Calcineurin Is Required for Pseudohyphal Growth, Virulence, and Drug Resistance in Candida lusitaniae

Abstract

Candida lusitaniae is an emerging fungal pathogen that infects immunocompromised patients including HIV/AIDS, cancer, and neonatal pediatric patients. Though less prevalent than other Candida species, C. lusitaniae is unique in its ability to develop resistance to amphotericin B. We investigated the role of the calcium-activated protein phosphatase calcineurin in several virulence attributes of C. lusitaniae including pseudohyphal growth, serum survival, and growth at 37°C. We found that calcineurin and Crz1, a C. albicans Crz1 homolog acting as a downstream target of calcineurin, are required for C. lusitaniae pseudohyphal growth, a process for which the underlying mechanism remains largely unknown in C. lusitaniae but hyphal growth is fundamental to C. albicans virulence. We demonstrate that calcineurin is required for cell wall integrity, ER stress response, optimal growth in serum, virulence in a murine systemic infection model, and antifungal drug tolerance in C. lusitaniae. To further examine the potential of targeting the calcineurin signaling cascade for antifungal drug development, we examined the activity of a calcineurin inhibitor FK506 in combination with caspofungin against echinocandin resistant C. lusitaniae clinical isolates. Broth microdilution and drug disk diffusion assays demonstrate that FK506 has synergistic fungicidal activity with caspofungin against echinocandin resistant isolates. Our findings reveal that pseudohyphal growth is controlled by the calcineurin signaling cascade, and highlight the potential use of calcineurin inhibitors and caspofungin for emerging drug-resistant C. lusitaniae infections.

Components of the Calcium-Calcineurin Signaling Pathway in Fungal Cells and Their Potential as Antifungal Targets

“…Calcineurin (CN) is a Ca 2ϩ /calmodulin (CaM)-activated protein phosphatase that is highly conserved from fungi to mammals (66) and transmits Ca 2ϩ signals to elicit downstream responses mainly by regulating various transcriptional factors, such as CRZ1 and PRZ1 (19,67,68). CN consists of two subunits, a catalytic subunit A (encoded by CNA1 and CNA2) (66,69) and a regulatory subunit B (encoded by CNB1) (70).…”

“…(82,83). A calcium cell survival (CCS) pathway may be involved in the survival of cells subjected to a variety of cellular stresses, because the activation of a variety of Ca 2ϩ channels, calmodulin, calcineurin, and other factors is necessary for the long-term survival of cells undergoing ER stress, and the genes involved in this pathway are known to be essential in many cell biological processes; these essential components of the whole pathway would likely make good targets for antifungal therapy (19,56,(84)(85)(86).…”

“…One of the regulators of calcium homeostasis, calcineurin (CN), has been identified as a virulence factor in filamentous fungi, and some calcium channel proteins have been found to be responsible for the filamentation of these pathogenic fungi (18)(19)(20)(21). Moreover, calcium-mediated and calcineurin-mediated azole resistance has frequently been documented (22)(23)(24).…”

Fungal Keratitis: Update for 2014

“…A recent study revealed a vital role of calcineurin in the virulence of Candida sp. [56] This makes the calcineurin inhibitor and immunomodulator Tacrolimus potentially effective at providing some antifungal activity while delivering the desired immunosuppression. This also supports the rationale for utilizing topical cyclosporine, another calcineurin inhibitor, as prophylaxis of transplant rejection in cases of therapeutic keratoplasty.…”

Calcineurin in fungal virulence and drug resistance: Prospects for harnessing targeted inhibition of calcineurin for an antifungal therapeutic approach

“…10,12 For example, combinations of calcium and calcineurin inhibitors with known antifungal compounds have been shown to inhibit the growth of drug resistant fungal strains. [13][14][15][16][17][18][19] Therefore, inhibition of calcineurin signaling is a novel antifungal strategy that both attenuates fungal virulence and increases the efficacy of the existing antifungals with concomitant suppression of antifungal resistance. 10,20,21 Calcineurin is a conserved Ca 2C -calmodulin (CaM) activated protein phosphatase 2B belonging to the phospho-protein phosphatase family of enzymes and is involved in calcium-dependent signaling and regulation of several important cellular processes in both yeasts and filamentous fungi (Fig.…”