Conserved and rewired MAPK networks in human fungal pathogens

Abstract

Mitogen-activated protein kinase (MAPK) cascades are among the most ancient and evolutionarily conserved signalling modules in eukaryotes, translating extracellular cues into adaptive transcriptional and metabolic outputs through a hierarchical three-tier kinase architecture. In yeasts, these pathways control osmotic and oxidative stress adaptation, mating, morphogenesis, and cell wall remodelling, while in fungal pathogens they have been adapted to support virulence-factor production, host adaptation, and antifungal tolerance. Here, we summarise the current understanding of MAPK signalling in the budding model yeast Saccharomyces cerevisiae, which serves throughout as a reference template, and in four human-pathogenic fungi that span the fungal kingdom—the ascomycetous yeast Candida albicans, the emerging multidrug-resistant pathogen Candidozyma (formerly Candida) auris, the filamentous mould Aspergillus fumigatus, and the basidiomycetous yeast Cryptococcus neoformans—emphasising how a conserved kinase framework has been functionally diversified to meet species-specific demands. We focus on the MAPK signalling system of C. neoformans, which is characterised by five MAPK homologues arising from paralogue retention and pervasive pathway crosstalk. We further discuss how recent systematic genetic dissection is prompting a reassessment of MAPK redundancy and network plasticity. Finally, we consider how the divergence between fungal and host MAPK signalling defines new conceptual and therapeutic directions in medical mycology.

This review compares how disease-causing fungi use related MAPK signaling pathways to survive stress and cause infection, revealing evolutionary differences that point toward promising new targets for antifungal drugs.

Introduction

Mitogen-activated protein kinases (MAPKs) are serine/threonine kinases that are central to eukaryotic signal transduction. They convert diverse extracellular stimuli into specific cellular responses through transcriptional, metabolic, and cytoskeletal reprogramming. Such stimuli include nutrients, mating pheromones, osmotic perturbation, oxidative insults, mechanical stress, and host-derived cues (Gustin et al. 1998). Their molecular architecture is highly uniform across eukaryotes: a MAP kinase kinase kinase (MAP3K) phosphorylates a MAP kinase kinase (MAP2K), which in turn dually phosphorylates a terminal MAPK on a conserved Thr–X–Tyr (TXY) activation-loop motif, triggering engagement of target substrates such as transcription factors and other protein kinases (Chen and Thorner 2007). Scaffold proteins, docking motifs and dual-specificity phosphatases add specificity and temporal control to this hierarchical cascade, allowing parallel MAPK pathways to coexist without spurious crosstalk (Garrenton et al. 2006).

Fungi were central to establishing this paradigm. The budding yeast Saccharomyces cerevisiae provided the first comprehensive genetic dissection of eukaryotic MAPK cascades, defining the mating, filamentous growth, cell wall integrity (CWI), high-osmolarity glycerol (HOG), and sporulation modules that continue to anchor our conceptual framework today (Chen and Thorner 2007). Notably, mammalian MAPKs can functionally substitute for their yeast counterparts: human CSBP1/p38α complements the osmosensitive phenotype of S. cerevisiae hog1Δ mutants (Kumar et al. 1995), while human ERK5 can substitute for the CWI MAPK Slt2/Mpk1 (Truman et al. 2006). These findings underscore the deep evolutionary conservation of the core MAPK machinery.

In human-pathogenic fungi, the MAPK toolkit has been adapted to support survival within the host, stress tolerance and virulence-associated traits. Cryptococcus neoformans, Candida albicans, Candidozyma auris (formerly Candida auris), and Aspergillus fumigatus constitute the critical priority group of the World Health Organization (WHO) fungal priority pathogens list (FPPL) and together account for a substantial share of the global burden of invasive fungal disease (WHO 2022). Among these, C. neoformans is ranked first and is the predominant cause of fungal meningoencephalitis, which affects an estimated 194 000 people and causes ∼147 000 deaths each year (case-fatality rate ∼76%), mainly among people with advanced HIV disease (Denning 2024). The central argument of this review is that fungal MAPK pathways are best understood not as conserved linear cascades but as adaptable signalling networks. Extensive crosstalk, shared upstream sensors, paralogue retention and species-specific pathway expansions create an interconnected network whose overall behaviour cannot be predicted from individual pathway studies alone (Bahn et al. 2006, So et al. 2018). This is most conspicuous in the upstream sensor layer, where the hybrid histidine kinase families that feed the HOG module are markedly expanded in pathogenic species. Together these features generate compensatory buffering. As a consequence, the phenotypic impact of losing any single MAPK depends on the state of the remaining network rather than on the nominal function of that pathway alone, and the apparent essentiality of an individual pathway may be conditional rather than absolute.

This review summarises current understanding of MAPK signalling in fungi, with an emphasis on human-pathogenic species. Because the network-level argument developed here is most fully documented in C. neoformans, that species receives the most detailed treatment; S. cerevisiae serves as a reference template, and C. albicans, A. fumigatus, and C. auris are discussed both individually and as comparative reference points. We first outline the canonical MAPK modules and their regulatory logic, then summarise the foundational architecture defined in S. cerevisiae. We next discuss how this template has diverged in each pathogen and how the resulting networks govern the production of canonical virulence factors. Finally, we consider MAPK pathway crosstalk, evolutionary plasticity and the prospects for MAPK-directed antifungal therapy. These sections draw on systematic genetic and transcriptomic dissections that are beginning to reshape our view of pathway redundancy and compensatory signalling capacity.

The canonical MAPK module: architecture, regulation, and evolutionary conservation

The canonical MAPK signalling cascade is an ancient and broadly conserved eukaryotic module that comprises three sequentially activated kinases (MAP3K → MAP2K → MAPK) that amplify and propagate signals while encoding specificity through tightly regulated phosphorylation events (Fig. 1A) (Chen and Thorner 2007, Keshet and Seger 2010). MAP3Ks, which sit at the apex of the cascade, become active in response to upstream stimuli via diverse mechanisms, including small GTPase binding (Ras/Rho family) and phosphorylation by sensor or scaffold-associated kinases (Keshet and Seger 2010). Once active, MAP3Ks phosphorylate MAP2Ks at a conserved S/T–X_3–5–S/T motif. MAP2Ks are dual-specificity kinases that in turn dually phosphorylate the terminal MAPK at the canonical TXY motif located in the activation loop between subdomains VII and VIII of the kinase domain; in classical fungal MAPKs, Fus3/Kss1- and Slt2/Mpk1-type kinases generally carry TEY motifs, whereas Hog1-type stress-activated MAPKs carry TGY motifs (Fig. 1B) (Chen and Thorner 2007).

Figure 1.

Architecture and conservation of the eukaryotic MAPK module. (A) Generalised organisation of a canonical mitogen-activated protein kinase (MAPK) cascade. Diverse external stimuli, including osmotic stress, oxidative stress (ROS), cell wall stress, and mating pheromone, are detected by upstream sensors and relayed to a three-tiered kinase core. The MAP3K is phosphorylated by diverse mechanisms, the MAP2K on Ser/Thr residues, and the terminal MAPK on the Thr and Tyr residues of its activation-loop motif; scaffold proteins organise the tiers to promote signalling fidelity. Activated MAPK phosphorylates substrates, including transcription factors (TFs), to drive cellular outputs such as stress adaptation, cell wall remodelling, cell morphology, and mating. Encircled “P” denotes a phosphorylation event. (B) Structural and regulatory features of MAPK signalling. Structural insets show the TEY-containing activation loop of non-phosphorylated S. cerevisiae Fus3 (PDB: 2B9F) and the recognition of a Ste7 D/KIM docking peptide by the Fus3 docking groove in the Fus3 bound to a Ste7 docking peptide (PDB: 2B9H) (Remenyi et al. 2005). These experimentally determined Fus3 structures are used to illustrate the TEY-containing activation loop and the docking groove that recognises D/KIM motif-containing partners. Scaffold proteins organise MAPK cascade components and promote ordered phosphotransfer from MAP3K to MAP2K and then to MAPK. MAPK phosphatases, including dual-specificity and tyrosine phosphatases, remove activation-loop phosphorylation to attenuate and reset signalling. Activated MAPKs can also phosphorylate upstream adaptors or pathway components, providing negative feedback that dampens further signalling input and limits inappropriate pathway activation. Created in BioRender (https://BioRender.com/6ykpizs).

Several additional features are essential for the fidelity of MAPK signalling. First, docking motifs on substrates and regulators, particularly D/KIM-type motifs that engage conserved MAPK docking surfaces, position interaction partners near the kinase domain and confer pathway selectivity (Tanoue et al. 2000, Kusari et al. 2004). Second, scaffold proteins such as Ste5 in the S. cerevisiae mating pathway physically organise the three kinase tiers, promoting efficient Fus3 activation while limiting inappropriate signalling through parallel pathways that share upstream kinases (Good et al. 2009). Finally, MAPK phosphatases and feedback phosphorylation events terminate or tune signalling. In yeasts, the dual-specificity phosphatase Msg5 and the tyrosine phosphatases Ptp2 and Ptp3 dephosphorylate MAPKs to attenuate responses once homeostasis is restored (Wurgler-Murphy et al. 1997, Mattison and Ota 2000, Andersson et al. 2004), while substrate-mediated feedback such as Hog1-dependent phosphorylation of Ste50 prevents inappropriately sustained or cross-pathway activation (Fig. 1B) (Hao et al. 2008, de Nadal and Posas 2022).

Despite this conserved core architecture, lineage-specific duplications and rewiring events have produced substantial functional diversification. In mammals, the MAPK repertoire expanded to fourteen kinases comprising four conventional subfamilies (ERK1/2, JNK1–3, p38α–δ and ERK5) (Cargnello and Roux 2011) and a smaller group of atypical MAPKs (ERK3/4, ERK7/8 and NLK) with non-canonical activation loops and regulatory mechanisms (Deleris et al. 2011). In fungi, a comparative analysis of MAPK cascade components across 231 species has shown that the present-day repertoire arose largely through gene duplication and subfunctionalisation, with marked lineage-specific expansion and loss that correlate with fungal phylogeny and lifestyle (Xu et al. 2017). This diversification is exemplified by paralogue retention and by lineage-specific variation in the upstream two-component phosphorelay systems that feed into MAPK cascades. Multiple hybrid histidine kinases (HHKs) are in fact the norm across the fungal kingdom: filamentous ascomycetes are especially rich (Neurospora crassa, for example, encodes eleven), and even the yeast pathogen C. albicans carries three (Sln1, Nik1/Cos1, and Chk1), whereas S. cerevisiae is the outlier in retaining only a single HHK, Sln1 (Kruppa et al. 2004). Against this background, C. neoformans lies at the upper end of the range seen in yeasts, employing seven HHKs (Tco1–Tco7) and two response regulators (Ssk1 and Skn7) that govern stress responses, drug sensitivity, sexual development and virulence largely through the Pbs2–Hog1 cascade (Bahn et al. 2006). More distinctive than their number is their architecture: all seven Cryptococcus HHKs are predicted to be cytosolic, and Tco2 is an atypical kinase carrying duplicated histidine kinase and receiver domains (Bahn et al. 2006). Because canonical two-component phosphorelay systems are nonetheless absent from animals, these upstream architectures—common to pathogenic and environmental fungi alike—have no direct counterpart in the mammalian host (Capra and Laub 2012).

MAPK signalling in S. cerevisiae: the foundational template

Saccharomyces cerevisiae harbours five MAPK pathways that together govern osmotic stress adaptation (Hog1), mating (Fus3), filamentous growth (Kss1), cell wall integrity (Slt2/Mpk1), and sporulation (Smk1) (Gustin et al. 1998).

The HOG pathway adapts cells to hyperosmotic stress through Sln1 and Sho1 upstream branches that converge on the MAP2K Pbs2. The Sln1 branch operates through the Sln1-Ypd1-Ssk1 phosphorelay to regulate MAP3K Ssk2/Ssk22, whereas the Sho1 branch employs the mucin-like sensors Msb2 and Hkr1, Opy2 and the Cdc42–Ste20 module to activate the MAP3K Ste11. Both branches converge on Pbs2-mediated dual phosphorylation of Hog1, which enters the nucleus to induce osmoadaptive genes such as GPD1 and GPP2. Hog1 signalling is later attenuated by Ptp2/Ptp3 phosphatases and by Hog1-dependent phosphorylation of Ste50, which limits inappropriate cross-activation of the filamentation pathway under osmotic stress (Hao et al. 2008, de Nadal and Posas 2022).

The pheromone-responsive MAPK pathway provides the most studied example of scaffold-mediated specificity. Binding of a- or α-factor pheromones to cognate G protein-coupled receptors releases Gβγ, which recruits the scaffold protein Ste5 to the plasma membrane. Ste5 assembles Ste11 (MAP3K), Ste7 (MAP2K) and the MAPKs Fus3 and Kss1 into a functional unit (Gustin et al. 1998, Sabbagh et al. 2001). Although Fus3 and Kss1 are activated by the same upstream kinases, their downstream outputs are distinct. Fus3 promotes Ste12-dependent transcription to induce mating genes such as FUS1 and KAR4 and enforces G1 arrest through Far1-mediated inhibition of Cln–Cdc28 activity, while Kss1 has a dual repressor/activator role through its interaction with Ste12 and Tec1 in the filamentous growth pathway (Gartner et al. 1992, Karunanithi and Cullen 2012, Winters and Pryciak 2019, Venta et al. 2020). Under basal conditions, unphosphorylated Kss1 binds and represses Ste12, but upon dual phosphorylation by Ste7, it switches conformation, releases Ste12 and partners with Tec1 to drive expression of filamentation genes such as FLO11, which encodes a cell-surface adhesin essential for invasive growth (Karunanithi and Cullen 2012, Pujari and Cullen 2024). The Ste12 repressors Dig1 and Dig2 are phosphorylated by Fus3 to relieve Ste12 inhibition, while Msg5 dephosphorylates Fus3 to terminate signalling, and Fus3 itself feeds back to phosphorylate Far1 and disrupt the Ste5–membrane interaction (Cherkasova et al. 1999, Andersson et al. 2004).

The CWI pathway senses cell wall damage through the Wsc1–Wsc3, Mid2 and Mtl1 sensors, which engage the GEF Rom2 to activate the small GTPase Rho1 and protein kinase C (Pkc1), driving sequential activation of Bck1 (MAP3K), Mkk1/Mkk2 (MAP2Ks), and Slt2/Mpk1. Slt2 phosphorylates Rlm1 and the SBF complex (Swi4/Swi6) to induce Rlm1 target genes such as the β-1,3-glucan synthase gene FKS1 and the Slt2 paralogue MLP1, and also intersects with the HOG pathway via the Ssk2–Pbs2–Hog1 module during SDS- and detergent-induced stress, illustrating context-dependent crosstalk between otherwise parallel pathways (Chen and Thorner 2007, Garcia et al. 2019). The CWI pathway is further fine-tuned by the dual-specificity phosphatases Msg5 and the stress-inducible Sdp1, which dephosphorylate Slt2 to terminate signalling (Hahn and Thiele 2002). Beyond canonical cell wall maintenance, CWI signalling participates in P-body assembly under stress and in lipid homeostasis, with slt2Δ mutants showing perturbed phosphatidylcholine and phosphatidylinositol metabolism (Garcia et al. 2019).

Finally, the meiosis-specific MAPK Smk1 illustrates the flexibility of the MAPK module. Rather than relying on a canonical MAP2K, Smk1 is activated by Cak1-mediated Thr207 phosphorylation, followed by cis-autophosphorylation of Tyr209 that is facilitated by the meiosis-specific activator Ssp2. This produces distinct mono- and dual-phosphorylated activity states, which sequentially deposit the inner glucan and outer chitosan layers of the spore wall (Krisak et al. 1994, Wagner et al. 1999, Tio et al. 2015).

Together, these five pathways established key concepts that continue to frame our understanding of MAPK signalling in pathogenic fungi, including scaffold-mediated insulation, branched upstream input, feedback-driven attenuation, threshold-dependent output, and quantitative encoding of signal duration. As we discuss next, however, fungal pathogens have substantially remodelled this template through paralogue retention, sensor expansion, and quantitative re-tuning of pathway activity in ways that have direct consequences for host adaptation and virulence.

MAPK pathways in human pathogenic fungi

Human pathogenic fungi retain the core MAPK toolkit of S. cerevisiae but have rewired and expanded individual modules to support survival in the mammalian host. Figure 2 provides a comparative overview of MAPK pathway architectures in S. cerevisiae and the major pathogenic fungi.

Figure 2.

Comparative architecture of fungal MAPK pathways. Schematic comparison of the major MAPK pathways in representative fungi, including S. cerevisiae, C. albicans, A. fumigatus, C. auris, and C. neoformans. (A) The high osmolarity glycerol (HOG), (B) mating, and (C) cell wall integrity (CWI) pathways are shown as modular kinase cascades; to aid comparison, each complex pathway is simplified to retain only the signal, upstream sensors, MAP3Ks, MAP2Ks, MAPKs, and cellular response. (D) The spore wall pathway, a meiosis-associated MAPK module, is present only in S. cerevisiae among the species compared. Solid boxes indicate relatively well-characterised components, whereas dashed boxes indicate poorly defined or predicted components. Solid arrows denote established signalling steps, whereas dashed arrows denote steps omitted for clarity or not yet defined; in particular, the sensor histidine kinases do not activate the Ssk2 MAP3K directly but signal through a Ypd1–Ssk1 phosphorelay that is collapsed into a dashed arrow. Bold arrows mark the branch acting through the paralogue that dominates under standard laboratory conditions, as for Cek1 relative to Cek2 in C. albicans and Cpk1 relative to Cpk2 in C. neoformans. Only sensors with assigned functions in the pathway depicted are shown; in several species the genome encodes additional hybrid histidine kinases (HHKs)—for example, Tco3-Tco7 in C. neoformans—whose roles have not been defined. In C. neoformans, Wml1 and Wml2 are shown as putative cell-surface stress sensors of the CWI pathway. Their combined loss reduces Mpk1 phosphorylation, but their relatively mild mutant phenotypes suggest that additional unidentified upstream sensors or inputs contribute to Mpk1 activation; these are indicated by a question mark. The connection between Wml1/Wml2 and the core CWI cascade remains unresolved. In C. auris, most pathway components remain incompletely characterised, particularly in the mating- and CWI-associated modules. In C. neoformans, Tco1 and Tco2 have been identified as sensors initiating the HOG pathway, rather than a single Sln1-type osmosensor, and in A. fumigatus the pheromone module (SteC–MkkB–MpkB) governs asexual development and secondary metabolism rather than mating. The figure highlights both conservation of the core MAPK architecture and lineage-specific diversification in upstream sensors, pathway wiring, and biological outputs. Created in BioRender (https://BioRender.com/6ykpizs).

Candida albicans

Candida albicans is a polymorphic commensal of human mucosal surfaces that becomes pathogenic when host defences are compromised. Its capacity to switch between yeast and filamentous forms, remodel its cell wall, form drug-tolerant biofilms, and persist in diverse host niches is closely linked to MAPK signalling (Roman et al. 2009, Kramara et al. 2024).

The HOG pathway is activated by osmotic and oxidative stress and is essential for glycerol accumulation, oxidative defence, cell cycle adaptation, and heavy metal tolerance. In contrast to S. cerevisiae, C. albicans uses three sensor histidine kinases (Sln1, Chk1, and Nik1) that converge on Ypd1 and Ssk1 to activate Ssk2–Pbs2–Hog1 (Cheetham et al. 2007, Day et al. 2024). Genetic dissection has revealed an unexpected division of labour within this module. Ssk1 acts as a master regulator that promotes Hog1 phosphorylation in response to most stresses except hyperosmotic shock, and does so at least partly in a two-component-independent manner by scaffolding the Ssk2–Pbs2 interaction; none of the histidine kinases relay oxidative-stress signals directly to Hog1 (Day et al. 2024). Activated Hog1 reprograms glycerol biosynthesis, oxidative defence and a broad heavy metal and metabolic stress response, while hog1Δ mutants display attenuated virulence in murine systemic infection models that cannot be explained by osmosensitivity alone (Cheetham et al. 2007, Day et al. 2024).

The CWI pathway in C. albicans is mediated by Mkc1, originally identified by functional complementation of S. cerevisiae slt2Δ mutants (55% sequence identity) (Navarro-Garcia et al. 1995). Although Mkc1 is dispensable under standard growth conditions, mkc1Δ mutants exhibit thermal sensitivity, increased susceptibility to cell-wall-targeting enzymes, impaired biofilm formation, and markedly attenuated virulence in murine systemic infection models, with reduced colonization of kidney and brain (Navarro-Garcia et al. 1995, Diez-Orejas et al. 1997). Mkc1 drives expression of β-1,3-glucan and chitin synthases, mannosyltransferases and cross-linking proteins, and regulates the spatial delivery of cell wall biosynthetic machinery to sites of active growth (Heilmann et al. 2013, da Silva Dantas et al. 2021). Mkc1 is functionally intertwined with calcineurin signalling. Pharmacological inhibition of calcineurin compensatorily activates Mkc1 and induces FKS1, while calcineurin mutations antagonize Mkc1-mediated chitin synthesis (da Silva Dantas et al. 2021). The CWI pathway also crosstalks with telomere and DNA damage networks. Deletion of the telomere-binding protein Rap1 co-activates Mkc1 and Cek1, suggesting a previously unappreciated link between cell wall integrity and genome maintenance (Wang et al. 2022).

The mating MAPK pathway in C. albicans couples sexual reproduction to white–opaque phenotypic switching, regulated by the mating-type-like (MTL) locus (Miller and Johnson 2002). Only MTL homozygous strains can transition to the mating-competent opaque state, which exhibits a million-fold increase in mating efficiency over white-phase cells (Miller and Johnson 2002, Sasse et al. 2013). The Cst20–Ste11–Hst7 cascade activates two terminal MAPKs, Cek1 and Cek2. As in the S. cerevisiae pheromone response, which engages both Fus3 and Kss1, two MAPKs act downstream of a shared upstream module; in C. albicans, however, Cek1 and Cek2 perform complementary, non-redundant roles in the pheromone response (Sasse et al. 2013, Rastghalam et al. 2019). In opaque cells, Cek1 and Cek2 drive Cph1-mediated pheromone responses, with cek1Δ mutants showing a near-complete mating defect (mating efficiency reduced to ∼0.3% of wild type) and severely impaired projection formation, whereas cek2Δ mutants show milder mating defects but specific impairment in pheromone-induced cell cycle adaptation (Sasse et al. 2013, Rastghalam et al. 2019). In white cells, the same Cst20–Ste11–Hst7 module activates Tec1 rather than Cph1, promoting biofilm formation instead of mating. This bifunctional output couples pheromone signalling to community-level behaviours that can support genetic exchange among neighbouring opaque cells embedded in biofilm matrix (Miller and Johnson 2002, Sasse et al. 2013, Rastghalam et al. 2019). The dual-specificity phosphatase Cpp1 fine-tunes Cek1/Cek2 activity, and cpp1Δ mutants exhibit pheromone hypersensitivity coupled with paradoxically reduced mating efficiency. This illustrates that precise temporal control of MAPK activation and inactivation, rather than peak activity alone, is essential for productive output (Rastghalam et al. 2019).

Aspergillus fumigatus

Aspergillus fumigatus is a saprophytic, airborne mould and the principal agent of invasive aspergillosis, a frequently fatal infection of immunocompromised patients that underlies its designation as a WHO critical-priority fungal pathogen. Unlike the yeasts discussed above it grows as filamentous hyphae and disperses as asexual conidia, yet it relies on a conserved MAPK toolkit to adapt to host-imposed stress and sustain pathogenicity. Its genome encodes four MAPKs—SakA and MpkC of the stress-activated high-osmolarity glycerol (HOG) module, MpkA of the cell wall integrity (CWI) module, and MpkB of the pheromone/development module—which together coordinate stress adaptation, cell wall remodelling, secondary metabolism and development (Du et al. 2006, Reyes et al. 2006).

The HOG module is organized around the Hog1 orthologue SakA and its paralogue MpkC, which govern responses to osmotic, oxidative, thermal and cell wall stress and influence developmental processes such as germination and conidiation. Both kinases translocate to the nucleus upon osmotic or cell wall stress to activate stress-responsive transcription, with SakA responding more rapidly, consistent with a leading role in the immediate response (Bruder Nascimento et al. 2016). The two kinases act both independently and cooperatively: deletion of either sakA or mpkC confers moderate stress sensitivity—more pronounced for ΔsakA—and slight reduction in pulmonary fungal burden, whereas the ΔsakA ΔmpkC double mutant is markedly more sensitive to osmotic, oxidative and cell wall stress and is strongly attenuated for virulence (Bruder Nascimento et al. 2016). Notably, MpkA phosphorylation under osmotic and cell wall stress is itself dependent on SakA and MpkC—reduced in ΔsakA and undetectable in the ΔsakA ΔmpkC double mutant—providing functional evidence of HOG-to-CWI signalling (Bruder Nascimento et al. 2016). Biochemically, SakA and MpkC physically associate under osmotic and cell wall stress, and under cell wall stress SakA additionally interacts with the CWI MAPK MpkA and the phosphatase PtcB, providing a direct molecular link between the HOG and CWI pathways (Manfiolli et al. 2019a). The HOG module is further integrated with cAMP/PKA signalling, as SakA forms a complex with the catalytic subunit PkaC1 and the regulatory subunit PkaR; dissociation of PkaR is proposed to release an active SakA–PkaC1 complex that mobilizes stored carbohydrates for cell wall biosynthesis, thereby coupling stress signalling to cell wall carbohydrate exposure (de Assis et al. 2018).

The CWI pathway is built on the terminal MAPK MpkA, activated by the canonical Bck1–Mkk2–MpkA cascade; deletion of any tier causes severe sensitivity to cell wall–perturbing agents and pronounced morphological defects, and Bck1 and Mkk2 directly activate MpkA during both vegetative growth and cell wall stress (Valiante et al. 2009). Beyond cell wall maintenance, transcriptome profiling revealed that MpkA coordinates a much broader adaptive programme spanning reactive oxygen species protection, secondary-metabolite biosynthesis (gliotoxin and pseurotin) and adaptation to iron starvation (Jain et al. 2011). The pathway also modulates pyomelanin biosynthesis derived from tyrosine degradation, further connecting MpkA to oxidative protection and secondary metabolism (Valiante et al. 2009). Consistent with the crosstalk noted above, the CWI and HOG modules are physically connected through the SakA–MpkA–PtcB interaction under cell wall stress, and such interactions are increasingly regarded as compensatory routes that buffer the fungus against cell wall–targeting antifungals (Valiante et al. 2015).

The pheromone-responsive module is centred on MpkB (Fus3 orthologue) which, despite the predominantly asexual life cycle of A. fumigatus, has been repurposed to control asexual development and secondary metabolism. MpkB assembles with SteC (MAP3K), MkkB (MAP2K), the SteD adaptor and the HamE scaffold into a cytoplasmic pentameric pheromone module; upon activation MpkB enters the nucleus, and disruption of the module impairs conidiation, vegetative growth and the production of secondary metabolites such as gliotoxin while increasing sensitivity to cell wall and oxidative stress (Frawley et al. 2020). MpkB also intersects with the CWI pathway: loss of MpkB elevates dihydroxynaphthalene (DHN)-melanin production, an effect normalized by simultaneous deletion of mpkA, and MpkB translocates to the nucleus under caspofungin stress in an MpkA-dependent manner. Upstream G-protein signalling feeds into this axis, since deletion of the Gα subunit GpaA or the G-protein-coupled receptor GprM, which physically interact, likewise elevates DHN-melanin, implicating GPCR input in MpkB-dependent control of this cell wall–associated virulence pigment (Manfiolli et al. 2019b).

Candidozyma (Candida) auris

Candidozyma auris is an emerging multidrug-resistant pathogen that has spread rapidly through healthcare settings since its first description in 2009. Its tolerance to environmental disinfection, persistent skin colonization, capacity for nosocomial transmission, and frequent resistance to fluconazole—with variable resistance to amphotericin B and emerging echinocandin resistance—have contributed to its designation by the WHO as a critical-priority fungal pathogen (WHO 2022).

The Hog1 MAPK cascade is currently the best-characterized MAPK pathway in C. auris. Unlike C. albicans, which carries three sensor histidine kinases (Sln1, Chk1, and Nik1), C. auris encodes only two predicted sensor histidine kinase homologues, Sln1 and Nik1, together with the phosphotransfer protein Ypd1, the response regulator Ssk1 and the canonical HOG cascade components Ssk2, Pbs2, and Hog1 (Yaakoub et al. 2025). These upstream histidine kinase and phosphorelay components are assigned by orthology and have been proposed to constitute a two-component signalling system linked to the HOG module, but the phosphotransfer relationships among them and their functional connection to the Ssk2–Pbs2–Hog1 MAPK cascade remain poorly resolved (Yaakoub et al. 2025). Genetic disruption of SSK1 or HOG1 alters antifungal susceptibility, cell wall architecture and stress adaptation (Shivarathri et al. 2020, 2024), and HOG1 deletion impairs biofilm formation, skin colonization and intradermal persistence (Shivarathri et al. 2020, 2024).

By contrast, the CWI- and pheromone/development-associated MAPK modules remain poorly defined in C. auris. Orthologues of the CWI MAPK Mkc1 and the Cek1-type MAPK are encoded in the genome, and both can be detected in a phosphorylated state (Shivarathri et al. 2020, 2024). Notably, these signals are not independent of the HOG module: loss of SSK1 or HOG1 elevates Cek1 phosphorylation while reducing that of Mkc1, indicating that Ssk1–Hog1 signalling positively supports the Mkc1 pathway and restrains Cek1 (Shivarathri et al. 2020). Beyond the crosstalk, however, their pathway architecture, upstream inputs and physiological outputs have not been resolved through systematic genetic dissection. Because these assignments rest on antibodies raised against phosphorylated mammalian Erk1/2, and because no mating cycle has been observed in C. auris, detection of a phosphorylated species does not by itself identify the activating cascade; placement of these kinases in the CWI- and pheromone/development modules therefore reflects orthology to C. albicans rather than direct functional evidence in C. auris. This is a notable gap, given the contribution of these modules to cell wall remodelling and drug tolerance in C. albicans and related species.

Cryptococcus neoformans

Cryptococcus neoformans is a basidiomycetous yeast that causes life-threatening cryptococcal meningoencephalitis, particularly in people with advanced HIV disease. Among fungi of medical importance, it possesses an expanded MAPK network: five MAPKs (Cpk1, Cpk2, Mpk1, Mpk2, and Hog1) operate within an architecture with broad paralogue expansion, an unusually complex upstream phosphorelay, and extensive crosstalk with the cAMP/PKA, calcineurin and unfolded protein response (UPR) pathways (Bahn et al. 2006, Jang et al. 2024). Figure 3 illustrates the integrated network; below we summarize the three best-characterized modules and the emerging roles of the minor paralogues.

Figure 3.

MAPK signalling network in model basidiomycetous yeast C. neoformans. Integrated model of the (A) mating, (B) HOG, and (C) cell wall integrity (CWI) MAPK pathways in C. neoformans. The mating pathway is activated by pheromone signalling through Ste3 receptors and transmits signals through Ste20/Pak1, Ste11–Ste50, Ste7, and Cpk1, with the paralogous MAPK Cpk2 contributing to mating output. Crg1 and Ras1 are shown as upstream modulators of pheromone signalling, with Crg1 acting as a negative regulator at the G-protein signalling level and Ras1 feeding into Ste20/Pak1-dependent activation. Downstream regulators, including Mat2, Sxi1α/Sxi2a, and Znf2, together with MFα1 expression, promote filamentation and mating. The HOG pathway responds to high osmolarity and environmental stimuli through Tco1/Tco2 histidine kinases, Ypd1, Ssk1, Ssk2, Pbs2, and Hog1. The Hog1-dependent network is linked to specific downstream outputs, including Hrk1–Gpd1-associated intracellular glycerol production and the consequent fludioxonil susceptibility, Ada2–Gat201–Gat204/Zfc3-associated capsule production, negative modulation of capsule production by Atf1, Atf1-mediated osmotic and oxidative stress responses, Lac1-associated melanin production, and Hog1-mediated repression of Erg11-associated ergosterol biosynthesis (which underlies the pathway’s influence on azole and polyene susceptibility). The inhibitory link from Hog1 to the mating pathway is drawn to the Ste11–Ste7–Cpk1 module rather than to Cpk1 itself, since the point of action upstream of Cpk1 has not been defined. Wml1 and Wml2 have been implicated as putative cell-surface stress sensors involved in Mpk1 activation. However, their relatively mild mutant phenotypes are consistent with the presence of additional unidentified upstream sensors or signalling inputs, and their precise connection to the core CWI cascade remains unresolved. The pathway also integrates cell wall stress, high temperature, lipid-mediated signalling through Ipc1, phosphatidylinositol (PI), diacylglycerol (DAG), and Pkh202, Rho GTPase signalling through Rho1, Rho10, and Rho11, and Pkc1-dependent signalling through Ste11- and Bck1-linked branches that converge on Mkk2 and the Mpk1/Mpk2 MAPKs. Within this pathway, Rom2 is shown as a positive regulator that promotes Rho GTPase activation as a guanine nucleotide exchange factor, whereas Ppg1 and Lrg1 are shown as negative regulators of CWI-associated signalling. These branches contribute to cell wall integrity and cell membrane integrity. Solid connectors denote relationships established experimentally; these include the cross-pathway links shown here, among them the reciprocal relationship between the Cpk1 pathway and the Mpk1/Mpk2 module revealed in higher-order deletion backgrounds. Dashed connectors denote relationships that are established but whose point of action has not been defined, as for the bracket spanning the Ste11–Ste7–Cpk1 module described above. Created in BioRender (https://BioRender.com/6ykpizs).

The HOG pathway

The C. neoformans HOG pathway is structurally distinct from its S. cerevisiae counterpart in several important respects. Upstream regulation involves a multi-component phosphorelay with a single phosphotransfer protein (Ypd1), two response regulators (Ssk1 and Skn7) and seven HHKs (Tco1–Tco7), of which only Tco1 and Tco2 have so far been assigned sensor functions in the pathway: both are required for fludioxonil susceptibility, whereas Tco1 negatively regulates melanin production and positively regulates mating, and Tco2 governs responses to osmotic stress, oxidative damage and toxic metabolites (Bahn et al. 2006). Tco2 is also structurally unusual, containing two histidine kinase and two response regulator domains in a single polypeptide—the first functionally characterized dual HHK (Bahn et al. 2006). Unlike S. cerevisiae Sln1, which contains N-terminal transmembrane regions, C. neoformans HHKs lack these domains and are likely cytosolic, raising mechanistic questions about how diverse environmental cues are routed into the same phosphorelay. Ypd1 is essential when Hog1 is functional, as its deletion is lethal in the H99 background. This finding indicates that the phosphorelay plays an important role in maintaining basal Hog1 regulation (Bahn et al. 2006, Lee et al. 2011). Ssk1 transmits signals to Hog1 itself, whereas Skn7 acts in parallel to control Na⁺ tolerance, antifungal sensitivity and melanin biosynthesis independently of Hog1, sometimes producing even more pronounced melanin phenotypes than hog1Δ or ssk1Δ mutants under specific conditions (Bahn et al. 2006).

A striking feature of C. neoformans Hog1 is its phosphorylation state under basal conditions. In the representative serotype A strain H99, as well as in other clinical and environmental serotype A isolates, Hog1 is constitutively phosphorylated under basal (unstressed) conditions, where it is distributed between the nucleus and the cytosol with a modest nuclear enrichment; osmotic stress triggers transient dephosphorylation accompanied by further nuclear translocation, after which Hog1 returns to its basal state. By contrast, this pattern is reversed in S. cerevisiae, the representative serotype D strain Cryptococcus deneoformans JEC21, and other clinical and environmental serotype D isolates, in which Hog1 is activated through stress-induced phosphorylation (Bahn et al. 2005). This inverted regulatory logic is thought to enable Hog1 to act simultaneously as a repressor of virulence-associated programmes, including capsule, melanin and mating, and as a stress-responsive switch that releases this repression upon environmental challenge. Consistent with this dual role, hog1Δ, pbs2Δ and ssk1Δ mutants show enhanced capsule and melanin production, elevated pheromone production, increased mating efficiency, and increased resistance to the phenylpyrrole antifungal drug fludioxonil, while also exhibiting growth defects under specific stresses (Bahn et al. 2006). A recent study using localization-restricted Hog1 variants further showed that Hog1 compartmentalization, rather than its phosphorylation status alone, is a key determinant of output specificity. Membrane-tethered Hog1 was sufficient to restore thermotolerance and antifungal resistance, whereas nuclear Hog1 was required for the osmotic and ER stress responses and for capsule–melanin biosynthesis, indicating that distinct subcellular pools of Hog1 drive distinct adaptive and virulence programmes (Jang and Bahn 2026).

The CWI pathway

Cell wall integrity is essential not only for general stress tolerance but also for growth at the mammalian body temperature of 37°C, a key virulence-associated trait that distinguishes pathogenic Cryptococcus species from their non-pathogenic relatives (Kraus et al. 2003). More recently, Wml1 and Wml2 were identified as putative cell-surface stress sensors in C. neoformans. Simultaneous deletion of WML1 and WML2, but not either single deletion, markedly reduced tunicamycin-induced Mpk1 phosphorylation, indicating that Wml1 and Wml2 make partially redundant contributions to Mpk1-mediated CWI signalling (Thak et al. 2022). However, the wml1Δ wml2Δ double mutant showed a growth defect only under NaCl stress, whereas the mpk1Δ mutant is broad stress-sensitive, suggesting that additional, as-yet-unidentified sensors contribute to CWI signalling. Downstream signalling converges on a core three-tier cascade: the PDK1 orthologue Pkh202 (but not its paralogue Pkh201) sustains basal activity of protein kinase C (Pkc1), generally regarded as the major upstream regulator, which in turn drives the MAP3K Bck1, the MAP2K Mkk2, and the MAPK Mpk1 (Kraus et al. 2003, Chabrier-Rosello et al. 2013). Loss of Pkh202 dampens Mpk1 activation under cell-wall, oxidative and antifungal stress (Chabrier-Rosello et al. 2013). mpk1Δ mutants are highly sensitive to calcofluor white, caspofungin, and nikkomycin Z, and they fail to grow at 37°C, a defect rescued by osmotic stabilization with sorbitol; this shows that thermal sensitivity is a downstream consequence of compromised cell wall integrity rather than a direct effect of temperature on protein folding (Kraus et al. 2003). Deletion of BCK1 or MKK2 produces severe cell integrity defects with phenotypes that differ in detail from mpk1Δ, suggesting additional pathway branches or non-linear MAP2K/MAP3K functions beyond classical Mpk1 activation.

Rho-family GTPase signalling also appears to be rewired in C. neoformans. Constitutive activation of Rho1 or deletion of RHO10 drives Mpk1 phosphorylation, while Rho10 and Rho11 appear to act in a coordinately regulated manner. Pkc1, generally regarded as the principal upstream regulator of the cascade, is nonetheless dispensable for heat-shock-induced Mpk1 activation. Thermal activation instead proceeds through a Pkc1-independent, Rho-driven route that still requires Bck1 and Mkk2. This suggests that Rho proteins engage the pathway via downstream or alternative components (Lam et al. 2013). The calcineurin pathway intersects with Mpk1 signalling at multiple points. Treatment with the calcineurin inhibitor FK506 compensatorily activates Mpk1, suggesting that calcineurin and CWI act as parallel safeguards of cell wall and membrane integrity (Kraus et al. 2003). Elevated CO₂, a hallmark of the host environment with arterial CO₂ tensions of ∼5%, suppresses temperature-induced Mpk1 phosphorylation in CO₂-tolerant strains, suggesting an antagonistic relationship between thermal and CO₂ adaptation programmes that converges on the CWI pathway (Ristow et al. 2023). Genetic evidence indicates functional interdependence between the CWI and HOG pathways: deletion of the sensors SHO1 or MSB2 in an mpk1Δ background substantially exacerbates cell wall defects, implicating the HOG and pheromone/development-responsive upstream components in CWI maintenance, although a direct biochemical connection has not been demonstrated (So et al. 2018). Mpk1-deficient mutants display markedly attenuated virulence in murine cryptococcosis, reflecting heightened susceptibility to host-imposed thermal, oxidative and membrane stresses, and increased susceptibility to echinocandin and azole antifungals (Kraus et al. 2003).

The mating MAPK pathway and minor MAPK paralogues

Sexual development in C. neoformans involves a dimorphic transition from unicellular yeast to dikaryotic hyphae and proceeds via both opposite-sex (MATα × MATa) and unisexual routes, the latter typically involving same-mating-type cells of α genotype under nitrogen-limited conditions (Lin et al. 2005). Pheromone/development signalling is initiated by binding of MFα or MFa peptide pheromones to the Ste3 receptor on partner cells, releasing the Gβγ complex and engaging the canonical Ste11α–Ste7–Cpk1 cascade (Davidson et al. 2003, Kim and Bahn 2022). Deletion of any of the core pathway components causes severe defects in pheromone production, cell fusion and filamentous growth, with sterility even in unilateral crosses. Importantly, cpk1Δ, ste7Δ, and ste11Δ mutants retain full virulence in animal models, dissociating mating from pathogenesis in this organism (Davidson et al. 2003). Two downstream transcription factors (Mat2 and Znf2) govern distinct phases of sexual development: Mat2 acts as the direct effector of Cpk1 signalling and is essential for pheromone-induced cell fusion, while Znf2 operates downstream of Mat2 to direct hyphal morphogenesis. Notably, znf2Δ mutants are locked in the yeast phase yet exhibit enhanced virulence, the opposite of the conventional pattern in which hyphal growth is associated with increased invasiveness (Lin et al. 2010). Completion of sexual reproduction in bisexual crosses requires the homeodomain transcription factors Sxi1α and Sxi2a, which form a heterodimeric complex regulating hyphal and sporulation gene programmes; these factors are dispensable for unisexual reproduction, indicating distinct molecular architectures for the two mating modes (Lin et al. 2010). The HOG pathway negatively regulates pheromone production, providing an environmental sensor by which stress and nutrient cues restrict mating to favourable conditions (Bahn et al. 2006).

Beyond the canonical Cpk1 and Mpk1 modules, C. neoformans retains two minor paralogues, Cpk2 (of the pheromone-responsive Cpk1) and Mpk2 (of the CWI MAPK Mpk1), whose functions have only recently been defined (Lee et al. 2016, Jang et al. 2024). Their roles emerge mainly under overexpression or in higher-order deletion backgrounds: CPK2 overexpression restores mating in cpk1Δ cells via Mat2, while MPK2 overexpression partially rescues the cell-wall defects of mpk1Δ by restoring chitin and capsule but cannot restore thermotolerance or membrane integrity, indicating roles that are both redundant with and opposed to Mpk1. Most strikingly, dual deletion of MPK1 and MPK2 restores wild-type mating in cpk1Δ cells, showing that Mpk1 and Mpk2 together negatively regulate the Cpk1 pheromone/development pathway (Jang et al. 2024). Because such inter-module crosstalk surfaces only in these sensitized backgrounds, it would be missed in single-gene studies. That these paralogues are retained and functionally specialized in C. neoformans but not in S. cerevisiae or C. albicans illustrates how lineage-specific MAPK expansion underlies the regulatory complexity of basidiomycete pathogens, motivating the systematic dissection of higher-order MAPK combinations considered next.

MAPK regulation of canonical virulence factors in C. neoformans

The pathogenicity of C. neoformans depends on a small number of major virulence factors, including the polysaccharide capsule, melanin pigment, thermotolerance at 37°C, and the inducible formation of large polyploid titan cells (Crabtree et al. 2012, Jung et al. 2015, Lee et al. 2016), as well as secondary factors such as urease, other hydrolytic enzymes, and extracellular vesicles (Cox et al. 2000, Rodrigues et al. 2007).

The polysaccharide capsule, composed primarily of glucuronoxylomannan (GXM) and galactoxylomannan (GalXM), is the most distinctive virulence factor of C. neoformans and expands greatly during infection to interfere with phagocytosis, complement deposition and antigen presentation (De Jesus et al. 2010). Systematic genetic screens first identified the GATA-family transcription factor Gat201 as a major regulator of capsule production and virulence in C. neoformans (Liu et al. 2008); subsequent transcription factor screens have identified Gat201, Yap1, Bzp4, and Ada2 as core regulators of capsule biosynthesis, acting downstream of upstream kinases including Hog1, Pka1, and Irk5 (Jang et al. 2022). Hog1 itself negatively regulates capsule production, consistent with the elevated capsule observed in hog1Δ, pbs2Δ, and ssk1Δ mutants, while the cAMP/PKA pathway acts in parallel to drive capsule induction in response to nutrient and host-derived cues.

Melanin synthesis from exogenous catecholamine precursors is catalyzed by the cell wall-associated enzyme laccase, encoded by LAC1, and protects cryptococcal cells from oxidative and nitrosative stress, ultraviolet radiation, antimicrobial peptides and certain antifungals (Eisenman et al. 2007, Samarasinghe et al. 2018). Four transcription factors (Bzp4, Usv101, Mbs1, and Hob1) form the core regulatory module driving LAC1 expression, and the HOG pathway negatively regulates both BZP4 induction and laccase activity (Lee et al. 2019). Notably, while Hog1 modulates this network, the cAMP/PKA pathway is not required for the activity of these four transcription factors, illustrating that capsule and melanin, although classically grouped as the major virulence factors, are regulated by overlapping but distinct kinase architectures.

Thermotolerance at 37°C, the third major virulence factor, is supported by several converging signalling pathways: the calcineurin–calmodulin axis, Ras, HOG, the CWI pathway (Mpk1), the UPR, and the Sch9–Hsf1 module all contribute, and mutants with severe 37°C growth defects are generally attenuated for virulence regardless of their capsule and melanin status (Yang et al. 2017, Stempinski et al. 2021). The redundancy and partial cooperation among these pathways is consistent with the fundamental role of thermotolerance in establishing mammalian infection. Titan cell formation, in which a subpopulation of cells can enlarge up to ∼50–100 μm in diameter with thickened, cross-linked cell walls and polyploid genomes, is promoted by signals including the Gpr4/Gpr5 GPCRs acting through cAMP/PKA, whereas transcription factors such as Pdr802 act as negative regulators that restrain it (Crabtree et al. 2012, Hommel et al. 2018, Reuwsaat et al. 2021). While the direct contribution of MAPK pathways to titan cell induction is less well defined than for capsule and melanin, the convergence of cAMP/PKA, Hog1 and Mpk1 signalling on cell wall and cytokinesis programmes suggests that MAPK pathways modulate titan biology indirectly through their effects on cell wall remodelling and stress adaptation.

Interconnected MAPK networks and evolutionary plasticity

A common theme from systematic genetic analyses across pathogenic fungi is that MAPK pathways do not operate as insulated linear circuits. In C. albicans, the HOG pathway crosstalks extensively with the CWI and morphogenetic pathways, and Mkc1 activity is intertwined with calcineurin signalling, telomere maintenance via Rap1, and the white–opaque switch (da Silva Dantas et al. 2021, Wang et al. 2022). In C. neoformans, the five MAPKs and their upstream phosphorelay components are entangled to an even greater extent: deletion of MPK1 alone increases sensitivity to cell-wall-perturbing agents, but additional loss of the osmosensors SHO1 or MSB2 in an mpk1Δ background substantially exacerbates these defects, implicating the HOG and pheromone-responsive MAPK pathways in CWI maintenance (So et al. 2018). Conversely, the constitutive phosphorylation state of Hog1 in clinical C. neoformans isolates means that the HOG pathway acts as a tonic regulator of capsule, melanin and mating programmes that are simultaneously held under negative control and primed for rapid release upon stress (Bahn et al. 2005).

Paralogue retention and divergence are particularly informative. C. albicans retains two ERK-like MAPKs (Cek1, Cek2) whose specialized roles in pheromone response, biofilm formation and stress tolerance illustrate how gene duplication can support functional diversification within a single signalling module. In C. albicans, the two terminal pheromone/development MAPKs invert the S. cerevisiae paradigm: Cek1 (the Kss1 orthologue) acts as the dominant positive effector for pheromone/development-associated outputs, whereas Cek2 (the Fus3 orthologue) contributes more modestly and modulates signalling intensity (Correia et al. 2016, Rastghalam et al. 2019), and additionally plays a cryptic, non-redundant role in cell-wall biogenesis (Correia et al. 2016, Rastghalam et al. 2019). Cryptococcus neoformans extends this principle by retaining minor paralogues of both the pheromone/development-associated (Cpk2) and CWI (Mpk2) MAPKs alongside the canonical Cpk1 and Mpk1, in addition to a single Hog1 (Bahn et al. 2006). Recent dissection of Cpk2 and Mpk2 has shown that these paralogues are not merely redundant duplicates: they contribute distinct, non-overlapping functions in stress adaptation, virulence-factor regulation and cell wall remodelling, with some phenotypes only revealed in higher-order deletion backgrounds where canonical pathway activity is compromised (Jang et al. 2024). The retention of these paralogues, together with the elaborate Tco1–Tco7 sensor expansion upstream, is consistent with functional specialization rather than neutral persistence.

These observations raise a fundamental question that remains unresolved: what is the minimal MAPK signalling capacity required to sustain fungal viability and host adaptation? A naïve additive model would predict that combinatorial deletion of multiple MAPKs should progressively compromise growth, stress tolerance and virulence-associated traits. However, the extent to which fungal MAPK networks can absorb large-scale perturbations through compensatory rewiring remains poorly defined. Accordingly, the central HOG-pheromone/development-CWI triangle in Fig. 4 should be read as a conceptual representation of buffering capacity and inter-module crosstalk, rather than as a complete wiring diagram.

Figure 4.

Interconnected MAPK networks and evolutionary plasticity in fungi. Conceptual model illustrating how conserved fungal MAPK modules are diversified and rewired in pathogenic fungi. The upper-left scheme depicts paralogue diversification from ancestral MAPK modules, with lineage-specific retention of pheromone/mating- and CWI-associated MAPK paralogues; grey dashed connectors in this scheme denote inferred evolutionary relationships rather than documented duplication events. The core HOG components (Ssk2-Pbs2-Hog1) are comparatively conserved in architecture, although terminal-kinase duplication also occurs in some lineages (e.g. SakA/MpkC in A. fumigatus). The central HOG–pheromone/mating–CWI triangle represents pathway crosstalk and network buffering rather than a complete direct-wiring diagram. In C. albicans, the Cek1/Cek2 module is shown as a partially overlapping pheromone-responsive branch in which Cek1 acts as the dominant positive effector for mating-associated outputs, whereas Cek2 contributes more modestly and plays a cryptic, non-redundant role in cell-wall biogenesis. In C. neoformans, Cpk2 and Mpk2 are conditionally revealed MAPK paralogues, whose individual deletion produces no more than a weak phenotype in an otherwise wild-type background. However, their contribution to the shared output becomes apparent in sensitized backgrounds—for example, when the paralogue is overexpressed in cells lacking its dominant counterpart, or in higher-order deletion mutants. Additional regulatory systems, including calcineurin, cAMP/PKA, Rim, RAM, UPR and TOR-associated pathways, are shown as intersecting inputs that modulate MAPK outputs rather than as linear upstream components. The upper-right inset contrasts canonical and non-canonical Hog1 phosphorylation. “Canonical” phosphorylation is low under basal conditions and rises upon stress, as in S. cerevisiae and C. albicans and the C. deneoformans strain JEC21; “non-canonical” refers to the reverse pattern, in which basal phosphorylation is constitutively high and is transiently reduced upon stress, as observed in most serotype A strains, including the reference strain H99, as well as in some serotype D isolates such as B-3501. Transient dephosphorylation should not be read as pathway inactivation: strains with constitutively phosphorylated Hog1 are more stress resistant, and in H99 Hog1 is nuclear-enriched even without stress. The distinction concerns the direction of the phosphorylation response, not the residues modified, which correspond to the conserved dual phosphorylation TGY motif in both cases. The lower-right inset highlights contextual rewiring of the CWI pathway, in which host-relevant CO₂ dampens CnMpk1 phosphorylation, whereas cell-wall stress and heat promote CWI-associated responses. Thermal adaptation is shown as a representative convergent host-adaptation output: in C. albicans, CaHog1, CaMkc1, and CaCek1 contribute to temperature-associated stress responses, whereas in C. neoformans, CnMpk1 is the major CWI-linked contributor, with weaker or indirect contributions from pheromone-associated inputs such as Cpk1 and possible HOG-associated buffering. Elsewhere in the figure, dashed connectors denote relationships that are indirect or inferred rather than demonstrated to be direct, whereas solid connectors denote direct relationships established experimentally; bold and thin arrows independently indicate dominant and subordinate contributions, as defined above. Overall, the figure emphasizes that pathogenic MAPK outputs arise from paralogue retention, feedback, and contextual rewiring of conserved signalling modules rather than from isolated linear pathways. Created in BioRender (https://BioRender.com/6ykpizs).

Evolutionary plasticity is also evident in the integration of MAPK signalling with environmental adaptation. The constitutive phosphorylation of Hog1 in H99 and other C. neoformans serotype A isolates is inverted relative to S. cerevisiae and to the serotype D reference strain JEC21 (Bahn et al. 2005). Because serotype A and D Hog1 are identical at the amino-acid level, this divergence lies not in the kinase itself but in a small number of coding substitutions in the upstream Ssk2 MAP3K that render the Pbs2-Hog1 cascade constitutively active, illustrating how evolutionary re-tuning of an upstream regulator can repurpose a conserved terminal kinase (Bahn et al. 2007). Analogous re-tuning is observed in the antagonistic interaction between thermal and CO₂ adaptation programmes in C. neoformans, in which host-relevant CO₂ concentrations dampen Mpk1 phosphorylation in CO₂-tolerant strains, suggesting that the CWI pathway integrates host-environment cues beyond simple cell wall stress (Ristow et al. 2023). This contextual integration is also consistent with the involvement of additional pathways connected to CWI and host adaptation, including Rim-mediated cell wall remodelling, morphology, CO₂ tolerance, UPR-dependent stress adaptation and TOR-associated growth control (O’Meara et al. 2013, Cheon et al. 2014, So et al. 2019, Chadwick et al. 2022). At the level of development output, the same conserved cascades can be deployed to qualitatively different ends depending on cellular state: in C. neoformans, bisexual and unisexual mating share the upstream Cpk1 pheromone/development cascade yet differ in their requirement of Sxi1α/Sxi2a homeodomain complex, while in C. albicans the shared Ste11-Hst7-Cek1/Cek2 cascade drives biofilm formation in white cells but mating in opaque cells. These examples reinforce the idea that fungal pathogenesis comes not from gaining fundamentally new MAPK pathways but from quantitative and contextual changes in how a deeply conserved network is deployed.

MAPK signalling as a therapeutic frontier in medical mycology

The convergence of MAPK pathways on stress adaptation, antifungal tolerance and virulence-associated traits makes them attractive targets for antifungal intervention, but their therapeutic tractability remains unclear. Genetic disruption of MAPK pathway components frequently attenuates virulence-associated phenotypes in pathogenic fungi, including defects in cell wall integrity, thermotolerance, virulence-factor production, and antifungal stress adaptation (Diez-Orejas et al. 1997, Kraus et al. 2003, So et al. 2018). However, they do not by themselves establish that individual MAPKs are clinically useful drug targets.

Potential selectivity may be easier to achieve at fungal-specific regulatory steps than at the conserved kinase active sites. The two-component phosphorelay upstream of Hog1, including HHKs and Ypd1–Ssk1/Skn7 response-regulator modules, has no direct counterpart in mammalian cells and therefore remains an attractive area for target exploration (Bahn et al. 2006, Lee et al. 2011). Similarly, fungal-specific docking interfaces, scaffold interactions and paralogue-specific regulatory surfaces may provide routes to selectivity, but these possibilities require structural, biochemical and chemical-genetic validation.

Several antifungals also engage MAPK-linked stress responses. Fludioxonil perturbs the HOG pathway, whereas echinocandins and azoles impose cell wall or membrane stress that can activate CWI and HOG signalling (Bahn et al. 2006, Kojima et al. 2006). Thus, MAPK-pathway perturbation may be most useful as part of combination strategies that weaken compensatory stress responses rather than as standalone MAPK inhibition. Important caveats remain: many MAPK components are kinases with conserved ATP-binding pockets, and experience with mammalian p38 inhibitors shows the difficulty of achieving selectivity and durable safety. Future work should therefore prioritize fungal-specific upstream sensors, non-orthosteric regulatory interfaces and infection-relevant combination testing before MAPK-directed antifungal therapy can be considered a realistic translational strategy.

Conclusions and outlook

MAPK cascades remain a central framework for understanding fungal biology and disease. The foundational pathways of S. cerevisiae continue to provide an essential reference point, but pathogenic fungi increasingly show that MAPK signalling cannot be understood only as a set of linear pathways.

Three priorities should guide the next phase of work. First, systematic analysis of MAPK pathway combinations will be needed to define how much signalling capacity is required for fungal viability, stress adaptation and virulence-associated traits. Higher-order genetics, adaptive evolution and integrative multi-omics may reveal whether loss of individual pathways can be buffered by compensatory rewiring, secondary mutations, chromosomal changes or dosage effects.

Second, structural and chemical biology should focus on fungal-specific regulatory features, including two-component sensors, scaffold proteins, docking surfaces and lineage-specific paralogue interfaces. These studies will be necessary to distinguish realistic antifungal target opportunities from conserved kinase features that are unlikely to provide adequate selectivity. Third, MAPK signalling should be integrated with broader regulatory programmes, including chromatin-based cell-state switching, host CO₂ and temperature adaptation, cell wall remodelling, stress metabolism and host-pathogen interactions. The next challenge is therefore not simply to assign each fungal MAPK to a pathway, but to define how combinations of MAPK modules generate stress tolerance, virulence traits and antifungal resilience at the network level.

Conflicts of interest

The authors declare no conflict of interest.

Funding

This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korea government (MSIT) (RS-2025-18362970, RS-2025-02215093, and RS-2025-00555365); by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (RS-2026-25518347); and by the Korea Institute for Advancement of Technology (KIAT), funded by the Ministry of Trade, Industry and Energy in 2026 (RS-2024-00418203).