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. 2019 Dec 10;10(6):e01952-19.
doi: 10.1128/mBio.01952-19.

A Mechanosensitive Channel Governs Lipid Flippase-Mediated Echinocandin Resistance in Cryptococcus Neoformans

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Free PMC article

A Mechanosensitive Channel Governs Lipid Flippase-Mediated Echinocandin Resistance in Cryptococcus Neoformans

Chengjun Cao et al. mBio. .
Free PMC article

Abstract

Echinocandins show fungicidal activity against common invasive mycoses but are ineffective against cryptococcosis. The underlying mechanism for echinocandin resistance in Cryptococcus neoformans remains poorly understood but has been shown to involve Cdc50, the regulatory subunit of lipid flippase. In a forward genetic screen for cdc50Δ suppressor mutations that are caspofungin resistant, we identified Crm1 (caspofungin resistant mutation 1), a homolog of mechanosensitive channel proteins, and showed that crm1Δ restored caspofungin resistance in cdc50Δ cells. Caspofungin-treated cdc50Δ cells exhibited abnormally high intracellular calcium levels ([Ca2+]c) and heightened activation of the calcineurin pathway. Deletion of CRM1 in the cdc50Δ background normalized the abnormally high [Ca2+]c. Cdc50 interacts with Crm1 to maintain cellular calcium homeostasis. Analysis of chitin/chitosan content showed that deleting CRM1 reversed the decreased chitosan production of cdc50Δ cells. Together, these results demonstrate that Cdc50 and Crm1 regulation of the calcineurin pathway and cytoplasmic calcium homeostasis may underlie caspofungin resistance in C. neoformans IMPORTANCE Cryptococcus neoformans is the leading cause of fungal meningitis, accounting for ∼15% of HIV/AIDS-related deaths, but treatment options for cryptococcosis are limited. Echinocandins are the newest fungicidal drug class introduced but are ineffective in treating cryptococcosis. Our previous study identified the lipid flippase subunit Cdc50 as a contributor to echinocandin resistance in C. neoformans Here, we further elucidated the mechanism of Cdc50-mediated caspofungin drug resistance. We discovered that Cdc50 interacts with the mechanosensitive calcium channel protein Crm1 to regulate calcium homeostasis and caspofungin resistance via calcium/calcineurin signaling. These results provide novel insights into echinocandin resistance in this pathogen, which may lead to new treatment options, as well as inform echinocandin resistance mechanisms in other fungal organisms and, hence, advance our understanding of modes of antifungal drug susceptibility and resistance.

Keywords: Cryptococcus neoformans; antifungal drug resistance; calcium signaling; fungi; lipid flippase.

Figures

FIG 1
FIG 1
Deleting CRM1 rescues caspofungin sensitivity in the cdc50Δ mutant. (A) An agar-based spotting assay measured caspofungin (CAS) sensitivity. Cultures were grown overnight in YPD medium and adjusted to a starting concentration at an A600 of 1.0. Tenfold serial dilutions were prepared, and 5 μl of each suspension was spotted on YPD agar supplemented with 0, 16, or 32 μg/ml caspofungin. Prior to being photographed, plates were incubated for 4 days at 30°C. (B) Relative expression levels of CRM1 in the wild type, cdc50Δ mutant, and CDC50 overexpression strain. Yeast cells collected from overnight culture in YPD medium were replated onto YPD medium containing 0 or 4 μg/ml of caspofungin. Cells were incubated for an additional 16 h at 30°C before RNA extraction for quantitative RT-PCR analysis. The GAPDH gene served as a reference. The expression level of CRM1 under the YPD condition was set as 1. The data shown are cumulated from three independent experiments. Statistical analysis was done by a two-tailed t test. *, P < 0.05; **, P < 0.01. (C) Predicted Crm1 membrane topology. White rods represent transmembrane domains. The black rod represents a predicted EF-hand Ca2+-binding motif. The thick black line indicates the predicted mechanosensitive (MS) channel. aa, amino acids.
FIG 2
FIG 2
Calcium levels influence cell growth and survival rate of C. neoformans. (A to C) Growth curves (left) and survival rates in YPD medium (middle) or YPD medium with 5 mM CaCl2 (right). Cultures of H99, cdc50Δ, crm1Δ, and crm1Δ cdc50Δ strains were grown on YPD medium containing 0, 4, or 16 μg/ml of caspofungin (CAS), as indicated, and incubated for 33 h at 30°C. Cell density was determined by measuring the optical density at 600 nm (OD600) at different time points, as indicated. The number of yeast CFU/ml was determined at different time points after incubation by plating samples onto drug-free medium. Triplicates were used for each measurement. *, P < 0.05; **, P < 0.01 (two-tailed t test).
FIG 3
FIG 3
Crm1 is required to activate the calcineurin pathway. (A to F) Relative expression levels of CCH1 , MID1, CAM1 , CNB1, CNA1, and CRZ1, as indicated, were determined. Cultures of H99, crm1Δ, and crm1Δ cdc50Δ strains were incubated overnight in YPD medium and replated onto YPD medium containing 0 or 4 μg/ml caspofungin (CAS). Cultures were incubated for 16 h at 30°C before RNA extraction for quantitative RT-PCR analysis. The data shown are cumulated from three independent experiments. The GAPDH gene served as a reference. The expression levels of each gene under the YPD-only condition was set as 1. *, P < 0.05; **, P < 0.01 (two-tailed t test).
FIG 4
FIG 4
Loss of Crm1 restores calcium homeostasis and decreases cell surface PS exposure and ROS production in the cdc50Δ mutant. Intracellular calcium levels, PS signal, and ROS signal were determined at the indicated time points by flow cytometry. Overnight cultures of the H99, cdc50Δ, crm1Δ, crm1Δ cdc50Δ and CRM1OE strains were resuspended in YPD containing 0 or 4 μg/ml caspofungin. (A) Time-lapse measurement of intracellular calcium levels in the presence and absence of caspofungin (CAS). (B) Intracellular calcium levels in the CRM1OE strain. (C) Fluorescent signal intensities of intracellular calcium levels after 16 h of incubation in the presence or absence of caspofungin treatment. (D) The fluorescent signal of fungal cells was detected by fluorescence microscopy after annexin V binding. Bar, 10 μm. (E and F) Quantification of fluorescent signal intensities of cell surface PS and ROS, as indicated, using flow cytometry for 100,000 yeast cells. Triplicates were used for each measurement. *, P < 0.05; **, P < 0.01 (two-tailed t test).
FIG 5
FIG 5
Cdc50 interacts with Crm1. (A) Colocalization of Crm1 and Cdc50. Fluorescent signals generated by Crm1-mCherry and Cdc50-GFP in cells grown in YPD medium. DIC, differential interference contrast. Bar, 5 μm. (B) Interactions between Crm1 and Cdc50 were observed in the split-ubiquitin system. The C-terminal half of ubiquitin (Cub) was fused to the N terminus of Cdc50 cDNA (Cub-Cdc50). The N-terminal half of ubiquitin (NubG) was fused to the N terminus of Crm1 cDNA (NubG-Crm1). Yeast transformants were grown on selective medium. β-Galactosidase activity assays were performed to verify the interaction. Values were averaged from two independent experiments. SD, synthetic dextrose. (C) Total proteins from cells expressing Crm1-mCherry, Cdc50-GFP, or both Crm1-mCherry and Cdc50-GFP were extracted. The potential Crm1-Cdc50 interaction was analyzed by coimmunoprecipitation (IP) with anti-mCherry or anti-GFP antibody and evaluated by immunoblotting.
FIG 6
FIG 6
Regulation of Crm1 in caspofungin uptake in C. neoformans. (A) Fungal cultures were coincubated with 5 μM BODIPY-labeled caspofungin for 30 min at 30°C. The fluorescent signal of fungal cells was detected by fluorescence microscopy. (B) Fluorescent signal intensity was quantified using flow cytometry for 100,000 yeast cells treated with 5 μM BODIPY-caspofungin. Triplicates were used for each measurement. *, P < 0.05. (C) Representative images of fluorescent signal quantification results using flow cytometry.
FIG 7
FIG 7
Cdc50 and Crm1 contribute to the regulation of chitin and chitosan production in C. neoformans. (A to C) Chitin and chitosan production of the H99, crm1Δ, cdc50Δ, and crm1Δ cdc50Δ strains in the presence or absence of caspofungin (CAS). Cells were grown on YPD medium overnight as described in Materials and Methods. *, P < 0.01.
FIG 8
FIG 8
Model of the caspofungin resistance mechanism in C. neoformans. Caspofungin (CAS) treatment activates the Ca2+-calcineurin pathway, which regulates downstream targets that regulate cell wall integrity and drug resistance. In the wild type (WT), Cdc50 may coordinate Ca2+ efflux, which in conjunction with Crm1 plays roles in Ca2+ influx processes to maintain intracellular calcium homeostasis. An excessive elevation of intracellular calcium levels the in cdc50Δ mutant induces cell death. Loss of Crm1 in the cdc50Δ mutant alleviates the increased calcium levels. TF, transcription factor.

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References

    1. Park BJ, Wannemuehler KA, Marston BJ, Govender N, Pappas PG, Chiller TA. 2009. Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS. AIDS 23:525–530. doi:10.1097/QAD.0b013e328322ffac. - DOI - PubMed
    1. Pukkila-Worley R, Mylonakis E. 2008. Epidemiology and management of cryptococcal meningitis: developments and challenges. Expert Opin Pharmacother 9:551–560. doi:10.1517/14656566.9.4.551. - DOI - PubMed
    1. Perfect JR, Dismukes WE, Dromer F, Goldman DL, Graybill JR, Hamill RJ, Harrison TS, Larsen RA, Lortholary O, Nguyen MH, Pappas PG, Powderly WG, Singh N, Sobel JD, Sorrell TC. 2010. Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the infectious diseases society of America. Clin Infect Dis 50:291–322. doi:10.1086/649858. - DOI - PMC - PubMed
    1. Kartsonis NA, Nielsen J, Douglas CM. 2003. Caspofungin: the first in a new class of antifungal agents. Drug Resist Updat 6:197–218. doi:10.1016/S1368-7646(03)00064-5. - DOI - PubMed
    1. Denning DW. 2003. Echinocandin antifungal drugs. Lancet 362:1142–1151. doi:10.1016/S0140-6736(03)14472-8. - DOI - PubMed

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