
  16. Bugrysheva JV, Sue D, Gee JE, Elrod MG, Hoffmaster AR et al. Antibiotic resistance markers in Burkholderia pseudomallei strain Bp1651 Identified by genome sequence analysis. Antimicrob Agents Chemother 2017; 61:e00010-17 [View Article][PubMed]
     [Google Scholar]
  17. Podin Y, Sarovich DS, Price EP, Kaestli M, Mayo M et al. Burkholderia pseudomallei isolates from Sarawak, Malaysian Borneo, are predominantly susceptible to aminoglycosides and macrolides. Antimicrob Agents Chemother 2014; 58:162–166 [View Article][PubMed]
     [Google Scholar]
  18. Trunck LA, Propst KL, Wuthiekanun V, Tuanyok A, Beckstrom-Sternberg SM et al. Molecular basis of rare aminoglycoside susceptibility and pathogenesis of Burkholderia pseudomallei clinical isolates from Thailand. PLoS Negl Trop Dis 2009; 3:e519 [View Article][PubMed]
     [Google Scholar]
  19. Sarovich DS, Webb JR, Pitman MC, Viberg LT, Mayo M et al. Raising the stakes: Loss of efflux-pump regulation decreases meropenem susceptibility in Burkholderia pseudomallei. Clin Infect Dis 2018243–250 [View Article][PubMed]
     [Google Scholar]
  20. Mima T, Schweizer HP. The BpeAB-OprB efflux pump of Burkholderia pseudomallei 1026b does not play a role in quorum sensing, virulence factor production, or extrusion of aminoglycosides but is a broad-spectrum drug efflux system. Antimicrob Agents Chemother 2010; 54:3113–3120 [View Article][PubMed]
     [Google Scholar]
  21. Hayden HS, Lim R, Brittnacher MJ, Sims EH, Ramage ER et al. Evolution of Burkholderia pseudomallei in recurrent melioidosis. PLoS One 2012; 7:e36507 [View Article][PubMed]
     [Google Scholar]
  22. Podnecky NL, Wuthiekanun V, Peacock SJ, Schweizer HP. The BpeEF-OprC efflux pump is responsible for widespread trimethoprim resistance in clinical and environmental Burkholderia pseudomallei isolates. Antimicrob Agents Chemother 2013; 57:4381–4386 [View Article][PubMed]
     [Google Scholar]
  23. Podnecky NL, Rhodes KA, Mima T, Drew HR, Chirakul S et al. Mechanisms of resistance to folate pathway inhibitors in Burkholderia pseudomallei: deviation from the norm. MBio 2017; 8:e01357-17 [View Article][PubMed]
     [Google Scholar]
  24. Randall LB, Georgi E, Genzel GH, Schweizer HP. Finafloxacin overcomes Burkholderia pseudomallei efflux-mediated fluoroquinolone resistance. J Antimicrob Chemother 2017; 72:1258–1260 [View Article][PubMed]
     [Google Scholar]
  25. Nair BM, Cheung KJ, Griffith A, Burns JL. Salicylate induces an antibiotic efflux pump in Burkholderia cepacia complex genomovar III (B. cenocepacia). J Clin Invest 2004; 113:464–473 [View Article][PubMed]
     [Google Scholar]
  26. Nair BM, Joachimiak LA, Chattopadhyay S, Montano I, Burns JL. Conservation of a novel protein associated with an antibiotic efflux operon in Burkholderia cenocepacia. FEMS Microbiol Lett 2005; 245:337–344 [View Article][PubMed]
     [Google Scholar]
  27. López CM, Rholl DA, Trunck LA, Schweizer HP. Versatile dual-technology system for markerless allele replacement in Burkholderia pseudomallei. Appl Environ Microbiol 2009; 75:6496–6503 [View Article][PubMed]
     [Google Scholar]
  28. Propst KL, Mima T, Choi KH, Dow SW, Schweizer HP. A Burkholderia pseudomallei ∆purM mutant is avirulent in immunocompetent and immunodeficient animals: candidate strain for exclusion from select-agent lists. Infect Immun 2010; 78:3136–3143 [View Article][PubMed]
     [Google Scholar]
  29. CLSI Performance Standards for Antimicrobial Susceptibility Testing: Twenty Fifth Informational Supplement M100-S25 Wayne, PA: 2015
     [Google Scholar]
  30. Kumar A, Mayo M, Trunck LA, Cheng AC, Currie BJ et al. Expression of resistance-nodulation-cell-division efflux pumps in commonly used Burkholderia pseudomallei strains and clinical isolates from northern Australia. Trans R Soc Trop Med Hyg 2008; 102:S145–S151 [View Article][PubMed]
     [Google Scholar]
  31. Chuanchuen R, Gaynor JB, Karkhoff-Schweizer R, Schweizer HP. Molecular characterization of MexL, the transcriptional repressor of the mexJK multidrug efflux operon in Pseudomonas aeruginosa. Antimicrob Agents Chemother 2005; 49:1844–1851 [View Article][PubMed]
     [Google Scholar]
  32. Choi KH, Schweizer HP. mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa. Nat Protoc 2006; 1:153–161 [View Article][PubMed]
     [Google Scholar]
  33. Choi KH, Mima T, Casart Y, Rholl D, Kumar A et al. Genetic tools for select-agent-compliant manipulation of Burkholderia pseudomallei. Appl Environ Microbiol 2008; 74:1064–1075 [View Article][PubMed]
     [Google Scholar]
  34. Miller JH. A Short Course in Bacterial Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1992
     [Google Scholar]
  35. Schlager B, Straessle A, Hafen E. Use of anionic denaturing detergents to purify insoluble proteins after overexpression. BMC Biotechnol 2012; 12:95 [View Article][PubMed]
     [Google Scholar]
  36. Rhodes KA. Complex Regulation of BpeEF-OprC Mediated Drug Efflux In Burkholderia pseudomallei. Dissertation Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado; 2016
     [Google Scholar]
  37. Maddocks SE, Oyston PC. Structure and function of the LysR-type transcriptional regulator (LTTR) family proteins. Microbiology 2008; 154:3609–3623 [View Article][PubMed]
     [Google Scholar]
  38. Momany C, Neidle EL. Defying stereotypes: the elusive search for a universal model of LysR-type regulation. Mol Microbiol 2012; 83:453–456 [View Article][PubMed]
     [Google Scholar]
  39. Gong W, Xiong G, Maser E. Oligomerization and negative autoregulation of the LysR-type transcriptional regulator HsdR from Comamonas testosteroni. J Steroid Biochem Mol Biol 2012; 132:203–211 [View Article][PubMed]
     [Google Scholar]
  40. Ezezika OC, Haddad S, Neidle EL, Momany C. Oligomerization of BenM, a LysR-type transcriptional regulator: structural basis for the aggregation of proteins in this family. Acta Crystallogr Sect F Struct Biol Cryst Commun 2007; 63:361–368 [View Article][PubMed]
     [Google Scholar]
  41. Ruangprasert A, Craven SH, Neidle EL, Momany C. Full-length structures of BenM and two variants reveal different oligomerization schemes for LysR-type transcriptional regulators. J Mol Biol 2010; 404:568–586 [View Article][PubMed]
     [Google Scholar]
  42. Vadlamani G, Thomas MD, Patel TR, Donald LJ, Reeve TM et al. The β-lactamase gene regulator AmpR is a tetramer that recognizes and binds the D-Ala-D-Ala motif of its repressor UDP-N-acetylmuramic acid (MurNAc)-pentapeptide. J Biol Chem 2015; 290:2630–2643 [View Article][PubMed]
     [Google Scholar]
  43. Deghmane AE, Giorgini D, Larribe M, Alonso JM, Taha MK. Down-regulation of pili and capsule of Neisseria meningitidis upon contact with epithelial cells is mediated by CrgA regulatory protein. Mol Microbiol 2002; 43:1555–1564 [View Article][PubMed]
     [Google Scholar]
  44. Alanazi AM, Neidle EL, Momany C. The DNA-binding domain of BenM reveals the structural basis for the recognition of a T-N11-A sequence motif by LysR-type transcriptional regulators. Acta Crystallogr D Biol Crystallogr 2013; 69:1995–2007 [View Article][PubMed]
     [Google Scholar]
  45. Knapp GS, Hu JC. Specificity of the E. coli LysR-type transcriptional regulators. PLoS One 2010; 5:e15189 [View Article][PubMed]
     [Google Scholar]
  46. Ooi WF, Ong C, Nandi T, Kreisberg JF, Chua HH et al. The condition-dependent transcriptional landscape of Burkholderia pseudomallei. PLoS Genet 2013; 9:e1003795 [View Article][PubMed]
     [Google Scholar]
  47. Tuanyok A, Kim HS, Nierman WC, Yu Y, Dunbar J et al. Genome-wide expression analysis of iron regulation in Burkholderia pseudomallei and Burkholderia mallei using DNA microarrays. FEMS Microbiol Lett 2005; 252:327–335 [View Article][PubMed]
     [Google Scholar]
  48. Winsor GL, Khaira B, van Rossum T, Lo R, Whiteside MD et al. The Burkholderia genome database: facilitating flexible queries and comparative analyses. Bioinformatics 2008; 24:2803–2804 [View Article][PubMed]
     [Google Scholar]
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Molecular determinants of Burkholderia pseudomallei BpeEF-OprC efflux pump expression
Microbiology 164, 1156 (2018); https://doi.org/10.1099/mic.0.000691
/content/journal/micro/10.1099/mic.0.000691
/content/journal/micro/10.1099/mic.0.000691
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