Objective

Candida glabrata is the second most prevalent cause of candidemia worldwide, which is largely due to its high intrinsic and rapidly acquired resistance to azole antifungals. Phylogenetically, C. glabrata is more closely related to Saccharomyces cerevisiae than to the most commonly isolated Candida species, C. albicans. Recent evolutionary studies show that the gene CgMIP1 may have been under positive selection during the evolution of C. glabrata as a human pathogen. The ortholog MIP1 in S. cerevisiae encodes a mitochondrial polymerase, and its deletion leads to defects in mitochondrial functions and respiratory deficiency, as described in petite mutants. In yeast, petite mutants show high resistance to fluconazole due to the upregulation of the transcription factor Pdr1 and the efflux pumps Cdr1 and Cdr2. The aim of this study is to elucidate whether the evident past selection pressure on MIP1 is due to its involvement in the adaptation of C. glabrata to the human host.

Methods

We created a Cgmip1∆ mutant and analyzed its growth under different stress conditions similar to those that the yeast might cope within the human host, such as oxidative, osmotic, endoplasmic reticulum or cell wall stress. We also performed an in vitro phagocytosis survival assay exposing the wild type and mutant strains to human macrophages. Finally we implemented a laboratory evolution experiment of C. glabrata under high fluconazole concentrations to analyze the role of CgMIP1 in the emergence of petite mutants as a possible first stage for adaptation to the drug.

Results

We found that Cgmip1∆ mutant shows a petite phenotype, it is surprisingly resistant to endoplasmic reticulum stresses, and it exhibits an unexpected increased survival rate upon phagocytosis by human macrophages. A possible explanation of the stress resistance phenotype was found in form of an upregulation of the genes PDR1, CDR1 and CDR2. In addition, the laboratory evolution experiment showed stages of C. glabrata adaptation to the presence of the drug: Petite mutants emerged only at the beginning of the adaptation process and were lost at a later time point.

Conclusion

These results show that the loss of MIP1 activity can be beneficial for C. glabrata under certain stress conditions. We thus hypothesize that CgMIP1 may have evolved to be more prone to lose its activity under high stress conditions, which would trigger a temporary decline of mitochondria and the emergence of a petite phenotype, helping in survival under high stress conditions like within the host. This would allow time for the opportunistic pathogen to acquire secondary mutations, such as gain-of-function mutations in PDR1. The mechanism of the presumed temporary loss of CgMIP1 function, genetic mutations or epigenetic silencing, is now under investigation.

4 / Studying the role of CgMIP1 in the evolution of Candida glabrata during adaption to the human host