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Role of the cAMP-PKA pathway on adhesion and pathogenicity in the human fungal pathogen Candida glabrata
Book - Dissertation
Candida glabrata is a human fungal pathogen that easily forms biofilms on medical devices, such as catheters and implants. These biofilms are difficult to treat, which often results in the necessity to physically remove the implant from the patient. Furthermore, C. glabrata is an important cause of invasive infections associated with high mortality rates, as it becomes easily resistant to the current antifungal drugs. Pathogens continuously encounter stress during the infection process. For instance, inside the human body the availability of nutrients is limited and the immune system is trying to eliminate the infection. Therefore, it is crucial for the pathogen to quickly adapt to the changing environment.C. glabrata senses extracellular glucose, fructose and sucrose which elicits a fast intracellular response through the cAMP-PKA signaling pathway. These carbohydrates are sensed by the Gpr1-Gpa2 receptor complex causing an intracellular cAMP increase. cAMP activates the protein kinase A (PKA) and this in turn affects several cellular properties among which growth, the stress resistance properties of cells, the mobilization of reserve carbohydrates and adhesion.In this thesis, we further investigated how the C. glabrata cAMP-PKA pathway affects trehalase activity and adhesion to polystyrene. Firstly, we showed that the trehalase activity of glycerol-grown wild type C. glabrata cells was very high and this was caused by the acid trehalase Ath1. A strain expressing only the neutral trehalase Nth1 showed a low basal trehalase activity and the Nth1 trehalase acitivity was activated rapidly by the cAMP-PKA pathway, similar to the situation in S. cerevisiae.Secondly, we investigated adherence as a downstream target of the cAMP-PKA pathway. Adhesion to surfaces is the first step in biofilm formation and therefore represents an important C. glabrata virulence factor. We showed that C. glabrata adhesion to polystyrene is low in presence of glucose, fructose or sucrose. We verified that this reduced adherence is caused by signaling through the cAMP-PKA pathway using different pathway mutants. Next, we investigated which adhesin(s) are downstream of the pathway by looking into the glucose-induced transcriptome of predicted adhesins. The deletion of CAGL0J00253g or AED2 resulted in strains with a similar adherence in presence of glucose and galactose, indicating that these genes are probably targets of the cAMP-PKA pathway, which needs to be further investigated. We also showed that both lab strains as well as clinical isolates have the lower adherence in presence of glucose. Therefore, further investigation of molecules activating the cAMP-PKA pathway are potentially interesting to treat C. glabrata.We also looked into the C. glabrata transcriptome after addition of glucose or galactose. Analysis of the RNA sequencing data provided a general overview of how the cells respond to the addition of these sugars: glucose induced the machinery for cell growth and fermentation, while the expression of genes involved in stress resistance was lowered, similar to the situation in S. cerevisiae. Unexpectedly, while C. glabrata is not able to use galactose, this sugar still affected the expression of a substantial number of genes among which genes involved in central metabolism.Finally, we characterized the three C. glabrata trehalases. We showed that the acid trehalase Ath1 is necessary to grow on trehalose as a carbon source and is present in the periplasm or cell wall of the cells. The neutral trehalase Nth1 is located in the cytoplasm and affects the stress properties of the cells. Finally, the trehalase deletion strains were tested in two in vivo models: in a mouse model of systemic infection, the deletion of the trehalases did not affect virulence. On the other hand, the triple trehalase deletion strain was unable to stably colonize the mouse gastrointestinal tract.