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Project

A fluorescence-based tool for protein-protein interactions analysis and identification of novel processes of fluconazole susceptibility in Candida species

Candida species at the same time represent the predominant commensal fungi in the human gut microbiome and are among top 6 most common causes of serious fungal infections worldwide. Our research imperatives are better understanding the biology of these opportunistic pathogens that drives their dual lifestyle as well as finding effective treatments for the eradication of Candida infections.

Mapping the complex protein-protein interactions (PPI) contributes to the understanding of virulence signalling and host-pathogen interactions at the molecular level, as well as to identification of novel drug targets at the clinical level. The parasexual mating and alternative codon usage of C. albicans, complicate the fast application of standard genetic methods. Thus, in the first part of this work, we have established an optimized bimolecular fluorescence complementation (BiFC) assay, a robust method for investigation of PPI in C. albicans. We have developed a set of optimized plasmids that enables N- and C-terminal tagging of proteins of interest and the analysis of their interactions in all fusion orientations. After its validation with a proof-of-principle setup, we have applied BiFC on the cAMP-protein kinase A (PKA) pathway, one of the central signalling pathways that governs morphogenesis and virulence in C. albicans. For the first time, we have visualized the in vivo interaction of the upstream components of cAMP-PKA pathway, Gpr1-Gpa2 module, as well as the interaction of Bcy1-Tpk1 and Bcy1-Tpk2, the regulatory and catalytic components of the PKA complex. The application of BiFC for in vivo visualization and investigation of PPI shows excellent promise in elucidating novel molecular mechanisms in C. albicans.

In the second part of this work, we sought to identify novel processes involved in the fluconazole susceptibility of C. glabrata. Given the observed epidemiological shifts from more azole-susceptible C. albicans towards more azole-resistant C. glabrata infections, our research focus has been placed on the molecular mechanisms of fluconazole resistance, the most widely used azole drug. By screening a collection of C. glabrata deletion mutants, we have identified two groups of mutants that enhance and suppress the fluconazole susceptibility in C. glabrata. Phenotypic profiling of the identified mutants provides insight into novel processes involved in fluconazole susceptibility and resistance in C. glabrata.

Date:1 Oct 2012 →  19 Sep 2018
Keywords:Micorbiology
Disciplines:Biomaterials engineering, Biological system engineering, Biomechanical engineering, Other (bio)medical engineering, Environmental engineering and biotechnology, Industrial biotechnology, Other biotechnology, bio-engineering and biosystem engineering, Genetics, Systems biology, Molecular and cell biology, Plant biology
Project type:PhD project