Critical Assessment of Cell Wall Integrity Factors Contributing to in vivo Echinocandin Tolerance and Resistance in Candida glabrata.

Fungal infections are on the rise, and emergence of drug-resistant Candida strains refractory to treatment is particularly alarming. Resistance to azole class antifungals, which have been extensively used worldwide for several decades, is so high in several prevalent fungal pathogens, that another drug class, the echinocandins, is now recommended as a first line antifungal treatment. However, resistance to echinocandins is also prominent, particularly in certain species, such as Candida glabrata. The echinocandins target 1,3-ß-glucan synthase (GS), the enzyme responsible for producing 1,3-ß-glucans, a major component of the fungal cell wall. Although echinocandins are considered fungicidal, C. glabrata exhibits echinocandin tolerance both in vitro and in vivo, where a subset of the cells survives and facilitates the emergence of echinocandin-resistant mutants, which are responsible for clinical failure. Despite this critical role of echinocandin tolerance, its mechanisms are still not well understood. Additionally, most studies of tolerance are conducted in vitro and are thus not able to recapitulate the fungal-host interaction. In this study, we focused on the role of cell wall integrity factors in echinocandin tolerance in C. glabrata. We identified three genes involved in the maintenance of cell wall integrity - YPS1, YPK2, and SLT2 - that promote echinocandin tolerance both in vitro and in a mouse model of gastrointestinal (GI) colonization. In particular, we show that mice colonized with strains carrying deletions of these genes were more effectively sterilized by daily caspofungin treatment relative to mice colonized with the wild-type parental strain. Furthermore, consistent with a role of tolerant cells serving as a reservoir for generating resistant mutations, a reduction in tolerance was associated with a reduction in the emergence of resistant strains. Finally, reduced susceptibility in these strains was due both to the well described FKS-dependent mechanisms and as yet unknown, FKS-independent mechanisms. Together, these results shed light on the importance of cell wall integrity maintenance in echinocandin tolerance and emergence of resistance and lay the foundation for future studies of the factors described herein.

Variants of the human RAD52 gene confer defects in ionizing radiation resistance and homologous recombination repair in budding yeast.

RAD52 is a structurally and functionally conserved component of the DNA double-strand break (DSB) repair apparatus from budding yeast to humans. We recently showed that expressing the human gene, HsRAD52 in rad52 mutant budding yeast cells can suppress both their ionizing radiation (IR) sensitivity and homologous recombination repair (HRR) defects. Intriguingly, we observed that HsRAD52 supports DSB repair by a mechanism of HRR that conserves genome structure and is independent of the canonical HR machinery. In this study we report that naturally occurring variants of HsRAD52, one of which suppresses the pathogenicity of BRCA2 mutations, were unable to suppress the IR sensitivity and HRR defects of rad52 mutant yeast cells, but fully suppressed a defect in DSB repair by single-strand annealing (SSA). This failure to suppress both IR sensitivity and the HRR defect correlated with an inability of HsRAD52 protein to associate with and drive an interaction between genomic sequences during DSB repair by HRR. These results suggest that HsRAD52 supports multiple, distinct DSB repair apparatuses in budding yeast cells and help further define its mechanism of action in HRR. They also imply that disruption of HsRAD52-dependent HRR in BRCA2-defective human cells may contribute to protection against tumorigenesis and provide a target for killing BRCA2-defective cancers.

Homologous recombination in budding yeast expressing the human RAD52 gene reveals a Rad51-independent mechanism of conservative double-strand break repair.

RAD52 is a homologous recombination (HR) protein that is conserved from bacteriophage to humans. Simultaneously attenuating expression of both the RAD52 gene, and the HR and tumor suppressor gene, BRCA2, in human cells synergistically reduces HR - indicating that RAD52 and BRCA2 control independent mechanisms of HR. We have expressed the human RAD52 gene (HsRAD52) in budding yeast strains lacking the endogenous RAD52 gene and found that HsRAD52 supports repair of DNA double-strand breaks (DSB) by a mechanism of HR that conserves genome structure. Importantly, this mechanism of HR is independent of RAD51, which encodes the central strand exchange protein in yeast required for conservative HR. In contrast, BRCA2 exerts its effect on HR in human cells together with HsRAD51, potentially explaining the synergistic effect of attenuating the expression of both HsRAD52 and BRCA2. This suggests that multiple mechanisms of conservative DSB repair may contribute to tumor suppression in human cells.