Pan-azole-resistant Meyerozyma guilliermondii clonal isolates harbouring a double F126L and L505F mutation in Erg11.

BACKGROUND: Meyerozyma guilliermondii is a yeast species responsible for invasive fungal infections. It has high minimum inhibitory concentrations (MICs) to echinocandins, the first-line treatment of candidemia. In this context, azole antifungal agents are frequently used. However, in recent years, a number of azole-resistant strains have been described. Their mechanisms of resistance are currently poorly studied. OBJECTIVE: The aim of this study was consequently to understand the mechanisms of azole resistance in several clinical isolates of M. guilliermondii. METHODS: Ten isolates of M. guilliermondii and the ATCC 6260 reference strain were studied. MICs of azoles were determined first. Whole genome sequencing of the isolates was then carried out and the mutations identified in ERG11 were expressed in a CTG clade yeast model (C. lusitaniae). RNA expression of ERG11, MDR1 and CDR1 was evaluated by quantitative PCR. A phylogenic analysis was developed and performed on M. guilliermondii isolates. Lastly, in vitro experiments on fitness cost and virulence were carried out. RESULTS: Of the ten isolates tested, three showed pan-azole resistance. A combination of F126L and L505F mutations in Erg11 was highlighted in these three isolates. Interestingly, a combination of these two mutations was necessary to confer azole resistance. An overexpression of the Cdr1 efflux pump was also evidenced in one strain. Moreover, the three pan-azole-resistant isolates were shown to be genetically related and not associated with a fitness cost or a lower virulence, suggesting a possible clonal transmission. CONCLUSION: In conclusion, this study identified an original combination of ERG11 mutations responsible for pan-azole-resistance in M. guilliermondii. Moreover, we proposed a new MLST analysis for M. guilliermondii that identified possible clonal transmission of pan-azole-resistant strains. Future studies are needed to investigate the distribution of this clone in hospital environment and should lead to the reconsideration of the treatment for this species.

Rapamycin and caspofungin show synergistic antifungal effects in caspofungin-susceptible and caspofungin-resistant Candida strains in vitro.

OBJECTIVES: Caspofungin is an echinocandin antifungal agent that inhibits synthesis of glucan required for the fungal cell wall. Resistance is mediated by mutation of Fks1 glucan synthase, among which S645P is the most common resistance-associated polymorphism. Rapamycin is a macrolide that inhibits the mechanistic target of rapamycin (mTOR) protein kinase activity. This study investigated the interaction between rapamycin and caspofungin in inhibiting the growth of WT Candida albicans and Fks1 S645P mutant clinical isolate, and WT Candida lusitaniae and genetically engineered isogenic strain with Fks1 S645P mutation at equivalent position. METHODS: Interactions between caspofungin and rapamycin were evaluated using the microdilution chequerboard method in liquid medium. The results were analysed using the Loewe additivity model (FIC index, FICI) and the Bliss independence model (response surface, RS, analysis). RESULTS: Synergy between rapamycin and caspofungin was shown for C. albicans and C. lusitaniae strains by RS analysis of the chequerboard tests. Synergy was observed in strains susceptible and resistant to caspofungin. Weak subinhibitory concentrations of rapamycin were sufficient to restore caspofungin susceptibility. CONCLUSIONS: We report here, for the first time, synergy between caspofungin and rapamycin in Candida species. Synergy was shown for strains susceptible and resistant to caspofungin. This study highlights the possible implication of the TOR pathway in sensing antifungal-mediated cell wall stress and in modulating the cellular response to echinocandins in Candida yeasts.

Challenging SNP impact on caspofungin resistance by full-length FKS1 allele replacement in Candida lusitaniae.

OBJECTIVES: A strain of the opportunistic pathogenic yeast Candida lusitaniae was genetically engineered for full-length replacement of the FKS1 gene encoding the target of echinocandin antifungals in order to assess the impact of FKS mutations on echinocandin resistance and reduced echinocandin susceptibility (RES). METHODS: FKS1 allelic exchange was achieved by transforming C. lusitaniae with two DNA fragments covering the entire FKS1 ORF. Both fragments overlap a 40 bp region where SNPs or small indels of interest were inserted. To target integration at the FKS1 locus, each DNA fragment was fused with split auxotrophic markers of which complementary truncated parts were previously inserted into the chromosomal regions flanking FKS1, allowing selection on minimal medium. RESULTS: Three SNPs described in the FKS1 hotspot (HS) regions HS1 or HS2 of clinical isolates of Candida albicans were expressed at an equivalent position in C. lusitaniae and were confirmed to confer either reduced susceptibility (F641V) or full resistance (S645P and R1361G) to caspofungin. The F659 deletion reported in an FKS2 allele of Candida glabrata and the naturally occurring P660A substitution in FKS1 of Candida parapsilosis were shown to confer a 256-fold and 6-fold increase in caspofungin MIC, respectively, when introduced into an FKS1 allele of C. lusitaniae. CONCLUSIONS: We have successfully developed a C. lusitaniae strain for the expression of full-length FKS1 alleles harbouring known mutations contributing to reduced susceptibility or resistance to caspofungin, thus opening the way for the screening of other FKS1/FKS2 mutations potentially involved in RES.

A CTG Clade Candida Yeast Genetically Engineered for the Genotype-Phenotype Characterization of Azole Antifungal Resistance in Human-Pathogenic Yeasts.

A strain of the opportunistic pathogenic yeast Candida lusitaniae was genetically modified for use as a cellular model for assessing by allele replacement the impact of lanosterol C14α-demethylase ERG11 mutations on azole resistance. Candida lusitaniae was chosen because it is susceptible to azole antifungals, it belongs to the CTG clade of yeast, which includes most of the Candida species pathogenic for humans, and it is haploid and easily amenable to genetic transformation and molecular modeling. In this work, allelic replacement is targeted at the ERG11 locus by the reconstitution of a functional auxotrophic marker in the 3' intergenic region of ERG11 Homologous and heterologous ERG11 alleles are expressed from the resident ERG11 promoter of C. lusitaniae, allowing accurate comparison of the phenotypic change in azole susceptibility. As a proof of concept, we successfully expressed in C. lusitaniae different ERG11 alleles, either bearing or not bearing mutations retrieved from a clinical context, from two phylogenetically distant yeasts, C. albicans and Kluyveromyces marxianusCandida lusitaniae constitutes a high-fidelity expression system, giving specific Erg11p-dependent fluconazole MICs very close to those observed with the ERG11 donor strain. This work led us to characterize the phenotypic effect of two kinds of mutation: mutation conferring decreased fluconazole susceptibility in a species-specific manner and mutation conferring fluconazole resistance in several yeast species. In particular, a missense mutation affecting amino acid K143 of Erg11p in Candida species, and the equivalent position K151 in K. marxianus, plays a critical role in fluconazole resistance.

Genome Sequence of the Yeast Clavispora lusitaniae Type Strain CBS 6936.

Clavispora lusitaniae, an environmental saprophytic yeast belonging to the CTG clade of Candida, can behave occasionally as an opportunistic pathogen in humans. We report here the genome sequence of the type strain CBS 6936. Comparison with sequences of strain ATCC 42720 indicates conservation of chromosomal structure but significant nucleotide divergence.

Two missense mutations, E123Q and K151E, identified in the ERG11 allele of an azole-resistant isolate of Candida kefyr recovered from a stem cell transplant patient for acute myeloid leukemia.

We report on the first cloning and nucleotide sequencing of an ERG11 allele from a clinical isolate of Candida kefyr cross-resistant to azole antifungals. It was recovered from a stem cell transplant patient, in an oncohematology unit exhibiting unexpected high prevalence of C. kefyr. Two amino acid substitutions were identified: K151E, whose role in fluconazole resistance was already demonstrated in Candida albicans, and E123Q, a new substitution never described so far in azole-resistant Candida yeast.

Deletion of the uracil permease gene confers cross-resistance to 5-fluorouracil and azoles in Candida lusitaniae and highlights antagonistic interaction between fluorinated nucleotides and fluconazole.

We characterized two additional membrane transporters (Fur4p and Dal4p) of the nucleobase cation symporter 1 (NCS1) family involved in the uptake transport of pyrimidines and related molecules in the opportunistic pathogenic yeast Candida lusitaniae. Simple and multiple null mutants were constructed by gene deletion and genetic crosses. The function of each transporter was characterized by supplementation experiments, and the kinetic parameters of the uptake transport of uracil were measured using radiolabeled substrate. Fur4p specifically transports uracil and 5-fluorouracil. Dal4p is very close to Fur4p and transports allantoin (glyoxyldiureide). Deletion of the FUR4 gene confers resistance to 5-fluorouracil as well as cross-resistance to triazoles and imidazole antifungals when they are used simultaneously with 5-fluorouracil. However, the nucleobase transporters are not involved in azole uptake. Only fluorinated pyrimidines, not pyrimidines themselves, are able to promote cross-resistance to azoles by both the salvage and the de novo pathway of pyrimidine synthesis. A reinterpretation of the data previously obtained led us to show that subinhibitory doses of 5-fluorocytosine, 5-fluorouracil, and 5-fluorouridine also were able to trigger resistance to fluconazole in susceptible wild-type strains of C. lusitaniae and of different Candida species. Our results suggest that intracellular fluorinated nucleotides play a key role in azole resistance, either by preventing azoles from targeting the lanosterol 14-alpha-demethylase or its catalytic site or by acting as a molecular switch for the triggering of efflux transport.

Cabanillasin, a new antifungal metabolite, produced by entomopathogenic Xenorhabdus cabanillasii JM26.

Since the early 1980s, fungi have emerged as a major cause of human disease. Fungal infections are associated with high levels of morbidity and mortality, and are now recognized as an important public health problem. Gram-negative bacterial strains of genus Xenorhabdus are known to form symbiotic associations with soil-dwelling nematodes of the Steinernematidae family. We describe here the discovery of a new antifungal metabolite, cabanillasin, produced by Xenorhabdus cabanillasii. We purified this molecule by cation-exchange chromatography and reverse-phase chromatography. We then determined the chemical structure of cabanillasin by homo- and heteronuclear NMR and MS-MS. Cabanillasin was found to be active against yeasts and filamentous fungi involved in opportunistic infections.

A disseminated infection with the antifungal-multiresistant teleomorphic fungus Neocosmospora vasinfecta in a patient with acute B-lymphoblastic leukemia.

We report on a fatal invasive infection due to the ascomycetous fungus Neocosmospora vasinfecta, in a 20-year-old European patient suffering from an acute lymphoblastic leukemia. The infection could not be controlled by a bitherapy combining liposomal amphotericin B and voriconazole. This is the second case of disseminated infection reported with this unusual fungus, which develops under its teleomorphic state, is fully resistant to all systemic antifungals, and which is known to live in tropical countries.

Nonsense and missense mutations in FCY2 and FCY1 genes are responsible for flucytosine resistance and flucytosine-fluconazole cross-resistance in clinical isolates of Candida lusitaniae.

The aim of this work was to elucidate the molecular mechanisms of flucytosine (5FC) resistance and 5FC/fluconazole (FLC) cross-resistance in 11 genetically and epidemiologically unrelated clinical isolates of Candida lusitaniae. We first showed that the levels of transcription of the FCY2 gene encoding purine-cytosine permease (PCP) in the isolates were similar to that in the wild-type strain, 6936. Nucleotide sequencing of the FCY2 alleles revealed that 5FC and 5FC/FLC resistance could be correlated with a cytosine-to-thymine substitution at nucleotide 505 in the fcy2 genes of seven clinical isolates, resulting in a nonsense mutation and in a putative nonfunctional truncated PCP of 168 amino acids. Reintroducing a FCY2 wild-type allele at the fcy2 locus of a ura3 auxotrophic strain derived from the clinical isolate CL38 fcy2(C505T) restored levels of susceptibility to antifungals comparable to those of the wild-type strains. In the remaining four isolates, a polymorphic nucleotide was found in FCY1 where the nucleotide substitution T26C resulted in the amino acid replacement M9T in cytosine deaminase. Introducing this mutated allele into a 5FC- and 5FC/FLC-resistant fcy1Delta strain failed to restore antifungal susceptibility, while susceptibility was obtained by introducing a wild-type FCY1 allele. We thus found a correlation between the fcy1 T26C mutation and both 5FC and 5FC/FLC resistances. We demonstrated that only two genetic events occurred in 11 unrelated clinical isolates of C. lusitaniae to support 5FC and 5FC/FLC resistance: either the nonsense mutation C505T in the fcy2 gene or the missense mutation T26C in the fcy1 gene.