Regulatory circuitry governing fungal development, drug resistance, and disease

Abstract

Pathogenic fungi have become a leading cause of human mortality due to the increasing frequency of fungal infections in immunocompromised populations and the limited armamentarium of clinically useful antifungal drugs. Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus are the leading causes of opportunistic fungal infections. In these diverse pathogenic fungi, complex signal transduction cascades are critical for sensing environmental changes and mediating appropriate cellular responses. For C. albicans, several environmental cues regulate a morphogenetic switch from yeast to filamentous growth, a reversible transition important for virulence. Many of the signaling cascades regulating morphogenesis are also required for cells to adapt and survive the cellular stresses imposed by antifungal drugs. Many of these signaling networks are conserved in C. neoformans and A. fumigatus, which undergo distinct morphogenetic programs during specific phases of their life cycles. Furthermore, the key mechanisms of fungal drug resistance, including alterations of the drug target, overexpression of drug efflux transporters, and alteration of cellular stress responses, are conserved between these species. This review focuses on the circuitry regulating fungal morphogenesis and drug resistance and the impact of these pathways on virulence. Although the three human-pathogenic fungi highlighted in this review are those most frequently encountered in the clinic, they represent a minute fraction of fungal diversity. Exploration of the conservation and divergence of core signal transduction pathways across C. albicans, C. neoformans, and A. fumigatus provides a foundation for the study of a broader diversity of pathogenic fungi and a platform for the development of new therapeutic strategies for fungal disease.

Fig. 1.

Antifungal drugs and their targets. (A) The azoles function by targeting the ergosterol biosynthetic enzyme lanosterol demethylase, encoded by ERG11 (C. albicans and C. neoformans) or cyp51A and cyp51B (A. fumigatus), causing a block in the production of ergosterol and the accumulation of a toxic sterol produced by Erg3. This toxic sterol exerts a severe membrane stress on the cell. (B) The fungicidal polyenes are amphipathic drugs that function by binding to ergosterol to create drug-lipid complexes, which intercalate into the fungal cell membrane to form a membrane-spanning channel. This causes cellular ions to leak out of the cell, destroying the proton gradient and culminating in osmotic cellular lysis. (C) Fungal cell walls are composed of (1,3)-β-d-glucans covalently linked to (1,6)-β-d-glucans as well as chitin, mannans, and cell wall proteins. The echinocandins act as noncompetitive inhibitors of (1,3)-β-d-glucan synthase (encoded by FKS1 in C. albicans, C. neoformans, and A. fumigatus and by FKS1 and FKS2 in C. glabrata and S. cerevisiae) and thereby cause a loss of cell wall integrity and severe cell wall stress. (Adapted from reference with permission of Nature Publishing Group.)

Fig. 2.

The distinct morphogenetic states of C. albicans, including yeast, filaments, and biofilms. C. albicans transitions between yeast and filamentous growth states and also forms biofilms, which are complex surface-associated communities composed of multiple cell types. Yeast cells are shown under differential interference contrast (DIC) microscopy (top left) or scanning electron microscopy (SEM) (bottom left) (644). Filaments are heterogeneous structures, which vary greatly depending on the signaling cue that induces them. The filaments depicted (middle panels) were induced by different environmental cues: cell cycle arrest upon the depletion of CDC5 (19), 10% serum at 37°C, compromised Hsp90 function by treatment with geldanamycin, and pseudohyphae produced in medium at pH 6.0 at 35°C (570). The biofilm image on the right is an SEM image of a mature (48-h) biofilm composed of yeast and filamentous cells (477). Scale bars indicate 10 μm. (Image of yeast cells reprinted from reference with permission from the Society for General Microbiology; image of cell cycle-arrested filaments reprinted from reference with permission from John Wiley and Sons; image of pseudohyphae reprinted from reference 570 with permission from Elsevier; image of biofilm reprinted from reference with permission.)

Fig. 3.

Environmental cues and corresponding pathways that mediate morphogenesis in C. albicans. C. albicans transitions between distinct morphogenetic states, including yeast, pseudohyphae, and hyphae, as depicted. Numerous environmental signals mediate the transitions between yeast and filamentous forms. Cues and pathways at the top mediate filament-to-yeast morphogenesis, and cues and pathways at the bottom mediate yeast-to-filament morphogenesis.

Fig. 4.

Key cellular signaling cascades regulating morphogenesis in C. albicans. Numerous signaling pathways regulate C. albicans morphogenesis; the six most well-characterized pathways along with many of the key proteins involved in each pathway are depicted.

Fig. 5.

C. albicans drug resistance mechanisms. (A) C. albicans can acquire resistance to the azoles through multiple mechanisms, including the upregulation or alteration of the drug target Erg11; the upregulation of the multidrug transporter Cdr1, Cdr2, or Mdr1 (fluconazole specific); or the induction of numerous cellular stress responses. (B) Although resistance to the polyenes is rare in C. albicans, resistance is acquired through loss-of-function mutations in ERG3, which block the production of ergosterol, inhibit the formation of the drug-lipid complex, and therefore prevent osmotic cellular lysis. The alteration of the drug transporters does not play a major role in polyene resistance, and cellular stress responses have not been implicated as major determinants of resistance. (C) Resistance to the echinocandins through mutations in two distinct hot-spot regions in FKS1, encoding the catalytic subunit of (1,3)-β-d-glucan synthase, has been widely found in C. albicans isolates. The upregulation of drug transporters does not play a major role in resistance; however, the induction of cellular stress responses is important for echinocandin resistance. The bullet points below each mechanism describe the manner in which resistance is acquired. Bright images represent those mechanisms important for that particular drug class, whereas dimmed images represent those mechanisms that do not play a key role. (Adapted from reference with permission of Nature Publishing Group.)

Fig. 6.

Role of Hsp90 in antifungal drug resistance in C. albicans. (A) Simplified schematic of the mechanisms by which Hsp90 regulates responses to antifungals important for basal tolerance and resistance. Hsp90 interacts with and stabilizes the catalytic subunit of calcineurin (Cna1) to enable calcineurin-dependent stress responses through the effector protein Crz1 and through an additional target. Drug-induced stress also activates signaling through the Pkc1-regulated MAPK cascade, where the terminal kinase Mkc1 is an Hsp90 client protein. Notably, Pkc1 also signals through a distinct pathway in common with calcineurin to regulate antifungal drug resistance and tolerance. Hsp90, calcineurin, and PKC signaling regulate resistance to drugs that target the cell membrane and the cell wall. (B) A C. albicans laboratory strain (CAI4) and a series of clinical isolates (CaCi) obtained from an HIV-1-infected individual who was undergoing fluconazole (FL) treatment over the course of 2 years were evaluated for fluconazole resistance using an MIC assay. Clinical isolates at the top were recovered early in treatment, and those at the bottom were recovered late in treatment. Growth differences are color coded, with the brightest green representing maximal growth, light green representing intermediate growth, and black representing no growth. The laboratory strain was unable to grow at any concentration of fluconazole tested, while the clinical isolates displayed robust growth. The Hsp90 inhibitor geldanamycin (GdA), the calcineurin inhibitor cyclosporine (CsA), or the PKC inhibitor staurosporine (STS) reduced the resistance of all clinical isolates but had a greater effect on isolates recovered early in treatment than on those recovered late. (Left three panels of panel B adapted from reference with permission of AAAS; right panel of panel B adapted from reference .)

Fig. 7.

The C. neoformans life cycle. C. neoformans exists most commonly as a budding yeast; however, it is capable of undergoing a dimorphic transition to filamentous growth by two main differentiation pathways: mating and monokaryotic fruiting. The mating pathway is initiated, under nutrient-limiting conditions, with the fusion of haploid cells of opposite mating types to produce dikaryotic filaments. During this time the two parental nuclei migrate coordinately in the hyphae, a septum forms to separate the cells, one nucleus is transferred to the penultimate hyphal cell via a clamp connection, and the clamp cell and hyphal cell fuse. During this hyphal growth, blastospores can bud from the hyphae and divide mitotically in the yeast form. Some hyphal cells can enlarge and form chlamydospores. As the basidium continues to develop, meiosis occurs, where eventually four chains of basidiospores are produced. The second pathway regulating morphogenesis, monokaryotic fruiting, occurs when haploid spores produce filaments and basidiospores in response to severe nitrogen starvation or water deprivation in the absence of a mating partner. Cells of one mating type form diploid monokaryotic hyphae, where rudimentary clamp connections are formed, which do not fuse to the preceding cell. Blastospores and chlamydospores can also form. At the stage of basidium development, meiosis occurs, and haploid basidiospores are produced in four chains. Finally, C. neoformans is capable of forming extremely large polyploid cells, referred to as “titan cells” or “giant cells,” in a human host. Microscopy images of the various stages of the C. neoformans life cycle are included beside their cartoon representations. Red circles represent MATα nuclei, blue circles represent MATa nuclei, purple circles represent diploid nuclei with a/α content, and gray circles represent either MATα or MATa nuclei. (Figure adapted from reference with permission from Annual Reviews; scanning electron microscopy images reprinted from reference with permission; image of titan cell courtesy of K. Nielsen [University of Minnesota], reproduced with permission; images of chlamydospores reprinted from reference with permission.)

Fig. 8.

Comparison of cAMP-PKA and MAPK signaling cascades in C. albicans, C. neoformans, and A. fumigatus. The cAMP-PKA signaling cascade and MAPK cascade represent two key morphogenetic signaling pathways conserved in these species. The MAPK signaling cascades modulating morphogenesis in A. fumigatus (the HOG-MAPK pathway and cell wall integrity MAPK pathway) are distinct from the MAPK module depicted in this figure. (Top) The cAMP-PKA pathway. (Bottom) The MAPK cascade. Corresponding colors between pathways indicate orthologous proteins between the species. Gray arrows indicate that links between certain factors in this pathway have not yet been established.

Fig. 9.

The calcineurin signaling pathway in C. albicans, C. neoformans, and A. fumigatus. Shown is a simplified schematic of how calcineurin regulates a myriad of responses in C. albicans, C. neoformans, and A. fumigatus. In C. albicans, the activation of the Cch1-Mid1 channel leads to the accumulation of intracellular Ca²⁺, which is bound by calmodulin (encoded by CAM1), leading to the activation of calcineurin. The molecular chaperone Hsp90 physically interacts with the catalytic subunit of calcineurin, Cna1, keeping it poised for activation. Once activated, calcineurin dephosphorylates the transcription factor Crz1 as well as other unknown effectors to regulate a myriad of cellular responses. In C. neoformans and A. fumigatus, homologues of this signaling pathway are depicted in identical colors. Components that have been identified only based on sequence homology are dimmed. Drugs and signaling molecules are depicted as stars, whereas proteins are depicted as circles. The cellular responses mediated by calcineurin are listed below the pathway. (Adapted from reference with permission of Macmillan Publishers Ltd.)

Fig. 10.

C. neoformans drug resistance mechanisms. C. neoformans can acquire resistance to the azoles through multiple mechanisms, including the upregulation or alteration of the drug target Erg11, the upregulation of the multidrug transporter Afr1, or the induction of numerous cellular stress responses. The bullet points below each mechanism describe the manner in which resistance is acquired. Notably, in C. neoformans, resistance to the polyenes is extremely rare. Furthermore, C. neoformans displays intrinsic resistance to the echinocandins. (Adapted from reference with permission of Nature Publishing Group.)

Fig. 11.

The life cycle and distinct morphological states of A. fumigatus. Like other filamentous fungi, A. fumigatus produces conidiophore structures (scale bar, 10 μm), which produce conidial spores (SEM images) (scale bar, 2 μm) through the process of conidiation. Under certain environmental conditions, conidia can germinate, develop, and begin to undergo polarized growth, ultimately becoming hyphal cells (scale bar, 20 μm). Hyphal cells will continue to grow, elongate, and branch and can eventually go on to form conidiophores. (Image of spores reprinted from reference with permission; image of hyphae courtesy of W. J. Steinbach [Duke University Medical Center], reproduced with permission; image of conidiophore courtesy of A. Beauvais and J. P. Latgé [Institut Pasteur, France], reproduced with permission.)

Fig. 12.

A. fumigatus drug resistance mechanisms. (A) A. fumigatus can acquire resistance to the azoles through multiple mechanisms, including the upregulation or alteration of the drug target Cyp51A or the upregulation of the multidrug transporters AtrF, Mdr3, and Mdr4. (B) Resistance to the echinocandins through mutations in fks1, encoding the catalytic subunit of (1,3)-β-d-glucan synthase, has been discovered in experimentally evolved populations but has not yet been found in the clinic. Numerous stress response pathways are also important for the basal tolerance and resistance of A. fumigatus to echinocandins. The bullet points below each mechanism describe the manner in which resistance is acquired. Bright images represent those mechanisms important for that particular drug class, whereas dimmed images represent those mechanisms that do not play a key role. (Adapted from reference with permission of Nature Publishing Group.)