1887

Abstract

Five hundred and thirty-four unrelated isolates from inanimate habitats, patients with cystic fibrosis (CF) and other human infections were sequenced in 19 genes that had been identified previously as the hot spots of genomic within-host evolution in serial isolates from 12 CF lungs. Amplicon sequencing confirmed a significantly higher sequence diversity of the 19 loci in isolates from CF patients compared to those from other habitats, but this overrepresentation was mainly due to the larger share of synonymous substitutions. Correspondingly, non-synonymous substitutions were either rare (, , ) or benign (, , ) in some loci. Other loci, however, showed an accumulation of non-neutral coding variants. Strains from the CF habitat were often mutated at evolutionarily conserved positions in the elements of stringent response (RelA, SpoT), LPS (PagL), polyamine transport (SpuE, SpuF) and alginate biosynthesis (AlgG, AlgU). The strongest skew towards the CF lung habitat was seen for amino acid sequence variants in AlgG that clustered in the carbohydrate-binding/sugar hydrolysis domain. The master regulators of quorum sensing and were frequent targets for coding variants in isolates from chronic and acute human infections. Unique variants in showed strong evidence of positive selection indicated by / values of ~4. The gene that encodes a multidomain enzyme involved in both the formation and dispersion of Pel biofilms carried the highest number of single-nucleotide variants among the 19 genes and was the only gene with a higher frequency of missense mutations in strains from non-CF habitats than in isolates from CF airways. PelA protein variants are widely distributed in the population. In conclusion, coding variants in a subset of the examined loci are indeed characteristic for the adaptation of to the CF airways, but for other loci the elevated mutation rate is more indicative of infections in human habitats () or global diversifying selection ().

Funding
This study was supported by the:
  • Deutsche Forschungsgemeinschaft (Award SFB 900/3 - 158989968 - A2, Z1)
    • Principle Award Recipient: BurkhardTümmler
  • This is an open-access article distributed under the terms of the Creative Commons Attribution NonCommercial License.
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2021-12-07
2022-01-28
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References

  1. Cutting GR. Cystic fibrosis genetics: from molecular understanding to clinical application. Nat Rev Genet 2015; 16:45–56 [View Article] [PubMed]
    [Google Scholar]
  2. Stoltz DA, Meyerholz DK, Welsh MJ. Origins of cystic fibrosis lung disease. N Engl J Med 2015; 372:351–362 [View Article] [PubMed]
    [Google Scholar]
  3. Ratjen F, Bell SC, Rowe SM, Goss CH, Quittner AL et al. Cystic fibrosis. Nat Rev Dis Primers 2015; 1:15010 [View Article] [PubMed]
    [Google Scholar]
  4. Elborn JS. Cystic fibrosis. Lancet 2016; 388:2519–2531 [View Article] [PubMed]
    [Google Scholar]
  5. Blanchard AC, Waters VJ. Microbiology of cystic fibrosis airway disease. Semin Respir Crit Care Med 2019; 40:727–736 [View Article] [PubMed]
    [Google Scholar]
  6. Cramer N, Wiehlmann L, Tümmler B. Clonal epidemiology of Pseudomonas aeruginosa in cystic fibrosis. Int J Med Microbiol 2010; 300:526–533 [View Article] [PubMed]
    [Google Scholar]
  7. Parkins MD, Somayaji R, Waters VJ. Epidemiology, biology, and impact of clonal Pseudomonas aeruginosa infections in cystic fibrosis. Clin Microbiol Rev 2018; 31:e00019-18 [View Article] [PubMed]
    [Google Scholar]
  8. Döring G, Flume P, Heijerman H, Elborn JS. Consensus Study Group Treatment of lung infection in patients with cystic fibrosis: current and future strategies. J Cyst Fibros 2012; 11:461–479 [View Article] [PubMed]
    [Google Scholar]
  9. Taccetti G, Francalanci M, Pizzamiglio G, Messore B, Carnovale V et al. Cystic Fibrosis: Recent insights into inhaled antibiotic treatment and future perspectives. Antibiotics (Basel) 2021; 10:338 [View Article] [PubMed]
    [Google Scholar]
  10. Rossi E, La Rosa R, Bartell JA, Marvig RL, Haagensen JAJ et al. Pseudomonas aeruginosa adaptation and evolution in patients with cystic fibrosis. Nat Rev Microbiol 2021; 19:331–342 [View Article] [PubMed]
    [Google Scholar]
  11. Camus L, Vandenesch F, Moreau K. From genotype to phenotype: adaptations of Pseudomonas aeruginosa to the cystic fibrosis environment. Microb Genom 2021; 7: [View Article] [PubMed]
    [Google Scholar]
  12. Smith EE, Buckley DG, Wu Z, Saenphimmachak C, Hoffman LR et al. Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients. Proc Natl Acad Sci U S A 2006; 103:8487–8492 [View Article] [PubMed]
    [Google Scholar]
  13. Cramer N, Klockgether J, Wrasman K, Schmidt M, Davenport CF et al. Microevolution of the major common Pseudomonas aeruginosa clones C and PA14 in cystic fibrosis lungs. Environ Microbiol 2011; 13:1690–1704 [View Article] [PubMed]
    [Google Scholar]
  14. Feliziani S, Marvig RL, Luján AM, Moyano AJ, Di Rienzo JA et al. Coexistence and within-host evolution of diversified lineages of hypermutable Pseudomonas aeruginosa in long-term cystic fibrosis infections. PLoS Genet 2014; 10:e1004651 [View Article] [PubMed]
    [Google Scholar]
  15. van Mansfeld R, de Been M, Paganelli F, Yang L, Bonten M et al. Within-host evolution of the Dutch high-prevalent Pseudomonas aeruginosa clone ST406 during chronic colonization of a patient with cystic fibrosis. PLoS One 2016; 11:e0158106 [View Article] [PubMed]
    [Google Scholar]
  16. Bianconi I, D’Arcangelo S, Esposito A, Benedet M, Piffer E et al. Persistence and microevolution of Pseudomonas aeruginosa in the cystic fibrosis lung: A single-patient longitudinal genomic study. Front Microbiol 2019; 9:3242 [View Article] [PubMed]
    [Google Scholar]
  17. Yang L, Jelsbak L, Marvig RL, Damkiær S, Workman CT et al. Evolutionary dynamics of bacteria in a human host environment. Proc Natl Acad Sci U S A 2011; 108:7481–7486 [View Article] [PubMed]
    [Google Scholar]
  18. Marvig RL, Johansen HK, Molin S, Jelsbak L. Genome analysis of a transmissible lineage of Pseudomonas aeruginosa reveals pathoadaptive mutations and distinct evolutionary paths of hypermutators. PLoS Genet 2013; 9:e1003741 [View Article] [PubMed]
    [Google Scholar]
  19. Marvig RL, Sommer LM, Molin S, Johansen HK. Convergent evolution and adaptation of Pseudomonas aeruginosa within patients with cystic fibrosis. Nat Genet 2015; 47:57–64 [View Article] [PubMed]
    [Google Scholar]
  20. Marvig RL, Dolce D, Sommer LM, Petersen B, Ciofu O et al. Within-host microevolution of Pseudomonas aeruginosa in Italian cystic fibrosis patients. BMC Microbiol 2015; 15:218 [View Article] [PubMed]
    [Google Scholar]
  21. Klockgether J, Cramer N, Fischer S, Wiehlmann L, Tümmler B. Long-term microevolution of Pseudomonas aeruginosa differs between mildly and severely affected cystic fibrosis lungs. Am J Respir Cell Mol Biol 2018; 59:246–256 [View Article] [PubMed]
    [Google Scholar]
  22. Ciofu O, Lee B, Johannesson M, Hermansen NO, Meyer P et al. Investigation of the algT operon sequence in mucoid and non-mucoid Pseudomonas aeruginosa isolates from 115 Scandinavian patients with cystic fibrosis and in 88 in vitro non-mucoid revertants. Microbiology (Reading) 2008; 154:103–113 [View Article] [PubMed]
    [Google Scholar]
  23. Hoffman LR, Kulasekara HD, Emerson J, Houston LS, Burns JL et al. Pseudomonas aeruginosa lasR mutants are associated with cystic fibrosis lung disease progression. J Cyst Fibros 2009; 8:66–70 [View Article] [PubMed]
    [Google Scholar]
  24. Bjarnsholt T, Jensen , Jakobsen TH, Phipps R, Nielsen AK et al. Quorum sensing and virulence of Pseudomonas aeruginosa during lung infection of cystic fibrosis patients. PLoS One 2010; 5:e10115 [View Article] [PubMed]
    [Google Scholar]
  25. Feltner JB, Wolter DJ, Pope CE, Groleau M-C, Smalley NE et al. LasR variant cystic fibrosis isolates reveal an adaptable quorum-sensing hierarchy in Pseudomonas aeruginosa. mBio 2016; 7:e01513-16 [View Article] [PubMed]
    [Google Scholar]
  26. Candido Caçador N, Paulino da Costa Capizzani C, Gomes Monteiro Marin Torres LA, Galetti R, Ciofu O et al. Adaptation of Pseudomonas aeruginosa to the chronic phenotype by mutations in the algTmucABD operon in isolates from Brazilian cystic fibrosis patients. PLoS One 2018; 13:e0208013 [View Article] [PubMed]
    [Google Scholar]
  27. Wiehlmann L, Cramer N, Tümmler B. Habitat-associated skew of clone abundance in the Pseudomonas aeruginosa population. Environ Microbiol Rep 2015; 7:955–960 [View Article] [PubMed]
    [Google Scholar]
  28. Fischer S, Dethlefsen S, Klockgether J, Tümmler B. Phenotypic and genomic comparison of the two most common ExoU-positive Pseudomonas aeruginosa clones, PA14 and ST235. mSystems 2020; 5:e01007-20 [View Article] [PubMed]
    [Google Scholar]
  29. Wiehlmann L, Wagner G, Cramer N, Siebert B, Gudowius P et al. Population structure of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 2007; 104:8101–8106 [View Article] [PubMed]
    [Google Scholar]
  30. Dale JW, Greenaway PJ. Preparation of chromosomal DNA from E. coli. Methods Mol Biol 1985; 2:197–200 [View Article] [PubMed]
    [Google Scholar]
  31. Bolger AM, Lohse M, Usadel B. Trimmomatic: A flexible trimmer for Illumina Sequence Data. Bioinformatics 2914btu170
    [Google Scholar]
  32. Li H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM; 2013
  33. Cingolani P, Platts A, Wang LL, Coon M, Nguyen T et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin) 2012; 6:80–92 [View Article] [PubMed]
    [Google Scholar]
  34. Cramer N, Wiehlmann L, Ciofu O, Tamm S, Høiby N et al. Molecular epidemiology of chronic Pseudomonas aeruginosa airway infections in cystic fibrosis. PLoS One 2012; 7:e50731 [View Article] [PubMed]
    [Google Scholar]
  35. Schurr MJ, Martin DW, Mudd MH, Deretic V. Gene cluster controlling conversion to alginate-overproducing phenotype in Pseudomonas aeruginosa: functional analysis in a heterologous host and role in the instability of mucoidy. J Bacteriol 1994; 176:3375–3382 [View Article] [PubMed]
    [Google Scholar]
  36. Williams P, Cámara M. Quorum sensing and environmental adaptation in Pseudomonas aeruginosa: a tale of regulatory networks and multifunctional signal molecules. Curr Opin Microbiol 2009; 12:182–191 [View Article] [PubMed]
    [Google Scholar]
  37. Schuster M, Sexton DJ, Diggle SP, Greenberg EP. Acyl-homoserine lactone quorum sensing: from evolution to application. Annu Rev Microbiol 2013; 67:43–63 [View Article] [PubMed]
    [Google Scholar]
  38. Smith EE, Sims EH, Spencer DH, Kaul R, Olson MV. Evidence for diversifying selection at the pyoverdine locus of Pseudomonas aeruginosa. J Bacteriol 2005; 187:2138–2147 [View Article] [PubMed]
    [Google Scholar]
  39. Ganne G, Brillet K, Basta B, Roche B, Hoegy F et al. Iron release from the siderophore pyoverdine in Pseudomonas aeruginosa involves three new actors: FpvC, FpvG, and FpvH. ACS Chem Biol 2017; 12:1056–1065 [View Article] [PubMed]
    [Google Scholar]
  40. Dayhoff MO. Atlas of Protein Sequence and Structure Washington, DC: National Biomedical Research Foundation; 1978
    [Google Scholar]
  41. Pausch P, Abdelshahid M, Steinchen W, Schäfer H, Gratani FL et al. Structural basis for regulation of the opposing (p)ppGpp synthetase and hydrolase within the stringent response orchestrator Rel. Cell Rep 2020; 32:108157 [View Article] [PubMed]
    [Google Scholar]
  42. Arenz S, Abdelshahid M, Sohmen D, Payoe R, Starosta AL et al. The stringent factor RelA adopts an open conformation on the ribosome to stimulate ppGpp synthesis. Nucleic Acids Res 2016; 44:6471–6481 [View Article] [PubMed]
    [Google Scholar]
  43. Winther KS, Roghanian M, Gerdes K. Activation of the stringent response by loading of RelA-tRNA complexes at the ribosomal A-site. Molecular Cell 2018; 70:95–105 [View Article]
    [Google Scholar]
  44. Pletzer D, Blimkie TM, Wolfmeier H, Li Y, Baghela A et al. The stringent stress response controls proteases and global regulators under optimal growth conditions in Pseudomonas aeruginosa. mSystems 2020; 5:e00495-20 [View Article] [PubMed]
    [Google Scholar]
  45. Wu D, Lim SC, Dong Y, Wu J, Tao F et al. Structural basis of substrate binding specificity revealed by the crystal structures of polyamine receptors SpuD and SpuE from Pseudomonas aeruginosa. J Mol Biol 2012; 416:697–712 [View Article] [PubMed]
    [Google Scholar]
  46. Zhang Y, Sun X, Qian Y, Yi H, Song K et al. A potent anti-SpuE antibody allosterically inhibits Type III secretion system and attenuates virulence of Pseudomonas aeruginosa. J Mol Biol 2019; 431:4882–4896 [View Article] [PubMed]
    [Google Scholar]
  47. Juhas M, Eberl L, Tümmler B. Quorum sensing: the power of cooperation in the world of Pseudomonas. Environ Microbiol 2005; 7:459–471 [View Article] [PubMed]
    [Google Scholar]
  48. Bottomley MJ, Muraglia E, Bazzo R, Carfì A. Molecular insights into quorum sensing in the human pathogen Pseudomonas aeruginosa from the structure of the virulence regulator LasR bound to its autoinducer. J Biol Chem 2007; 282:13592–13600 [View Article] [PubMed]
    [Google Scholar]
  49. Qiu H, Li Y, Dai W. Codon-usage frequency mediated SNPs selection in lasR gene of cystic fibrosis Pseudomonas aeruginosa isolates. Microbiol Res 2019137–143 [PubMed]
    [Google Scholar]
  50. Govan JR, Deretic V. Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol Rev 1996; 60:539–574 [View Article] [PubMed]
    [Google Scholar]
  51. Damron FH, Goldberg JB. Proteolytic regulation of alginate overproduction in Pseudomonas aeruginosa. Mol Microbiol 2012; 84:595–607 [View Article] [PubMed]
    [Google Scholar]
  52. Sherbrock-Cox V, Russell NJ, Gacesa P. The purification and chemical characterisation of the alginate present in extracellular material produced by mucoid strains of Pseudomonas aeruginosa. Carbohydr Res 1984; 135:147–154 [View Article] [PubMed]
    [Google Scholar]
  53. Franklin MJ, Chitnis CE, Gacesa P, Sonesson A, White DC et al. Pseudomonas aeruginosa AlgG is a polymer level alginate C5-mannuronan epimerase. J Bacteriol 1994; 176:1821–1830 [View Article] [PubMed]
    [Google Scholar]
  54. Wolfram F, Kitova EN, Robinson H, Walvoort MTC, Codée JDC et al. Catalytic mechanism and mode of action of the periplasmic alginate epimerase AlgG. J Biol Chem 2014; 289:6006–6019 [View Article] [PubMed]
    [Google Scholar]
  55. Douthit SA, Dlakic M, Ohman DE, Franklin MJ. Epimerase active domain of Pseudomonas aeruginosa AlgG, a protein that contains a right-handed beta-helix. J Bacteriol 2005; 187:4573–4583 [View Article] [PubMed]
    [Google Scholar]
  56. Jennings LK, Storek KM, Ledvina HE, Coulon C, Marmont LS et al. Pel is a cationic exopolysaccharide that cross-links extracellular DNA in the Pseudomonas aeruginosa biofilm matrix. Proc Natl Acad Sci U S A 2015; 112:11353–11358 [View Article] [PubMed]
    [Google Scholar]
  57. Colvin KM, Alnabelseya N, Baker P, Whitney JC, Howell PL et al. PelA deacetylase activity is required for Pel polysaccharide synthesis in Pseudomonas aeruginosa. J Bacteriol 2013; 195:2329–2339 [View Article] [PubMed]
    [Google Scholar]
  58. Cherny KE, Sauer K. Untethering and degradation of the polysaccharide matrix are essential steps in the dispersion response of Pseudomonas aeruginosa biofilms. J Bacteriol 2020; 202: [View Article] [PubMed]
    [Google Scholar]
  59. Geurtsen J, Steeghs L, Hove JT, van der Ley P, Tommassen J. Dissemination of lipid A deacylases (pagL) among gram-negative bacteria: identification of active-site histidine and serine residues. J Biol Chem 2005; 280:8248–8259 [View Article] [PubMed]
    [Google Scholar]
  60. Rutten L, Geurtsen J, Lambert W, Smolenaers JJM, Bonvin AM et al. Crystal structure and catalytic mechanism of the LPS 3-O-deacylase PagL from Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 2006; 103:7071–7076 [View Article] [PubMed]
    [Google Scholar]
  61. Han M-L, Velkov T, Zhu Y, Roberts KD, Le Brun AP et al. Polymyxin-induced Lipid A deacylation in Pseudomonas aeruginosa perturbs polymyxin penetration and confers high-level resistance. ACS Chem Biol 2018; 13:121–130 [View Article] [PubMed]
    [Google Scholar]
  62. Hilker R, Munder A, Klockgether J, Losada PM, Chouvarine P et al. Interclonal gradient of virulence in the Pseudomonas aeruginosa pangenome from disease and environment. Environ Microbiol 2015; 17:29–46 [View Article] [PubMed]
    [Google Scholar]
  63. Fischer S, Klockgether J, Morán Losada P, Chouvarine P, Cramer N et al. Intraclonal genome diversity of the major Pseudomonas aeruginosa clones C and PA14. Environ Microbiol Rep 2016; 8:227–234 [View Article] [PubMed]
    [Google Scholar]
  64. Tümmler B. Clonal variations in Pseudomonas aeruginosa. In Ramos JL, Levesque RC. eds Pseudomonas. Volume 4. Molecular Biology of Emerging Issues New York: Springer; 2006 pp 35–68
    [Google Scholar]
  65. Kostylev M, Kim DY, Smalley NE, Salukhe I, Greenberg EP et al. Evolution of the Pseudomonas aeruginosa quorum-sensing hierarchy. Proc Natl Acad Sci U S A 2019; 116:7027–7032 [View Article] [PubMed]
    [Google Scholar]
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