Regulation of translation initiation during amino acid starvation and oxidative stress in Candida albicans

Candida albicans is a human commensal organism which can cause superficial, systemic and deep tissue infections in otherwise healthy individuals. It is well-known that eukaryotic cells including C. albicans regulate essential gene expression programs during stress and infection. Although several studies have investigated the transcriptional and proteomic changes during stress conditions in C. albicans, few studies have assessed the role of protein synthesis, which is the focus of this thesis. This study has examined the regulation of translation initiation mediated by the Gcn2 and Caf20 translation factors during amino acid starvation and oxidative stress conditions. Gcn2 is the sole C. albicans kinase that phosphorylates eIF2alpha, which is an essential translation initiation factor. Caf20 is a putative eIF4E-binding protein (BP) that can competitively inhibit the interaction of eIF4E with eI4G and reduces eIF4F complex formation. We show that amino acid starvation induced by 3-amino-1,2,4-triazole (3AT) and oxidative stress (H2O2, cadmium and diamide) cause an inhibition of translation initiation in C. albicans. Translation initiation is predominantly blocked via the action of GCN2 during 3AT, H2O2 and cadmium stresses, whereas, diamide inhibits initiation in a Gcn2-independent manner. During this global inhibition of protein synthesis, a major transcriptional activator, CaGcn4, is activated. We mapped the transcriptional start site of GCN4 and identified three upstream open reading frames (uORFs) present in the 5'-untranslated region (5'-UTR) of GCN4. The 5'-UTR of the GCN4 mRNA was cloned upstream of a Renilla luciferase reporter gene and used to study translational control of GCN4 under stress conditions. During amino acid starvation conditions, GCN4 is regulated both at the transcriptional and translational levels. Transcriptional regulation is mediated in a Gcn2-independent manner, whilst translational regulation is activated via Gcn2-mediated phosphorylation of eIF2alpha. A single uORF3 is shown to be necessary and sufficient for translational regulation of GCN4. This novel translational control mechanism involves leaky scanning past uORF3 under stress conditions. During hydrogen peroxide and cadmium stress conditions, GCN4 is also predominately regulated at the translational level in a Gcn2-dependent manner. Additionally Gcn4 is required for tolerance and adaptation to H2O2 stress. In contrast, Gcn4 and Gcn2 are not required during diamide stress and this correlates with a low level of translation regulation of GCN4. We discuss the similarities and differences in oxidant-specific regulation of translation initiation between S. cerevisiae and C. albicans. In the final part of the thesis, we studied the single putative eIF4E-BP identified by the C. albicans genome project, namely CAF20. CAF20 was successfully deleted in C. albicans and wild-type CAF20 and its 4E-BP mutant (CAF20m) reintegrated in the mutant. Caf20 was directly confirmed to be a 4E-BP using cap affinity chromatography. Caf20 expression was also shown to be elevated in response to an increase in growth temperature. Despite a number of experimental approaches being used, including growth analysis, polysome analysis, stress sensitivity tests and hyphal growth studies, no phenotypic differences were detected in the caf20 mutants compared with a wild-type strain. Thus, it is unclear, what if any functional roles Caf20 plays in C. albicans. We suggest that future studies could be performed to assess the levels of eIF4F complex formation, and to investigate stress sensitivity and hyphal formation during diverse stress conditions such as nutritional limitations, osmotic stress, endoplasmic stress and pH alterations.