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Sphingolipids modulate echinocandin susceptibility in opportunistic fungi
Dissertation   Open access

Sphingolipids modulate echinocandin susceptibility in opportunistic fungi

Kelley R. Healey
Doctor of Philosophy (Ph.D.), Drexel University
Oct 2013
DOI:
https://doi.org/10.17918/00000971
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Abstract

Allergy and Immunology Microbiology
Fungal infections caused by Candida yeast and Aspergillus molds have significantly increased over the past 30 years as immunocompromised populations expanded and hospitalization rates increased. Candida species, specifically C. glabrata, are becoming refractory to the widely-used azole antifungals. New echinocandin antifungals, including caspofungin and micafungin, are now approved for therapy of invasive candidiasis. Resistance (MICs >0.5[mu]g/ml) to these lipopeptide inhibitors of cell wall synthesis is rare and characterized by echinocandin cross-resistance and mutations in integral membrane proteins Fks1/Fks2. In search of alternate mechanisms of reduced susceptibility, we discovered a differential, echinocandin phenotype in C. glabrata following selection on low-level (0.2.0.3[mu]g/ml) caspofungin. Mutants exhibited 4- to 32-fold reduced susceptibility to caspofungin (CRS) and surprisingly, 4- to 32-fold increased susceptibility to micafungin (MIS). Sequencing and gene deletion studies demonstrated 'CRS-MIS' was Fks-independent. Saccharomyces cerevisiae was used as a genetic model which revealed CRS was conferred by multiple mutations (fen1[delta], sur4[delta], cka2[delta], tsc10-ts) disrupting sphingolipid biosynthesis. Disruption of homologous genes in C. glabrata resulted in CRS-MIS. Sphingolipid analysis of both laboratory-generated mutants and clinical isolates demonstrated increased levels of long-chain bases (LCBs). Consistent with the lipid data, fen1, sur4, ifa38, and sur2 mutations were identified. Furthermore, exogenous LCBs conferred CRS-MIS on wild-type species of Candida and Aspergillus. C. albicans tsc13 mutants that underwent a loss-of-heterozygosity and an A. nidulans sur2 mutant all contained elevated LCBs and displayed CRS-MIS. The phenotype of a C. albicans mutant was established in vivo using the wax moth larvae model, Galleria mellonella. We also analyzed the connection between Cka2 protein kinase and the sphingolipid pathway. Previous studies implicated Cka2-mediated ceramide synthase regulation, and subsequent ceramide synthase deletion (lag1[delta]lac1[delta] ) also yielded CRS-MIS. Mutation of three, putative Cka2 phosphorylation sites (Lag1:S400A/S402A/S404A) retained CRS-MIS in a reconstituted lag1[delta]lac1[delta] strain, while mutation to phosphorylation-mimicking residues (Lag1:S400D/S402D/S404D) reverted CRS-MIS in cka2[delta] , implying direct phosphorylation. In our model, elevated LCBs within the plasma membrane weaken interactions between caspofungin and Fks but strengthen interactions with micafungin. This thesis highlights the importance of the lipid environment in echinocandin effectiveness and suggests therapy can be enhanced through multi-echinocandin use or with sphingolipid pathway inhibitors/LCBs plus micafungin.

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