New Mechanisms of Flucytosine Resistance in C. glabrata Unveiled by a Chemogenomics Analysis in S. cerevisiae

Abstract

5-Flucytosine is currently used as an antifungal drug in combination therapy, but fungal pathogens are rapidly able to develop resistance against this drug, compromising its therapeutic action. The understanding of the underlying resistance mechanisms is crucial to deal with this problem. In this work, the S. cerevisiae deletion mutant collection was screened for increased resistance to flucytosine. Through this chemogenomics analysis, 183 genes were found to confer resistance to this antifungal agent. Consistent with its known effect in DNA, RNA and protein synthesis, the most significant Gene Ontology terms over-represented in the list of 5-flucytosine resistance determinants are related to DNA repair, RNA and protein metabolism. Additional functional classes include carbohydrate and nitrogen—particularly arginine—metabolism, lipid metabolism and cell wall remodeling. Based on the results obtained for S. cerevisiae as a model system, further studies were conducted in the pathogenic yeast Candida glabrata. Arginine supplementation was found to relieve the inhibitory effect exerted by 5-flucytosine in C. glabrata. Lyticase susceptibility was found to increase within the first 30min of 5-flucytosine exposure, suggesting this antifungal drug to act as a cell wall damaging agent. Upon exponential growth resumption in the presence of 5-flucytosine, the cell wall exhibited higher resistance to lyticase, suggesting that cell wall remodeling occurs in response to 5-flucytosine. Additionally, the aquaglyceroporin encoding genes CgFPS1 and CgFPS2, from C. glabrata, were identified as determinants of 5-flucytosine resistance. CgFPS1 and CgFPS2 were found to mediate 5-flucytosine resistance, by decreasing 5-flucytosine accumulation in C. glabrata cells.

Membrane Proteomics Analysis of the Candida glabrata Response to 5-Flucytosine: Unveiling the Role and Regulation of the Drug Efflux Transporters CgFlr1 and CgFlr2

“…Indeed, Liu et al found an up-regulation of several genes involved in RNA metabolism and translation (Liu et al, 2005), while Zhang and co-workers showed a down regulation of a few ribosomal protein encoding genes in these conditions (Zhang et al, 2002). It is also in agreement with a previous chemogenomic analysis of the determinants of 5-FC resistance in the model yeast S. cerevisiae , in which about one fourth of the determinants of resistance to this drug were found to be related to RNA and protein metabolism (Costa et al, 2015). It appears that C. glabrata cells try to compensate, with the increased expression of translation associated proteins, the detrimental effect that 5-FC exerts in this process.…”

“…These two transporters constitute, thus, two additional players in the antifungal drug resistance phenomenon. Our group had previously shown that the acquaglyceroporins CgFps1 and CgFps2 (Costa et al, 2015), as well as the DHA transporters CgAqr1 (Costa et al, 2013a) and CgTpo1_1 and CgTpo1_2 (Pais et al, 2016) are determinants of flucytosine resistance as well, suggesting that 5-FC extrusion is an important mechanism of resistance against this antifungal drug and showing this phenomenon to be the consequence of the additive contribution of several players.…”

“…Some epidemiological studies suggest, however, that resistance mechanisms, independent of the Fcy2-Fcy1-Fur1 pathway, may play an important role in this phenomenon (Zhang et al, 2002). For example, reduction of 5-FC intracellular accumulation, mediated by the Drug:H+ Antiporter (DHA) CgAqr1 (Costa et al, 2013a), or by the acquaglyceroporins CgFps1 and CgFps2 (Costa et al, 2015) were recently registered.…”

Evolutionary Emergence of Drug Resistance in Candida Opportunistic Pathogens

“…Lower abundance of the enzyme has been observed in polyene-resistant species, which has been attributed to mutations in ERG2 [ 61 , 112 ], ERG3 [ 51 ], ERG5 [ 60 ], ERG6 [ 113 ], and ERG11 [ 60 ] genes, which encode enzymes involved in ergosterol synthesis. Decreased susceptibility towards flucytosine has been associated with point mutations in FCY1 , FCY2 , and FUR1 genes and the deletion of FPS1 and FPS2 genes [ 36 , 114 , 115 , 116 , 117 ]. Changes in FCY2 interfere with the drug uptake and alterations in FCY1 and FUR1 inactivate enzymes involved in the pyrimidine pathway, while the absence of FPS1 and FPS2 reduced the accumulation of the drug in the cell.…”

An Overview on Conventional and Non-Conventional Therapeutic Approaches for the Treatment of Candidiasis and Underlying Resistance Mechanisms in Clinical Strains

“…Up to now, increased activity of drug-efflux pumps has not been identified as a relevant mechanism by which clinical isolates acquire resistance to echinocandins. Concerning flucytosine, resistance in clinical isolates has been linked to modifications on the coding sequence of the Fcy cytosine permease or in the uracil phosphotransferase Fur1 (Table 4 and Figure 1) [105][106][107][108][109][110]. Resistance of some C. tropicalis isolates was linked to the emergence of mutations in CtURA3 gene, encoding the enzyme involved in the metabolization of UMP, the natural substrate of thymidylate synthase (Figure 1) [108,109].…”