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

Abstract

Fludioxonil is employed as an agricultural fungicide to control plant-pathogenic fungi such as Botrytis cinerea. Cryptococcus neoformans is a basidiomycetous human fungal pathogen that causes fatal disease in immunocompromised hosts. This paper demonstrates that three different signalling cascades regulate sensitivity of C. neoformans 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 hog1D 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 hog1D 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 C. neoformans growth. mpk1D 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.

Regulatory Circuitry Governing Fungal Development, Drug Resistance, and Disease

“…Moreover, fludioxonil exerts fungicidal activity against calcineurin mutants and acts synergistically with FK506 (280). Finally, mpk1 mutants from the PKC cell wall integrity pathway exhibit fludioxonil sensitivity (280).…”

“…The two-component sensor kinases Tco1 and Tco2 play distinct and redundant roles in fludioxonil sensitivity through the Hog1 MAPK signaling cascade (28). Moreover, fludioxonil exerts fungicidal activity against calcineurin mutants and acts synergistically with FK506 (280). Finally, mpk1 mutants from the PKC cell wall integrity pathway exhibit fludioxonil sensitivity (280).…”

“…Specifically, three distinct signaling pathways have been shown to contribute to the tolerance of C. neoformans to fludioxonil. This antifungal has been shown to activate the Hog1 MAPK pathway, and hog1 mutants are resistant to fludioxonil (280). The two-component sensor kinases Tco1 and Tco2 play distinct and redundant roles in fludioxonil sensitivity through the Hog1 MAPK signaling cascade (28).…”

Eca1, a Sarcoplasmic/Endoplasmic Reticulum Ca2+-ATPase, Is Involved in Stress Tolerance and Virulence inCryptococcus neoformans

“…The serotype A MAT␣ strain H99 and congenic MAT␣ KN99␣ and MATa KN99a strains were used as the wild type (53,57). The calcineurin subunit A cna1⌬ deletion strains were as reported previously (40). The collection of ϳ590 insertional mutants was generated previously by biolistic transformation of strain H99 with plasmid pCH233 conferring resistance to nourseothricin (38).…”

Calcineurin in fungal virulence and drug resistance: Prospects for harnessing targeted inhibition of calcineurin for an antifungal therapeutic approach

“…10,12 For example, combinations of calcium and calcineurin inhibitors with known antifungal compounds have been shown to inhibit the growth of drug resistant fungal strains. [13][14][15][16][17][18][19] Therefore, inhibition of calcineurin signaling is a novel antifungal strategy that both attenuates fungal virulence and increases the efficacy of the existing antifungals with concomitant suppression of antifungal resistance. 10,20,21 Calcineurin is a conserved Ca 2C -calmodulin (CaM) activated protein phosphatase 2B belonging to the phospho-protein phosphatase family of enzymes and is involved in calcium-dependent signaling and regulation of several important cellular processes in both yeasts and filamentous fungi (Fig.…”