Identification and Functional Ascertainment of the Pneumocystis jirovecii Potential Drug Targets Gsc1 and Kre6 Involved in Glucan Synthesis

Abstract

The most efficient drug against the human pathogenic fungus Pneumocystis jirovecii is cotrimoxazole targeting the folate biosynthesis. However, resistance toward it is emerging and adverse effects occur in some patients. Studies in rodent models suggested that echinocandins could be useful to treat Pneumocystis pneumonia. Echinocandins inhibit the catalytic subunit Gsc1 of the enzymatic complex ensuring the synthesis of 1,3-β glucan, an essential constituent of cell walls of most fungi. Besides, inhibitors of the enzyme Kre6 involved in the synthesis of 1,6-β glucan, another essential component of fungal walls, were recently described. We identified and functionally characterized these two potential drug targets in the human pathogen P. jirovecii by rescue of the null allele of the orthologous gene in Saccharomyces cerevisiae. The P. jirovecii proteins Gsc1 and Kre6 identified using those of the relative Pneumocystis carinii as the query sequence showed high sequence identity to the putative fungal orthologs (53-97% in conserved functional domains). The expression of their encoding genes on plasmid rescued the increased sensitivity to, respectively, caspofungin or calcofluor white of the corresponding S. cerevisiae null allele. The uniqueness and likely essentiality of these proteins suggest that they are potential good drug targets.

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

“…Functional ascertainment of the P. carinii and P. murina gsc1 genes by complementation of the S. cerevisiae gsc1 deletant. As previously reported for P. carinii (11) and P. jirovecii (12), we identified a single Gsc1 protein within the P. murina proteome by a homology search using the Gsc1 protein of P. carinii as the query sequence. To ascertain the function of the P. carinii and P. murina gsc1 genes, recombinant plasmids expressing them were introduced into the S. cerevisiae gsc1 deletant.…”

“…The P. murina gene sequence encoding the Gsc1 protein was then retrieved from the European Nucleotide Archive (http://www.ebi.ac.uk/ena). The cloning of the P. jirovecii and S. cerevisiae gsc1 genes was previously described (12). Since the P. carinii and P. murina gsc1 genes include each three introns, their cDNAs were synthesized and cloned into the p416GPD vector (41) by GeneCust Europe (Ellange Luxemburg).…”

“…Transformation with plasmids containing the P. jirovecii gsc1 or the S. cerevisiae GSC1 gene were previously described (12). The S. cerevisiae GSC1 gene could not be cloned in the p416GPD plasmid because of restriction sites issues, but into p415GPD (leu marker instead of ura).…”

“…Before studying the sensitivity to echinocandins, we had to assess the function of the P. carinii and the P. murina gsc1 genes, as we previously carried out for the P. jirovecii gsc1 gene (12). Functional complementation of the gsc1 deletant was proven by the spot dilution test on YNB selective medium lacking uracil and supplemented with or without 150 ng/ml CAS (Fluka, Chemie AG).…”

“…The Gsc1 protein of Pneumocystis carinii , the species infecting rats, was reported to be inhibited by the compound L-733,560, a molecule structurally close to echinocandins (11). Recently, we identified and functionally ascertained the function of the Gsc1 subunit of P. jirovecii using complementation of the orthologous gene of Saccharomyces cerevisiae (12). The presence of a unique gsc1 gene in the genome of P. jirovecii , as in that of P. carinii , further suggests that the Gsc1 subunit is a potential new drug target to fight PCP.…”