The monothiol glutaredoxin Grx4 influences thermotolerance, cell wall integrity, and Mpk1 signaling in Cryptococcus neoformans

Abstract

Monothiol glutaredoxins are important regulators of iron homeostasis that play conserved roles in the sensing and trafficking of iron-sulfur clusters. We previously characterized the role of the monothiol glutaredoxin Grx4 in iron homeostasis, the interaction with the iron regulator Cir1, and virulence in Cryptococcus neoformans. This important fungal pathogen causes cryptococcal meningoencephalitis in immunocompromised individuals worldwide. Here we demonstrate that Grx4 is required for proliferation at elevated temperatures (both 37 °C and 39 °C) and under stress conditions. In particular, the grx4Δ mutant was hypersensitive to SDS, calcofluor white, and caffeine, suggesting that Grx4 is required for membrane and cell wall integrity (CWI). In this context, we found that Grx4 regulated the phosphorylation of the Mpk1 mitogen-activated protein kinase (MAPK) of the cell wall integrity pathway in cells grown at elevated temperature or upon treatment with calcofluor white, caffeine, or SDS. The grx4Δ mutant also displayed increased sensitivity to FK506 and cyclosporin A, two inhibitors of the calcineurin pathway, indicating that Grx4 may influence growth at higher temperatures in parallel with calcineurin signaling. Upon thermal stress or calcium treatment, loss of Grx4 also caused partial mis-localization of Crz1, the transcription factor that is a calcineurin substrate. The phenotypes of the grx4Δ, crz1Δ and cna1Δ (calcineurin) mutants suggest shared contributions to the regulation of temperature, cell wall and other stresses. In summary we show that Grx4 is also a key regulator of the responses to a variety of stress conditions in addition to its roles in iron homeostasis in C. neoformans.

A critical role of calcineurin in stress responses, hyphal formation, and virulence of the pathogenic fungus Trichosporon asahii

“…In Cryptococcus gattii and C. neoformans , cna1 gene-deficient mutants are sensitive to sodium dodecyl sulfate (SDS), which damages the cell membrane, and Congo red, which damages the cell wall 21 . The cna1 gene-deficient mutant of C. neoformans is also sensitive to caffeine, which inhibits the cell wall integrity (CWI) signaling pathway, which promotes cell wall integrity; and sorbitol, which is an osmotic stressor 22 . Growth was delayed in cna1 and cnb1 gene-deficient T. asahii mutants treated with SDS and Congo red, but not affected by treatment with caffeine or sorbitol (Fig.…”

“…3 ). In C. neoformans , the cna1 gene-deficient mutant is sensitive to dithiothreitol (DTT) and tunicamycin (TM), which stress the ER; brefeldin A (BFA), which inhibits intracellular vesicle formation and protein trafficking between the ER and the Golgi apparatus; and monensin, which is involved in intracellular transport 22 . Growth was delayed in cna1 and cnb1 gene-deficient T. asahii mutants treated with DTT and TM, but not in those treated with BFA and monensin (Fig.…”

“…In Cryptococcus gattii and C. neoformans , calcineurin plays an important role in cell membrane homeostasis, cell wall integrity, the ER stress response, cation homeostasis, and fluconazole tolerance 21 , 22 . Calcineurin in T. asahii is related to resistance against cell membrane and cell wall damage, and ER stress, but not cell wall integrity dependent on the CWI pathway, osmotic stress resistance, cation homeostasis, or antifungal drug resistance.…”

“…It is essential for the growth of Cryptococcus neoformans , a pathogenic basidiomycete yeast like T. asahii , at 37 °C, as well as its virulence in a rabbit model of cryptococcal meningitis and cation homeostasis 19 , 20 . In addition, calcineurin contributes to resistance to cell membrane damage, cell wall damage, osmotic stress, and endoplasmic reticulum (ER) stress 21 , 22 . The role of calcineurin in the virulence of T. asahii , however, has remained unclear.…”

Response and regulatory mechanisms of heat resistance in pathogenic fungi

“…In addition, the loss of Grx4 would decrease the heat resistance and damage cell wall integrity in C. neoformans . At the same time, Grx4 and calcineurin signaling jointly affect the heat tolerance of C. neoformans (Hu et al 2021 ).…”