1887

Abstract

Some species of fusaria are well-known pathogens of humans, animals and plants. and (formerly ) cause human infections that range from onychomycosis or keratitis to severe disseminated infections. In general, these infections are difficult to treat due to poor therapeutic responses in immunocompromised patients. Despite that, little is known about the molecular mechanisms and transcriptional changes responsible for the antifungal resistance in fusaria. To shed light on the transcriptional response to antifungals, we carried out the first reported high-throughput RNA-seq analysis for and that had been exposed to amphotericin B (AMB) and posaconazole (PSC). We detected significant differences between the transcriptional profiles of the two species and we found that some oxidation-reduction, metabolic, cellular and transport processes were regulated differentially by both fungi. The same was found with several genes from the ergosterol synthesis, efflux pumps, oxidative stress response and membrane biosynthesis pathways. A significant up-regulation of the C-22 sterol desaturase (), the sterol 24-C-methyltransferase () gene, the glutathione S-transferase () gene and of several members of the major facilitator superfamily () was demonstrated in this study after treating with AMB. These results offer a good overview of transcriptional changes after exposure to commonly used antifungals, highlights the genes that are related to resistance mechanisms of these fungi, which will be a valuable tool for identifying causes of failure of treatments.

Funding
This study was supported by the:
  • , Colciencias , (Award This work was supported by the Faculty of Sciences at Universidad de los Andes and the Departamento Administrativo de Ciencia, Tecnología e Innovación (Colciencias) agreement Nº RC 268-2010.)
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2020-07-09
2020-11-30
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References

  1. Al-Hatmi AMS, Meis JF, de Hoog GS. Fusarium: molecular diversity and intrinsic drug resistance. PLoS Pathog 2016; 12:e1005464 [CrossRef]
    [Google Scholar]
  2. Peres NTA, Persinoti GF, Lang EAS, Rossi A, Martinez-Rossi NM. Transcriptome in Human Mycoses. In Passos GA. editor Transcriptomics in Health and Disease Cham: Springer International Publishing; pp 227–263
    [Google Scholar]
  3. Nucci M, Anaissie E. Fusarium infections in immunocompromised patients. Clin Microbiol Rev 2007; 20:695–704 [CrossRef]
    [Google Scholar]
  4. Sandoval-Denis M, Crous PW. Removing chaos from confusion: assigning names to common human and animal pathogens in Neocosmospora . Persoonia 2018; 41:109–129 [CrossRef]
    [Google Scholar]
  5. Scheel CM, Hurst SF, Barreiros G, Akiti T, Nucci M et al. Molecular analyses of Fusarium isolates recovered from a cluster of invasive mold infections in a Brazilian hospital. BMC Infect Dis 2013; 13:49 [CrossRef]
    [Google Scholar]
  6. Espinel-Ingroff A, Colombo AL, Cordoba S, Dufresne PJ, Fuller J et al. International Evaluation of MIC Distributions and Epidemiological Cutoff Value (ECV) Definitions for Fusarium Species Identified by Molecular Methods for the CLSI Broth Microdilution Method. Antimicrob Agents Chemother 2016; 60:1079–1084 [CrossRef]
    [Google Scholar]
  7. Lortholary O, Obenga G, Biswas P, Caillot D, Chachaty E et al. International retrospective analysis of 73 cases of invasive fusariosis treated with voriconazole. Antimicrob Agents Chemother 2010; 54:4446–4450 [CrossRef]
    [Google Scholar]
  8. Tortorano AM, Prigitano A, Esposto MC, Arsic Arsenijevic V, Kolarovic J et al. European Confederation of Medical Mycology (ECMM) epidemiological survey on invasive infections due to Fusarium species in Europe. Eur J Clin Microbiol Infect Dis 2014; 33:1623–1630 [CrossRef][PubMed]
    [Google Scholar]
  9. Raad II, Hachem RY, Herbrecht R, Graybill JR, Hare R et al. Posaconazole as salvage treatment for invasive fusariosis in patients with underlying hematologic malignancy and other conditions. Clin Infect Dis 2006; 42:1398–1403 [CrossRef]
    [Google Scholar]
  10. Campo M, Lewis RE, Kontoyiannis DP. Invasive fusariosis in patients with hematologic malignancies at a cancer center: 1998–2009. J Infect 2010; 60:331–337 [CrossRef]
    [Google Scholar]
  11. Revie NM, Iyer KR, Robbins N, Cowen LE. Antifungal drug resistance: evolution, mechanisms and impact. Curr Opin Microbiol 2018; 45:70–76 [CrossRef]
    [Google Scholar]
  12. Sokol-Anderson ML, Brajtburg J, Medoff G. Amphotericin B-induced oxidative damage and killing of Candida albicans . J Infect Dis 1986; 154:76–83 [CrossRef]
    [Google Scholar]
  13. Vandeputte P, Tronchin G, Bergès T, Hennequin C, Chabasse D et al. Reduced susceptibility to polyenes associated with a missense mutation in the ERG6 gene in a clinical isolate of Candida glabrata with pseudohyphal growth. Antimicrob Agents Chemother 2007; 51:982–990 [CrossRef]
    [Google Scholar]
  14. Araujo R, Espinel-Ingroff A. Antifungal resistance: Cellular and molecular mechanisms. Combating Fungal Infections: Problems and Remedy 2010 pp 125–145
    [Google Scholar]
  15. Young LY, Hull CM, Heitman J. Disruption of ergosterol biosynthesis confers resistance to amphotericin B in Candida lusitaniae . Antimicrob Agents Chemother 2003; 47:2717–2724 [CrossRef]
    [Google Scholar]
  16. Nolte FS, Parkinson T, Falconer DJ, Dix S, Williams J et al. Isolation and characterization of fluconazole- and amphotericin B-resistant Candida albicans from blood of two patients with leukemia. Antimicrob Agents Chemother 1997; 41:196–199 [CrossRef]
    [Google Scholar]
  17. Alcazar-Fuoli L, Mellado E. Ergosterol biosynthesis in Aspergillus fumigatus: its relevance as an antifungal target and role in antifungal drug resistance. Front Microbiol 2013; 3:439 [CrossRef]
    [Google Scholar]
  18. Marichal P, Koymans L, Willemsens S, Bellens D, Verhasselt P et al. Contribution of mutations in the cytochrome P450 14α-demethylase (Erg11p, Cyp51p) to azole resistance in Candida albicans . Microbiology 1999; 145:2701–2713 [CrossRef]
    [Google Scholar]
  19. Fan J, Urban M, Parker JE, Brewer HC, Kelly SL et al. Characterization of the sterol 14α-demethylases of Fusarium graminearum identifies a novel genus-specific CYP51 function. New Phytol 2013; 198:821–835 [CrossRef]
    [Google Scholar]
  20. Slaven JW, Anderson MJ, Sanglard D, Dixon GK, Bille J et al. Increased expression of a novel Aspergillus fumigatus ABC transporter gene, atrF, in the presence of itraconazole in an itraconazole resistant clinical isolate. Fungal Genetics and Biology 2002; 36:199–206 [CrossRef]
    [Google Scholar]
  21. Morschhäuser J. Regulation of multidrug resistance in pathogenic fungi. Fungal Genetics and Biology 2010; 47:94–106 [CrossRef]
    [Google Scholar]
  22. Abou Ammar G, Tryono R, Döll K, Karlovsky P, Deising HB et al. Identification of ABC transporter genes of Fusarium graminearum with roles in azole tolerance and/or virulence. PLoS One 2013; 8:e79042–13 [CrossRef]
    [Google Scholar]
  23. Berthiller F, Crews C, Dall'Asta C, Saeger SD, Haesaert G et al. Masked mycotoxins: a review. Mol Nutr Food Res 2013; 57:165–186 [CrossRef]
    [Google Scholar]
  24. Kulik T, Łojko M, Jestoi M, Perkowski J. Sublethal concentrations of azoles induce tri transcript levels and trichothecene production in Fusarium graminearum . FEMS Microbiol Lett 2012; 335:58–67 [CrossRef]
    [Google Scholar]
  25. Shor E, Perlin DS. Coping with stress and the emergence of multidrug resistance in fungi. PLoS Pathog 2015; 11:e1004668 [CrossRef]
    [Google Scholar]
  26. Azor Mónica, Gené J, Cano J, Guarro J. Universal in vitro antifungal resistance of genetic clades of the Fusarium solani species complex. Antimicrob Agents Chemother 2007; 51:1500–1503 [CrossRef]
    [Google Scholar]
  27. Azor M, Cano J, Gené J, Guarro J. High genetic diversity and poor in vitro response to antifungals of clinical strains of Fusarium oxysporum . J Antimicrob Chemother 2009; 63:1152–1155 [CrossRef]
    [Google Scholar]
  28. Wang B, Guo G, Wang C, Lin Y, Wang X et al. Survey of the transcriptome of Aspergillus oryzae via massively parallel mRNA sequencing. Nucleic Acids Res 2010; 38:5075–5087 [CrossRef]
    [Google Scholar]
  29. Krieg PA. A Laboratory Guide To RNA: Isolation, Analysis, and Synthesis New York: Wiley-Liss; 1996
    [Google Scholar]
  30. Andrews S. FastQC: A quality control tool for high throughput sequence data. http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ (2010).
  31. Arvidsson S, Kwasniewski M, Riaño-Pachón DM, Mueller-Roeber B. QuantPrime - a flexible tool for reliable high-throughput primer design for quantitative PCR. BMC Bioinformatics 2008; 9:465 [CrossRef]
    [Google Scholar]
  32. Trapnell C, Pachter L, Salzberg SL. TopHat: discovering splice junctions with RNA-seq. Bioinformatics 2009; 25:1105–1111 [CrossRef]
    [Google Scholar]
  33. LJ M, Van Der Does HC, Borkovich KA, Coleman JJ, Daboussi MJ et al. Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium . Nature 2010; 464:367–373
    [Google Scholar]
  34. Coleman JJ, Rounsley SD, Rodriguez-Carres M, Kuo A, Wasmann CC et al. The genome of Nectria haematococca: contribution of supernumerary chromosomes to gene expansion. PLoS Genet 2009; 5:e1000618 [CrossRef]
    [Google Scholar]
  35. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G et al. Transcript assembly and quantification by RNA-seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 2010; 28:511–515 [CrossRef]
    [Google Scholar]
  36. Trapnell C, Hendrickson DG, Sauvageau M, Goff L, Rinn JL et al. Differential analysis of gene regulation at transcript resolution with RNA-seq. Nat Biotechnol 2013; 31:46–53 [CrossRef]
    [Google Scholar]
  37. Götz S, García-Gómez JM, Terol J, Williams TD, Nagaraj SH et al. High-Throughput functional annotation and data mining with the Blast2GO suite. Nucleic Acids Res 2008; 36:3420–3435 [CrossRef]
    [Google Scholar]
  38. Supek F, Bošnjak M, Škunca N, Šmuc T. REVIGO summarizes and visualizes long Lists of gene ontology terms. PLoS One 2011; 6:e21800 [CrossRef][PubMed]
    [Google Scholar]
  39. De Backer MD, Ilyina T, Ma X-J, Vandoninck S, Luyten WHML et al. Genomic profiling of the response of Candida albicans to itraconazole treatment using a DNA microarray. Antimicrob Agents Chemother 2001; 45:1660–1670 [CrossRef]
    [Google Scholar]
  40. Gasch AP, Spellman PT, Kao CM, Carmel-Harel O, Eisen MB et al. Genomic expression programs in the response of yeast cells to environmental changes. Mol Biol Cell 2000; 11:4241–4257 [CrossRef]
    [Google Scholar]
  41. Vandeputte P, Ferrari S, Coste AT. Antifungal resistance and new strategies to control fungal infections. Int J Microbiol 2012; 2012:1–26 [CrossRef]
    [Google Scholar]
  42. Gautam P, Shankar J, Madan T, Sirdeshmukh R, Sundaram CS et al. Proteomic and transcriptomic analysis of Aspergillus fumigatus on exposure to amphotericin B. Antimicrob Agents Chemother 2008; 52:4220–4227 [CrossRef]
    [Google Scholar]
  43. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001; 29:45e–45 [CrossRef]
    [Google Scholar]
  44. Wang Z, Gerstein M, Snyder M. Rna-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 2009; 10:57–63 [CrossRef]
    [Google Scholar]
  45. Nagalakshmi U, Wang Z, Waern K, Shou C, Raha D et al. The transcriptional landscape of the yeast genome defined by RNA sequencing. Science 2008; 320:1344–1349 [CrossRef]
    [Google Scholar]
  46. Bullard JH, Purdom E, Hansen KD, Dudoit S. Evaluation of statistical methods for normalization and differential expression in mRNA-seq experiments. BMC Bioinformatics 2010; 11:94 [CrossRef]
    [Google Scholar]
  47. Lombard L, van der Merwe NA, Groenewald JZ, Crous PW. Generic concepts in Nectriaceae . Stud Mycol 2015; 80:189–245 [CrossRef]
    [Google Scholar]
  48. Sandoval-Denis M, Guarnaccia V, Polizzi G, Crous PW. Symptomatic Citrus trees reveal a new pathogenic lineage in Fusarium and two new Neocosmospora species. Persoonia 2018; 40:1–25 [CrossRef]
    [Google Scholar]
  49. Sharma C, Chowdhary A. Molecular bases of antifungal resistance in filamentous fungi. Int J Antimicrob Agents 2017; 50:607–616 [CrossRef]
    [Google Scholar]
  50. Lotfali E, Ghajari A, Kordbacheh P, Zaini F, Mirhendi H et al. Regulation of ERG3, ERG6, and ERG11 Genes in Antifungal-Resistant isolates of Candida parapsilosis . Iran Biomed J 2017; 21:275–281 [CrossRef]
    [Google Scholar]
  51. Konecna A, Toth Hervay N, Bencova A, Morvova M, Sikurova L et al. Erg6 gene is essential for stress adaptation in Kluyveromyces lactis . FEMS Microbiol Lett 2018; 365: [CrossRef]
    [Google Scholar]
  52. Song J, Zhai P, Zhang Y, Zhang C, Sang H et al. The Aspergillus fumigatus Damage Resistance Protein Family Coordinately Regulates Ergosterol Biosynthesis and Azole Susceptibility. MBio 2016; 7:e01919-15 [CrossRef]
    [Google Scholar]
  53. Nakayama N, Takemae A, Shoun H, Hirofumi S. Cytochrome P450foxy, a Catalytically Self-Sufficient Fatty Acid Hydroxylase of the Fungus Fusarium oxysporum . J Biochem 1996; 119:435–440 [CrossRef]
    [Google Scholar]
  54. Kitazume T, Tanaka A, Takaya N, Nakamura A, Matsuyama S et al. Kinetic analysis of hydroxylation of saturated fatty acids by recombinant P450foxy produced by an Escherichia coli expression system. Eur J Biochem 2002; 269:2075–2082 [CrossRef]
    [Google Scholar]
  55. Venkateswarlu K, Kelly DE, Manning NJ, Kelly SL. Nadph cytochrome P-450 oxidoreductase and susceptibility to ketoconazole. Antimicrob Agents Chemother 1998; 42:1756–1761 [CrossRef]
    [Google Scholar]
  56. Shin J, Kim J-E, Lee Y-W, Son H. Fungal cytochrome P450s and the P450 complement (CYPome) of Fusarium graminearum . Toxins 2018; 10:112–191 [CrossRef]
    [Google Scholar]
  57. Ferreira MEdaS, Malavazi I, Savoldi M, Brakhage AA, Goldman MHS et al. Transcriptome analysis of Aspergillus fumigatus exposed to voriconazole. Curr Genet 2006; 50:32–44 [CrossRef]
    [Google Scholar]
  58. Diao Y, Zhao R, Deng X, Leng W, Peng J, Diao Y, Zhao R et al. Transcriptional profiles of Trichophyton rubrum in response to itraconazole. Med Mycol 2009; 47:237–247 [CrossRef][PubMed]
    [Google Scholar]
  59. Hu C, Zhou M, Wang W, Sun X, Yarden O et al. Abnormal ergosterol biosynthesis activates transcriptional responses to antifungal azoles. Front Microbiol 2018; 9:1–14 [CrossRef]
    [Google Scholar]
  60. Liu TT, Lee REB, Barker KS, Lee RE, Wei L et al. Genome-Wide expression profiling of the response to azole, polyene, echinocandin, and pyrimidine antifungal agents in Candida albicans. Antimicrob Agents Chemother 2005; 49:2226–2236 [CrossRef]
    [Google Scholar]
  61. Vincent BM, Lancaster AK, Scherz-Shouval R, Whitesell L, Lindquist S. Fitness trade-offs restrict the evolution of resistance to amphotericin B. PLoS Biol 2013; 11:e1001692 [CrossRef]
    [Google Scholar]
  62. Ibe C, Walker LA, NAR G, Munro CA. Unlocking the Therapeutic Potential of the Fungal Cell Wall: Clinical Implications and Drug Resistance.. In Prasad R. editor Candida albicans: Cellular and Molecular Biology, 2nd ed. Cham: Springer International Publishing; 1990 pp 313–346
    [Google Scholar]
  63. Singh S, Brocker C, Koppaka V, Chen Y, Jackson BC et al. Aldehyde dehydrogenases in cellular responses to oxidative/electrophilicstress. Free Radical Biology and Medicine 2013; 56:89–101 [CrossRef]
    [Google Scholar]
  64. Fradin C, Kretschmar M, Nichterlein T, Gaillardin C, D’Enfert C et al. Stage-specific gene expression of Candida albicans in human blood. Mol Microbiol 2003; 47:1523–1543 [CrossRef]
    [Google Scholar]
  65. Sugui JA, Kim HS, Zarember KA, Chang YC, Gallin JI et al. Genes differentially expressed in conidia and hyphae of Aspergillus fumigatus upon exposure to human neutrophils. PLoS One 2008; 3:e2655 [CrossRef]
    [Google Scholar]
  66. Florio A, Ferrari S, De Carolis E, Torelli R, Fadda G et al. Genome-wide expression profiling of the response to short-term exposure to fluconazole in Cryptococcus neoformans serotype A. BMC Microbiol 2011; 11:97 [CrossRef]
    [Google Scholar]
  67. Berne S, Podobnik B, Zupanec N, Novak M, Kraševec N et al. Virtual screening yields inhibitors of novel antifungal drug target, benzoate 4-monooxygenase. J Chem Inf Model 2012; 52:3053–3063 [CrossRef]
    [Google Scholar]
  68. Korošec B, Sova M, Turk S, Kraševec N, Novak M et al. Antifungal activity of cinnamic acid derivatives involves inhibition of benzoate 4-hydroxylase (CYP53). J Appl Microbiol 2014; 116:955–966 [CrossRef]
    [Google Scholar]
  69. Podobnik B, Stojan J, Lah L, Kras̆evec N, Selis̆kar M et al. CYP53A15 of Cochliobolus lunatus, a Target for Natural Antifungal Compounds . J Med Chem 2008; 51:3480–3486 [CrossRef]
    [Google Scholar]
  70. Purkait B, Kumar A, Nandi N, Sardar AH, Das S et al. Mechanism of amphotericin B resistance in clinical isolates of Leishmania donovani . Antimicrob Agents Chemother 2012; 56:1031–1041 [CrossRef]
    [Google Scholar]
  71. Garcia-Effron G, Dilger A, Alcazar-Fuoli L, Park S, Mellado E et al. Rapid detection of triazole antifungal resistance in Aspergillus fumigatus . J Clin Microbiol 2008; 46:1200–1206 [CrossRef]
    [Google Scholar]
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