1887

Abstract

Purpose. Burkholderia pseudomallei , the tier 1 agent of melioidosis, is a saprophytic microbe that causes endemic infections in tropical regions such as South-East Asia and Northern Australia. It is globally distributed, challenging to diagnose and treat, infectious by several routes including inhalation, and has potential for adversarial use. B. pseudomallei strain MSHR5848 produces two colony variants, smooth (S) and rough (R), which exhibit a divergent range of morphological, biochemical and metabolic phenotypes, and differ in macrophage and animal infectivity. We aimed to characterize two major phenotypic differences, analyse gene expression and study the regulatory basis of the variation.

Methodology. Phenotypic expression was characterized by DNA and RNA sequencing, microscopy, and differential bacteriology. Regulatory genes were identified by cloning and bioinformatics.

Results/Key findings. Whereas S produced larger quantities of extracellular DNA, R was upregulated in the production of a unique chromosome 1-encoded Siphoviridae-like bacteriophage, φMSHR5848. Exploratory transcriptional analyses revealed significant differences in variant expression of genes encoding siderophores, pili assembly, type VI secretion system cluster 4 (T6SS-4) proteins, several exopolysaccharides and secondary metabolites. A single 3 base duplication in S was the only difference that separated the variants genetically. It occurred upstream of a cluster of bacteriophage-associated genes on chromosome 2 that were upregulated in S. The first two genes were involved in regulating expression of the multiple phenotypes distinguishing S and R.

Conclusion. Bacteriophage-associated proteins have a major role in the phenotypic expression of MSHR5848. The goals are to determine the regulatory basis of this phenotypic variation and its role in pathogenesis and environmental persistence of B. pseudomallei .

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.000908
2019-01-10
2019-10-18
Loading full text...

Full text loading...

References

  1. 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 [CrossRef][PubMed]
    [Google Scholar]
  2. Dance D. Treatment and prophylaxis of melioidosis. Int J Antimicrob Agents 2014;43:310–318 [CrossRef][PubMed]
    [Google Scholar]
  3. Currie BJ, Fisher DA, Anstey NM, Jacups SP. Melioidosis: acute and chronic disease, relapse and re-activation. Trans R Soc Trop Med Hyg 2000;94:301–304 [CrossRef][PubMed]
    [Google Scholar]
  4. Currie BJ, Ward L, Cheng AC. The epidemiology and clinical spectrum of melioidosis: 540 cases from the 20 year Darwin prospective study. PLoS Negl Trop Dis 2010;4:e900 [CrossRef][PubMed]
    [Google Scholar]
  5. Wiersinga WJ, Currie BJ, Peacock SJ. Melioidosis. N Engl J Med 2012;367:1035–1044 [CrossRef][PubMed]
    [Google Scholar]
  6. Dance DA, Wuthiekanun V, Chaowagul W, Suputtamongkol Y, White NJ. Development of resistance to ceftazidime and co-amoxiclav in Pseudomonas pseudomallei. J Antimicrob Chemother 1991;28:321–324 [CrossRef][PubMed]
    [Google Scholar]
  7. Shea AA, Bernhards RC, Cote CK, Chase CJ, Koehler JW et al. Two stable variants of Burkholderia pseudomallei strain MSHR5848 express broadly divergent in vitro phenotypes associated with their virulence differences. PLoS One 2017;12:e0171363 [CrossRef][PubMed]
    [Google Scholar]
  8. FSAP Occupational Health Program Atlanta, GA:: Centers for Disease Control and Prevention;; 2017;www.selectagents.gov/ohp-app1.html
    [Google Scholar]
  9. Stanton AT, Fletcher W, Kanagarayer K. Two cases of melioidosis. J Hyg (Lond) 1924;23:268–276 [CrossRef][PubMed]
    [Google Scholar]
  10. Nicholls L. Melioidosis with special reference to the dissociation of Bacillus whittori. Brit J Exptl Pathol 1930;XI:393–399
    [Google Scholar]
  11. Rogul M, Carr SR. Variable ammonia production among smooth and rough strains of Pseudomonas pseudomallei: resemblance to bacteriocin production. J Bacteriol 1972;112:372–380[PubMed]
    [Google Scholar]
  12. Vial L, Groleau MC, Lamarche MG, Filion G, Castonguay-Vanier J et al. Phase variation has a role in Burkholderia ambifaria niche adaptation. ISME J 2010;4:49–60 [CrossRef][PubMed]
    [Google Scholar]
  13. van der Woude MW, Bäumler AJ. Phase and antigenic variation in bacteria. Clin Microbiol Rev 2004;17:581–611 [CrossRef][PubMed]
    [Google Scholar]
  14. Wisniewski-Dyé F, Vial L. Phase and antigenic variation mediated by genome modifications. Antonie Van Leeuwenhoek 2008;94:493–515 [CrossRef][PubMed]
    [Google Scholar]
  15. Chantratita N, Wuthiekanun V, Boonbumrung K, Tiyawisutsri R, Vesaratchavest M et al. Biological relevance of colony morphology and phenotypic switching by Burkholderia pseudomallei. J Bacteriol 2007;189:807–817 [CrossRef][PubMed]
    [Google Scholar]
  16. Velapatiño B, Limmathurotsakul D, Peacock SJ, Speert DP. Identification of differentially expressed proteins from Burkholderia pseudomallei isolated during primary and relapsing melioidosis. Microbes Infect 2012;14:335–340 [CrossRef][PubMed]
    [Google Scholar]
  17. Tandhavanant S, Thanwisai A, Limmathurotsakul D, Korbsrisate S, Day NP et al. Effect of colony morphology variation of Burkholderia pseudomallei on intracellular survival and resistance to antimicrobial environments in human macrophages in vitro. BMC Microbiol 2010;10:303 [CrossRef][PubMed]
    [Google Scholar]
  18. Gierok P, Kohler C, Steinmetz I, Lalk M. Burkholderia pseudomallei colony norphotypes show a synchronized metabolic pattern after acute infection. PLoS Negl Trop Dis 2016;10:e0004483 [CrossRef][PubMed]
    [Google Scholar]
  19. Austin CR, Goodyear AW, Bartek IL, Stewart A, Sutherland MD et al. A Burkholderia pseudomallei colony variant necessary for gastric colonization. MBio 2015;6::e02462-14 [CrossRef][PubMed]
    [Google Scholar]
  20. Wikraiphat C, Saiprom N, Tandhavanant S, Heiss C, Azadi P et al. Colony morphology variation of Burkholderia pseudomallei is associated with antigenic variation and O-polysaccharide modification. Infect Immun 2015;83:2127–2138 [CrossRef][PubMed]
    [Google Scholar]
  21. Bernhards RC, Cote CK, Amemiya K, Waag DM, Klimko CP et al. Characterization of in vitro phenotypes of Burkholderia pseudomallei and Burkholderia mallei strains potentially associated with persistent infection in mice. Arch Microbiol 2017;199:277–301 [CrossRef]
    [Google Scholar]
  22. Ronning CM, Losada L, Brinkac L, Inman J, Ulrich RL et al. Genetic and phenotypic diversity in Burkholderia: contributions by prophage and phage-like elements. BMC Microbiol 2010;10:202 [CrossRef][PubMed]
    [Google Scholar]
  23. Nierman WC, Deshazer D, Kim HS, Tettelin H, Nelson KE et al. Structural flexibility in the Burkholderia mallei genome. Proc Natl Acad Sci USA 2004;101:14246–14251 [CrossRef][PubMed]
    [Google Scholar]
  24. Srinivasan A, Kraus CN, Deshazer D, Becker PM, Dick JD et al. Glanders in a military research microbiologist. N Engl J Med 2001;345:256–258 [CrossRef][PubMed]
    [Google Scholar]
  25. Ashdown LR. An improved screening technique for isolation of Pseudomonas pseudomallei from clinical specimens. Pathology 1979;11:293–297 [CrossRef][PubMed]
    [Google Scholar]
  26. Smith PB, Hancock GA, Rhoden DL. Improved medium for detecting deoxyribonuclease-producing bacteria. Appl Microbiol 1969;18:991–993[PubMed]
    [Google Scholar]
  27. Deshazer D, Waag DM, Fritz DL, Woods DE. Identification of a Burkholderia mallei polysaccharide gene cluster by subtractive hybridization and demonstration that the encoded capsule is an essential virulence determinant. Microb Pathog 2001;30:253–269 [CrossRef][PubMed]
    [Google Scholar]
  28. Fritz DL, Vogel P, Brown DR, Deshazer D, Waag DM. Mouse model of sublethal and lethal intraperitoneal glanders (Burkholderia mallei). Vet Pathol 2000;37:626–636 [CrossRef][PubMed]
    [Google Scholar]
  29. Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnetJ 2011;17:10–12 [CrossRef]
    [Google Scholar]
  30. Schmieder R, Edwards R. Quality control and preprocessing of metagenomic datasets. Bioinformatics 2011;27:863–864 [CrossRef][PubMed]
    [Google Scholar]
  31. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods 2012;9:357–359 [CrossRef][PubMed]
    [Google Scholar]
  32. Anders S, Pyl PT, Huber W. HTSeq – a Python framework to work with high-throughput sequencing data. Bioinformatics 2015;31:166–169 [CrossRef][PubMed]
    [Google Scholar]
  33. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 2014;15:550 [CrossRef][PubMed]
    [Google Scholar]
  34. Deshazer D. Genomic diversity of Burkholderia pseudomallei clinical isolates: subtractive hybridization reveals a Burkholderia mallei-specific prophage in B. pseudomallei 1026b. J Bacteriol 2004;186:3938–3950 [CrossRef][PubMed]
    [Google Scholar]
  35. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J et al. The sequence alignment/map format and SAMtools. Bioinformatics 2009;25:2078–2079 [CrossRef][PubMed]
    [Google Scholar]
  36. R Core Team A Language and Environment for Statistical Computing Vienna: R Foundation for Statistical Computing; 2016
    [Google Scholar]
  37. Khan SR, Gaines J, Roop RM, Farrand SK. Broad-host-range expression vectors with tightly regulated promoters and their use to examine the influence of TraR and TraM expression on Ti plasmid quorum sensing. Appl Environ Microbiol 2008;74:5053–5062 [CrossRef][PubMed]
    [Google Scholar]
  38. Deshazer D, Brett PJ, Carlyon R, Woods DE. Mutagenesis of Burkholderia pseudomallei with Tn5-OT182: isolation of motility mutants and molecular characterization of the flagellin structural gene. J Bacteriol 1997;179:2116–2125 [CrossRef][PubMed]
    [Google Scholar]
  39. Fokine A, Rossmann MG. Molecular architecture of tailed double-stranded DNA phages. Bacteriophage 2014;4:e28281 [CrossRef][PubMed]
    [Google Scholar]
  40. Conway T, Cohen P. Metabolism and Bacterial Pathogenesis Washington, DC: ASM Press; 2015
    [Google Scholar]
  41. Essex-Lopresti AE, Boddey JA, Thomas R, Smith MP, Hartley MG et al. A type IV pilin, PilA, contributes to adherence of Burkholderia pseudomallei and virulence in vivo. Infect Immun 2005;73:1260–1264 [CrossRef][PubMed]
    [Google Scholar]
  42. Brown NF, Boddey JA, Flegg CP, Beacham IR. Adherence of Burkholderia pseudomallei cells to cultured human epithelial cell lines is regulated by growth temperature. Infect Immun 2002;70:974–980 [CrossRef][PubMed]
    [Google Scholar]
  43. 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 [CrossRef][PubMed]
    [Google Scholar]
  44. Chen Y, Wong J, Sun GW, Liu Y, Tan GY et al. Regulation of type VI secretion system during Burkholderia pseudomallei infection. Infect Immun 2011;79:3064–3073 [CrossRef][PubMed]
    [Google Scholar]
  45. Marahiel MA. Working outside the protein-synthesis rules: insights into non-ribosomal peptide synthesis. J Pept Sci 2009;15:799–807 [CrossRef][PubMed]
    [Google Scholar]
  46. Esmaeel Q, Pupin M, Kieu NP, Chataigné G, Béchet M et al. Burkholderia genome mining for nonribosomal peptide synthetases reveals a great potential for novel siderophores and lipopeptides synthesis. Microbiology Open 2016;5:512–526 [CrossRef][PubMed]
    [Google Scholar]
  47. Alice AF, López CS, Lowe CA, Ledesma MA, Crosa JH. Genetic and transcriptional analysis of the siderophore malleobactin biosynthesis and transport genes in the human pathogen Burkholderia pseudomallei K96243. J Bacteriol 2006;188:1551–1566 [CrossRef][PubMed]
    [Google Scholar]
  48. Franke J, Ishida K, Hertweck C. Evolution of siderophore pathways in human pathogenic bacteria. J Am Chem Soc 2014;136:5599–5602 [CrossRef][PubMed]
    [Google Scholar]
  49. Biggins JB, Kang HS, Ternei MA, Deshazer D, Brady SF. The chemical arsenal of Burkholderia pseudomallei is essential for pathogenicity. J Am Chem Soc 2014;136:9484–9490 [CrossRef][PubMed]
    [Google Scholar]
  50. Adler C, Corbalán NS, Seyedsayamdost MR, Pomares MF, de Cristóbal RE et al. Catecholate siderophores protect bacteria from pyochelin toxicity. PLoS One 2012;7:e46754 [CrossRef]
    [Google Scholar]
  51. Sokol PA, Woods DE. Effect of pyochelin on Pseudomonas cepacia respiratory infections. Microb Pathog 1988;5:197–205 [CrossRef][PubMed]
    [Google Scholar]
  52. Klaus JR, Deay J, Neuenswander B, Hursh W, Gao Z et al. Malleilactone is a Burkholderia pseudomallei virulence factor regulated by antibiotics and quorum sensing. J Bacteriol 2018;200::e00008-18 [CrossRef][PubMed]
    [Google Scholar]
  53. Mathews D, Payne J. Transmembrane transport of small peptides. In: Current Topics in Membrane and Transport, vol. 14 New York: Academic Press; 1980; pp.331–425
    [Google Scholar]
  54. Abu Kwaik Y, Bumann D. Microbial quest for food in vivo: 'nutritional virulence' as an emerging paradigm. Cell Microbiol 2013;15:882–890 [CrossRef][PubMed]
    [Google Scholar]
  55. Pletzer D, Lafon C, Braun Y, Köhler T, Page MG et al. High-throughput screening of dipeptide utilization mediated by the ABC transporter DppBCDF and its substrate-binding proteins DppA1-A5 in Pseudomonas aeruginosa. PLoS One 2014;9:e111311 [CrossRef][PubMed]
    [Google Scholar]
  56. Alkhuder K, Meibom KL, Dubail I, Dupuis M, Charbit A. Glutathione provides a source of cysteine essential for intracellular multiplication of Francisella tularensis. PLoS Pathog 2009;5:e1000284 [CrossRef][PubMed]
    [Google Scholar]
  57. Niu H, Yamaguchi M, Rikihisa Y. Subversion of cellular autophagy by Anaplasma phagocytophilum. Cell Microbiol 2008;10:593–605 [CrossRef][PubMed]
    [Google Scholar]
  58. Welkos SL, Klimko CP, Kern SJ, Bearss JJ, Bozue JA et al. Characterization of Burkholderia pseudomallei strains using a murine intraperitoneal infection model and in vitro macrophage assays. PLoS One 2015;10:e0124667 [CrossRef][PubMed]
    [Google Scholar]
  59. Cuccui J, Milne TS, Harmer N, George AJ, Harding SV et al. Characterization of the Burkholderia pseudomallei K96243 capsular polysaccharide I coding region. Infect Immun 2012;80:1209–1221 [CrossRef][PubMed]
    [Google Scholar]
  60. Borlee GI, Plumley BA, Martin KH, Somprasong N, Mangalea MR et al. Genome-scale analysis of the genes that contribute to Burkholderia pseudomallei biofilm formation identifies a crucial exopolysaccharide biosynthesis gene cluster. PLoS Negl Trop Dis 2017;11:e0005689 [CrossRef][PubMed]
    [Google Scholar]
  61. Reckseidler-Zenteno SL, Viteri DF, Moore R, Wong E, Tuanyok A et al. Characterization of the type III capsular polysaccharide produced by Burkholderia pseudomallei. J Med Microbiol 2010;59:1403–1414 [CrossRef][PubMed]
    [Google Scholar]
  62. Sarkar-Tyson M, Thwaite JE, Harding SV, Smither SJ, Oyston PC et al. Polysaccharides and virulence of Burkholderia pseudomallei. J Med Microbiol 2007;56:1005–1010 [CrossRef][PubMed]
    [Google Scholar]
  63. Tuanyok A, Stone JK, Mayo M, Kaestli M, Gruendike J et al. The genetic and molecular basis of O-antigenic diversity in Burkholderia pseudomallei lipopolysaccharide. PLoS Negl Trop Dis 2012;6:e1453 [CrossRef][PubMed]
    [Google Scholar]
  64. Woods DE. The use of animal infection models to study the pathogenesis of melioidosis and glanders. Trends Microbiol 2002;10:483–484 [CrossRef][PubMed]
    [Google Scholar]
  65. Zulkefli NJ, Mariappan V, Vellasamy KM, Chong CW, Thong KL et al. Molecular evidence of Burkholderia pseudomallei genotypes based on geographical distribution. PeerJ 2016;4:e1802 [CrossRef][PubMed]
    [Google Scholar]
  66. Daligault HE, Davenport KW, Minogue TD, Bishop-Lilly KA, Broomall SM et al. Whole-genome assemblies of 56 Burkholderia species. Genome Announc 2014;2::e01106-14 [CrossRef][PubMed]
    [Google Scholar]
  67. Amemiya K, Dankmeyer JL, Fetterer DP, Worsham PL, Welkos SL et al. Comparison of the early host immune response to two widely diverse virulent strains of Burkholderia pseudomallei that cause acute or chronic infections in BALB/c mice. Microb Pathog 2015;86:53–63 [CrossRef][PubMed]
    [Google Scholar]
  68. van Zandt KE, Tuanyok A, Keim PS, Warren RL, Gelhaus HC. An objective approach for Burkholderia pseudomallei strain selection as challenge material for medical countermeasures efficacy testing. Front Cell Infect Microbiol 2012;2:120 [CrossRef][PubMed]
    [Google Scholar]
  69. Chantratita N, Tandhavanant S, Wikraiphat C, Trunck LA, Rholl DA et al. Proteomic analysis of colony morphology variants of Burkholderia pseudomallei defines a role for the arginine deiminase system in bacterial survival. J Proteomics 2012;75:1031–1042 [CrossRef][PubMed]
    [Google Scholar]
  70. Deitsch KW, Lukehart SA, Stringer JR. Common strategies for antigenic variation by bacterial, fungal and protozoan pathogens. Nat Rev Microbiol 2009;7:493–503 [CrossRef][PubMed]
    [Google Scholar]
  71. Dubnau D, Losick R. Bistability in bacteria. Mol Microbiol 2006;61:564–572 [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.000908
Loading
/content/journal/jmm/10.1099/jmm.0.000908
Loading

Data & Media loading...

Supplements

Supplementary File 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error