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. 2023 Aug 31;14(4):e0007423.
doi: 10.1128/mbio.00074-23. Epub 2023 Jun 28.

Caspofungin-induced β(1,3)-glucan exposure in Candida albicans is driven by increased chitin levels

Andrew S Wagner  1 , Stephen W Lumsdaine  1 , Mikayla M Mangrum  1 , Todd B Reynolds  1
Affiliations

Caspofungin-induced β(1,3)-glucan exposure in Candida albicans is driven by increased chitin levels

Andrew S Wagner et al. mBio. .

Abstract

To successfully induce disease, Candida albicans must effectively evade the host immune system. One mechanism used by C. albicans to achieve this is to mask immunogenic β(1,3)-glucan epitopes within its cell wall under an outer layer of mannosylated glycoproteins. Consequently, induction of β(1,3)-glucan exposure (unmasking) via genetic or chemical manipulation increases fungal recognition by host immune cells in vitro and attenuates disease during systemic infection in mice. Treatment with the echinocandin caspofungin is one of the most potent drivers of β(1,3)-glucan exposure. Several reports using murine infection models suggest a role for the immune system, and specifically host β(1,3)-glucan receptors, in mediating the efficacy of echinocandin treatment in vivo. However, the mechanism by which caspofungin-induced unmasking occurs is not well understood. In this report, we show that foci of unmasking co-localize with areas of increased chitin within the yeast cell wall in response to caspofungin, and that inhibition of chitin synthesis via nikkomycin Z attenuates caspofungin-induced β(1,3)-glucan exposure. Furthermore, we find that both the calcineurin and Mkc1 mitogen-activated protein kinase pathways work synergistically to regulate β(1,3)-glucan exposure and chitin synthesis in response to drug treatment. When either of these pathways are interrupted, it results in a bimodal population of cells containing either high or low chitin content. Importantly, increased unmasking correlates with increased chitin content within these cells. Microscopy further indicates that caspofungin-induced unmasking correlates with actively growing cells. Collectively, our work presents a model in which chitin synthesis induces unmasking within the cell wall in response to caspofungin in growing cells. IMPORTANCE Systemic candidiasis has reported mortality rates ranging from 20% to 40%. The echinocandins, including caspofungin, are first-line antifungals used to treat systemic candidiasis. However, studies in mice have shown that echinocandin efficacy relies on both its cidal impacts on Candida albicans, as well as a functional immune system to successfully clear invading fungi. In addition to direct C. albicans killing, caspofungin increases exposure (unmasking) of immunogenic β(1,3)-glucan moieties. To evade immune detection, β(1,3)-glucan is normally masked within the C. albicans cell wall. Consequently, unmasked β(1,3)-glucan renders these cells more visible to the host immune system and attenuates disease progression. Therefore, discovery of how caspofungin-induced unmasking occurs is needed to elucidate how the drug facilitates host immune system-mediated clearance in vivo. We report a strong and consistent correlation between chitin deposition and unmasking in response to caspofungin and propose a model in which altered chitin synthesis drives increased unmasking during drug exposure.

Keywords: Candida albicans; caspofungin; chitin; unmasking; β-glucan.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig 1
Fig 1
Areas of increased chitin co-localize with unmasked foci in growing yeast cells in response to caspofungin. (A–B) SC5314-derived LEU2/leu2∆ WT cells (AWY006) grown to mid-log phase were exposed to 46.9 ng/mL of caspofungin for 30 minutes and then stained with anti-β(1,3)-glucan antibody and a phycoerythrin-conjugated secondary antibody, along with calcofluor white (CFW) to assess the levels of β(1,3)-glucan exposure and total chitin by flow cytometry, respectively. (A) β(1,3)-glucan unmasking in WT cells treated with sublethal concentrations of caspofungin. (B) CFW staining of WT cells treated with sublethal concentrations of caspofungin (****P < 0.0001 by Student’s t-test; n = 3 biological replicates). (C) Representative confocal microscopy of mid-log phase cells treated with an ethanol solvent control or caspofungin. White arrows highlight examples of unmasking across images (scale bar indicates 5 µm). (D–I) CFW staining and β(1,3)-glucan unmasking quantifications of microscopy images of mother cells, daughter cells, and septa of budding yeast (n = 38 budding cells analyzed per treatment). (D) CFW staining of WT cells treated with sublethal concentrations of caspofungin or an ethanol solvent control [**P < 0.005, ****P < 0.0001 by one-way analysis of variance (ANOVA)]. (E) CFW staining of WT cells treated with ethanol only. These data are the same data as were presented in Fig. 1D, but were highlighted here for specific analysis of total chitin levels in basal growth conditions (****P < 0.0001 by Student’s t-test). (F) Unmasking of WT cells treated with sublethal concentrations of caspofungin or an ethanol solvent control (****P < 0.0001 by Kruskal–Wallis test). (G) Unmasking of WT cells treated with ethanol only. These data are the same data as were presented in Fig. 1F, but were highlighted here for specific analysis of β(1,3)-glucan exposure levels in basal growth conditions (*P < 0.05 by Student’s t-test). (H) CFW staining of the septa of cells treated with caspofungin or an ethanol solvent control (****P < 0.0001 by Student’s t-test). (I) Unmasking of the septa of cells treated with caspofungin or an ethanol solvent control. MFI, median fluorescent intensity; ns, not significant by Mann–Whitney test.
Fig 2
Fig 2
Nikkomycin Z treatment attenuates caspofungin-induced unmasking in yeast cells. SC5314-derived LEU2/leu2∆ (AWY006) WT cells grown to mid-log phase were exposed to 46.9 ng/mL of caspofungin and varying concentrations of nikkomycin Z (6.5 µg/mL or 16.5 µg/mL), or appropriate solvent controls, for 30 minutes. β(1,3)-glucan exposure and total chitin levels were then assessed by flow cytometry. (A) CFW staining of nikkomycin Z- and caspofungin-treated samples (*P < 0.05, **P < 0.005, ****P < 0.0001, by one-way ANOVA; n = 3 biological replicates). (B) Representative histogram of impact that nikkomycin Z treatment has on caspofungin-induced chitin synthesis. Colors of each sample match those shown in Fig. 2A. (C) β(1,3)-glucan exposure of nikkomycin Z- and caspofungin-treated samples (****P < 0.0001 by one-way ANOVA; n = 3 biological replicates). (D) Representative histogram of impact that nikkomycin Z treatment has on caspofungin-induced unmasking. Colors of each sample match those shown in Fig. 2C. (E) Representative microscopy images of nikkomycin Z- and caspofungin-treated cells (scale bar indicates 5 µm).
Fig 3
Fig 3
Calcineurin inhibition attenuates both unmasking and chitin synthesis in yeast cells in response to caspofungin exposure. SC5314-derived LEU2/leu2∆ (AWY006) WT cells grown to mid-log phase in the presence of 100 µg/mL of cyclosporine A, or an appropriate volume of the DMSO solvent control, were exposed to 46.9 ng/mL of caspofungin (or EtOH solvent control) for 30 minutes. β(1,3)-glucan exposure and total chitin levels were then assessed by flow cytometry. (A) β(1,3)-glucan exposure of cyclosporine A- and caspofungin-treated cells (****P < 0.0001 by one-way ANOVA; n = 3 biological replicates). (B) Representative histogram of the impact that calcineurin inhibition has on caspofungin-induced unmasking. Colors of each sample match those shown in Fig. 2A. (C) CFW staining of cyclosporine A- and caspofungin-treated cells (*P < 0.01, **P < 0.005; n = 3 biological replicates). (D) Representative histogram of the impact that calcinuerin inhibition has on caspofungin-induced chitin synthesis. Colors of each sample match those shown in Fig. 2A and C.
Fig 4
Fig 4
Chitin levels correlate with β(1,3)-glucan exposure following caspofungin treatment in calcineurin-inhibited yeast cells. SC5314-derived LEU2/leu2∆ (AWY006) WT cells grown to mid-log phase in the presence of 100 µg/mL of cyclosporine A, or an appropriate volume of the DMSO solvent control, were exposed to 46.9 ng/mL of caspofungin (or EtOH solvent control) for 30 minutes, and β(1,3)-glucan exposure and total chitin levels were assessed by flow cytometry. (A) Representative scatter plot and adjunct histograms of cyclosporine A- and caspofungin-treated cells when assessing unmasking and total chitin. Gates represent populations of high and low chitin within the sample. (B) Percentage of the total population within low and high chitin gates for cyclosporine A- and caspofungin-treated samples (n = 3 biological replicates). (C) Representative scatter plot with adjunct histograms for CFW staining and exposed β(1,3)-glucan when plotting low (blue) and high (red) chitin populations independently. (D) β(1,3)-glucan unmasking of cells within the low and high chitin populations following caspofungin addition to cyclosporine A-treated cells. Fluorescence intensity was normalized to median cell size for each of the two populations (***P = 0.0001 by Student’s t-test; n = 3 biological replicates).
Fig 5
Fig 5
Loss of MKC1 attenuates caspofungin-driven unmasking and chitin synthesis in yeast cells. (A–D) mkc1Δ/Δ cells grown to mid-log phase were exposed to 46.9 ng/mL of caspofungin (or EtOH solvent control) for 30 minutes, and β(1,3)-glucan exposure and total chitin levels were assessed by flow cytometry. (A) β(1,3)-glucan unmasking in mkc1Δ/Δ cells treated with sublethal concentrations of caspofungin (****P < 0.0001 by one-way ANOVA; n = 3 biological replicates). (B) Representative histogram of the impact that loss of MKC1 has on caspofungin-induced unmasking. Colors of each sample match those shown in Fig. 5A. (C) CFW staining of cells treated with sublethal concentrations of caspofungin (*P < 0.01, **P < 0.01, ***P < 0.0005, ****P < 0.0001 by one-way ANOVA; n = 3 biological replicates). (D) Representative histogram of the impact that loss of MKC1 has on caspofungin-induced chitin synthesis. Colors of each sample match those shown in 5A and C. (E) Representative scatter plot and adjunct histograms for CFW staining and exposed β(1,3)-glucan of mkc1Δ/Δ cells treated with caspofungin. Gates represent populations of high and low chitin within the sample. (F) Representative scatter plot with adjunct histograms for CFW staining and exposed β(1,3)-glucan when plotting low (blue) and high (red) chitin populations independently. (G) β(1,3)-glucan unmasking of cells within the low and high chitin populations following caspofungin addition to mkc1Δ/Δ cells. Fluorescence intensity was normalized to median cell size for each of the two populations (***P = 0.0003 by Student’s t-test; n = 3 biological replicates).
Fig 6
Fig 6
Calcineurin and Mkc1 work in parallel to mediate caspofungin-induced chitin synthesis and unmasking. (A–D) mkc1Δ/Δ cells grown to mid-log phase in either the presence of 100 µg/mL of cyclosporine A, or the appropriate solvent control, were exposed to 46.9 ng/mL of caspofungin (or EtOH solvent control) for 30 minutes. β(1,3)-glucan exposure and total chitin levels were then assessed by flow cytometry. (A) β(1,3)-glucan exposure of mkc1Δ/Δ cells treated with cyclosporine A and caspofungin (****P < 0.0001; n = 3 biological replicates). (B) Representative histogram of the impact that calcineurin inhibition has on caspofungin-induced unmasking in an mkc1Δ/Δ background. Colors of each sample match those shown in Fig. 6A. (C) CFW staining of mkc1Δ/Δ cells treated with cyclosporine A (calcineurin-inhibited) and caspofungin (**P < 0.01, ***P < 0.001,****P < 0.0001, by one-way ANOVA; n = 3 biological replicates). (D) Representative histogram of the impact that calcineurin inhibition has on caspofungin-induced chitin synthesis in an mkc1Δ/Δ background. Colors of each sample match those shown in Fig. 6A and C. (E) Representative scatter plot and adjunct histograms for CFW staining and exposed β(1,3)-glucan of mkc1Δ/Δ cells treated with cyclosporine A and caspofungin. Gates represent populations of high and low chitin within the sample. (F) Representative scatter plot with adjunct histograms for CFW staining and exposed β(1,3)-glucan when plotting low (blue) and high (red) chitin populations independently. (G) β(1,3)-glucan unmasking of cells within the low and high chitin populations following caspofungin exposure and cyclosporine A treatment of mkc1Δ/Δ cells. Fluorescence intensity was normalized to median cell size for each of the two populations (**P = 0.0019 by Student’s t-test; n = 3 biological replicates). (H) Western blot analysis of Mkc1 activation in response to cyclosporine A and caspofungin exposure. Cells were grown to mid-log phase in the presence of 100 µg/mL of cyclosporine A or an appropriate volume of the DMSO solvent control, and were exposed to 46.9 ng/mL of caspofungin (or EtOH solvent control) for 30 minutes, and proteins were harvested for western blot analysis. Membranes were blotted using an anti-P44/42 antibody for phosphorylated (active) Mkc1 detection. An anti-tubulin antibody was used as a loading control.
Fig 7
Fig 7
Inhibition of calcineurin in an mkc1Δ/Δ mutant attenuates caspofungin-induced unmasking and chitin synthesis. Representative microscopy images of calcineurin-inhibited mkc1Δ/Δ cells exposed to caspofungin or an ethanol solvent control. mkc1Δ/Δ cells grown to mid-log phase in either the presence of 100 µg/mL of cyclosporine A, or the appropriate solvent control, were exposed to 46.9 ng/mL of caspofungin (or EtOH solvent control) for 30 minutes, and β(1,3)-glucan exposure and total chitin levels were assessed (scale bar indicates 5 µm).
Fig 8
Fig 8
Mkc1 regulates CHS3 levels in yeast cells in response to caspofungin. CHS3-GFP WT cells grown to mid-log phase were exposed to 46.9 ng/mL of caspofungin for 30 minutes, stained with CFW, and the corresponding GFP and CFW emission was assessed via microscopy and flow cytometry. (A) Representative images of CHS3-GFP cells exposed to caspofungin or an ethanol solvent control (scale bar indicates 5 µm). (B) Quantification of GFP emission in ethanol-treated and caspofungin-treated cells (n = 75 cells per treatment; ****P < 0.0001 by Mann–Whitney test). (C) Chs3-GFP quantifications of microscopy images in mother cells, daughter cells, and septa of budding yeast (n = 42 budding cells analyzed per treatment; ****P < 0.0001 by one-way ANOVA). (D) GFP emission of CHS3-GFP WT cells treated with cyclosporine A and caspofungin (****P < 0.0001 by one-way ANOVA; n = 3 biological replicates). (E) GFP emission of mkc1Δ/Δ CHS3-GFP cells treated with caspofungin (***P < 0.0005, ****P < 0.0001 by one-way ANOVA; n = 3 biological replicates).
Fig 9
Fig 9
Proposed model for caspofungin-induced unmasking in growing yeast cells. (A) Cell wall organization of a budding daughter cell in exponentially growing cells. (B) The cell wall organization of budding daughter cells following exposure to caspofungin. Inhibition of the Fks enzymes by caspofungin leads to activation of both Mkc1 and calcineurin. In turn, both pathways work independently to regulate expression and proper localization of chitin synthase enzymes to the pole of budding daughter cells. This in turn increases chitin synthesis at this location, leading to a disruption of the cell wall architecture and ultimate exposure of central β(1,3)-glucan moieties to the surrounding environment.
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