Elevated Cell Wall Chitin in Candida albicans Confers Echinocandin Resistance In Vivo

Abstract

Candida albicans cells with increased cell wall chitin have reduced echinocandin susceptibility in vitro. The aim of this study was to investigate whether C. albicans cells with elevated chitin levels have reduced echinocandin susceptibility in vivo. BALB/c mice were infected with C. albicans cells with normal chitin levels and compared to mice infected with high-chitin cells. Caspofungin therapy was initiated at 24 h postinfection. Mice infected with chitin-normal cells were successfully treated with caspofungin, as indicated by reduced kidney fungal burdens, reduced weight loss, and decreased C. albicans density in kidney lesions. In contrast, mice infected with high-chitin C. albicans cells were less susceptible to caspofungin, as they had higher kidney fungal burdens and greater weight loss during early infection. Cells recovered from mouse kidneys at 24 h postinfection with highchitin cells had 1.6-fold higher chitin levels than cells from mice infected with chitin-normal cells and maintained a significantly reduced susceptibility to caspofungin when tested in vitro. At 48 h postinfection, caspofungin treatment induced a further increase in chitin content of C. albicans cells harvested from kidneys compared to saline treatment. Some of the recovered clones had acquired, at a low frequency, a point mutation in FKS1 resulting in a S645Y amino acid substitution, a mutation known to confer echinocandin resistance. This occurred even in cells that had not been exposed to caspofungin. Our results suggest that the efficacy of caspofungin against C. albicans was reduced in vivo due to either elevation of chitin levels in the cell wall or acquisition of FKS1 point mutations.

Echinocandin resistance due to simultaneous FKS mutation and increased cell wall chitin in a Candida albicans bloodstream isolate following brief exposure to caspofungin

“…(Walker et al, 2008), but has been less well studied in clinical isolates. In an experimental murine model, infection with C. albicans with elevated chitin levels was associated with reduced survival following treatment with caspofungin (Lee et al, 2012).…”

“…The exact contribution made by these two independent mechanisms is undefined at present although recent data suggest that elevated cell wall chitin in C. albicans could provide cells with a window of opportunity to acquire an FKS1 mutation, even without exposure to caspofungin (Lee et al, 2012). Clinical isolates with reduced echinocandin susceptibility should be investigated for both FKS mutations and cell wall alterations.…”

“…The dried cell wall was acid-hydrolysed, washed and resuspended in deionized water. Chitin, glucan and mannan levels were measured by acid-hydrolysing the cell wall polymers and quantifying the amounts of the released monosaccharide (glucosamine, glucose and mannose) by high performance anion-exchange chromatography with pulsed amperometric detection (Lee et al, 2012). The results are presented as a percentage of total dried cell wall.…”

“…2a). Analysis of the cell wall composition by high performance anion-exchange chromatography with pulsed amperometric detection (Lee et al, 2012) revealed elevated chitin levels approximately four times higher than that of a caspofungin-sensitive strain, SC5314 (Fig. 2b, c).…”

“…As the MIC for caspofungin was .2 mg l 21 (non-susceptible), we reported the strain as clinically resistant to all echinocandins as per the laboratory protocol. DNA sequencing (Lee et al, 2012) of the FKS1 gene from the strain detected a welldescribed mutation (Park et al, 2005) at the hotspot 1 region of the gene resulting in an amino acid substitution, serine to proline, at position 645 (Fig. 2a).…”