Role of trehalose biosynthesis enzymes in the virulence and stress tolerance of the human fungal pathogens Candida glabrata and Candida auris

There is an increase in infections caused by human fungal pathogens. The underlying reason is that there is an increased resistance towards the few antifungal drug classes that are used in the clinic. In addition, the advancement of medicine makes that people become older but their immune system gets weaker, which makes them vulnerable for fungal infections. Also the increased use of various implant materials causes an increase of biofilm-related infections as fungal cells attach to such implants and then produce a biofilm. Cells in such a biofilm are much more difficult to treat. The most common-isolated species is Candida albicans but there is a steady increase in the incidence of Candida glabrata infections. The reasons for this is that this species is more tolerant to various types of stress, including antifungals. Next to C. glabrata, there is a new kid on the block, which is C. auris. This species only appeared recently (first case in Japan in 2009), but it can be considered as the MRSA among fungal pathogens as it can cause hospital outbreaks.This species is often multiresistant. In our previous work we have focused a lot on trehalose metabolism and it possible role as novel antifungal drug targets. Trehalose is a disaccharide of two glucose monomers, but non-reducing and is well-known for its stress-protecting characterisics. As an example, dry yeast contains 20 % trehalose. Trehalose is produced in a two-step reaction where UDPglucose and glucose-6P are used to produce trehalose-6P (by the activity of trehalose-6P synthase, Tps1) which is then dephosphorylated to trehalose (by the activity of trehalose-6P phosphatase, Tps2). We have previously shown that the Tps2 enzyme is a very interesting antifungal drug target as its deletion prevents the production of the stress protecting molecule trehalose and results in the accumulation of Tre-6P, removing free Pi from central metabolism.Indeed, a tps2 mutant is strongly affected in its virulence (Van Dijck et al. 2002). So far, trehalose biosynthesis enzymes have not been investigated in C.glabrata, and indications are that trehalose metabolism may be very important as there even exists an assay to identify C. glabrata, using a trehalase assay. Also in C. auris, trehalose metabolism is not yet investigated.  In this project we plan to: 1. Generate deletions in the C. glabrata and C. auris TPS1 and TPS2 genes and generate reconstituted strains. We developed CRISPR-Cas9 for use in C. glabrata and we also developed strains with defect in non-homologous endjoining, enhancing the chance to identify a correct deleletion mutant (Cen et al.,2015; Cen et al., 2017). 2. Phenotypic characterization of the deletion mutants. We will determine trehalose levels, osmotic and temperature stress tolerance, antifungal drug tolerance. We will also determine virulence characteristics such as the capacity to form biofilms, adhesion and virulence assays in appropriate animal model systems 3. We will investigate the role of trehalose metabolism in the control of glycolysis and determine whether, similar to S. cerevisiae (Peeters et al., 2017 Nat Commun) there is a connection with the Ras pathway and apoptosis. 4. We will screen our inhouse compound libraries (plant essential oil collection and 360 soil microbe fermentation products) to identify compounds that may inhibit the Tps2 enzymes of both species. This could result in lead compounds for further drug development. At the end of the project we should have a clear view on the role of trehalose metabolism in both species and whether these enzymes are important for virulence. We may also already have identified molecules that would inhibit these enzymes.