Abstract

In the life cycle of the fungal pathogen Candida albicans, the formation of filamentous cells is a differentiation process that is critically involved in host tissue invasion and in adaptation to host cell and environmental stresses. Here we have used the GRACETM library to identify genes controlling invasiveness and filamentation; conditional repression of the library revealed 69 mutants that triggered these processes. Intriguingly, the genes encoding the SUMO E3 ligase Mms21 and all other tested members of the sumoylation pathway were both non-essential and capable of triggering filamentation upon repression, suggesting an important role for sumoylation in controlling filamentation in C. albicans. Both Mms21 nulls (mms21Δ/Δ) and SP domain (SUMO E3 ligase domain) deleted mutants displayed filamentation and abnormal nuclear segregation. Transcriptional analysis of mms21Δ/Δ showed an increase in expression from 2 to 8 fold above WT for hyphal specific genes (HSGs) including ECE1, PGA13, PGA26, HWP1, ALS1, ALS3, SOD4, SOD5, UME6 and HGC1. The Mms21 deleted mutants were unable to recover from DNA damaging agents like methyl methane sulfonate (MMS), hydroxyurea, hydrogen peroxide and UV radiation, suggesting that the protein is important for genotoxic stress responses.
Mating in C. albicans requires switching from mating sterile white form to mating competent opaque form and this is only achieved in mating type-like (MTL) homozygous strain. In a heterozygous MTL (MTLa/α), protein heterodimer of a1-α2 will bind and repress Wor1, a master regulator of white-opaque switching. We have screened the GRACE library for genes that can be involved in MTL a/α mating and found 10 mating positive strains but half of them had become homozygous at MTL. Among the heterozygous mating positive mutants, Mms21 was identified which upon deletion in other strain background did mate with 3315αα testers. By performing phenotypic and genetic analysis, we have found that Mms21 deleted mutants, due to increased cellular and genotoxic stress, have increased homozygosis at MTLaa followed by increased white-opaque switching and subsequent mating. This analysis provides a direction of how natural Candida MTL a/α cells inside a stressful host environment can achieve mating.
We have performed a large scale GRACE library screen for MMS sensitivity and have identified 56 genes sensitive to MMS upon tetracycline shut off. About half of those sensitive mutants were involved in DNA replication, DNA damage and cytokinesis. Among the cytokinesis sensitive mutants was Hof1. Deletion of CaHof1 renders cells sensitive to MMS and generates genome instability that partially activates checkpoint kinase CaRad53. CaRad53 down-regulates CaHof1 in response to MMS. Genetic interaction analysis shows that CaHof1 is involved in the CaRad53 pathway and epistatic to nuclear excision repair pathway. Our data suggests a novel function of Hof1 in DNA damage response.