Fitness and virulence costs of Candida albicans FKS1 hot spot mutations associated with echinocandin resistance

Abstract

The identification of FKS1 mutations in Candida albicans associated with echinocandin resistance has raised concerns over the spread of drug-resistant strains. We studied the impact of fks1 mutations on C. albicans virulence and fitness. Compared with wild-type strains for FKS1, echinocandin-resistant C. albicans strains with homozygous fks1 hot-spot mutations had reduced maximum catalytic capacity of their glucan synthase complexes and thicker cell walls attributable to increased cell wall chitin content. The fks1 mutants with the highest chitin contents had reduced growth rates and impaired filamentation capacities. Fks1 mutants were hypovirulent in fly and mouse models of candidiasis, and this phenotype correlated with the cell wall chitin content. In addition, we observed reduced fitness of echinocandin-resistant C. albicans in competitive mixed infection models. We conclude that fks1 mutations that confer echinocandin resistance come at fitness and virulence costs, which may limit their epidemiological and clinical impact.

Figure 1.

C. albicans fks1 mutants have thickened cell walls and increased chitin content. Transmission electron microscopic images of (A and B) FKS1 wild-type C. albicans strain SC5314 and the (C and D) clonal fks1 mutant C42 (F641S) showing the thickened cell walls of the mutant strains (A and C, ×5000 magnification; B and D, ×50,000 magnification). (E) Mean cell wall thicknesses in all of the wild-type and mutant strains. (F) The chitin content of the fks1 mutants was significantly higher than that of the clonal wild-type strains. (G) Minor differences in 1,3-β-glucan content among the different strains were noted. Group designation refers to clonal groups described in Table 1. Open bars: FKS1 wild-type strains; Black bars: fks1 mutants. Statistical significance determined using one-sided ANOVA with post hoc Dunn’s test (*P < .05; **P < .01). RFU, relative fluorescence units.

Figure 2.

C. albicans fks1 mutants have attenuated virulence in Toll-deficient D. melanogaster flies. We used an invertebrate model of invasive candidiasis in Toll-deficient D. melanogaster flies to screen for variations in virulence among echinocandin-resistant and -susceptible Candida isolates. (A–C) Kaplan-Meier survival curves for three clonal groups of C. albicans strains. *P < .05; log-rank test.

Figure 3.

C. albicans fks1 mutants have attenuated virulence in a murine model of hematogenously disseminated candidiasis. The virulence of a FKS1 wild-type C. albicans strain and two clonal fks1 mutant strains was assessed in nonimmunosuppressed BALB/c mice after intravenous injection of blastospores. (A) Kaplan Meier survival curves of 2 fks1 mutants and the clonal wild-type strain (log rank test P < .0001 for comparison among all 3 strains; dashed lines show intergroup comparisons; **P < .01). Tissue fungal burden in the (B) kidneys and (C) spleen 48 hours after inoculation (horizontal lines represent median values). One-sided ANOVA P values were <0.0001 for the kidney fungal burdens and 0.01 for the spleen fungal burdens. P values in the figure represent post hoc comparisons between wild-type and fks1 mutant fungal burdens. (D and E) Macroscopic appearance of kidneys (left) and findings of histopathologic examination of GMS-stained kidney tissue sections (right) are shown for (D) the wild-type strain and (E) an fks1 mutant (×100 magnification; inset, ×400 magnification). Whereas multiple fungal abscesses were observed on the surface of kidneys excised from wild-type–infected mice, those excised from fks1 mutant-infected mice appeared smooth. Moreover, in contrast with the extensive hyphal growth observed in wild-type–infected kidney tissue, fungal elements were rare in fks1 mutant-infected kidneys, and only yeast cells were observed.

Figure 4.

Competition experiments reveal reduced fitness of an fks1 mutant relative to the matched FKS1 wild-type strain. The relative fitness of 2 matched C. albicans strains, CLY19229 (FKS1 WT) and CLY19230 (fks1 S645F), was determined in competition experiments (A) in vitro and (B) in vivo. (A) Bars represent the growth of each strain in a mixed population during a single daily cycle. (B) Bars represent arithmetic means of six experiments performed using DNA extracted from 6 individual mouse kidneys infected with equal inoculums (10⁶ CFU/mouse) of the fks1 mutant and the wild-type strain via tail injection. All mice were euthanized on day +4 post infection. Ct values and genome equivalents are beacon-specific. The value shown for the mixed infection experiment represents the signal for the FKS1 wild-type molecular beacon. Only one of the 6 dually infected mice showed a positive signal for the fks1 mutant molecular beacon.

Figure 5.

Growth defects and attenuated virulence of C. albicans fks1 mutants correlate strongly with cell wall chitin content. A significant linear correlation was observed between the survival rates in Toll-deficient D. melanogaster flies infected with fks1 mutant C. albicans strains and the chitin content of those strains (r = 0.91; P = .009). In addition, defects in growth rate and morphotype-switching capacity were detected in the fks1 mutant strains with the highest chitin content. G, impaired growth rate; H, impaired transition to hyphal growth; F, reduced fitness in the mixed infection model; M, reduced virulence in the mouse model; #, virulence not assessed in the mouse model.

Figure 6.

Inflammatory response to FKS1 wild-type and mutant C. albicans strains. The inflammatory response to 3 C. albicans was assessed using the murine macrophage RAW264.7 cell line containing a secreted alkaline phosphatase (SEAP) reporter construct indicative of NF-kB/AP-1 transcription factor activity (see methods). Both CLY16998 (FKS1-WT) and CLY16996 (fks1-S645F not associated with increased cell-wall chitin content) induced a robust inflammatory response. In contrast, CLY16997 (fks1-S645P), a mutant with markedly increased cell-wall chitin content (Figure 1), induced less than 50% of SEAP levels of the wild-type strain, indicating a dampened inflammatory response. All responses were reversed by anti-Dectin-1 antibodies. Values represent the means of 3 separate experiments. *P < .05; one-sided ANOVA for comparison among controls. #P < .01; Students’s t-test for comparison between control and anti-Dectin-1 pretreated cells.