Candida haemulonii echinocandin reduced susceptibility is linked with Fks1p amino acid substitutions.

DUDIUK, CATIANA; ISLA, GUILLERMINA; GAMARRA, SOLEDAD; CORDOBA, SUSANA; GARCIA, GUILLERMO

Santiago de Chile

Congreso; XIII Infocus 2016 ? Forum on Fungal Infection in the clinical practice in Latin America; 2016

Candida haemulonii is an emerging yeast pathogen that is increasingly reported as candidemia agent worldwide. This species is usually misidentified since it is phenotypically similar to C. famata and C. guilliermondii. Reports showing its reduced fluconazole and amphotericin B susceptibility were published. Taken this data into account, echinocandin drugs became the drug of choice to treat C. haemulonii infections. However, it was shown that this yeast exhibited reduced echinocandin susceptibility similar to which was observed for C. parapsilosis complex. Also, echinocandin clinical resistance was reported in pediatric patients. Echinocandin drugs interact with the Fksp subunits of the β-1,3-glucan synthase complex inhibiting the biosynthesis of the principal cell wall glucan. Both clinical resistance (in Candida spp.) and intrinsic reduced susceptibility (in C. parapsilosis complex) phenotypes were linked with hot spot substitutions in Fksp. Nevertheless, the molecular mechanism underlying C. haemulonii echinocandin reduced susceptibility phenotype is not known. The objective of this work was to study the molecular mechanism of echinocandin reduced susceptibility observed in C. haemulonii. To reach this goal, we construct a Saccharomyces cerevisiae strain with a hybrid FKS1 gene where a hot spot 1 region of its FKS1 gene was replaced with that from C. haemulonii. A clinical C. haemulonii strain (named 1290) showing elevated echinocandin MIC values (0.5 µg/ml for caspofungin and anidulafungin) was used to obtain the DNA. A partial C. haemulonii FKS1 sequence was PCR-amplified and sequenced by Sanger methodology by using FKS universal primers. This sequence was used to generate the S. cerevisiae mutant harboring a chimeric FKS1 that was obtained by a two-steps PCR-based mutagenesis. Briefly, the first step consisted in the partial deletion of the FKS1 gene of S. cerevisiae BY4742 using an URA3 cassette. The deletion includes the region from the amino acid residues 453 to 649 (includes the Fks1p hot spot 1 region). This gene disruption leads to FK506 (tacrolimus) and echinocandin hypersensitivity. The second step of the method comprised the replacement of the partially deleted S. cerevisiae FKS1 gene (fks1Δ453-649::URA3) by a gene construction that included a 695 nt. portion of the C. haemulonii FKS1 gene surrounded by S. cerevisiae FKS1 regions from nt. 1123 to 1611 and nt. 2308 to 2548 (S. cerevisiae FKS1 sequence no. U12893.1). This construction was designed to obtain, after an homologous recombination, a functional FKS1 gene encoding a chimeric Fks1p which included a C. haemulonii Fks1p region comprising the hot spot 1 region and the C- and N-terminus of the Fks1p from S. cerevisiae. Transformations were performed by a lithium acetate-based procedure. Caspofungin susceptibility testing was performed following the CLSI reference documents M27-A3/M27-S4. C. haemulonii Fks1p hot spot 1 region transcribed sequence was FMALSLRDP. It showed amino acids differences when compared with echinocandin susceptible species e.g. C. albicans 641-FLTLSLRDP. C. haemulonii 1290 showed caspofungin MIC values of 0.5 µg/ml while for the C. albicans ATCC 90028 the caspofungin MIC was 0.06 µg/ml. The chimeric S. cerevisiae strain showed 32?fold increase in caspofungin MIC values when compared with its parental strain S. cerevisiae BY4742 (0.25 µg/ml and 0.007 µg/ml, respectively). We can conclude that the described Fks1p´s hot spot 1 amino acid substitutions in C. haemulonii (FMALSLRDP vs. C. albicans 641-FLTLSLRDP) are necessary and sufficient to explain the reduced echinocandin susceptibility observed in C. haemulonii.