Harnessing calcineurin as a novel anti-infective agent against invasive fungal infections

The number of immunocompromised patients with invasive fungal infections continues to increase and new antifungal therapies are not keeping pace with the growing incidence of these infections and their associated mortality. Calcineurin inhibition is currently used to exert effective immunosuppression following organ transplantation and in treating various other conditions. However, the calcineurin pathway is also intricately involved in the growth and pathogenesis of the three major fungal pathogens of humans, Cryptococcus neoformans, Candida albicans and Aspergillus fumigatus, and the exploitation of fungal calcineurin pathways holds great promise for the future development of novel antifungal agents. This Review summarizes our current understanding of calcineurin biology in these fungal species, and its exciting potential role in treating invasive fungal infections.     

On the Roles of Calcineurin in Fungal Growth and Pathogenesis

Calcineurin is a calcium-activated phosphatase that controls morphogenesis and stress responses in eukaryotes. Fungal pathogens have adopted the calcineurin pathway to survive and effectively propagate within the host. The difficulty in treating fungal infections stems from similarities between pathogen and host eukaryotic cells. Using calcineurin inhibitors such as cyclosporin A or tacrolimus (FK506) in combination with antifungal drugs, including azoles or echinocandins, renders these drugs fungicidal, even towards drug-resistant species or strains, making calcineurin a promising drug target. This article summarizes the current understanding of the calcineurin pathway and its roles in governing the growth and virulence of pathogenic fungi, and compares and contrasts the roles of calcineurin in fungal pathogens that infect humans (Candida albicans and Cryptococcus neoformans) or plants (Magnaporthe oryzae and Ustilago maydis). Further investigation of calcineurin biology will advance opportunities to develop novel antifungal therapeutic approaches and provide insight into the evolution of virulence.     

Good fungi gone bad: the corruption of calcineurin

Calcineurin is a Ca(2+)/calmodulin-activated protein phosphatase that is conserved in eukaryotes, from yeast to humans, and is the conserved target of the immunosuppressive drugs cyclosporin A (CsA) and FK506. Genetic studies in yeast and fungi established the molecular basis of calcineurin inhibition by the cyclophilin A-CsA and FKBP12-FK506 complexes. Calcineurin also functions in fungi to control a myriad of physiological processes including cell cycle progression, cation homeostasis, and morphogenesis. Recent investigations into the molecular mechanisms of pathogenesis in Candida albicans and Cryptococcus neoformans, two fungi that cause life-threatening infections in humans, have revealed an essential role for calcineurin in morphogenesis, virulence, and antifungal drug action. Novel non-immunosuppressive analogs of the calcineurin inhibitors CsA and FK506 that retain antifungal activity have been identified and hold promise as candidate antifungal drugs. In addition, comparisons of calcineurin function in both fungi and humans may identify fungal-specific components of calcineurin-signaling pathways that could be targeted for therapy, as well as conserved elements of calcium signaling events.     

Comparative genomics of MAP kinase and calcium–calcineurin signalling components in plant and human pathogenic fungi

Mitogen-activated protein kinase (MAPK) cascades and the calcium–calcineurin pathway control fundamental aspects of fungal growth, development and reproduction. Core elements of these signalling pathways are required for virulence in a wide array of fungal pathogens of plants and mammals. In this review, we have used the available genome databases to explore the structural conservation of three MAPK cascades and the calcium–calcineurin pathway in ten different fungal species, including model organisms, plant pathogens and human pathogens. While most known pathway components from the model yeast Saccharomyces cerevisiae appear to be widely conserved among taxonomically and biologically diverse fungi, some of them were found to be restricted to the Saccharomycotina. The presence of multiple paralogues in certain species such as the zygomycete Rhizopus oryzae and the incorporation of new functional domains that are lacking in S. cerevisiae signalling proteins, most likely reflect functional diversification or adaptation as filamentous fungi have evolved to occupy distinct ecological niches.     

A calcineurin antifungal strategy with analogs of FK506

A novel antifungal strategy targeting the inhibition of calcineurin is described. To develop a calcineurin based inhibitor of pathogenic fungi, analogs of FK506 were synthesized that were able to permeate mammalian but not fungal cells. Antagonists in combination with FK506 were not immunosuppressive and retained antifungal activity in A. fumigatus. To reduce the dosage burden of the antagonist, murine oral PK was improved an order of magnitude relative to previous FK506 antagonists.     

Dendritic cells in antifungal immunity and vaccine design

Life-threatening fungal infections have increased in recent years while treatment options remain limited. The development of vaccines against fungal pathogens represents a key advance sorely needed to combat the increasing fungal disease threat. Dendritic cells (DC) are uniquely able to shape antifungal immunity by initiating and modulating naive T?cell responses. Targeting DC may allow for the generation of potent vaccines against fungal pathogens. In the context of antifungal vaccine design, we describe the characteristics of the varied DC subsets, how DC recognize fungi, their function in immunity against fungal pathogens, and how DC can be targeted in order to create new antifungal vaccines. Ongoing studies continue to highlight the critical role of DC in antifungal immunity and will help guide DC-based vaccine strategies.     

PKC Signaling Regulates Drug Resistance of the Fungal Pathogen Candida albicans via Circuitry Comprised of Mkc1, Calcineurin,and Hsp90

Fungal pathogens exploit diverse mechanisms to survive exposure to antifungal drugs. This poses concern given the limited number of clinically useful antifungals and the growing population of immunocompromised individuals vulnerable to life-threatening fungal infection. To identify molecules that abrogate resistance to the most widely deployed class of antifungals, the azoles, we conducted a screen of 1,280 pharmacologically active compounds. Three out of seven hits that abolished azole resistance of a resistant mutant of the model yeast Saccharomyces cerevisiae and a clinical isolate of the leading human fungal pathogen Candida albicans were inhibitors of protein kinase C (PKC), which regulates cell wall integrity during growth, morphogenesis, and response to cell wall stress. Pharmacological or genetic impairment of Pkc1 conferred hypersensitivity to multiple drugs that target synthesis of the key cell membrane sterol ergosterol, including azoles, allylamines, and morpholines. Pkc1 enabled survival of cell membrane stress at least in part via the mitogen activated protein kinase (MAPK) cascade in both species, though through distinct downstream effectors. Strikingly, inhibition of Pkc1 phenocopied inhibition of the molecular chaperone Hsp90 or its client protein calcineurin. PKC signaling was required for calcineurin activation in response to drug exposure in S. cerevisiae. In contrast, Pkc1 and calcineurin independently regulate drug resistance via a common target in C. albicans. We identified an additional level of regulatory control in the C. albicans circuitry linking PKC signaling, Hsp90, and calcineurin as genetic reduction of Hsp90 led to depletion of the terminal MAPK, Mkc1. Deletion of C. albicans PKC1 rendered fungistatic ergosterol biosynthesis inhibitors fungicidal and attenuated virulence in a murine model of systemic candidiasis. This work establishes a new role for PKC signaling in drug resistance, novel circuitry through which Hsp90 regulates drug resistance, and that targeting stress response signaling provides a promising strategy for treating life-threatening fungal infections.     

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

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.     

The contribution of the Toll-like/IL-1 receptor superfamily to innate and adaptive immunity to fungal pathogens in vivo

In vitro studies have indicated the importance of Toll-like receptor (TLR) signaling in response to the fungal pathogens Candida albicans and Aspergillus fumigatus. However, the functional consequences of the complex interplay between fungal morphogenesis and TLR signaling in vivo remain largely undefined. In this study we evaluate the impact of the IL-1R/TLR/myeloid differentiation primary response gene 88 (MyD88)-dependent signaling pathway on the innate and adaptive Th immunities to C. albicans and A. fumigatus in vivo. It was found that 1) the MyD88-dependent pathway is required for resistance to both fungi; 2) the involvement of the MyD88 adapter may occur through signaling by distinct members of the IL-1R/TLR superfamily, including IL-1R, TLR2, TLR4, and TLR9, with the proportional role of the individual receptors varying depending on fungal species, fungal morphotypes, and route of infection; 3) individual TLRs and IL-1R activate specialized antifungal effector functions on neutrophils, which correlates with susceptibility to infection; and 4) MyD88-dependent signaling on dendritic cells is crucial for priming antifungal Th1 responses. Thus, the finding that the innate and adaptive immunities to C. albicans and A. fumigatus require the coordinated action of distinct members of the IL-1R/TLR superfamily acting through MyD88 makes TLR manipulation amenable to the induction of host resistance to fungi.     

Functional characterization of the regulators of calcineurin in Candida glabrata

The serine-threonine-specific protein phosphatase calcineurin is a key mediator of various stress responses in fungi. Herein, we characterized functions of the endogenous regulators of calcineurin (RCNs), Rcn1 and Rcn2, in the pathogenic fungus Candida glabrata. Rcn1 exerted both inhibitory and stimulatory effects on calcineurin signaling, but Rcn2 displayed only inhibitory activity. Phenotypic analyses of C. glabrata strains lacking either RCNs, calcineurin, or both revealed that calcineurin requires Rcn1, but not Rcn2, for antifungal tolerance in C. glabrata.     

pH Signaling in Human Fungal Pathogens: a New Target for Antifungal Strategies

Fungi are exposed to broadly fluctuating environmental conditions, to which adaptation is crucial for their survival. An ability to respond to a wide pH range, in particular, allows them to cope with rapid changes in their extracellular settings. PacC/Rim signaling elicits the primary pH response in both model and pathogenic fungi and has been studied in multiple fungal species. In the predominant human pathogenic fungi, namely, Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans, this pathway is required for many functions associated with pathogenesis and virulence. Aspects of this pathway are fungus specific and do not exist in mammalian cells. In this review, we highlight recent advances in our understanding of PacC/Rim-mediated functions and discuss the growing interest in this cascade and its factors as potential drug targets for antifungal strategies. We focus on both conserved and distinctive features in model and pathogenic fungi, highlighting the specificities of PacC/Rim signaling in C. albicans, A. fumigatus, and C. neoformans. We consider the role of this pathway in fungal virulence, including modulation of the host immune response. Finally, as now recognized for other signaling cascades, we highlight the role of pH in adaptation to antifungal drug pressure. By acting on the PacC/Rim pathway, it may therefore be possible (i) to ensure fungal specificity and to limit the side effects of drugs, (ii) to ensure broad-spectrum efficacy, (iii) to attenuate fungal virulence, (iv) to obtain additive or synergistic effects with existing antifungal drugs through tolerance inhibition, and (v) to slow the emergence of resistant mutants.     

Evolution of antifungal-drug resistance: mechanisms and pathogen fitness

Like other microorganisms, fungi exist in populations that are adaptable. Under the selection imposed by antifungal drugs, drug-sensitive fungal pathogens frequently evolve resistance. Although the molecular mechanisms of resistance are well-characterized, there are few measurements of the impact of these mechanisms on pathogen fitness in different environments. To predict resistance before a new drug is prescribed in the clinic, the full spectrum of potential resistance mutations and the interactions among combinations of divergent mechanisms can be determined in evolution experiments. In the search for new strategies to manage drug resistance, measuring the limits of adaptation might reveal methods for trapping fungal pathogens in evolutionary dead ends.     

The RAM network in pathogenic fungi

The regulation of Ace2 and morphogenesis (RAM) network is a protein kinase signaling pathway conserved among eukaryotes from yeasts to humans. Among fungi, the RAM network has been most extensively studied in the model yeast Saccharomyces cerevisiae and has been shown to regulate a range of cellular processes, including daughter cell-specific gene expression, cell cycle regulation, cell separation, mating, polarized growth, maintenance of cell wall integrity, and stress signaling. Increasing numbers of recent studies on the role of the RAM network in pathogenic fungal species have revealed that this network also plays an important role in the biology and pathogenesis of these organisms. In addition to providing a brief overview of the RAM network in S. cerevisiae, we summarize recent developments in the understanding of RAM network function in the human fungal pathogens Candida albicans, Candida glabrata, Cryptococcus neoformans, Aspergillus fumigatus, and Pneumocystis spp.     

钙调磷酸酶信号调控真菌生长代谢、毒力及抗逆性能

钙调磷酸酶是一种丝氨酸/苏氨酸(Ser/Thr)蛋白磷酸酶,在真菌中普遍保守,上游信号途径由Ca2+通道(Cch1)、转运蛋白(Mid1)、钙离子感应蛋白(CaM)、钙调蛋白依赖性磷酸酶等组成。钙调磷酸酶受钙离子和钙调蛋白调节,在调控真菌Ca2+稳态的钙信号级联途径中发挥着中心作用,通过钙信号级联途径参与生物学过程,调控真菌生长、发育和毒力形成来响应外界环境因素的变化,使真菌能够适应不同环境,维持正常的生命活动。本文综述了真菌钙调磷酸酶信号的组成和上下游信号转导途径、调控细胞生长代谢、毒力形成以及抗逆性能调控的研究进展;结合对真菌代谢产物合成的调控作用,对钙调磷酸酶信号作为重要合成生物学元件及调控开关进行了展望。     

Structural and biological characterization of chromofungin, the antifungal chromogranin A-(47-66)-derived peptide.

Vasostatin-I, the natural fragment of chromogranin A-(1-76), is a neuropeptide able to kill a large variety of fungi and yeast cells in the micromolar range. We have examined the antifungal properties of synthetic vasostatin-I-related peptides. The most active shortest peptide, named chromofungin, corresponds to the sequence Arg(47)-Leu(66). Extensive (1)H NMR analysis revealed that it adopts a helical structure. The biophysical mechanism implicated in the interaction of chromofungin with fungi and yeast cells was studied, showing the penetration of this peptide with different lipid monolayers. In order to examine thoroughly the antifungal activity of chromofungin, confocal laser microscopy was used to demonstrate the ability of the rhodamine-labeled peptide to interact with the fungal cell wall, to cross the plasma membrane, and to accumulate in Aspergillus fumigatus, Alternaria brassicola, and Candida albicans. Our present data reveal that chromofungin inhibits calcineurin activity, extending a previous observation that the N-terminal region of chromogranin A interacts with calmodulin in the presence of calcium. Therefore, the destabilization of fungal wall and plasma membrane, together with the possible intracellular inhibition of calmodulin-dependent enzymes, is likely to represent the mechanism by which vasostatin-I and chromofungin exert antifungal activity.     

Tissue Diagnosis of Invasive Fungal Infections: Current Limitations and the Emerging Use of Molecular Techniques

Invasive fungal diseases (IFD) due to opportunistic fungi are commonly treated using empirical antifungal therapy. Therefore, a comprehensive study of organisms associated with IFD is essential to define successful empiric therapies in each setting. Current diagnostic tests, such as culture, histology and serology are suboptimal, leading to delays in the initiation of antifungal therapies and resulting in high mortality rates despite the availability of several new antifungal agents. Using molecular methods to identify fungal pathogens directly from formalin-fixed, paraffin-embedded tissues is emerging as a diagnostic approach. The goal of this molecular approach is to complement conventional diagnostic tests through the reliable detection and identification of fungal nucleic acids or antigens in tissues so as to direct antiinfective therapies and improve patient outcomes. Here we review challenges and recent advances in the identification of fungal pathogens from tissue samples by conventional and molecular methods.