Lrg1 regulates ß (1,3)-glucan masking in Candida albicans through the Cek1 MAP kinase pathway

Candida albicans is among the most prevalent opportunistic human fungal pathogens. The ability to mask the immunogenic polysaccharide ß (1,3)-glucan from immune detection via a layer of mannosylated proteins is a key virulence factor of C. albicans. We previously reported that hyperactivation of the Cek1 MAP kinase pathway promotes ß (1,3)-glucan exposure. In this communication, we report a novel upstream regulator of Cek1 activation and characterize the impact of Cek1 activity on fungal virulence. Lrg1 encodes a GTPase activating protein (GAP) that has been suggested to inhibit the GTPase Rho1. We find that disruption of LRG1 causes Cek1 hyperactivation and ß (1,3)-glucan unmasking. However, when GTPase activation is measured for a panel of GTPases, the lrg1?? mutant exhibits increased activation of Cdc42 and Ras1, but not Rho1 or Rac1. Unmasking and Cek1 activation in the lrg1?? mutant can be blocked by inhibition of the Ste11 MAPKKK, indicating that lrg1?? acts through the canonical Cek1 MAP kinase cascade. In order to determine how Cek1 hyper-activation specifically impacts virulence, a doxycycline repressible hyperactive STE11?N467 mutant was generated in C. albicans. In the absence of doxycycline, this allele overexpresses STE11?N467, which induces production of pro-inflammatory TNF-a from murine macrophages. This in vitro phenotype correlates with decreased colonization and virulence in a mouse model of systemic infection. The mechanism by which Ste11?N467 causes unmasking was explored with RNA-seq. Overexpression of Ste11?N467 causes upregulation of the Cph1 transcription factor and a group of cell wall modifying proteins, which are predicted to impact cell wall architecture.