PKC1 Is Essential for Protection against both Oxidative and Nitrosative Stresses, Cell Integrity, and Normal Manifestation of Virulence Factors in the Pathogenic Fungus Cryptococcus neoformans

Abstract

Cell wall integrity is crucial for fungal growth, survival, and pathogenesis. Responses to environmental stresses are mediated by the highly conserved Pkc1 protein and its downstream components. In this study, we demonstrate that both oxidative and nitrosative stresses activate the PKC1 cell integrity pathway in wild-type cells, as measured by phosphorylation of Mpk1, the terminal protein in the PKC1 phosphorylation cascade. Furthermore, deletion of PKC1 shows that this gene is essential for defense against both oxidative and nitrosative stresses; however, other genes involved directly in the PKC1 pathway are dispensable for protection against these stresses. This suggests that Pkc1 may have multiple and alternative functions other than activating the mitogen-activated protein kinase cascade from a "top-down" approach. Deletion of PKC1 also causes osmotic instability, temperature sensitivity, severe sensitivity to cell wall-inhibiting agents, and alterations in capsule and melanin. Furthermore, the vital cell wall components chitin and its deacetylated form chitosan appear to be mislocalized in a pkc1⌬ strain, although this mutant contains wild-type levels of both of these polymers. These data indicate that loss of Pkc1 has pleiotropic effects because it is central to many functions either dependent on or independent of PKC1 pathway activation. Notably, this is the first time that Pkc1 has been implicated in protection against nitrosative stress in any organism.

Regulatory Circuitry Governing Fungal Development, Drug Resistance, and Disease

“…In the absence of calcineurin, Mpk1 is able to partially protect cells from caspofungin-induced cell wall stress (285). Further studies have also shown that pkc1 mutants exhibit osmotic instability, alterations in capsule and melanin production, as well as sensitivity to temperature and cell wall-inhibiting agents (202).…”

Two Major Inositol Transporters and Their Role in Cryptococcal Virulence

“…The importance of sphingolipids for pathogenicity has also been recognized in some parasitic pathogens, including Leishmania and Trypanosoma species, and related enzymes have been proposed as potential drug targets (36,37,45,60,67). The diacyglycerol (DAG)-protein kinase C1 (Pkc1) signaling pathway is critical for virulence factor production and pathogenicity in C. neoformans (18,19,24,25). The glycophosphatidylinositol (GPI)-anchored glycolipid is derived from inositol and is necessary for pathogenicity in Candida species (61).…”

Cryptococcus neoformans Phosphoinositide-Dependent Kinase 1 (PDK1) Ortholog Is Required for Stress Tolerance and Survival in Murine Phagocytes

“…The CWI pathway is conserved across many fungi (41) and is also an important regulator of the cell wall stress response in C. neoformans (12,37,44). We therefore hypothesized that Pkc1 activity may be reduced in pkh2-02⌬ mutants compared to in the WT.…”

“…Western blot analysis was performed as described previously (37). Briefly, cultures were grown overnight in YPD or YNB at 30°C with shaking and then diluted to an OD 660 of 0.2 in 50 ml of medium.…”

“…The cell suspensions (5 l) were spotted on YPD plates containing calcofluor white (CFW) (1.5 mg/ml), caffeine (10 mM), Congo red (5 mg/ml), and rapamycin (200 ng/ml). For oxidative and nitrosative stress phenotypes (37), strains (3 l each dilution) were plated on yeast nitrogen base (YNB) (pH 4.0 with succinate) containing H 2 O 2 (1 mM), diamide (1 mM), or NaNO 2 (1.5 mM). All plates were incubated at 30°C for 3 to 7 days.…”