Screening for natural polymorphisms in genes involved in Candida albicans azoles and echinocandins resistance and description of new mutations for azole resistance in clinical isolates

International audienceBackground: Azoles and echinocandins are the 2 main classes of antifungal agents used to treat candidiasis. Resistance of Candida albicans isolates is a major cause of breakthrough infections. C. albicans is a diploid yeast which displays a high genetic polymorphism. Point mutations found in genes involved in the resistance of C. albicans to antifungals could either be natural polymorphisms or can confer phenotypic resistance. Objective: Our objective was to establish a comprehensive repertoire of the non-synonymous polymorphisms (natural polymorphisms and/ or mutations of resistance) in genes involved in resistance to azoles and echinocandins. Methods: Two collections of C. albicans clinical isolates were used. The first one consists of 151 epidemiologically-unrelated strains susceptible to antifungal agents. The second collection consists of 24 isolates with a high level of resistance to fluconazole (MIC > 256 μg/mL, n = 21/24) and/or caspofungin (n = 5/24). The resistant isolates were obtained sequentially over a period of up to 8 years from 7 Chronic Mucocutaneous Candidiasis patients. The whole genome sequencing of these 2 strain collections was performed on an Illumina HiSeq 2000, generating 100 bp reads (roughly 100× coverage on average). The reads were mapped to the SC5314 reference genome (Assembly 22) using the BWA alignment tool. Then SNPs were detected by GATK and selected with the recommended filters. Using homemade scripts we analyzed the sequences of 5 genes involved in the resistance to azoles (ERG11, TAC1, MRR1 and UPC2) and echinocandins (FKS1) and compared them to the sequences of reference strain SC5314. Results: Among the 151 antifungal susceptible strains we identified 126 distinct natural amino acid substitutions, including 38 substitutions in Tac1p, 20 in Erg11p, 15 in Upc2p, 33 in Mrr1p and 20 in Fks1p. Novel substitutions were found; respectively 37% (14/38), 50% (10/20), 67% (10/15) and 73% (24/33). Whereas, from the 24 resistant strains we identified 20 amino acid substitutions, in addition to the above-mentioned natural polymorphisms, affecting Erg11p (n = 9), Tac1p (n = 7), Upc2p (n = 3) and Fks1p (n = 1). From the latter, 9 of these mutations have already been associated to azoles or echinocandins resistance. However, the remaining 11 substitutions are novel putative azoles resistance mutations. To confirm that these mutations are indeed responsible for the resistance phenotype, direct mutagenesis experiments are in progress. Conclusion: Whole genome sequencing of numerous antifungal susceptible C. albicans strains allowed us to determine a repertoire of the natural polymorphisms within the main genes involved in the resistance to azoles and echinocandins. Using this repertoire as a guide, 11 new putative mutations conferring resistance where identified. Thus this repertoire can serve as a major tool helping to rapidly unveil mutations potentially linked to antifungal resistance

Large-scale genome mining allows identification of neutral polymorphisms and novel resistance mutations in genes involved in Candida albicans resistance to azoles and echinocandins.

The genome of Candida albicans displays significant polymorphism. Point mutations in genes involved in resistance to antifungals may either confer phenotypic resistance or be devoid of phenotypic consequences. To catalogue polymorphisms in azole and echinocandin resistance genes occurring in susceptible strains in order to rapidly pinpoint relevant mutations in resistant strains. Genome sequences from 151 unrelated C. albicans strains susceptible to fluconazole and caspofungin were used to create a catalogue of non-synonymous polymorphisms in genes involved in resistance to azoles (ERG11, TAC1, MRR1 and UPC2) or echinocandins (FKS1). The potential of this catalogue to reveal putative resistance mutations was tested in 10 azole-resistant isolates, including 1 intermediate to caspofungin. Selected mutations were analysed by mutagenesis experiments or mutational prediction effect. In the susceptible strains, we identified 126 amino acid substitutions constituting the catalogue of phenotypically neutral polymorphisms. By excluding these neutral substitutions, we identified 22 additional substitutions in the 10 resistant strains. Among these substitutions, 10 had already been associated with resistance. The remaining 12 were in Tac1p (n = 6), Upc2p (n = 2) and Erg11p (n = 4). Four out of the six homozygous substitutions in Tac1p (H263Y, A790V, H839Y and P971S) conferred increases in azole MICs, while no effects were observed for those in Upc2p. Additionally, two homozygous substitutions (Y64H and P236S) had a predicted conformation effect on Erg11p. By establishing a catalogue of neutral polymorphisms occurring in genes involved in resistance to antifungal drugs, we provide a useful resource for rapid identification of mutations possibly responsible for phenotypic resistance in C. albicans

Génomique des populations des bactéries du complexe <em>Borrelia burgdorferi sl</em>

National audienc

The intraspecies diversity of C. albicans triggers qualitatively and temporally distinct host responses that determine the balance between commensalism and pathogenicity

The host immune status is critical for preventing opportunistic infections with Candida albicans. Whether the natural fungal diversity that exists between C. albicans isolates also influences disease development remains unclear. Here, we used an experimental model of oral infection to probe the host response to diverse C. albicans isolates in vivo and found dramatic differences in their ability to persist in the oral mucosa, which inversely correlated with the degree and kinetics of immune activation in the host. Strikingly, the requirement of interleukin (IL)-17 signaling for fungal control was conserved between isolates, including isolates with delayed induction of IL-17. This underscores the relevance of IL-17 immunity in mucosal defense against C. albicans. In contrast, the accumulation of neutrophils and induction of inflammation in the infected tissue was strictly strain dependent. The dichotomy of the inflammatory neutrophil response was linked to the capacity of fungal strains to cause cellular damage and release of alarmins from the epithelium. The epithelium thus translates differences in the fungus into qualitatively distinct host responses. Altogether, this study provides a comprehensive understanding of the antifungal response in the oral mucosa and demonstrates the relevance of evaluating intraspecies differences for the outcome of fungal–host interactions in vivo