1887

Abstract

Burkholderia pseudomallei, the aetiological agent of melioidosis, is an inhabitant of soil and water in many tropical and subtropical regions worldwide. It possesses six distinct type VI secretion systems (T6SS-1 to T6SS-6), but little is known about most of them, as they are poorly expressed in laboratory culture media. A genetic screen was devised to locate a putative repressor of the T6SS-2 gene cluster and a MarR family transcriptional regulator, termed TctR, was identified. The inactivation of tctR resulted in a 50-fold increase in the expression of an hcp2lacZ transcriptional fusion, indicating that TctR is a negative regulator of the T6SS-2 gene cluster. Surprisingly, the tctR mutation resulted in a significant decrease in the expression of an hcp6–lacZ transcriptional fusion. B. pseudomallei K96243 and a tctR mutant were grown to logarithmic phase in rich culture medium and RNA was isolated and sequenced in order to identify other genes regulated by TctR. The results identified seven gene clusters that were repressed by TctR, including T6SS-2, and three gene clusters that were significantly activated. A small molecule library consisting of 1120 structurally defined compounds was screened to identify a putative ligand (or ligands) that might bind TctR and derepress transcription of the T6SS-2 gene cluster. Seven compounds, six fluoroquinolones and one quinolone, activated the expression of hcp2–lacZ. Subinhibitory ciprofloxacin also increased the expression of the T6SS-3, T6SS-4 and T6SS-6 gene clusters. This study highlights the complex layers of regulatory control that B. pseudomallei utilizes to ensure that T6SS expression only occurs under very defined environmental conditions.

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2018-07-27
2024-03-28
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References

  1. Fhogartaigh C, Dance D. Glanders & Melioidosis: a Zoonosis and a Sapronosis – “Same Same, but Different”. In Sing A. (editor) Zoonoses – Infections Affecting Humans and Animals Dordrecht: Springer; 2015 pp. 859–888
    [Google Scholar]
  2. Kuris AM, Lafferty KD, Sokolow SH. Sapronosis: a distinctive type of infectious agent. Trends Parasitol 2014; 30:386–393 [View Article][PubMed]
    [Google Scholar]
  3. 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]
  4. Centers for Disease Control and Prevention (CDC), Department of Health and Human Services (HHS) Possession, use, and transfer of select agents and toxins; biennial review. Final rule. Fed Regist 2012; 77:61083–61115[PubMed]
    [Google Scholar]
  5. Perumal Samy R, Stiles BG, Sethi G, Lim LHK. Melioidosis: clinical impact and public health threat in the tropics. PLoS Negl Trop Dis 2017; 11:e0004738 [View Article][PubMed]
    [Google Scholar]
  6. Willcocks SJ, Denman CC, Atkins HS, Wren BW. Intracellular replication of the well-armed pathogen Burkholderia pseudomallei. Curr Opin Microbiol 2016; 29:94–103 [View Article][PubMed]
    [Google Scholar]
  7. Burtnick MN, Brett PJ, DeShazer D. Proteomic analysis of the Burkholderia pseudomallei type II secretome reveals hydrolytic enzymes, novel proteins, and the deubiquitinase TssM. Infect Immun 2014; 82:3214–3226 [View Article][PubMed]
    [Google Scholar]
  8. Tan KS, Chen Y, Lim YC, Tan GY, Liu Y et al. Suppression of host innate immune response by Burkholderia pseudomallei through the virulence factor TssM. J Immunol 2010; 184:5160–5171 [View Article][PubMed]
    [Google Scholar]
  9. vander Broek CW, Stevens JM. Type III secretion in the melioidosis pathogen Burkholderia pseudomallei. Front Cell Infect Microbiol 2017; 7:255 [View Article][PubMed]
    [Google Scholar]
  10. Benanti EL, Nguyen CM, Welch MD. Virulent Burkholderia species mimic host actin polymerases to drive actin-based motility. Cell 2015; 161:348–360 [View Article][PubMed]
    [Google Scholar]
  11. Stevens MP, Stevens JM, Jeng RL, Taylor LA, Wood MW et al. Identification of a bacterial factor required for actin-based motility of Burkholderia pseudomallei. Mol Microbiol 2005; 56:40–53 [View Article][PubMed]
    [Google Scholar]
  12. Burtnick MN, Brett PJ, Harding SV, Ngugi SA, Ribot WJ et al. The cluster 1 type VI secretion system is a major virulence determinant in Burkholderia pseudomallei. Infect Immun 2011; 79:1512–1525 [View Article][PubMed]
    [Google Scholar]
  13. Toesca IJ, French CT, Miller JF. The Type VI secretion system spike protein VgrG5 mediates membrane fusion during intercellular spread by pseudomallei group Burkholderia species. Infect Immun 2014; 82:1436–1444 [View Article][PubMed]
    [Google Scholar]
  14. Hachani A, Wood TE, Filloux A. Type VI secretion and anti-host effectors. Curr Opin Microbiol 2016; 29:81–93 [View Article][PubMed]
    [Google Scholar]
  15. Lien YW, Lai EM. Type VI secretion effectors: methodologies and biology. Front Cell Infect Microbiol 2017; 7:254 [View Article][PubMed]
    [Google Scholar]
  16. Wong J, Chen Y, Gan YH. Host cytosolic glutathione sensing by a membrane histidine kinase activates the Type VI secretion system in an intracellular bacterium. Cell Host Microbe 2015; 18:38–48 [View Article][PubMed]
    [Google Scholar]
  17. Burtnick MN, DeShazer D, Nair V, Gherardini FC, Brett PJ. Burkholderia mallei cluster 1 type VI secretion mutants exhibit growth and actin polymerization defects in RAW 264.7 murine macrophages. Infect Immun 2010; 78:88–99 [View Article][PubMed]
    [Google Scholar]
  18. Schwarz S, West TE, Boyer F, Chiang WC, Carl MA et al. Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions. PLoS Pathog 2010; 6:e1001068 [View Article][PubMed]
    [Google Scholar]
  19. Bernard CS, Brunet YR, Gueguen E, Cascales E. Nooks and crannies in type VI secretion regulation. J Bacteriol 2010; 192:3850–3860 [View Article][PubMed]
    [Google Scholar]
  20. Brett PJ, DeShazer D, Woods DE. Burkholderia thailandensis sp. nov., a Burkholderia pseudomallei-like species. Int J Syst Bacteriol 1998; 48:317–320 [View Article][PubMed]
    [Google Scholar]
  21. Majerczyk C, Schneider E, Greenberg EP. Quorum sensing control of Type VI secretion factors restricts the proliferation of quorum-sensing mutants. eLife 2016; 5:e14712 [View Article][PubMed]
    [Google Scholar]
  22. Russell AB, Singh P, Brittnacher M, Bui NK, Hood RD et al. A widespread bacterial type VI secretion effector superfamily identified using a heuristic approach. Cell Host Microbe 2012; 11:538–549 [View Article][PubMed]
    [Google Scholar]
  23. Hamad MA, Zajdowicz SL, Holmes RK, Voskuil MI. An allelic exchange system for compliant genetic manipulation of the select agents Burkholderia pseudomallei and Burkholderia mallei. Gene 2009; 430:123–131 [View Article][PubMed]
    [Google Scholar]
  24. Logue CA, Peak IR, Beacham IR. Facile construction of unmarked deletion mutants in Burkholderia pseudomallei using sacB counter-selection in sucrose-resistant and sucrose-sensitive isolates. J Microbiol Methods 2009; 76:320–323 [View Article][PubMed]
    [Google Scholar]
  25. Simons RW, Houman F, Kleckner N. Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene 1987; 53:85–96 [View Article][PubMed]
    [Google Scholar]
  26. Miller JH. Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1972
    [Google Scholar]
  27. Dennis JJ, Zylstra GJ. Plasposons: modular self-cloning minitransposon derivatives for rapid genetic analysis of Gram-negative bacterial genomes. Appl Environ Microbiol 1998; 64:2710–2715[PubMed]
    [Google Scholar]
  28. Deochand DK, Grove A. MarR family transcription factors: dynamic variations on a common scaffold. Crit Rev Biochem Mol Biol 2017; 52:595–613 [View Article][PubMed]
    [Google Scholar]
  29. Lefebre MD, Valvano MA. Construction and evaluation of plasmid vectors optimized for constitutive and regulated gene expression in Burkholderia cepacia complex isolates. Appl Environ Microbiol 2002; 68:5956–5964 [View Article][PubMed]
    [Google Scholar]
  30. 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]
  31. Gutiérrez-Corona JF, Romo-Rodríguez P, Santos-Escobar F, Espino-Saldaña AE, Hernández-Escoto H. Microbial interactions with chromium: basic biological processes and applications in environmental biotechnology. World J Microbiol Biotechnol 2016; 32:191 [View Article][PubMed]
    [Google Scholar]
  32. Bochner BR, Gadzinski P, Panomitros E. Phenotype MicroArrays for high-throughput phenotypic testing and assay of gene function. Genome Res 2001; 11:1246–1255 [View Article][PubMed]
    [Google Scholar]
  33. Cohen SP, Hächler H, Levy SB. Genetic and functional analysis of the multiple antibiotic resistance (mar) locus in Escherichia coli. J Bacteriol 1993; 175:1484–1492 [View Article][PubMed]
    [Google Scholar]
  34. Hächler H, Cohen SP, Levy SB. marA, a regulated locus which controls expression of chromosomal multiple antibiotic resistance in Escherichia coli. J Bacteriol 1991; 173:5532–5538 [View Article][PubMed]
    [Google Scholar]
  35. Basler M. Type VI secretion system: secretion by a contractile nanomachine. Philos Trans R Soc Lond B Biol Sci 2015; 370:20150021 [View Article][PubMed]
    [Google Scholar]
  36. Goh EB, Yim G, Tsui W, McClure J, Surette MG et al. Transcriptional modulation of bacterial gene expression by subinhibitory concentrations of antibiotics. Proc Natl Acad Sci USA 2002; 99:17025–17030 [View Article][PubMed]
    [Google Scholar]
  37. 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]
    [Google Scholar]
  38. DeShazer D, Woods DE. Animal models of melioidosis. In Zak O, Sande M. (editors) Handbook of Animal Models of Infection London: Academic Press Ltd; 1999 pp. 199–203
    [Google Scholar]
  39. Grove A. MarR family transcription factors. Curr Biol 2013; 23:R142–R143 [View Article][PubMed]
    [Google Scholar]
  40. Holden MT, Titball RW, Peacock SJ, Cerdeño-Tárraga AM, Atkins T et al. Genomic plasticity of the causative agent of melioidosis, Burkholderia pseudomallei. Proc Natl Acad Sci USA 2004; 101:14240–14245 [View Article][PubMed]
    [Google Scholar]
  41. Grove A. Urate-responsive MarR homologs from Burkholderia. Mol Biosyst 2010; 6:2133–2142 [View Article][PubMed]
    [Google Scholar]
  42. Gupta A, Fuentes SM, Grove A. Redox-sensitive MarR homologue BifR from Burkholderia thailandensis regulates biofilm formation. Biochemistry 2017; 56:2315–2327 [View Article][PubMed]
    [Google Scholar]
  43. Gupta A, Grove A. Ligand-binding pocket bridges DNA-binding and dimerization domains of the urate-responsive MarR homologue MftR from Burkholderia thailandensis. Biochemistry 2014; 53:4368–4380 [View Article][PubMed]
    [Google Scholar]
  44. Hantrakun V, Rongkard P, Oyuchua M, Amornchai P, Lim C et al. Soil nutrient depletion is associated with the presence of Burkholderia pseudomallei. Appl Environ Microbiol 2016; 82:7086–7092 [View Article][PubMed]
    [Google Scholar]
  45. Davies J, Spiegelman GB, Yim G. The world of subinhibitory antibiotic concentrations. Curr Opin Microbiol 2006; 9:445–453 [View Article][PubMed]
    [Google Scholar]
  46. Yim G, Wang HH, Davies J. Antibiotics as signalling molecules. Philos Trans R Soc Lond B Biol Sci 2007; 362:1195–1200 [View Article][PubMed]
    [Google Scholar]
  47. Jones C, Allsopp L, Horlick J, Kulasekara H, Filloux A. Subinhibitory concentration of kanamycin induces the Pseudomonas aeruginosa type VI secretion system. PLoS One 2013; 8:e81132 [View Article][PubMed]
    [Google Scholar]
  48. Okada BK, Wu Y, Mao D, Bushin LB, Seyedsayamdost MR. Mapping the trimethoprim-induced secondary metabolome of Burkholderia thailandensis. ACS Chem Biol 2016; 11:2124–2130 [View Article][PubMed]
    [Google Scholar]
  49. Chaowagul W, Suputtamongkol Y, Dance DA, Rajchanuvong A, Pattara-Arechachai J et al. Relapse in melioidosis: incidence and risk factors. J Infect Dis 1993; 168:1181–1185[PubMed]
    [Google Scholar]
  50. Limmathurotsakul D, Chaowagul W, Chantratita N, Wuthiekanun V, Biaklang M et al. A simple scoring system to differentiate between relapse and re-infection in patients with recurrent melioidosis. PLoS Negl Trop Dis 2008; 2:e327 [View Article][PubMed]
    [Google Scholar]
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