Two Rac paralogs regulate polarized growth in the human fungal pathogen Cryptococcus neoformans

Abstract

A genome wide analysis of the human fungal pathogen Cryptococcus neoformans var. grubii has revealed a number of duplications of highly conserved genes involved in morphogenesis. Previously, we reported that duplicate Cdc42 paralogs provide C. neoformans with niche-specific responses to environmental stresses: Cdc42 is required for thermotolerance, while Cdc420 supports the formation of titan cells. The related Rho-GTPase Rac1 has been shown in C. neoformans var. neoformans to play a major role in filamentation and to share Cdc42/Cdc420 binding partners. Here we report the characterization of a second Rac paralog in C. neoformans, Rac2, and describe its overlapping function with the previously described CnRac, Rac1. Further, we demonstrate that the Rac paralogs play a primary role in polarized growth via the organization of reactive oxygen species and play only a minor role in the organization of actin. Finally, we provide preliminary evidence that pharmacological inhibitors of Rac activity and actin stability have synergistic activity.

Restricted Substrate Specificity for the Geranylgeranyltransferase-I Enzyme in Cryptococcus neoformans: Implications for Virulence

“…Temperature sensitivity often results from dysregulation of the cell cycle at high temperature, leading to specific morphological defects in C. neoformans cells (22,(40)(41)(42). Consistent with this hypothesis, there were notable morphological changes in the cdc43⌬ cells compared to wild-type cells when they were incubated at 37°C.…”

“…In C. neoformans, Ras1 also terminates in a leucine, designating it as a potential Ggtase-I substrate, even though Ras proteins are generally farnesylated in other eukaryotes (19). These three GTPases are required for mating, morphology, and high-temperature growth in C. neoformans (19,21,22,41). Since Ras, Rac, and Cdc42 proteins in C. neoformans regulate different aspects of morphogenesis, mating, and stress responses, elucidating their roles in the cdc43⌬ mutant phenotype could potentially provide insight into which of these proteins require geranylgeranylation for their full activity.…”

“…For example, the duplicate C. neoformans Cdc42 and Cdc420 paralogs help to direct septin protein localization and cytokinesis (21). The Rac proteins, Rac1 and Rac2, play a more specialized role in cell polarity, reactive oxygen species localization, and endocytic vesicular trafficking (22)(23)(24). C. neoformans Rho proteins are important for cell wall synthesis and integrity (25).…”

Fungal Morphogenesis

“…For example, the CnRac proteins are required for the highly polar state of filamentous growth. Deletion of either the RAC1 or RAC2 gene results in the production of dysmorphic hyphae during mating (Ballou et al 2013b). Moreover, overexpression of RAC genes complements the ras1 mutant defect in mating hyphal development (Ballou et al 2013a).…”

“…Also, CnRac proteins are required for intracellular trafficking events, which are also important in the polarization process before cell division (Shen et al 2012). The double rac1/rac2 mutation appears to be synthetically lethal, suggesting a central role for Rac proteins in cell division and basic growth (Ballou et al 2013b).…”