#PAGE_PARAMS# #ADS_HEAD_SCRIPTS# #MICRODATA#

Global Analysis of the Fungal Microbiome in Cystic Fibrosis Patients Reveals Loss of Function of the Transcriptional Repressor Nrg1 as a Mechanism of Pathogen Adaptation


Microbial cells vastly outnumber human cells in our bodies, yet we are only beginning to understand how these microbes influence human health and disease. One disease for which microbial communities are especially important is cystic fibrosis, where persistent lung infections can be lethal. Fungi are associated with poor respiratory function, but how fungal communities change with disease progression or treatment remains enigmatic. Here, we assess the dynamics of fungal communities by combining high-throughput sequencing of sputum samples from 28 patients with detailed analysis of phenotypes and genotypes of 1,603 fungal isolates. We found stable communities dominated by Candida and Aspergillus, and diversity in traits important for host adaptation. Antifungal drug resistance varied largely between species, while morphogenesis varied within species. For Candida species, the capacity to transition between yeast and filaments is a key virulence trait that is normally regulated by inducing cues, however, 28 isolates grew as filaments without such cues. Filamentation was due to loss-of-function mutations in the transcriptional regulator NRG1 in most isolates, which conferred resistance to the filament-repressive effects of a common bacterial pathogen. This work provides a portrait of the fungal microbiome associated with a lethal disease, and illuminates a genetic basis of pathogen adaptation.


Vyšlo v časopise: Global Analysis of the Fungal Microbiome in Cystic Fibrosis Patients Reveals Loss of Function of the Transcriptional Repressor Nrg1 as a Mechanism of Pathogen Adaptation. PLoS Pathog 11(11): e32767. doi:10.1371/journal.ppat.1005308
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1005308

Souhrn

Microbial cells vastly outnumber human cells in our bodies, yet we are only beginning to understand how these microbes influence human health and disease. One disease for which microbial communities are especially important is cystic fibrosis, where persistent lung infections can be lethal. Fungi are associated with poor respiratory function, but how fungal communities change with disease progression or treatment remains enigmatic. Here, we assess the dynamics of fungal communities by combining high-throughput sequencing of sputum samples from 28 patients with detailed analysis of phenotypes and genotypes of 1,603 fungal isolates. We found stable communities dominated by Candida and Aspergillus, and diversity in traits important for host adaptation. Antifungal drug resistance varied largely between species, while morphogenesis varied within species. For Candida species, the capacity to transition between yeast and filaments is a key virulence trait that is normally regulated by inducing cues, however, 28 isolates grew as filaments without such cues. Filamentation was due to loss-of-function mutations in the transcriptional regulator NRG1 in most isolates, which conferred resistance to the filament-repressive effects of a common bacterial pathogen. This work provides a portrait of the fungal microbiome associated with a lethal disease, and illuminates a genetic basis of pathogen adaptation.


Zdroje

1. Nguyen LDN, Viscogliosi E, Delhaes L. The lung mycobiome: an emerging field of the human respiratory microbiome. Front Microbiol. 2015;6: 1–9. doi: 10.3389/fmicb.2015.00089 25762987

2. Surette MG. The cystic fibrosis lung microbiome. Ann Am Thorac Soc. 2014;11 Suppl 1: S61–5. doi: 10.1513/AnnalsATS.201306-159MG 24437409

3. LiPuma JJ. The changing microbial epidemiology in cystic fibrosis. Clinical Microbiology Reviews. 2010. pp. 299–323. doi: 10.1128/CMR.00068-09 20375354

4. Sibley CD, Parkins MD, Rabin HR, Duan K, Norgaard JC, Surette MG. A polymicrobial perspective of pulmonary infections exposes an enigmatic pathogen in cystic fibrosis patients. Proc Natl Acad Sci U S A. 2008;105: 15070–15075. doi: 10.1073/pnas.0804326105 18812504

5. Brown GD, Denning DW, Gow N a R, Levitz SM, Netea MG, White TC. Hidden killers: human fungal infections. Sci Transl Med. 2012;4: 165rv13. doi: 10.1126/scitranslmed.3004404 23253612

6. Nagano Y, Elborn JS, Millar BC, Walker JM, Goldsmith CE, Rendall J, et al. Comparison of techniques to examine the diversity of fungi in adult patients with cystic fibrosis. Med Mycol Off Publ Int Soc Hum Anim Mycol. 2010;48: 166–176.e1.

7. Delhaes L, Monchy S, Fréalle E, Hubans C, Salleron J, Leroy S, et al. The airway microbiota in cystic fibrosis: A complex fungal and bacterial community-implications for therapeutic management. PLoS One. 2012;7. doi: 10.1371/journal.pone.0036313

8. Iversen M, Burton CM, Vand S, Skovfoged L, Carlsen J, Milman N, et al. Aspergillus infection in lung transplant patients: Incidence and prognosis. Eur J Clin Microbiol Infect Dis. 2007;26: 879–886. doi: 10.1007/s10096-007-0376-3 17874329

9. Skov M, Koch C, Reimert CM, Poulsen LK. Diagnosis of allergic bronchopulmonary aspergillosis (ABPA) in cystic fibrosis. Allergy. 2000;55: 50–58. doi: 10.1034/j.1398-9995.2000.00342.x 10696856

10. Chowdhary A, Agarwal K, Kathuria S, Gaur SN, Randhawa HS, Meis JF. Allergic bronchopulmonary mycosis due to fungi other than Aspergillus: a global overview. Crit Rev Microbiol. 2014;40: 30–48. doi: 10.3109/1040841X.2012.754401 23383677

11. Chotirmall SH, O’Donoghue E, Bennett K, Gunaratnam C, O’Neill SJ, McElvaney NG. Sputum Candida albicans presages FEV₁ decline and hospital-treated exacerbations in cystic fibrosis. Chest. 2010;138: 1186–1195. doi: 10.1378/chest.09-2996 20472859

12. Charlson ES, Diamond JM, Bittinger K, Fitzgerald AS, Yadav A, Haas AR, et al. Lung-enriched organisms and aberrant bacterial and fungal respiratory microbiota after lung transplant. Am J Respir Crit Care Med. 2012;186: 536–545. doi: 10.1164/rccm.201204-0693OC 22798321

13. Willger SD, Grim SL, Dolben EL, Shipunova A, Hampton TH, Morrison HG, et al. Characterization and quantification of the fungal microbiome in serial samples from individuals with cystic fibrosis. Microbiome. 2014;2: 40. doi: 10.1186/2049-2618-2-40 25408892

14. Hill JA, O’Meara TR, Cowen LE. Fitness trade-offs associated with the evolution of resistance to antifungal drug combinations. Cell Rep. The Authors; 2015;10: 809–819. doi: 10.1016/j.celrep.2015.01.009

15. Singh-Babak SD, Babak T, Diezmann S, Hill JA, Xie JL, Chen YL, et al. Global analysis of the evolution and mechanism of echinocandin resistance in Candida glabrata. PLoS Pathog. 2012;8. doi: 10.1371/journal.ppat.1002718

16. Ford CB, Funt JM, Abbey D, Issi L, Guiducci C, Martinez D a, et al. The evolution of drug resistance in clinical isolates of Candida albicans. Elife. 2015;4: 1–27. doi: 10.7554/eLife.00662

17. Shapiro RS, Robbins N, Cowen LE. Regulatory circuitry governing fungal development, drug resistance, and disease. Microbiol Mol Biol Rev. 2011;75: 213–267. doi: 10.1128/MMBR.00045-10 21646428

18. Hogan DA, Kolter R. Pseudomonas-Candida interactions: an ecological role for virulence factors. Science. 2002;296: 2229–2232. doi: 10.1126/science.1070784 12077418

19. Hogan DA, Vik A, Kolter R. A Pseudomonas aeruginosa quorum-sensing molecule influences Candida albicans morphology. Mol Microbiol. 2004;54: 1212–1223. doi: 10.1111/j.1365-2958.2004.04349.x 15554963

20. Morales DK, Grahl N, Okegbe C, Dietrich LEP, Jacobs NJ, Hogan DA. Control of Candida albicans metabolism and biofilm formation by Pseudomonas aeruginosa phenazines. MBio. 2013;4: e00526–12. doi: 10.1128/mBio.00526-12 23362320

21. Boon C, Deng Y, Wang L-H, He Y, Xu J-L, Fan Y, et al. A novel DSF-like signal from Burkholderia cenocepacia interferes with Candida albicans morphological transition. ISME J. 2008;2: 27–36. doi: 10.1038/ismej.2007.76 18049456

22. Harriott MM, Noverr MC. Candida albicans and Staphylococcus aureus form polymicrobial biofilms: effects on antimicrobial resistance. Antimicrob Agents Chemother. 2009;53: 3914–3922. doi: 10.1128/AAC.00657-09 19564370

23. Peters BM, Noverr MC. Candida albicans-Staphylococcus aureus polymicrobial peritonitis modulates host innate immunity. Infect Immun. 2013;81: 2178–89. doi: 10.1128/IAI.00265-13 23545303

24. Kusenbach G, Skopnik H, Haase G, Friedrichs F, Döhmen H. Exophiala dermatitidis pneumonia in cystic fibrosis. Eur J Pediatr. 1992;151: 344–346. doi: 10.1007/BF02113255 1396889

25. Giraud S, Pihet M, Razafimandimby B, Carrère J, Degand N, Mely L, et al. Geosmithia argillacea: An emerging pathogen in patients with cystic fibrosis. J Clin Microbiol. 2010;48: 2381–2386. doi: 10.1128/JCM.00047-10 20463155

26. Revankar SG, Sutton DA. Melanized fungi in human disease. Clinical Microbiology Reviews. 2010. pp. 884–928. doi: 10.1128/CMR.00019-10 20930077

27. Gomez-Lopez A, Pan D, Cuesta I, Alastruey-Izquierdo A, Rodriguez-Tudela JL, Cuenca-Estrella M. Molecular identification and susceptibility profile in vitro of the emerging pathogen Candida kefyr. Diagn Microbiol Infect Dis. 2010;66: 116–119. doi: 10.1016/j.diagmicrobio.2009.06.007 19709840

28. Odds FC, Bernaerts R. CHROMagar Candida, a new differential isolation medium for presumptive identification of clinically important Candida species. J Clin Microbiol. 1994;32: 1923–1929. 7989544

29. Iwen PC, Hinrichs SH, Rupp ME. Utilization of the internal transcribed spacer regions as molecular targets to detect and identify human fungal pathogens. Med Mycol Off Publ Int Soc Hum Anim Mycol. 2002;40: 87–109.

30. Balajee SA, Gribskov JL, Hanley E, Nickle D, Marr KA. Aspergillus lentulus sp. nov., a new sibling species of A. fumigatus. Eukaryot Cell. 2005;4: 625–632. doi: 10.1128/EC.4.3.625–632.2005 15755924

31. Velegraki A, Alexopoulos EC, Kritikou S, Gaitanis G. Use of fatty acid RPMI 1640 media for testing susceptibilities of eight Malassezia species to the new triazole posaconazole and to six established antifungal agents by a modified NCCLS M27-A2 microdilution method and Etest. J Clin Microbiol. 2004;42: 3589–3593. doi: 10.1128/JCM.42.8.3589–3593.2004 15297502

32. Horré R, Schaal KP, Siekmeier R, Sterzik B, De Hoog GS, Schnitzler N. Isolation of fungi, especially Exophiala dermatitidis, in patients suffering from cystic fibrosis: A prospective study. Respiration. 2004;71: 360–366. doi: 10.1159/000079640 15316209

33. O’Meara TR, Veri AO, Ketela T, Jiang B, Roemer T, Cowen LE. Global analysis of fungal morphology exposes mechanisms of host cell escape. Nat Commun. 2015;6: 6741. doi: 10.1038/ncomms7741 25824284

34. Noble SM, French S, Kohn LA, Chen V, Johnson AD. Systematic screens of a Candida albicans homozygous deletion library decouple morphogenetic switching and pathogenicity. Nat Genet. 2010;42: 590–598. doi: 10.1038/ng.605 20543849

35. Van het Hoog M, Rast TJ, Martchenko M, Grindle S, Dignard D, Hogues H, et al. Assembly of the Candida albicans genome into sixteen supercontigs aligned on the eight chromosomes. Genome Biol. 2007;8: R52. doi: 10.1186/gb-2007-8-4-r52 17419877

36. Cibulskis K, Lawrence MS, Carter SL, Sivachenko A, Jaffe D, Sougnez C, et al. Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples. Nat Biotechnol. 2013;31: 213–9. doi: 10.1038/nbt.2514 23396013

37. Murad AMA, Leng P, Straffon M, Wishart J, Macaskill S, MacCallum D, et al. NRG1 represses yeast-hypha morphogenesis and hypha-specific gene expression in Candida albicans. EMBO J. 2001;20: 4742–4752. doi: 10.1093/emboj/20.17.4742 11532938

38. Noble SM, Johnson AD. Strains and strategies for large-scale gene deletion studies of the diploid human fungal pathogen Candida albicans. Eukaryot Cell. 2005;4: 298–309. doi: 10.1128/EC.4.2.298–309.2005 15701792

39. Homann OR, Dea J, Noble SM, Johnson AD. A phenotypic profile of the Candida albicans regulatory network. PLoS Genet. 2009;5. doi: 10.1371/journal.pgen.1000783 20041210

40. LiPuma JJ, Spilker T, Gill LH, Campbell PW, Liu L, Mahenthiralingam E. Disproportionate distribution of Burkholderia cepacia complex species and transmissibility markers in cystic fibrosis. Am J Respir Crit Care Med. 2001;164: 92–96. doi: 10.1164/ajrccm.164.1.2011153 11435245

41. Price KE, Hampton TH, Gifford AH, Dolben EL, Hogan D a, Morrison HG, et al. Unique microbial communities persist in individual cystic fibrosis patients throughout a clinical exacerbation. Microbiome. 2013;1: 27. doi: 10.1186/2049-2618-1-27 24451123

42. Coburn B, Wang PW, Diaz Caballero J, Clark ST, Brahma V, Donaldson S, et al. Lung microbiota across age and disease stage in cystic fibrosis. Sci Rep. Nature Publishing Group; 2015;5: 10241. doi: 10.1038/srep10241 25974282

43. Cox MJ, Allgaier M, Taylor B, Baek MS, Huang YJ, Daly RA, et al. Airway microbiota and pathogen abundance in age-stratified cystic fibrosis patients. PLoS One. 2010;5. doi: 10.1371/journal.pone.0011044

44. Burgel PR, Baixench MT, Amsellem M, Audureau E, Chapron J, Kanaan R, et al. High prevalence of azole-resistant Aspergillus fumigatus in adults with cystic fibrosis exposed to itraconazole. Antimicrob Agents Chemother. 2012;56: 869–874. doi: 10.1128/AAC.05077-11 22123701

45. Mortensen KL, Jensen RH, Johansen HK, Skov M, Pressler T, Howard SJ, et al. Aspergillus species and other molds in respiratory samples from patients with cystic fibrosis: A laboratory-based study with focus on Aspergillus fumigatus azole resistance. J Clin Microbiol. 2011;49: 2243–2251. doi: 10.1128/JCM.00213-11 21508152

46. Gow NAR, van de Veerdonk FL, Brown AJP, Netea MG. Candida albicans morphogenesis and host defence: discriminating invasion from colonization. Nature Reviews Microbiology. 2011. doi: 10.1038/nrmicro2711

47. Kadosh D, Johnson AD. Induction of the Candida albicans filamentous growth program by relief of transcriptional repression: a genome-wide analysis. Mol Biol Cell. 2005;16: 2903–2912. doi: 10.1091/mbc.E05-01-0073 15814840

48. Nantel A, Dignard D, Bachewich C, Harcus D, Marcil A, Bouin A-P, et al. Transcription profiling of Candida albicans cells undergoing the yeast-to-hyphal transition. Mol Biol Cell. 2002;13: 3452–3465. doi: 10.1091/mbc.E02-05-0272 12388749

49. Clark ST, Diaz Caballero J, Cheang M, Coburn B, Wang PW, Donaldson SL, et al. Phenotypic diversity within a Pseudomonas aeruginosa population infecting an adult with cystic fibrosis. Sci Rep. Nature Publishing Group; 2015;5: 10932. doi: 10.1038/srep10932 26047320

50. Lieberman TD, Flett KB, Yelin I, Martin TR, McAdam AJ, Priebe GP, et al. Genetic variation of a bacterial pathogen within individuals with cystic fibrosis provides a record of selective pressures. Nat Genet. 2014;46: 82–7. doi: 10.1038/ng.2848 24316980

51. Workentine ML, Sibley CD, Glezerson B, Purighalla S, Norgaard-Gron JC, Parkins MD, et al. Phenotypic heterogeneity of Pseudomonas aeruginosa populations in a cystic fibrosis patient. PLoS One. 2013;8. doi: 10.1371/journal.pone.0060225

52. Silva IN, Ferreira AS, Becker JD, Zlosnik JEA, Speert DP, He J, et al. Mucoid morphotype variation of Burkholderia multivorans during chronic cystic fibrosis lung infection is correlated with changes in metabolism, motility, biofilm formation and virulence. Microbiology. 2011;157: 3124–3137. doi: 10.1099/mic.0.050989–0 21835880

53. Marvig RL, Sommer LM, Molin S, Johansen HK. Convergent evolution and adaptation of Pseudomonas aeruginosa within patients with cystic fibrosis. 2015;47. doi: 10.1038/ng.3148

54. Feliziani S, Marvig RL, Luján AM, Moyano AJ, Di Rienzo J a., Krogh Johansen H, 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. doi: 10.1371/journal.pgen.1004651 25330091

55. Gutierrez JP, Grimwood K, Armstrong DS, Carlin JB, Carzino R, Olinsky A, et al. Interlobar differences in bronchoalveolar lavage fluid from children with cystic fibrosis. Eur Respir J. 2001;17: 281–6. 11334132

56. Smith DL, Smith EG, Pitt TL, Stableforth DE. Regional microbiology of the cystic fibrosis lung: a post-mortem study in adults. J Infect. 1998;37: 41–43. doi: 10.1016/S0163-4453(98)90475-3 9733377

57. Willner D, Haynes MR, Furlan M, Schmieder R, Lim YW, Rainey PB, et al. Spatial distribution of microbial communities in the cystic fibrosis lung. The ISME Journal. 2012. pp. 471–474. doi: 10.1038/ismej.2011.104 21796216

58. Markussen T, Marvig RL, Gómez-lozano M, Aanæs K, Burleigh AE. Environmental heterogeneity drives within-host diversification and evolution of Pseudomonas aeruginosa. 2014;5: 1–22. doi: 10.1128/mBio.01592-14

59. Blount ZD, Borland CZ, Lenski RE. Historical contingency and the evolution of a key innovation in an experimental population of Escherichia coli. Proc Natl Acad Sci U S A. 2008;105: 7899–7906. doi: 10.1073/pnas.0803151105 18524956

60. Kinnersley M, Wenger J, Kroll E, Adams J, Sherlock G, Rosenzweig F. Ex Uno Plures: Clonal reinforcement drives evolution of a simple microbial community. PLoS Genet. 2014;10. doi: 10.1371/journal.pgen.1004430

61. Rainey PB, Rainey K. Evolution of cooperation and conflict in experimental bacterial populations. Nature. 2003;425: 72–74. doi: 10.1038/nature01906 12955142

62. Braun BR, Johnson AD. Control of filament formation in Candida albicans by the transcriptional repressor TUP1. Science. 1997;277: 105–109. doi: 10.1126/science.277.5322.105 9204892

63. Khalaf RA, Zitomer RS. The DNA binding protein Rfg1 is a repressor of filamentation in Candida albicans. Genetics. 2001;157: 1503–1512. 11290707

64. Edgar RC. UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat Methods. 2013;10: 996–8. doi: 10.1038/nmeth.2604 23955772

65. Dannemiller KC, Reeves D, Bibby K, Yamamoto N, Peccia J. Fungal high-throughput taxonomic identification tool for use with next-generation sequencing (FHiTINGS). J Basic Microbiol. 2014;54: 315–321. doi: 10.1002/jobm.201200507 23765392

66. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215: 403–410. doi: 10.1006/jmbi.1990.9999\nS0022283680799990 [pii] 2231712

67. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, et al. QIIME allows analysis of high-throughput community sequencing data. Nature methods. 2010. pp. 335–336. doi: 10.1038/nmeth.f.303 20383131

68. Bokulich NA, Subramanian S, Faith JJ, Gevers D, Gordon JI, Knight R, et al. Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing. Nat Methods. 2013;10: 57–59. doi: 10.1038/nmeth.2276 23202435

69. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nature Methods. 2012. pp. 357–359. doi: 10.1038/nmeth.1923 22388286

70. Fiume M, Williams V, Brook A, Brudno M. Savant: genome browser for high-throughput sequencing data. Bioinformatics. 2010;26: 1938–1944. doi: 10.1093/bioinformatics/btq332 20562449

71. Cowen LE, Singh SD, Köhler JR, Collins C, Zaas AK, Schell WA, et al. Harnessing Hsp90 function as a powerful, broadly effective therapeutic strategy for fungal infectious disease. Proc Natl Acad Sci U S A. 2009;106: 2818–2823. doi: 10.1073/pnas.0813394106 19196973

72. Zhao J, Schloss PD, Kalikin LM, Carmody LA, Foster BK, Petrosino JF, et al. Decade-long bacterial community dynamics in cystic fibrosis airways. Proceedings of the National Academy of Sciences. 2012. pp. 5809–5814. doi: 10.1073/pnas.1120577109

Štítky
Hygiena a epidemiológia Infekčné lekárstvo Laboratórium

Článok vyšiel v časopise

PLOS Pathogens


2015 Číslo 11
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle
Kurzy

Zvýšte si kvalifikáciu online z pohodlia domova

Získaná hemofilie - Povědomí o nemoci a její diagnostika
nový kurz

Eozinofilní granulomatóza s polyangiitidou
Autori: doc. MUDr. Martina Doubková, Ph.D.

Všetky kurzy
Prihlásenie
Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.

Prihlásenie

Nemáte účet?  Registrujte sa

#ADS_BOTTOM_SCRIPTS#