1887

Abstract

grows in extracellular DNA (eDNA)-enriched biofilms and infection sites. eDNA is generally considered to be a structural biofilm polymer required for aggregation and biofilm maturation. In addition, eDNA can sequester divalent metal cations, acidify growth media and serve as a nutrient source.

We wanted to determine the genome-wide influence on the transcriptome of planktonic PAO1 grown in the presence of eDNA.

RNA-seq analysis was performed to determine the genome-wide effects on gene expression of PAO1 grown with eDNA. Transcriptional fusions were used to confirm eDNA regulation and to validate phenotypes associated with growth in eDNA.

The transcriptome of eDNA-regulated genes included 89 induced and 76 repressed genes (FDR<0.05). A large number of eDNA-induced genes appear to be involved in utilizing DNA as a nutrient. Several eDNA-induced genes are also induced by acidic pH 5.5, and eDNA/acidic pH promoted an acid tolerance response in . The terminal oxidase is induced by both eDNA and pH 5.5, and contributed to the acid tolerance phenotype. Quantitative metal analysis confirmed that DNA binds to diverse metals, which helps explain why many genes involved in a general uptake of metals were controlled by eDNA. Growth in the presence of eDNA also promoted intracellular bacterial survival and influenced virulence in the acute infection model of fruit flies.

The diverse functions of the eDNA-regulated genes underscore the important role of this extracellular polymer in promoting antibiotic resistance, virulence, acid tolerance and nutrient utilization; phenotypes that contribute to long-term survival.

Funding
This study was supported by the:
  • Shawn Lewenza , Cystic Fibrosis Canada , (Award na)
  • Heidi Mulcahy-O'Grady , Cystic Fibrosis Canada , (Award na)
Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.001184
2020-04-03
2020-06-04
Loading full text...

Full text loading...

/deliver/fulltext/jmm/10.1099/jmm.0.001184/jmm001184.html?itemId=/content/journal/jmm/10.1099/jmm.0.001184&mimeType=html&fmt=ahah

References

  1. Lewenza S. Extracellular DNA-induced antimicrobial peptide resistance mechanisms in Pseudomonas aeruginosa . Front Microbiol 2013; 4: 21 [CrossRef] [PubMed]
    [Google Scholar]
  2. Okshevsky M, Meyer RL. The role of extracellular DNA in the establishment, maintenance and perpetuation of bacterial biofilms. Crit Rev Microbiol 2015; 41: 1040 1841 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  3. Halverson TWR, Wilton M, Poon KKH, Petri B, Lewenza S. DNA is an antimicrobial component of neutrophil extracellular traps. PLoS Pathog 2015; 11: e1004593 [CrossRef] [PubMed]
    [Google Scholar]
  4. Marcos V, Zhou Z, Yildirim AO, Bohla A, Hector A et al. CXCR2 mediates NADPH oxidase-independent neutrophil extracellular trap formation in cystic fibrosis airway inflammation. Nat Med 2010; 16: 1018 1023 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  5. Manzenreiter R, Kienberger F, Marcos V, Schilcher K, Krautgartner WD et al. Ultrastructural characterization of cystic fibrosis sputum using atomic force and scanning electron microscopy. J Cyst Fibros 2012; 11: 84 92 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  6. Shan Q, Dwyer M, Rahman S, Gadjeva M. Distinct susceptibilities of corneal Pseudomonas aeruginosa clinical isolates to neutrophil extracellular trap-mediated immunity. Infect Immun 2014; 82: 4135 4143 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  7. Jakubovics NS, Shields RC, Rajarajan N, Burgess JG. Life after death: the critical role of extracellular DNA in microbial biofilms. Lett Appl Microbiol 2013; 57: 467 475 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  8. Gloag ES, Turnbull L, Huang A, Vallotton P, Wang H et al. Self-Organization of bacterial biofilms is facilitated by extracellular DNA. Proc Natl Acad Sci U S A 2013; 110: 11541 11546 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  9. Mulcahy H, Charron-Mazenod L, Lewenza S. Extracellular DNA chelates cations and induces antibiotic resistance in Pseudomonas aeruginosa biofilms. PLoS Pathog 2008; 4: e1000213 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  10. Johnson L, Mulcahy H, Kanevets U, Shi Y, Lewenza S. Surface-localized spermidine protects the Pseudomonas aeruginosa outer membrane from antibiotic treatment and oxidative stress. J Bacteriol 2012; 194: 813 826 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  11. Wilton M, Charron-Mazenod L, Moore R, Lewenza S. Extracellular DNA Acidifies Biofilms and Induces Aminoglycoside Resistance in Pseudomonas aeruginosa . Antimicrob Agents Chemother 2016; 60: 544 553 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  12. Palchevskiy V, Finkel SE. Escherichia coli competence gene homologs are essential for competitive fitness and the use of DNA as a nutrient. J Bacteriol 2006; 188: 3902 3910 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  13. Pinchuk GE, Ammons C, Culley DE, Li S-MW, McLean JS et al. Utilization of DNA as a sole source of phosphorus, carbon, and energy by Shewanella spp.: ecological and physiological implications for dissimilatory metal reduction. Appl Environ Microbiol 2008; 74: 1198 1208 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  14. Mulcahy H, Charron-Mazenod L, Lewenza S. Pseudomonas aeruginosa produces an extracellular deoxyribonuclease that is required for utilization of DNA as a nutrient source. Environ Microbiol 2010; 12: 1621-9 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  15. Wilton M, Halverson TWR, Charron-Mazenod L, Parkins MD, Lewenza S. Secreted Phosphatase and Deoxyribonuclease Are Required by Pseudomonas aeruginosa To Defend against Neutrophil Extracellular Traps. Infect Immun 2018; 86: 22 08 2018 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  16. Lewenza S, Falsafi RK, Winsor G, Gooderham WJ, McPhee JB et al. Construction of a mini-Tn5-luxCDABE mutant library in Pseudomonas aeruginosa PAO1: a tool for identifying differentially regulated genes. Genome Res 2005; 15: 583 589 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  17. Winsor GL, Van Rossum T, Lo R, Khaira B, Whiteside MD et al. Pseudomonas genome database: facilitating user-friendly, comprehensive comparisons of microbial genomes. Nucleic Acids Res 2009; 37: D483 D488 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  18. Anders S, Huber W. Differential expression analysis for sequence count data. Genome Biol 2010; 11: R106 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  19. Lee CK, Roberts AL, Finn TM, Knapp S, Mekalanos JJ. A new assay for invasion of HeLa 229 cells by Bordetella pertussis: effects of inhibitors, phenotypic modulation, and genetic alterations. Infect Immun 1990; 58: 2516 2522 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  20. Mead CG. A deoxyribonucleic acid-associated ribonucleic acid from Drosophila melanogaster. J Biol Chem 1964; 239: 550 554 [PubMed] [PubMed]
    [Google Scholar]
  21. Mulcahy H, Sibley CD, Surette MG, Lewenza S. Drosophila melanogaster as an animal model for the study of Pseudomonas aeruginosa biofilm infections in vivo. PLoS Pathog 2011; 7: e1002299 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  22. Arai H, Kawakami T, Osamura T, Hirai T, Sakai Y et al. Enzymatic characterization and in vivo function of five terminal oxidases in Pseudomonas aeruginosa . J Bacteriol 2014; 196: 4206 4215 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  23. Hirai T, Osamura T, Ishii M, Arai H. Expression of multiple cbb 3 cytochrome c oxidase isoforms by combinations of multiple isosubunits in Pseudomonas aeruginosa . Proc Natl Acad Sci U S A 2016; 113: 12815 12819 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  24. Jo JTH, Brinkman FSL, Hancock REW. Aminoglycoside efflux in Pseudomonas aeruginosa: involvement of novel outer membrane proteins. Antimicrob Agents Chemother 2003; 47: 1101 1111 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  25. Perron K, Caille O, Rossier C, Van Delden C, Dumas J-L et al. CzcR-CzcS, a two-component system involved in heavy metal and carbapenem resistance in Pseudomonas aeruginosa . J Biol Chem 2004; 279: 8761 8768 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  26. Caille O, Rossier C, Perron K. A copper-activated two-component system interacts with zinc and imipenem resistance in Pseudomonas aeruginosa . J Bacteriol 2007; 189: 4561 4568 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  27. Hauser AR. The type III secretion system of Pseudomonas aeruginosa: infection by injection. Nat Rev Microbiol 2009; 7: 654 665 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  28. Sana TG, Berni B, Bleves S. The T6SSs of Pseudomonas aeruginosa Strain PAO1 and Their Effectors: Beyond Bacterial-Cell Targeting. Front Cell Infect Microbiol 2016; 6: 61 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  29. Wilton M, Wong MJQ, Tang L, Liang X, Moore R et al. Chelation of Membrane-Bound Cations by Extracellular DNA Activates the Type VI Secretion System in Pseudomonas aeruginosa . Infect Immun 2016; 84: 2355 2361 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  30. Horsman SR, Moore RA, Lewenza S. Calcium chelation by alginate activates the type III secretion system in mucoid Pseudomonas aeruginosa biofilms. PLoS One 2012; 7: e46826 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  31. Foster JW, Hall HK. Adaptive acidification tolerance response of Salmonella typhimurium . J Bacteriol 1990; 172: 771 778 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  32. Kanjee U, Houry WA. Mechanisms of acid resistance in Escherichia coli . Annu Rev Microbiol 2013; 67: 65 81 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  33. Whitchurch CB, Tolker-Nielsen T, Ragas PC, Mattick JS. Extracellular DNA required for bacterial biofilm formation. Science 2002; 295: 1487 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  34. Teitzel GM, Parsek MR. Heavy metal resistance of biofilm and planktonic Pseudomonas aeruginosa . Appl Environ Microbiol 2003; 69: 2313 2320 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  35. Mulcahy H, Lewenza S. Magnesium limitation is an environmental trigger of the Pseudomonas aeruginosa biofilm lifestyle. PLoS One 2011; 6: e23307 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  36. Goodman AL, Kulasekara B, Rietsch A, Boyd D, Smith RS et al. A signaling network reciprocally regulates genes associated with acute infection and chronic persistence in Pseudomonas aeruginosa . Dev Cell 2004; 7: 745 754 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.001184
Loading
/content/journal/jmm/10.1099/jmm.0.001184
Loading

Data & Media loading...

Supplements

Supplementary material 1

EXCEL
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