Characterization of the Candida glabrata trehalase enzymes

Fungal infections pose a serious threat to immunocompromised people. Both superficial and systemic fungal infections can be caused by Candida glabrata, which is an opportunistic fungal pathogen. Several virulence factors support this pathogen to attack the human host: enzyme production, adhesion and biofilm formation, stress resistance, etc. During stress resistance, trehalose plays an important role. This disaccharide helps to maintain the integrity of proteins and membrane structures during periods of stress. It is of importance that trehalose is broken down after stress removal, in order for the cells to properly recover growth. This role is fulfilled by trehalases, the enzymes responsible for trehalose hydrolysis. Even though most fungal species contain trehalase enzymes, fast extracellular trehalose hydrolysis is a feature used in hospitals to identify C. glabrata. Therefore, we reasoned that the trehalose metabolism must be regulated differently in this pathogen. We observed that the trehalase inhibitor Validamycin A could affect C. glabrata growth. To further investigate a potential role for the trehalases as antifungal drug targets, we aimed to identify and characterize the trehalases in this pathogen. Three trehalases were identified and further investigated for their role in virulence and stress resistance: Nth1, Nth2 and Ath1. The neutral trehalases Nth1 and Nth2 are paralogs of one another, most probably due to the whole genome duplication event. In our experiments, the only observed phenotype for the nth2∆ mutant strain was a reduced C. glabrata survival in human macrophages. In contrast, Nth1 seems to be of much bigger importance for C. glabrata. Nth1 is important for stress resistance and nth1∆ strains also showed a decreased survival in human macrophages. Unexpectedly, the NTH1 deletion strains were more virulent in a mouse model of systemic infection, leading to a higher mortality rate of the mice. The Nth1 enzyme is regulated both transcriptionally and posttranscriptionally. The N-terminal region, responsible for S. cerevisiae ScNth1 posttranslational regulation is conserved in C. glabrata: the region contains 4 serines surrounded by a PKA consensus motif, Bmh1(A) and Bmh1(B) binding motifs and an EF-hand like motif, possibly responsible for Ca2+ binding. In C. glabrata, Bmh1(A) and Bmh1(B) were able to bind Nth1 and also most of the phosphorylated serines are conserved. Moreover, an additional phosphorylation site was identified in Nth1. The responsible kinase remains to be investigated but PKA, Ypk1, Sch9 and Cpk10 make good candidates. Furthermore, there were indications that PP2A or calcineurin could be the phosphatase, responsible for dephosphorylation of Nth1. The acid trehalase Ath1 was not involved in stress resistance or in survival in a mouse model of systemic infection. Unexpectedly, deletion of ATH1 in C. glabrata resulted in a decreased survival in human macrophages. Furthermore, Ath1 is present in the periplasm and the vacuole but also secreted to the extracellular environment via extracellular vesicles. This localization allows the cells to hydrolyze extracellular trehalose, thereby providing surrounding cells with glucose. This thesis shows that the trehalase enzymes are important for stress resistance, survival in human macrophages and extracellular trehalose hydrolysis. Based on our observations, we propose to search for a competitive drug that binds all three trehalases and suspect that this could be beneficial in fighting C. glabrata infections.

Publication year:2022

Accessibility:Embargoed