Site-directed mutagenesis of the 1,3-β glucan synthase catalytic subunit ofPneumocystis jiroveciiand susceptibility assays suggest its sensitivity to caspofungin

Abstract

AbstractThe echinocandin caspofungin inhibits the catalytic subunit Gsc1 of the enzymatic complex synthetizing 1,3-β glucan, an essential compound of the fungal wall. Studies in rodents showed that caspofungin can treatPneumocystisinfections. However, its efficacy againstPneumocystis jirovecii, the species infecting exclusively humans, remains controversial. The aim of this study was to assess the sensitivity to caspofungin of theP. jiroveciiGsc1 subunit, as well as of those ofPneumocystis cariniiandPneumocystis murinainfecting respectively rats and mice. In absence of an establishedin vitroculture method forPneumocystisspecies, we used functional complementation of theSaccharomyces cerevisiae gsc1deletant. In the fungal pathogenCandida albicans, mutations leading to amino acid substitutions in Gsc1 confer resistance to caspofungin. We introduced the corresponding mutations into thePneumocystis gsc1genes using site-directed mutagenesis. In spot dilution tests, the sensitivity to caspofungin of the complemented strains decreased with the number of mutations introduced, suggesting that the wild-type enzymes are sensitive. The minimum inhibitory concentrations of caspofungin determined by E-test®and Yeastone®for strains complemented withPneumocystisenzymes (respectively 0.125 and 0.12 microg/ml) were identical to those upon complementation with the enzyme ofC. albicansthat is sensitive to caspofungin. However, they were lower than the MICs upon complementation with the enzyme of the resistant speciesCandida parapsilosis(0.19 and 0.25). Sensitivity levels of Gsc1 enzymes of the threePneumocystisspecies were similar. Our results suggest thatP. jiroveciiis sensitive to caspofungin during infections, asP. cariniiandP. murina.