FKS1 mutation associated with decreased echinocandin susceptibility of Aspergillus fumigatus following anidulafungin exposure

Invasive aspergillosis (IA) is a potentially lethal infection that affects mostly immunocompromised patients caused by Aspergillus fumigatus. Echinocandins are a second-line therapy against IA, used as a salvage therapy as well as for empirical or prophylactic therapy. Although they cause lysis of growing hyphal tips, they are considered fungistatic against molds. In vivo echinocandins resistance is uncommon; however, its wide clinical use could shortly lead to the emergence of A. fumigatus resistance. The aims of the present work was to assess the development of reduced echinocandins susceptibility phenotype by a A. fumigatus strain and to unveil the molecular mechanism underlying such phenotype. We induced in vitro cross-resistance to echinocandins following exposure of A. fumigatus to anidulafungin. Stability of the resistant phenotype was confirmed after removal of anidulafungin pressure. The FKS1 gene was partially sequenced and a E671Q mutation was found. A computational approach suggests that it can play an important role in echinocandin resistance. Given the emerging importance of this mechanism for clinical resistance in pathogenic fungi, it would be prudent to be alert to the potential evolution of this resistant mechanism in Aspergillus spp infecting patients under echinocandins therapeutics.


Results and discussion
Dynamics of in vitro acquisition of resistance by A. fumigatus exposed to AFG is detailed in Table 1. After 30 days of exposure, resistance to AFG and cross-resistance to caspofungin (CAS) and micafungin (MFG) was developed. Exposure to AFG triggered macroscopic modification of morphology of A. fumigatus colonies, changing from the original green blue color to white ( Fig. 1), becoming notably smaller. Microscopy showed absence of conidiation (data not shown). AF R0 and AF R1 showed the same macroscopic and microscopic phenotype. Similar changes have been reported in A. fumigatus exposed to antifungals during long periods [8][9][10] . RAPD analysis exhibits high discriminatory power for analysis of A. fumigatus strains when using this set of primers 11 . RAPD patterns obtained were 100% identical for the three strains (Fig. 2). A point mutation was found in AF R0 corresponding to replacement of glutamine by glutamate at position 671 of Fks1p (E671Q); similar mutation was found in AF R1 . Since the resistant phenotype emerged abruptly, remaining stable following antifungal removal, it is highly plausible that this hot spot FKS1 mutation E671Q might be responsible for the reduced susceptibility of AF R0 and AF R1 . A mutation in A. fumigatus FKS1 gene with potential to reduce echinocandin susceptibility is hereby described. Such mutation was never reported among Candida spp. An S678P amino acid change, equivalent to a mutation found in a resistant Candida isolate was described in a laboratory mutant of A. fumigatus and associated with resistance to CAS 12,13 . A mutation resulting in a F675S amino acid change was found in a chronic pulmonary aspergillosis isolate from a patient in whom micafungin treatment failed 10 . Point mutations in FKS1 genes are the main mechanism that is implicated in decreased echinocandin susceptibility, however, Arendrup and colleagues found no mutations in FKS1 gene in two clinical isolates of A. fumigatus with MIC > 32 µg/mL to CAS 14 . Instead, an increased in FKS1 gene expression was observed 14 . This mechanism may be implicated in tolerance to echinocandin therapy. The E671Q mutation replaces an amino acid with a negatively charged side chain (glutamate) by an amino acid with a polar but uncharged side chain (glutamine). This position is conserved among several fungi (Fig. 3a). PROVEAN software considers an amino acid alteration at this position deleterious, suggesting that this region might have a relevant functional and/or structural role 15 . According to the three-dimensional (3D) structure obtained, E671 establishes polar contacts with K668 and T677 amino acids. Substitution by a glutamine would disrupt two of the three contacts with T677, which could distort conformation of the protein (Fig. 3b), given the proximity to a transmembrane domain (amino acids 679-699 in S. cerevisiae). Therefore, E671 may be www.nature.com/scientificreports/ necessary to maintain protein's three-dimensional structure, supporting the assumption that such substitution could impair its function. Ultimately, two approaches might be taken: FKS1 gene deletion in resistant strain to determine whether reversion to the susceptible phenotype occurs and site-directed mutagenesis in wild-type strain to observe whether resistant phenotype arises. Nevertheless, other mechanisms might also be involved in the development of echinocandin resistance, such as remodeling of cell wall components namely chitin levels, production of reactive oxygen species, alteration of the composition of plasma membrane lipids or expression levels of echinocandin target enzyme genes 16,17 .
Our results suggest that modification of Fks1p in A. fumigatus might confer echinocandins resistance. Given the emerging importance of clinical resistance among pathogenic fungi, it would be advisable to monitor the potential evolution of this mechanism in Aspergillus isolates from patients under echinocandin therapy.

Material and methods
A suspension of 5 × 10 4 conidia/mL of A. fumigatus (clinical brochoalveolar lavage isolate) was prepared in YEPD broth (0,3% yeast extract, 1% peptone, 2% dextrose) supplemented with sub-Minimal Effective Concentration (sub-MEC) (0.06 µg/mL) of AFG (Pfizer, Inc.) and incubated overnight, at 35 °C, 180 rpm. One mL was daily transferred to fresh YEPD supplemented with AFG. In parallel, 1 mL aliquot of was frozen (− 80 °C) but also cultured on YEPD agar at 35 °C for 72 h, to confirm viability and purity of culture. AFG concentration was increased to double whenever fungal growth was prominent, reaching a final concentration of 8 µg/mL. In vitro induction was carried out up to 30 days. Every 5 days, MEC values of the 3 echinocandin were determined according to CLSI 18 . A MEC value ≥ 1 µg/mL was considered resistance 19 . In order to assess the stability of echinocandin MEC values increments, the induced strain was daily sub-cultured for an additional 30 days in the absence of antifungal and MEC values re-determined. The resistant pattern remained stable. At the end of the assay, three strains were characterized: the initial susceptible strain (AF S ), the induced strain (AF R0 ) and the strain obtained following additional 30 days without antifungal exposure (AF R1 ). Genotyping by random amplification of polymorphic DNA (RAPD) of strains AF S, AF R0, AF R1 using primers R108 (5′-GTA TTG CCCT-3′), OPAX (5′-AGT GCA CACC-3′), OPQ6 (5′-GAG CGC CTTG-3′) and Primer 2 (5′-GCT GGT GG-3′) was performed 11 .
Following PCR with primers 5-GCT GAA GGA TGT CGT CTG GA and 5-CGG CAA GTG ATG GTC TCG TG, hot spot regions (between 1,875 and 4,318 bp) of FKS1 gene (GenBank accession no. AFU79728) from AF S , AF R0 and AF R1 strains were amplified and sequenced were sequenced by Sanger method. The sequences were analyzed using BLAST Sequence Analysis Tool of NCBI.
The three-dimensional model for the Fks1 protein structure was obtained by modeling using the I-TASSER online server as previously described 20,21 . Structures were visualized in PYMOL v1.1r1. In the predicted structural model for this domain of the Fks1 protein (amino acids 400-900), the E671Q substitution would result in the loss of polar contacts with T677, disrupting the contacts between α-helices. AF S , the initial susceptible strain is shown in green, and AF R0 , the resistant induced strain is shown in blue.