DNA stress induced filamentous growth in Candida albicans

Abstract

Candida albicans is the most prominent fungal pathogen in immuno-compromised patients, and systematic infections are often fatal. One of the most important features of C. albicans is its ability to switch between different morphological forms such as yeast, pseudo-hyphae, and hyphae. A diverse range of growth conditions can induce the growth switch from yeast to hyphae via several regulatory pathways, while some other conditions and perturbation of the cell cycle progression can lead to pseudohyphal growth. In this thesis, we report that a range of genotoxic insults that disturb cell cycle progression induced pseudohyphal growth of C. albicans, including the inhibition of DNA synthesis by hydroxyurea (HU) or aphidicolin (AC), the depletion of ribonucleotide reductase subunit Rnr2, and DNA damages by methylmethane sulphonate (MMS) and ultraviolet (UV). In spite of all the knowledge of filamentous growth, the pathways which are required for the pseudohyphal growth in C. albicans are still unknown. All the genotoxic insults mentioned above activate DNA replication or DNA damage checkpoint pathways. In order to test whether DNA checkpoint pathways are responsible for the genotoxic-stress-induced filamentous growth, we first characterized DNA checkpoint components in C. albicans. Based on sequence homology and shared domain organization, othologues of ScRad53, ScRad9, and ScMrc1 were found in C. albicans, and their roles in DNA replication and damage were investigated. Unlike its S. cerevisiae orthologue, CaRAD53 can be deleted without affecting cell viability. The deletion mutants grew much more slowly than wide-type cells, were unable to arrest cell cycle progression and lost viability in response to HU and MMS treatment. CaRAD9 deletion mutants were sensitive to MMS but not HU. rad9I? cells can slow down S-phase progression but cannot arrest G2/M transition under MMS treatment. Deleting CaMRC1 led to filamentous growth under normal growth conditions, slower DNA replication, HU and MMS sensitivity, and constitutive phosphorylation of CaRad53. Defects of these mutants confirm that CaRad53, CaRad9, and CaMrc1 are functional orthologues of their S. cerevisiae counterparts. By constructing mutants deleted for each of the checkpoint genes, the relationship between DNA checkpoint pathways and the genotoxic-stress-induced filamentous growth was then examined. Deleting CaRAD53 which is the downstream effector kinase for both DNA damage and replication checkpoint pathways completely abolished the filamentous growth caused by all the genotoxins used. Deleting CaRAD9 that encodes the signal transducer of the DNA damage checkpoint partially compromised the filamentous growth induced by MMS and UV but not that by HU and AC. Deleting CaMRC1, the counterpart of CaRAD9 in the DNA replication checkpoint, impaired DNA synthesis and caused cell elongation even in the absence of external genotoxic insults. Together, the results indicate that the DNA replication and DNA damage checkpoints are critically required for the genotoxic-stress-induced filamentous growth. Furthermore, mutation of amino acid, G65A or N104A, in the FHA1 domain of CaRad53 nearly completely blocked the filamentous growth but had no significant deleterious effect on cell cycle arrest. The results indicate that the FHA domain, known for its ability to bind phosphopeptides, has an important role in mediating the genotoxic-stress-induced filamentous growth. The functional separation by the FHA domain mutations of cell cycle arrest and filamentous growth suggests the possibility that the later may be one of the CaRad53-regulated cellular responses in C. albicans.