Lrg1 regulates β (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 β (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 β (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 β (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-α 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.