Calcineurin as a multifunctional regulator: Unraveling novel functions in fungal stress responses,hyphal growth,drug resistance,and pathogenesis

Calcineurin signaling plays diverse roles in fungi in regulating stress responses, morphogenesis and pathogenesis. Although calcineurin signaling is conserved among fungi, recent studies indicate important divergences in calcineurin-dependent cellular functions among different human fungal pathogens. Fungal pathogens utilize the calcineurin pathway to effectively survive the host environment and cause life-threatening infections. The immunosuppressive calcineurin inhibitors (FK506 and cyclosporine A) are active against fungi, making targeting calcineurin a promising antifungal drug development strategy. Here we summarize current knowledge on calcineurin in yeasts and filamentous fungi, and review the importance of understanding fungal-specific attributes of calcineurin to decipher fungal pathogenesis and develop novel antifungal therapeutic approaches.     

Calcineurin orchestrates dimorphic transitions,antifungal drug responses and host–pathogen interactions of the pathogenic mucoralean fungus Mucor circinelloides

Calcineurin plays essential roles in virulence and growth of pathogenic fungi and is a target of the natural products FK506 and Cyclosporine A. In the pathogenic mucoralean fungus Mucor circinelloides, calcineurin mutation or inhibition confers a yeast‐locked phenotype indicating that calcineurin governs the dimorphic transition. Genetic analysis in this study reveals that two calcineurin A catalytic subunits (out of three) are functionally diverged. Homology modeling illustrates modes of resistance resulting from amino substitutions in the interface between each calcineurin subunit and the inhibitory drugs. In addition, we show how the dimorphic transition orchestrated by calcineurin programs different outcomes during host–pathogen interactions. For example, when macrophages phagocytose Mucor yeast, subsequent phagosomal maturation occurs, indicating host cells respond appropriately to control the pathogen. On the other hand, upon phagocytosis of spores, macrophages fail to form mature phagosomes. Cytokine production from immune cells differs following exposure to yeast versus spores (which germinate into hyphae). Thus, the morphogenic transition can be targeted as an efficient treatment option against Mucor infection. In addition, genetic analysis (including gene disruption and mutational studies) further strengthens the understanding of calcineurin and provides a foundation to develop antifungal agents targeting calcineurin to deploy against Mucor and other pathogenic fungi.     

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.     

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.     

Fungal Secondary Metabolites and Their Fundamental Roles in Human Mycoses

Understanding which fungal factors allow colonization and infection of a human host is critical to lowering the incidence of human mycoses and related mortalities. In the pathogen Aspergillus fumigatus, secondary metabolites, small bioactive molecules produced by many opportunistic fungal pathogens, have important roles in suppressing and providing protection from host defenses. Deletion of LaeA, a global regulator of secondary metabolism in fungi, significantly decreases A. fumigatus virulence, in part owing to loss of gliotoxin and hydrophobin production. In addition to gliotoxin, dihydroxynaphthalene (DHN) melanin and siderophores are other A. fumigatus virulence factors; all three metabolites are derived from hallmark secondary metabolite gene clusters. Many of the gene clusters producing toxin metabolites have yet to be deciphered, and the study of secondary metabolites and their role in the virulence of human pathogens is a nascent field.     

Fungal and host protein persulfidation are functionally correlated and modulate both virulence and antifungal response

Aspergillus fumigatus is a human fungal pathogen that can cause devastating pulmonary infections, termed “aspergilloses,” in individuals suffering immune imbalances or underlying lung conditions. As rapid adaptation to stress is crucial for the outcome of the host–pathogen interplay, here we investigated the role of the versatile posttranslational modification (PTM) persulfidation for both fungal virulence and antifungal host defense. We show that an A. fumigatus mutant with low persulfidation levels is more susceptible to host-mediated killing and displays reduced virulence in murine models of infection. Additionally, we found that a single nucleotide polymorphism (SNP) in the human gene encoding cystathionine γ-lyase (CTH) causes a reduction in cellular persulfidation and correlates with a predisposition of hematopoietic stem cell transplant recipients to invasive pulmonary aspergillosis (IPA), as correct levels of persulfidation are required for optimal antifungal activity of recipients’ lung resident host cells. Importantly, the levels of host persulfidation determine the levels of fungal persulfidation, ultimately reflecting a host–pathogen functional correlation and highlighting a potential new therapeutic target for the treatment of aspergillosis.

This study reveals that the post-translational modification persulfidation is important for both fungal virulence and the host antifungal response. The level of persulfidation in the host, which correlates with its antifungal potency, impacts the level required in the fungus to counteract host attack, reflecting a functional correlation. Thus modulating persulfidation may be a promising strategy to target both pathogens and immune responses.     

The diverse roles of autophagy in medically important fungi

Autophagy is a highly conserved eukaryotic mechanism whereby cells recycle cellular elements to survive under adverse conditions. Surprisingly, of the three fungal pathogens of greatest relevance to human health, only Cryptococcus neoformans has been shown to require this process during infection. In contrast, autophagy is dispensable for the virulence of both Candida albicans and Aspergillus fumigatus. The divergent roles for autophagy in these opportunistic species underscore the uniqueness of the host infection niche occupied by each fungus and provide insights into the evolutionary pressures that may have influenced the need for autophagy during infection. Further study of fungal autophagy may reveal the host signals which induce this protective response and determine if these signals differ between host cells or tissues. In addition, a comprehensive understanding of the autophagy machinery in fungal pathogens may provide a rational basis for the design of future therapeutic interventions to improve outcome in patients who are at risk for these infections.     

The Cryptococcus neoformans MAP kinase Mpk1 regulates cell integrity in response to antifungal drugs and loss of calcineurin function

Cell wall integrity is crucial for fungal growth, development and stress survival. In the model yeast Saccharomyces cerevisiae, the cell integrity Mpk1/Slt2 MAP kinase and calcineurin pathways monitor cell wall integrity and promote cell wall remodelling under stress conditions. We have identified the Cryptococcus neoformans homologue of the S. cerevisiae Mpk1/Slt2 MAP kinase and have characterized its role in the maintenance of cell integrity in response to elevated growth temperature and in the presence of cell wall synthesis inhibitors. C. neoformans Mpk1 is required for growth at 37 degrees C in vitro, and this growth defect is suppressed by osmotic stabilization. C. neoformans mutants lacking Mpk1 are attenuated for virulence in the mouse model of cryptococcosis. Phosphorylation of Mpk1 is induced in response to perturbations of cell wall biosynthesis by the antifungal drugs nikkomycin Z (a chitin synthase inhibitor), caspofungin (a beta-1,3-glucan synthase inhibitor), or FK506 (a calcineurin inhibitor), and mutants lacking Mpk1 display enhanced sensitivity to nikkomycin Z and caspofungin. Lastly, we show that calcineurin and Mpk1 play complementing roles in regulating cell integrity in C. neoformans. Our studies demonstrate that pharmacological inhibition of the cell integrity pathway would enhance the activity of antifungal drugs that target the cell wall.     

Virulence phenotypes result from interactions between pathogen ploidy and genetic background

Studying fungal virulence is often challenging and frequently depends on many contexts, including host immune status and pathogen genetic background. However, the role of ploidy has often been overlooked when studying virulence in eukaryotic pathogens. Since fungal pathogens, including the human opportunistic pathogen Candida albicans, can display extensive ploidy variation, assessing how ploidy impacts virulence has important clinical relevance. As an opportunistic pathogen, C. albicans causes nonlethal, superficial infections in healthy individuals, but life‐threatening bloodstream infections in individuals with compromised immune function. Here, we determined how both ploidy and genetic background of C. albicans impacts virulence phenotypes in healthy and immunocompromised nematode hosts by characterizing virulence phenotypes in four near‐isogenic diploid and tetraploid pairs of strains, which included both laboratory and clinical genetic backgrounds. We found that C. albicans infections decreased host survival and negatively impacted host reproduction, and we leveraged these two measures to survey both lethal and nonlethal virulence phenotypes across the multiple C. albicans strains. In this study, we found that regardless of pathogen ploidy or genetic background, immunocompromised hosts were susceptible to fungal infection compared to healthy hosts. Furthermore, for each host context, we found a significant interaction between C. albicans genetic background and ploidy on virulence phenotypes, but no global differences between diploid and tetraploid pathogens were observed.     

Cryptococcal therapies and drug targets: the old,the new and the promising

Half a century after the introduction of Amphotericin B the management of cryptococcosis remains unsatisfactory. The disease, caused primarily by the two fungal species Cryptococcus neoformans and Cryptococcus gattii, remains responsible for considerable morbidity and mortality despite standard medical care. Current therapeutic options are limited to Amphotericin B, azoles and 5‐flucytosine. However, this organism has numerous well‐characterized virulence mechanisms that are amenable to pharmacological interference and are thus potential therapeutic targets. Here, we discuss existing approved antifungal drugs, resistance mechanisms to these drugs and non‐standard antifungal drugs that have potential in treatment of cryptococcosis, including immunomodulatory strategies that synergize with antifungal drugs, such as cytokine administration or monoclonal antibodies. Finally, we summarize attempts to target well‐described virulence factors of Cryptococcus, the capsule or fungal melanin. This review emphasizes the pressing need for new therapeutic alternatives for cryptococcosis.     

Calcium: a central player in Cryptococcus biology

Adaptation to the host environment is crucial for fungal pathogenesis. Calcium (Ca²⁺) signals are essential for fungal cells to respond rapidly to stress stimuli. In eukaryotic cells, Ca²⁺ is the main intracellular secondary messenger and regulates a myriad of processes, including the cellular fitness of the fungal pathogen Cryptococcus neoformans. In this minireview, we highlight the main cryptococcal processes regulated by Ca²⁺. Moreover, we underline all the characterized proteins responsible for intracellular calcium homeostasis in this yeast, such as Ca²⁺ transporters and binding proteins. These elements, in general, are essential for C. neoformans’ growth and adaptation to the host environment, as well as to virulence mechanisms. We also revisit the specific traits of the calcineurin signaling pathway in C. neoformans, which is the major pathway regulated by calcium and is crucial for yeast pathogenesis, adaptation, and growth at 37 °C. Notably, several Ca²⁺-related functions are highly conserved throughout fungal cells. Moreover, C. neoformans exhibits exclusive, significant features that are required for disease progression, thus attracting attention as feasible targets for antifungal drug development. Collectively, all the available data related to Ca²⁺ processes clarify the complex role that Ca²⁺ plays within cryptococcal cells, participating in host adaptation, transmigration, antifungal resistance, cell growth, and more.     

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.     

Fungicide effects on human fungal pathogens: Cross-resistance to medical drugs and beyond

Fungal infections are underestimated threats that affect over 1 billion people, and Candida spp., Cryptococcus spp., and Aspergillus spp. are the 3 most fatal fungi. The treatment of these infections is performed with a limited arsenal of antifungal drugs, and the class of the azoles is the most used. Although these drugs present low toxicity for the host, there is an emergence of therapeutic failure due to azole resistance. Drug resistance normally develops in patients undergoing azole long-term therapy, when the fungus in contact with the drug can adapt and survive. Conversely, several reports have been showing that resistant isolates are also recovered from patients with no prior history of azole therapy, suggesting that other routes might be driving antifungal resistance. Intriguingly, antifungal resistance also happens in the environment since resistant strains have been isolated from plant materials, soil, decomposing matter, and compost, where important human fungal pathogens live. As the resistant fungi can be isolated from the environment, in places where agrochemicals are extensively used in agriculture and wood industry, the hypothesis that fungicides could be driving and selecting resistance mechanism in nature, before the contact of the fungus with the host, has gained more attention. The effects of fungicide exposure on fungal resistance have been extensively studied in Aspergillus fumigatus and less investigated in other human fungal pathogens. Here, we discuss not only classic and recent studies showing that environmental azole exposure selects cross-resistance to medical azoles in A. fumigatus, but also how this phenomenon affects Candida and Cryptococcus, other 2 important human fungal pathogens found in the environment. We also examine data showing that fungicide exposure can select relevant changes in the morphophysiology and virulence of those pathogens, suggesting that its effect goes beyond the cross-resistance.     

The contribution of <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> stress responses to virulence and antifungal resistance

Invasive aspergillosis has emerged as one of the most common life-threatening fungal disease of humans. The emergence of antifungal resistant pathogens represents a current and increasing threat to society. In turn, new strategies to combat fungal infection are urgently required. Fungal adaptations to stresses experienced within the human host are a prerequisite for the survival and virulence strategies of the pathogen. Here, we review the latest information on the signalling pathways in Aspergillus fumigatus that contribute to stress adaptations and virulence, while highlighting their potential as targets for the development of novel combinational antifungal therapies.     

Inhibition of Candida albicans growth by brominated furanones

Candida albicans is the most virulent Candida species of medical importance, which presents a great threat to immunocompromised individuals such as HIV patients. Currently, there are only four classes of antifungal agents available for treating fungal infections: azoles, polyenes, pyrimidines, and echinocandins. The fast spread of multidrug resistant C. albicans strains has increased the demand for new antifungal drugs. In this study, we demonstrate the antifungal activity of brominated furanones on C. albicans. Studying the structure and activity of this class of furanones reveals that the exocyclic vinyl bromide conjugated with the carbonyl group is the most important structural element for fungal inhibition. Furthermore, gene expression analysis using DNA microarrays showed that 3 μg/mL of 4-bromo-5Z-(bromomethylene)-3-butylfuran-2-one (BF1) upregulated 32 C. albicans genes with functions of stress response, NADPH dehydrogenation, and small-molecule transport, and repressed 21 genes involved mainly in cell-wall maintenance. Interestingly, only a small overlap is observed between the gene expression changes caused by the representative brominated furanone (BF1) in this study and other antifungal drugs reported in literature. This result suggests that brominated furanones and other antifungal drugs may target different fungal proteins or genes. The existence of such new targets provides an opportunity for developing new agents to control fungal pathogens which are resistant to currently available drugs.