Genetic analysis of mannosylphosphorylation and investigation of its role in interactions between Candida albicans and macrophages
This project aimed to improve the understanding of the genetic control of mannosylphosphorylation in Candida albicans, and to examine the role of mannosylphosphate in certain interactions between C. albicans and macrophages, and in the virulence of C. albicans. A putative homologue of the ScMNN4 gene, which s a mannosylphosphorylation gene in S. cerevisiae, was identified in the C. albicans genome by bioinformatic analysis, and named CaMNN4. A set of Dmnn4 C. albicans strains was constructed, using the URA-blaster methodology. The CaMNN4 gene was cloned and sequenced. A reintegration vector was constructed by ligation of the MNN4 gene into the C. albicans integrating vector, Clp10. Clp10 and the reintegration vector were used to construct a set of parental, Dmnn4 and MNN4 reintegrant strains that were isogenic for the selectable marker, URA3. Phenotypic analysis revealed that disruption of CaMNN4 results in an absence of cell wall mannosylphosphate and acid-labile N-linked mannan. Otherwise, Dmnn4 C. albicans strains were remarkably similar to parental strains. Reintegration of CaMNN4 was only partially effective in restoring CaMNN4 expression to the level of parent strains. Assays of macrophage binding and/or phagocytosis, and nitric oxide (NO) production, revealed that there was no difference in phagocytosis of Dmnn4 and control strains, or in their inhibitory effect on macrophage NO production. Virulence in a murine model of systemic candidiasis was attenuated in a Dmnn4 strain, and restored in a reintegrant strain. However, virulence was also restored in a Dmnn4 strain transformed with Clp10 alone. The results of the project provided the first steps in gaining an understanding of the genetic control of mannosylphosphorylation in C. albicans. Although the results did not confirm a role for mannosylphosphate in macrophage interactions or virulence, they provided an insight into the potential pitfalls of the identification of virulence genes by reverse-genetic techniques.