Abstract
The HOG (High Osmolarity Glycerol response) pathway plays a central role in controlling stress response, ergosterol biosynthesis, virulence factor production, and differentiation of Cryptococcus neoformans, which causes fatal fungal meningoencephalitis. Recent transcriptome analysis of the HOG pathway discovered a Hog1-regulated gene (CNAG_00130.2), encoding a putative protein kinase orthologous to Rck1/2 in Saccharomyces cerevisiae and Srk1 in Schizosaccharomyces pombe. Its function is not known in C. neoformans. The present study functionally characterized the role of Hrk1 in C. neoformans. Northern blot analysis confirmed that HRK1 expression depends on the Hog1 MAPK. Similar to the hog1Δ mutant, the hrk1Δ mutant exhibited almost complete resistance to fludioxonil, which triggers glycerol biosynthesis via the HOG pathway. Supporting this, the hrk1Δ mutant showed reduced intracellular glycerol accumulation and swollen cell morphology in response to fludioxonil, further suggesting that Hrk1 works downstream of the HOG pathway. However, Hrk1 also appeared to have Hog1-independent functions. Mutation of HRK1 not only further increased osmosensitivity of the hog1Δ mutant, but also suppressed increased azole-resistance of the hog1Δ mutant in an Erg11-independent manner. Furthermore, unlike the hog1Δ mutant, Hrk1 was not involved in capsule biosynthesis. Hrk1 was slightly involved in melanin production but dispensable for virulence of C. neoformans. These findings suggest that Hrk1 plays both Hog1-dependent and –independent roles in stress and antifungal drug susceptibility and virulence factor production in C. neoformans. Particularly, the finding that inhibition of Hrk1 substantially increases azole drug susceptibility provides a novel strategy for combination antifungal therapy.
Highlights
The HOG (High Osmolarity Glycerol response) pathway plays central roles in several cellular functions in filamentous and nonfilamentous fungi, encompassing both pathogens and nonpathogens
The prior transcriptome analysis for elucidation of the downstream signaling network governed by the HOG pathway revealed that a C. neoformans gene (CNAG_00130.2), which encodes a putative member of the Ca2+/calmodulin-dependent protein kinase superfamily, is significantly downregulated even under unstressed condition in both ssk1D and hog1D mutants, but not in the skn7D mutant [9] (Fig. 1A)
C. neoformans Hog1-regulated kinase 1 (Hrk1) is highly homologous to S. pombe Srk1/Mkp1, S. cerevisiae Rck2, and mammalian MAPKAPK-2, all of which are known to be p38/Hog1 mitogen-activated protein kinase (MAPK)-dependent protein kinases [11,15,16,18](Fig. 1C)
Summary
The HOG (High Osmolarity Glycerol response) pathway plays central roles in several cellular functions in filamentous and nonfilamentous fungi, encompassing both pathogens and nonpathogens. This pathway is important in maintaining normal cellular homeostasis under a plethora of environmental stresses, but is required for development and differentiation of fungi (see reviews, [1,2]). Downstream of the phosphorelay system, Ssk further activates a mitogen-activated protein kinase (MAPK) module, which comprises a Hog MAPK, Pbs MAPK kinase (MAPKK), and Ssk MAPKK kinase (MAPKKK) This signaling cascade controls cellular responses against a variety of environmental cues, including osmotic shock, high temperature, oxidative stress, UV irradiation, heavy metal, antifungal drugs, and toxic metabolites [2,5,6,7,8,9]. The Skn response regulator is involved in Na+ stress response, oxidative stress response, and antifungal drug resistance, but mostly in a Hog1-independent manner [6]
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