CRS–MIS in Candida glabrata: sphingolipids modulate echinocandin–Fks interaction

Abstract

Summary Infections with the azole-refractory yeast Candida glabrata are now commonly treated with the echinocandins caspofungin (CSF) or micafungin (MCF). True resistance (>32-fold decreased susceptibility) to these lipopeptide inhibitors of cell wall synthesis is rare and strictly associated with mutations in integral membrane proteins Fks1 or Fks2. In contrast, mutants exhibiting 4 to 32-fold CSF reduced susceptibility (CRS) were readily selected in vitro, and surprisingly demonstrated 4 to 32-fold MCF increased susceptibility (MIS). Sequencing and gene deletion demonstrated that CRS-MIS is Fks-independent. To explore alternative mechanisms, we initially employed Saccharomyces cerevisiae, and observed that CRS was conferred by multiple mutations (fen1Δ, sur4Δ, cka2Δ, and tsc10-ts) disrupting sphingolipid biosynthesis. Following this lead, C. glabrata fen1Δ and cka2Δ deletants were constructed, and shown to exhibit CRS-MIS. Sphingolipid analysis of CRS-MIS laboratory mutants and clinical isolates demonstrated elevated dihydrosphingosine (DHS) and phytosphingosine (PHS) levels, and consistent with this sequencing revealed fen1, sur4, ifa38, and sur2 mutations. Moreover, exogenous DHS or PHS conferred a CRS-MIS phenotype on wild-type C. glabrata. Exogenous PHS failed, however, to suppress CRS-MIS in a sur2 mutant blocked in conversion of DHS to PHS, implying that accumulation of these intermediates confers CRS-MIS. We conclude that membrane sphingolipids modulate echinocandin-Fks interaction.

Evaluation of Polymorphic Locus Sequence Typing for Candida glabrata Epidemiology

“…As a final example of how typing can provide surrogate parents for mutational analysis of antifungal resistance, we recently described three C. glabrata clinical strains that exhibit caspofungin reduced susceptibility but micafungin increased susceptibility (CRS-MIS), and we subsequently identified substitutions (again relative to CBS138) in their sphingolipid biosynthesis genes (42). For two of these, complementation experiments confirmed that their Fen1 substitutions were mutations responsible for their CRS-MIS phenotype.…”

“…For two of these, complementation experiments confirmed that their Fen1 substitutions were mutations responsible for their CRS-MIS phenotype. For the third, strain 4743, tentative support for the role of an Ile339Met substitution in Ifa38 was obtained by sequencing its gene from 7 diverse C. glabrata strains, all of which encoded Ile339 as in CBS138 (42). However, in retrospect, none of the sequenced strains were from cluster V, which includes 4743 ( Fig.…”

“…The strains used in this study and their sources are listed in Table 1. From frozen glycerol stocks, strains were streaked for isolation on yeast extract-peptone-dextrose (YPD) (1% yeast extract, 2% peptone, 2% dextrose) agar plates, with incubation at 35°C for 48 h. Fluconazole susceptibility or resistance (MIC of Յ32 g/ml or Ն64 g/ml, respectively) and the CRS-MIS phenotype (Ն4-fold caspofungin reduced susceptibility and micafungin increased susceptibility) were determined by broth microdilution as previously described (15,42).…”

“…To characterize the 2,055-bp Ptp2 gene, the procedure above was modified by the use of primers CgPTP2uF (5=-TGGGATG CTGGATGTAGTGA) and CgPTP2dR (5=-CATGCTAGCTTTTCGAG CGA) for amplification and these and additional internal primers for sequencing. Amplification and sequencing of the Ifa38 gene was as previously described (42). Bioinformatics.…”

Use of Micafungin as a Surrogate Marker To Predict Susceptibility and Resistance to Caspofungin among 3,764 Clinical Isolates of Candida by Use of CLSI Methods and Interpretive Criteria

“…Although many of the resistant isolates and cases of breakthrough IC were due to Candida glabrata, a number of Candida albicans, Candida tropicalis, Candida krusei, and Candida lusitaniae isolates have also been observed to have reduced susceptibility or resistance to both micafungin and caspofungin (17, 22-24, 26, 28, 29). It should be noted that isolates of C. glabrata with paradoxical reduced caspofungin susceptibility but increased micafungin susceptibility have been reported (30,31). Although the clinical importance of such differential susceptibilities to the echinocandins is not clear, two recent studies found such differences to be meaningful in murine models of IC, irrespective of the presence or absence of fks mutations (19,21).…”