1887

Microbiology Society logo

Volume 152, Issue 3

Calcineurin, Mpk1 and Hog1 MAPK pathways independently control fludioxonil antifungal sensitivity in Free

Abstract

Fludioxonil is employed as an agricultural fungicide to control plant-pathogenic fungi such as . is a basidiomycetous human fungal pathogen that causes fatal disease in immunocompromised hosts. This paper demonstrates that three different signalling cascades regulate sensitivity of to fludioxonil. Fludioxonil inhibited growth of the serotype A sequence reference strain H99 but not that of the sequenced serotype D strain JEC21. In the drug-sensitive wild-type strain, fludioxonil exposure activated the Hog1 osmosensing pathway, and Δ mutations conferred fludioxonil resistance. Fludioxonil treatment caused cell growth inhibition following cell swelling and cytokinesis defects in the sensitive wild-type but not in a Δ mutant strain, suggesting that Hog1 activation results in morphological cellular defects. Fludioxonil exerted a fungistatic effect on the wild-type strain H99, but exhibited fungicidal activity against calcineurin mutant strains, indicating that the calcineurin pathway contributes to drug resistance in this fungus. Combination of fludioxonil and the calcineurin inhibitor FK506 synergistically inhibited growth. Δ MAPK mutant strains exhibited fludioxonil hypersensitivity, indicating that this pathway also contributes to drug resistance. These studies provide evidence that the broad-spectrum antifungal drug fludioxonil exerts its action via activation of the Hog1 MAPK pathway and provide insight into novel targets for synergistic antifungal drug combinations.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): 5-FOA, 5-fluoroorotic acid , dH2O, distilled water , FIC, fractional inhibitory concentration , MFC, minimal fungicidal concentration and WT, wild-type
SGM
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.28571-0
2006-03-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/micro/152/3/591.html?itemId=/content/journal/micro/10.1099/mic.0.28571-0&mimeType=html&fmt=ahah

References

  1. Albertyn J, Hohmann S, Thevelein J. M, Prior B. A. 1994; GPD1 , which encodes glycerol-3-phosphate dehydrogenase, is essential for growth under osmotic stress in Saccharomyces cerevisiae , and its expression is regulated by the high-osmolarity glycerol response pathway. Mol Cell Biol 14:4135–4144
     [Google Scholar]
  2. Bahn Y. S, Kojima K, Cox G. M, Heitman J. 2005; Specialization of the HOG pathway and its impact on differentiation and virulence of Cryptococcus neoformans . Mol Biol Cell 16:2285–2300 [CrossRef]
     [Google Scholar]
  3. Borel J. F. 1976; Comparative study of in vitro and in vivo drug effects on cell-mediated cytotoxicity. Immunology 31:631–641
     [Google Scholar]
  4. Cameron M. L, Schell W. A, Bruch S, Bartlett J. A, Waskin H. A, Perfect J. R. 1993; Correlation of in vitro fluconazole resistance of Candida isolates in relation to therapy and symptoms of individuals seropositive for human immunodeficiency virus type 1. Antimicrob Agents Chemother 37:2449–2453 [CrossRef]
     [Google Scholar]
  5. Cruz M. C, Del Poeta M, Wang P. 7 other authors 2000; Immunosuppressive and nonimmunosuppressive cyclosporine analogs are toxic to the opportunistic fungal pathogen Cryptococcus neoformans via cyclophilin-dependent inhibition of calcineurin. Antimicrob Agents Chemother 44:143–149 [CrossRef]
     [Google Scholar]
  6. Cruz M. C, Fox D. S, Heitman J. 2001; Calcineurin is required for hyphal elongation during mating and haploid fruiting in Cryptococcus neoformans . EMBO J 20:1020–1032 [CrossRef]
     [Google Scholar]
  7. Cruz M. C, Goldstein A. L, Blankenship J. R, Del Poeta M, Davis D, Cardenas M. E, Perfect J. R, McCusker J. H, Heitman J. 2002; Calcineurin is essential for survival during membrane stress in Candida albicans . EMBO J 21:546–559 [CrossRef]
     [Google Scholar]
  8. Davidson R. C, Blankenship J. R, Kraus P. R, de Jesus Berrios M, Hull C. M, D'Souza C, Wang P, Heitman J. 2002; A PCR-based strategy to generate integrative targeting alleles with large regions of homology. Microbiology 148:2607–2615
     [Google Scholar]
  9. Del Poeta M, Cruz M. C, Cardenas M. E, Perfect J. R, Heitman J. 2000; Synergistic antifungal activities of bafilomycin A(1), fluconazole, and the pneumocandin MK-0991/caspofungin acetate (L-743,873) with calcineurin inhibitors FK506 and L-685,818 against Cryptococcus neoformans . Antimicrob Agents Chemother 44:739–746 [CrossRef]
     [Google Scholar]
  10. Fox D. S, Cruz M. C, Sia R. A, Ke H, Cox G. M, Cardenas M. E, Heitman J. 2001; Calcineurin regulatory subunit is essential for virulence and mediates interactions with FKBP12-FK506 in Cryptococcus neoformans . Mol Microbiol 39:835–849 [CrossRef]
     [Google Scholar]
  11. Fraser J. A, Subaran R. L, Nichols C. B, Heitman J. 2003; Recapitulation of the sexual cycle of the primary fungal pathogen Cryptococcus neoformans var. gattii : implications for an outbreak on Vancouver Island, Canada. Eukaryot Cell 2:1036–1045 [CrossRef]
     [Google Scholar]
  12. Fujimura M, Ochiai N, Ichiishi A, Usami R, Horikoshi K, Yamaguchi I. 2000a; Fungicide resistance and osmotic stress sensitivity in os mutants of Neurospora crassa . Pestic Biochem Physiol 67:125–133 [CrossRef]
     [Google Scholar]
  13. Fujimura M, Ochiai N, Ichiishi A, Usami R, Horikoshi K, Yamaguchi I. 2000b; Sensitivity to phenylpyrrole fungicides and abnormal glycerol accumulation in os and cut mutant strains of Neurospora crassa . J Pestic Sci 25:31–36 [CrossRef]
     [Google Scholar]
  14. Gehmann K, Nyfeler R, Leadbeater A. J, Nevill D, Sozzi D. 1990; CGA 173506: a new phenylpyrrole fungicide for broad-spectrum disease control. Brighton Crop Prot Conf Pests Dis 2:399–406
     [Google Scholar]
  15. Giaever G, Chu A. M, Ni L. 71 other authors 2002; Functional profiling of the Saccharomyces cerevisiae genome. Nature 418:387–391 [CrossRef]
     [Google Scholar]
  16. Heitman J, Allen B, Alspaugh J. A, Kwon-Chung K. J. 1999; On the origins of congenic MAT α and MATa strains of the pathogenic yeast Cryptococcus neoformans . Fungal Genet Biol 28:1–5 [CrossRef]
     [Google Scholar]
  17. Hensel M, Shea J. E, Gleeson C, Jones M. D, Dalton E, Holden D. W. 1995; Simultaneous identification of bacterial virulence genes by negative selection. Science 269:400–403 [CrossRef]
     [Google Scholar]
  18. High K. P. 1994; The antimicrobial activities of cyclosporine, FK506, and rapamycin. Transplantation 57:1689–1700 [CrossRef]
     [Google Scholar]
  19. Hull C. M, Heitman J. 2002; Genetics of Cryptococcus neoformans . Annu Rev Genet 36:557–615 [CrossRef]
     [Google Scholar]
  20. Kino T, Hatanaka H, Hashimoto M, Nishiyama M, Goto T, Okuhara M, Kohsaka M, Aoki H, Imanaka H. 1987; FK-506, a novel immunosuppressant isolated from a Streptomyces . I. Fermentation, isolation, and physico-chemical and biological characteristics. J Antibiot 40:1249–1255 [CrossRef]
     [Google Scholar]
  21. Kojima K, Takano Y, Yoshimi A, Tanaka C, Kikuchi T, Okuno T. 2004; Fungicide activity through activation of a fungal signalling pathway. Mol Microbiol 53:1785–1796 [CrossRef]
     [Google Scholar]
  22. Kraus P. R, Fox D. S, Cox G. M, Heitman J. 2003; The Cryptococcus neoformans MAP kinase Mpk1 regulates cell integrity in response to antifungal drugs and loss of calcineurin function. Mol Microbiol 48:1377–1387 [CrossRef]
     [Google Scholar]
  23. Kwon-Chung K. J, Edman J. C, Wickes B. L. 1992; Genetic association of mating types and virulence in Cryptococcus neoformans . Infect Immun 60:602–605
     [Google Scholar]
  24. Lee B. N, Elion E. A. 1999; The MAPKKK Ste11 regulates vegetative growth through a kinase cascade of shared signaling components. Proc Natl Acad Sci U S A 96:12679–12684 [CrossRef]
     [Google Scholar]
  25. Lengeler K. B, Cox G. M, Heitman J. 2001; Serotype AD strains of Cryptococcus neoformans are diploid or aneuploid and are heterozygous at the mating-type locus. Infect Immun 69:115–122 [CrossRef]
     [Google Scholar]
  26. Moore T. D, Edman J. C. 1993; The alpha-mating type locus of Cryptococcus neoformans contains a peptide pheromone gene. Mol Cell Biol 13:1962–1970
     [Google Scholar]
  27. Motoyama T, Ohira T, Kadokura K, Ichiishi A, Fujimura M, Yamaguchi I, Kudo T. 2005; An Os-1 family histidine kinase from a filamentous fungus confers fungicide-sensitivity to yeast. Curr Genet 47:298–306 [CrossRef]
     [Google Scholar]
  28. Mrsa V, Tanner W. 1999; Role of NaOH-extractable cell wall proteins Ccw5p, Ccw6p, Ccw7p and Ccw8p (members of the Pir protein family) in stability of the Saccharomyces cerevisiae cell wall. Yeast 15:813–820 [CrossRef]
     [Google Scholar]
  29. Mrsa V, Seidl T, Gentzsch M, Tanner W. 1997; Specific labelling of cell wall proteins by biotinylation. Identification of four covalently linked O -mannosylated proteins of Saccharomyces cerevisiae . Yeast 13:1145–1154 [CrossRef]
     [Google Scholar]
  30. Ochiai N, Fujimura M, Oshima M, Motoyama T, Ichiishi A, Yamada-Okabe H, Yamaguchi I. 2002; Effects of iprodione and fludioxonil on glycerol synthesis and hyphal development in Candida albicans . Biosci Biotechnol Biochem 66:2209–2215 [CrossRef]
     [Google Scholar]
  31. Odom A, Del Poeta M, Perfect J, Heitman J. 1997a; The immunosuppressant FK506 and its nonimmunosuppressive analog L-685,818 are toxic to Cryptococcus neoformans by inhibition of a common target protein. Antimicrob Agents Chemother 41:156–161
     [Google Scholar]
  32. Odom A, Muir S, Lim E, Toffaletti D. L, Perfect J, Heitman J. 1997b; Calcineurin is required for virulence of Cryptococcus neoformans . EMBO J 16:2576–2589 [CrossRef]
     [Google Scholar]
  33. Onyewu C, Blankenship J. R, Del Poeta M, Heitman J. 2003; Ergosterol biosynthesis inhibitors become fungicidal when combined with calcineurin inhibitors against Candida albicans , Candida glabrata , and Candida krusei . Antimicrob Agents Chemother 47:956–964 [CrossRef]
     [Google Scholar]
  34. Orth A. B, Rzhetskaya M, Pell E. J, Tien M. 1995; A serine (threonine) protein kinase confers fungicide resistance in the phytopathogenic fungus Ustilago maydis . Appl Environ Microbiol 61:2341–2345
     [Google Scholar]
  35. Paugam A, Dupouy-Camet J, Blanche P, Gangneux J. P, Tourte-Schaefer C, Sicard D. 1994; Increased fluconazole resistance of Cryptococcus neoformans isolated from a patient with AIDS and recurrent meningitis. Clin Infect Dis 19:975–976
     [Google Scholar]
  36. Perfect J. R, Durack D. T. 1985; Effects of cyclosporine in experimental cryptococcal meningitis. Infect Immun 50:22–26
     [Google Scholar]
  37. Perfect J. R, Ketabchi N, Cox G. M, Ingram C. W, Beiser C. L. 1993; Karyotyping of Cryptococcus neoformans as an epidemiological tool. J Clin Microbiol 12:3305–3309
     [Google Scholar]
  38. Pillonel C, Mayer T. 1997; Effect of phenylpyrroles on glycerol accumulation and protein kinase activity of Neurospora crassa . Pestic Sci 49:229–236 [CrossRef]
     [Google Scholar]
  39. Ramesh M. A, Laidlaw R. D, Durrenberger F, Orth A. B, Kronstad J. W. 2001; The cAMP signal transduction pathway mediates resistance to dicarboximide and aromatic hydrocarbon fungicides in Ustilago maydis . Fungal Genet Biol 32:183–193 [CrossRef]
     [Google Scholar]
  40. Shitamukai A, Hirata D, Sonobe S, Miyakawa T. 2004; Evidence for antagonistic regulation of cell growth by the calcineurin and high osmolarity glycerol pathways in Saccharomyces cerevisiae . J Biol Chem 279:3651–3661
     [Google Scholar]
  41. Steinbach W. J, Schell W. A, Blankenship J. R, Onyewu C, Heitman J, Perfect J. R. 2004; In vitro interactions between antifungals and immunosuppressants against Aspergillus fumigatus . Antimicrob Agents Chemother 48:1664–1669 [CrossRef]
     [Google Scholar]
  42. Teparic R, Stuparevic I, Mrsa V. 2004; Increased mortality of Saccharomyces cerevisiae cell wall protein mutants. Microbiology 150:3145–3150 [CrossRef]
     [Google Scholar]
  43. Viaud M. C, Balhadere P. V, Talbot N. J. 2002; A Magnaporthe grisea cyclophilin acts as a virulence determinant during plant infection. Plant Cell 14:917–930 [CrossRef]
     [Google Scholar]
  44. Viaud M, Brunet-Simon A, Brygoo Y, Pradier J. M, Levis C. 2003; Cyclophilin A and calcineurin functions investigated by gene inactivation, cyclosporin A inhibition and cDNA arrays approaches in the phytopathogenic fungus Botrytis cinerea . Mol Microbiol 50:1451–1465 [CrossRef]
     [Google Scholar]
  45. White T. C, Marr K. A, Bowden R. A. 1998; Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clin Microbiol Rev 11:382–402
     [Google Scholar]
  46. Zhang Y, Lamm R, Pillonel C, Lam S, Xu J. R. 2002; Osmoregulation and fungicide resistance: the Neurospora crassa os-2 gene encodes a HOG1 mitogen-activated protein kinase homologue. Appl Environ Microbiol 68:532–538 [CrossRef]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.28571-0
Loading
Calcineurin, Mpk1 and Hog1 MAPK pathways independently control fludioxonil antifungal sensitivity in Cryptococcus neoformans
Microbiology 152, 591 (2006); https://doi.org/10.1099/mic.0.28571-0
/content/journal/micro/10.1099/mic.0.28571-0
/content/journal/micro/10.1099/mic.0.28571-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited Most Cited RSS feed

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