Unraveling the veiled function of mitogen-activated protein kinases Cpk2 and Mpk2 in Cryptococcus neoformans

In basidiomycetous human fungal pathogen, Cryptococcus neoformans, has five mitogen-activated protein kinases (MAPKs), of which Mpk1, Cpk1, and Hog1 play central roles in various physiological functions. Apart from these three major MAPKs, C. neoformans has Cpk2 and Mpk2, which are paralogs of Cpk1 and Mpk1, respectively, but their roles remain elusive. Our previous genome-wide functional analysis of kinases revealed that Cpk2 plays minor roles in osmotic and genotoxic stress response and melanin production but is dispensable for the mating process, unlike Cpk1. Unlike Mpk1, Mpk2 plays minor roles in cell membrane stress response, resistance to fludioxonil and fluconazole, and melanin as well as urease production. This study aimed to elucidate the functional connection of Cpk2 and Mpk2 to Cpk1- and Mpk1-dependent signaling pathways in C. neoformans. In support of their phylogenetic relationship, here we provide the following experimental evidence showing that Cpk2 and Mpk2 play redundant roles with Cpk1 and Mpk1, respectively. Overexpression of CPK2 could almost completely restore the mating defect of cpk1Δ, including mating pheromone production, filamentation, and sporulation. Cpk2 was shown to regulate the expression of Mat2, which is known as a downstream transcription factor of the Cpk1 mating pathway. Then, overexpression of MPK2 could also partially restore the cell wall integrity of mpk1Δ under cell wall destabilizing conditions. The fungal cell wall component, chitin, is reduced in mpk1Δ and more decreased in mpk1Δ mpk2Δ and overexpression of MPK2 can restore it. Surprisingly, mpk1Δ cpk1Δ mpk2Δ displayed complete restoration of sexual development including pheromone production to sporulation.