Characterization of echinocandin-resistant mutants of Candida albicans: genetic, biochemical, and virulence studies

Abstract

The pneumocandins are potent antifungal agents of the echinocandin class which are under development for use as broad-spectrum antimycotic therapy. One important consideration for any new therapeutic class for treating serious fungal infections is the potential for drug resistance development. In this study we have isolated and characterized four independent spontaneous Candida albicans mutants resistant to the potent semisynthetic pneumocandin L-733,560. These mutants have many of the properties of FKS1/ETG1 echinocandin-resistant mutants of Saccharomyces cerevisiae, including (i) cross-resistance to other 1,3-␤-D-glucan synthase inhibitors, such as papulacandin and echinocandins, but no change in sensitivity to other antifungal agents; (ii) in vitro glucan synthase activity that is more resistant to pneumocandins than the wild-type parent enzyme; and (iii) semidominant drug resistance in spheroplast fusion strains. The mutants were compared with C. albicans echinocandin-resistant mutants isolated by mutagenesis by L. Beckford and D. Kerridge (mutant M-2) (abstr. PS3.11, in Proceedings of the XI Congress of the International Society for Human and Animal Mycology, Montreal, Canada, 1992) and by A. Cassone, R. E. Mason, and D. Kerridge (mutant CA-2) (Sabouraudia 19:97-110, 1981). All of the strains had resistant enzyme activity in vitro. M-2 grew poorly and had low levels of enzyme activity. In contrast, CA-2 and the spontaneous mutants grew as well as the parents and had normal levels of glucan synthase activity. These results suggest that these resistant mutants may have alterations in glucan synthase. CA-2 was unable to form germ tubes, an ability retained by the spontaneous mutants. The virulence of the spontaneous mutants was unimpaired in a mouse model of disseminated candidiasis, while M-2 and CA-2 were 2 orders of magnitude less virulent than their parent strains. Significantly, mice challenged with the spontaneous mutant CAI4R1 responded therapeutically to lower levels of L-733,560 than would be predicted by the increase in in vitro susceptibility.

Progressive Esophagitis Caused byCandida albicanswith Reduced Susceptibility to Caspofungin

“…Our isolate was consistent with laboratory and clinical strains resistant to caspofungin due to a mutation in a single gene (FKS1). 8,9 Echinocandins inhibit 1,3-␤-D-glucan synthase, which is required for formation of 1,3-␤-D-glucan in the cell walls of fungi. 9 The FKS1 gene encodes a large integral membrane protein that is presumed to be the catalytic subunit of the (echinocandin target) glucan synthase complex.…”

“…9,10 The gene is cell-cycle regulated and is required for vegetative growth. 8,10 A recent investigation of C. albicans strains with reduced susceptibility to caspofungin identified serine-645 as the amino acid most often altered in FKS1 mutants. 9 This corresponds to the alteration in our isolate, which had a serine-to-proline substitution at position 645.…”

Herpes simplex virus type 1 dysregulates anti‐fungal defenses preventing monocyte activation and downregulating toll‐like receptor‐2

“…The non-germinative low-virulence strain PCa2 of Ca was used (25). This strain was a kind gift from Dr Kerridge, Department of Biochemistry, University of Cambridge, UK.…”

Characterization of the CaNAG3, CaNAG4, and CaNAG6 genes of the pathogenic fungus Candida albicans: possible involvement of these genes in the susceptibilities of cytotoxic agents

“…Because some of the genes a¡ecting drug sensitivity have been shown to be involved in virulence, we asked whether a null mutation of CaNAG3, CaNAG4, or CaN-AG6 also a¡ects the virulence of C. albicans [11,12]. Fourweek-old male CD-1 mice were intravenously infected with 10 6 or 10 7 cells of the wild-type CAF2-1, the canag3v, canag4v, and canag6v null mutants, as well as the can-ag3vcanag4v double disruptant.…”