Calcium transport systems as virulence determinants: Mechanistic insights into fungal pathogenicity and antifungal resistance

ABSTRACT

This review examines the molecular mechanisms through which calcium transport proteins modulate virulence and drug resistance in human pathogenic fungi, synthesizing recent advances in calcium homeostasis research. Emerging evidence from calcium signaling pathway analyses reveals that fungal calcium transporters (including PMC1, VCX1, and CCH1/MID1 complexes) orchestrate critical stress adaptation processes through calcineurin-dependent and calcineurin-independent pathways, highlighting their critical involvement in fungal physiology, adaptive stress responses, and pathogenicity. Through a systematic evaluation of genetic, biochemical, and clinical studies, we elucidate how these transport systems mediate fungal cell wall integrity, biofilm formation, and efflux pump regulation, which are key determinants of virulence evolution and the development of azole resistance. The mechanistic framework presented not only advances our understanding of calcium-mediated fungal pathogenesis but also identifies these transport systems as promising targets for antifungal development, particularly for overcoming multidrug resistance in Candida, Cryptococcus and Aspergillus species.

Introduction

Fungal infections pose significant health risks, particularly in immunocompromised patients, where pathogens such as Candida species, Cryptococcus species and Aspergillus species can lead to severe morbidity and mortality [1]. Globally, more than 90% of reported deaths from fungal infections are attributed to these fungal pathogens [2]. Candida albicans causes nosocomial infections with high mortality rates, Aspergillus fumigatus causes invasive aspergillosis, and Cryptococcus species cause cryptococcosis, leading to severe central nervous system infections in vulnerable individuals such as HIV/AIDS patients, cancer patients undergoing chemotherapy or radiotherapy, patients with Sjogren’s syndrome and diabetic patients [3–5]. Additionally, hospitalized patients and those with medical devices such as catheters or ventilators are at increased risk [3,6,7]. The gradual rise in temperature due to climate change has allowed these fungi to adapt and survive at relatively high temperatures, including those close to the human body, increasing their ability to survive in human hosts; for example, Candida auris has shown increased thermal tolerance, possibly linked to climate change [8,9]. The increasing prevalence of these infections has been linked to the ability of fungi to adapt to and develop resistance to antifungal therapies. A critical factor influencing the pathogenicity and resistance mechanisms of fungi is the regulation of calcium levels within their cellular environments. In fungi such as Saccharomyces cerevisiae, calcium is often required for regulating the cell wall integrity pathway [10]. However, in many other fungal pathogens, such as Candida albicans and Aspergillus fumigatus, the CWI pathway is largely regulated by calcium-independent mechanisms, including the PKC-MAPK cascade, although calcium may still influence parallel stress responses [11,12]. Calcium ions (Ca²⁺) act as vital second messengers in various cellular processes, including the modulation of virulence factors and the response to environmental stressors. Hence, improper regulation of Ca²⁺ can cause significant cell damage and lead to cell death [13].

Recent studies have highlighted the role of calcium transport proteins in maintaining calcium homeostasis in fungi, which is crucial for their survival and pathogenicity. Calcineurin plays a key role in calcium signaling and cell stress responses in eukaryotes, influencing neuronal growth, ion channel regulation, heart failure, Alzheimer’s disease, and various other cellular processes [14]. Calcineurin helps fungal pathogens, such as Aspergillus fumigatus, survive at high calcium levels [13]. In Cryptococcus neoformans and Candida albicans, it regulates growth at alkaline pH, elevated temperatures, membrane stress, and virulence [15]. Calcium signaling pathways are conserved across different fungal species and play pivotal roles in regulating processes such as growth, development, and virulence. For example, the transcription factor Crz1, which is activated by calcium signaling, has been shown to regulate the expression of genes related to cell wall integrity, ion homeostasis, and virulence in pathogenic fungi [16,17]. Climate change imposes heat stress that selects for fungi that can tolerate extreme temperatures, which is necessary for human infection. In species such as Cryptococcus neoformans, Candida albicans, and Aspergillus fumigatus, calcineurin, which regulates the stress response, mediates this response to thermal stress and is crucial for growth and survival under host-like conditions, as well as virulence [18]. In C. neoformans, calcineurin is necessary for growth at 37°C, a temperature typical of the human body. In C. albicans, calcineurin is essential for survival in serum, whereas in Aspergillus fumigatus, calcineurin inactivation leads to reduced virulence and diminished filamentation [19]. Calcium also acts as a messenger inside cells to control processes such as autophagy and apoptosis, which are also activated by calcium signaling [20]. Additionally, VCX1, a vacuolar calcium exchanger, is responsible for regulating intracellular calcium levels during calcium stress in C. neoformans [21]. Furthermore, specific calcium transporters, such as Cch1 and Mid1, are essential for calcium influx and signaling, influencing the ability of fungi to withstand environmental stresses and penetrate host tissues [22,23]. While calcium transport proteins such as Cch1/Mid1, TRPY1/Yvc1, and Vcx1 are found in many species, their roles in growth, virulence, stress adaptation, and antifungal resistance vary significantly among species, and further specific studies are needed [21,24,25].

The interplay between calcium homeostasis and fungal virulence is further complicated by the development of antifungal resistance, which often arises from mutations in genes associated with calcium transport and signaling pathways. Understanding the molecular mechanisms by which calcium transport proteins influence fungal pathogenicity and resistance to antifungal treatment is critical for developing effective therapeutic strategies. Although some calcium-regulating proteins, such as Pmr1, Eca1, and Spf1, have remained conserved throughout fungal evolution, others, such as Mid1 and TRPY1/Yvc1, have evolved differently among species [24].

This review examines evidence suggesting that calcium transport proteins are primary regulators of virulence and antifungal resistance, and how disruptions in calcium signaling and transport can alter these traits in key human fungal pathogens, including Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans. While interest in calcium signaling is increasing, the translational possibilities of targeting these transport systems remain unexplored. To facilitate a complete and reproducible summary, we conducted a systematic search across the literature, as available from PubMed and Web of Science. In the search, combinations of the terms “calcium transport,” “Cch1,” “Mid1,” “calcineurin,” “fungal virulence,” and “antifungal resistance” were included. Elucidating these dynamics will inform targeted strategies to circumvent antifungal resistance.

Classification and function of calcium transport proteins

Calcium transport proteins are critical for maintaining cellular calcium homeostasis, which is essential for various physiological functions, including muscle contraction, neurotransmitter release, and cellular signaling. These proteins can be broadly classified into three main categories: calcium channel proteins, calcium pump proteins, and calcium exchanger proteins.

Calcium channel proteins

Calcium channel proteins are integral membrane proteins that facilitate the influx of calcium ions into cells (Figure 1). They are pivotal in generating action potentials in excitable cells, such as neurons and muscle cells. The primary types of calcium channels include voltage-gated calcium channels (VGCCs) [26], which open in response to membrane depolarization, and ligand-gated calcium channels, which open upon the binding of specific ligands. These channels are important for calcium entry and are essential for converting electrical activity into biochemical events in excitable cells [27]. Among VGCCs, L-type and T-type channels play significant roles in cardiac and smooth muscle contraction as well as neurotransmitter release in neurons. Dysfunction of these homologous fungal calcium channels contributes to stress sensitivity, virulence attenuation, and antifungal resistance. Other calcium channels include the Cch1-Mid1 complex (HACS), which serves as the primary calcium influx system in fungal cells and responds to osmotic, oxidative, and alkaline stress, whereas the low-affinity Ca^{2 +} influx system (LACS) is shown in Fig. 1 (fungal integral membrane protein 1) [28], which also regulates calcium influx and functions under specific growth conditions to supplement HACS [23,29,30] (Figure 1). For example, in C. neoformans, the Cch1 channel is directly activated when intercellular calcium stores are depleted or reduced [31].

Figure 1.

Calcium transport mechanisms in fungi: channels, pumps and exchangers. this diagram illustrates a schematic representation of calcium transport proteins in a fungal cell, showing their subcellular localization and the direction of calcium movement. Influx pathways (arrows pointing into the cystol) include plasma membrane channels (e.g. Cch1–mid1) and internal stores such as the ER and mitochondria (via NCLX) [29,50]. Efflux pathways (arrows pointing out the cytosol) include calcium pumps and exchangers targeting the ER (SERCA), Golgi (SPCA), vacuole (Vcx1), mitochondria (MCU), and extracellular space (NCX, PMCA, NCKX) [45]. These proteins regulate calcium homeostasis, which is important for fungal growth, the stress response, and virulence.

Recent advancements in understanding the interactions between calcium channels and their partner proteins have opened new avenues for therapeutic interventions targeting these channels in various diseases [32,33]. For example, L-type calcium channels (CaV1.2 and CaV1.3) are being studied for the treatment of Parkinson’s disease by blocking them, as they contribute to calcium toxicity. However, blocking CaV1.2 with isradipine causes side effects such as hypotension; therefore, the aim is to develop a drug that selectively blocks CaV1.3 instead [34].

Calcium pump proteins

Calcium pump proteins, such as sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) [26,35,36], plasma membrane calcium ATPase (PMCA) [37] and secretory pathway calcium ATPase (SPCA) [38–40] are responsible for extruding calcium ions from the cytosol, thereby maintaining low intracellular calcium concentrations [41] (Table 1). SERCA pumps calcium ions back into the sarcoplasmic reticulum after muscle contraction, which is crucial for muscle relaxation. SPCA takes up calcium from the cytosol into the Golgi complex PMCA [38]; on the other hand, it extrudes calcium ions from the cell to prevent calcium overload, which can lead to cellular toxicity and apoptosis. Dysregulation of these pumps has been implicated in numerous diseases, including neurodegenerative disorders and cancer. For example, alterations in SERCA function have been associated with Alzheimer’s disease, bipolar disorder, schizophrenia and even Parkinson’s disease [10], where impaired calcium handling contributes to neuronal dysfunction [10,42]. Muscular disruption caused by prolonged calcium overload is associated with dysfunction of this SERCA pump [43]. Defects in PMCAs are associated with numerous diseases, particularly those linked to oxidative stress in the brain, including ischemia, diabetes, atherosclerosis, and aging [41]. Additionally, the role of calumenin, a calcium-binding protein, in regulating SERCA activity further underscores the complexity of calcium homeostasis [44].

Table 1.

Functions of calcium transport proteins.

Functions	Transporters/Channels/Pumps	Fungi
1. Calcium Homeostasis [37] [38] [26,50,54]	SERCA (pump) PMCA (pump) SPCA (pump) NCX, VCX1(exchangers) Cch1/Mid1(Channels)	C. albicans [29] C. neoformans C. neoformans C. neoformans C. albicans [31]
2.Stress Response [21]	Cch1/Mid1(Channels) VCX1(exchanger)	C. albicans [22] C. neoformans
3. Pathogenicity. [108] &virulence	Cch1 (Channels)	C. neoformans
4.Cell growth [23] &division [35] [37] [38] VCX1(exchanger) C.neoformans [54]	Cch1/Mid1(channels) SERCA (pump) PMCA (pump) SPCA (pump)	C. neoformans C. neoformans C.neoformans C. neoformans
5.Host interaction [122] &immune evasion	Cch1/Mid1	C.neoformans
6. Enzyme [50] Regulation	SERCA (pump)	A.fumigatus

Calcium exchanger proteins

Calcium exchanger proteins, such as the sodium/calcium exchanger (NCX), play a crucial role in calcium homeostasis by facilitating the exchange of sodium ions for calcium ions across the plasma membrane (Table 1). This mechanism allows for the fine-tuning of intracellular calcium levels, particularly in cardiac and neuronal tissues. NCX can operate in both forward and reverse modes, depending on the electrochemical gradients of sodium and calcium, thus adapting to cellular needs [26,40,45,46]. NCX regulation is complex and involves various intracellular signaling pathways and posttranslational modifications. Dysregulation of NCX has been linked to several pathological conditions, including heart failure and ischemic injury, making it a potential therapeutic target [42,47]. This sodium/calcium exchanger activity can be altered by mitochondrial dysfunction, leading to a disruption of calcium balance and stress adaptation, thereby influencing antifungal resistance mechanisms in C. neoformans [48,49]. Another exchanger protein is NCLX, the mitochondrial sodium-calcium exchanger [50,51]. Mitochondrial matrix Ca^{2 +} levels control ATP production and are maintained by a balance between Ca^{2 +} influx (via the MCU and other pathways) and Ca^{2 +} efflux (mainly through the Na⁺/Ca^{2 +} exchanger) [40,50]. The cytosolic Na⁺-dependent mitochondrial Ca²⁺ efflux transporter NCLX was also shown to regulate B lymphocyte chemotaxis [52]. Vcx is a vacuolar calcium/proton (Ca^{2 +}/H⁺) exchanger that balances calcium levels by swapping calcium ions for protons across the vacuolar membrane [53,54]. In addition, another exchanger called (NCKX), which is a K±dependent Na⁺/Ca²⁺ exchanger, removes excess calcium from the cell while transporting it into sodium and potassium ions [39,55]. The deletion of these transporters disrupts fungal growth, weakens resistance to stress and antifungal drugs, affects ion balance, damages the cell wall, interferes with cell division, and reduces virulence, highlighting the crucial role of calcium (Ca^{2 +}) in fungal pathogens [56].

Impact of calcium signalling pathways on fungal virulence

Regulation of virulence factor synthesis

Calcium (Ca²⁺) is a pivotal signaling molecule that orchestrates various cellular processes, including the synthesis of virulence factors in fungi. The transient increase in intracellular calcium levels activates a cascade of signaling pathways crucial for fungal growth and stress tolerance (Table 1). One of the key players in this process is the transcription factor Calcineurin-Responsive Zinc finger 1 (CRZ1), which regulates approximately 300 genes; however, the exact number and targets involved in stress responses and virulence vary among fungal species [57]. In S. cerevisiae, Crz1 directly regulates approximately 300 genes, whereas in pathogenic fungi such as C. albicans or C. neoformans, the number of regulated genes varies (typically 100–200) depending on the degree of environmental stress [58,59]. Research indicates that CRZ1 modulates pathways associated with osmotic and oxidative stress, enabling pathogenic fungi to adapt and thrive in hostile environments. In addition, calcium signaling regulates fungal virulence through calcineurin, which controls not only CRZ1 but also RNA-binding proteins such as Pbp1 and Puf4, influencing stress adaptation, mRNA stability, and fungal resistance [60]. This regulation is particularly important in host‒pathogen interactions, where the ability to synthesize virulence factors rapidly can determine the outcome of infection. For example, in Candida albicans, the deletion of PHR1, a gene induced by calcium stress, leads to increased sensitivity to calcium and reduced virulence, highlighting the intricate relationship between calcium signaling and the regulation of virulence factors in pathogenic fungi [61,62]. However, the expression of PHR1 is modulated indirectly through calcium influx but directly by the regulation of CRZ1. Specific experimental evidence has shown that PHR1 expression is induced by calcium stress in a CRZ1-dependent manner. Crz1 directly binds a CalcineurinDependent Response Element (CDRE) in the PHR1 promoter, leading to the upregulation of PHR1 transcription during calcium challenge [62]. Thus, PHR1 is directly regulated by CRZ1, not merely indirectly via calcium influx.

In C. neoformans, the Ca^{2 +}/calcineurin pathway is dependent on the catalytic subunit Cna1 and regulatory subunit Cnb1, which regulate thermotolerance, high CO₂ (5%) resistance, alkaline pH adaptation, and cell wall integrity, as the inhibition of calcineurin disrupts survival under these conditions (37°C, elevated CO₂, or alkaline pH [63,64]). Overall, calcineurin is essential for the virulence of C. neoformans and C. albicans, although its role differs between them. In C. neoformans, calcineurin is essential for growth at body temperature, whereas in C. albicans, it is needed for survival in the bloodstream and the spread of infection within the host [65]. Calcium-dependent signaling pathways not only control fungal adaptation and virulence but also contribute to major clinical challenges. Disruption of calcineurin in C. albicans and C. neoformans increases susceptibility to azole and echinocandin antifungals, demonstrating its critical role in drug tolerance and treatment resistance [66]. These pathways also facilitate immune evasion and survival under host-like pressures (high temperature, alkaline pH) through the regulation of mRNA stability and cell wall maintenance by proteins such as Puf4 and Pbp [67]. Understanding these pathways will provide the necessary information to develop targeted therapeutics that overcome drug resistance and improve therapeutic outcomes.

Stability of the cell wall and membrane

The stability of fungal cell walls and membranes is essential for maintaining structural integrity during environmental stress (Table 1). For example, the C. albicans cell wall comprises distinct layers: an inner core enriched with chitin and glucans and an outer layer of mannosylated proteins. This structure provides both mechanical stability and resilience to environmental stressors [68]. Calcium plays a critical role in this process by influencing the synthesis and organization of cell wall components. For example, the calcium-dependent signaling pathway regulates the production of chitin and glucans, which are vital for cell wall stability. Disruptions in calcium homeostasis can lead to compromised cell walls, increasing fungal susceptibility to environmental stresses and antifungal agents. Studies have shown that the calcineurin signaling pathway, which is activated by increased intracellular calcium levels, is crucial for maintaining cell wall integrity in response to stressors. In Magnaporthe oryzae, the deletion of the calcineurin regulator MoRCN1 resulted in reduced virulence due to impaired appressorium formation, further illustrating the importance of calcium signaling in cell wall stability and pathogenicity [61, 69]. Additionally, calcium signaling through CrzA and ZipD is essential for Aspergillus fumigatus cell wall stability, with ZipD regulating chitin and β-glucan synthesis to maintain integrity, reduce immune detection, and enhance virulence and antifungal resistance [70].

Ability to adapt to environmental changes

Fungi exhibit remarkable adaptability to environmental changes, and calcium signaling is central to this process. The ability to sense and respond to fluctuations in calcium levels allows fungi to modulate their growth and pathogenicity in response to external stimuli. Fungi use calcium as a signaling molecule to adapt to various environmental stresses, and this adaptation is mediated through CRZ1 signaling [71,72]. When the cytosolic calcium concentration increases, calcium binds to calmodulin, which activates the phosphatase calcineurin. Calcineurin then dephosphorylates the transcription factor CRZ1, enabling it to enter the nucleus and regulate genes involved in the stress response, cell wall integrity, and ion balance. This signaling pathway enables fungi to adapt rapidly and maintain homeostasis in response to environmental stress [57]. Calcium ions serve as secondary messengers that trigger adaptive responses to various stressors, including changes in osmotic pressure and temperature. The activation of the calcineurin pathway facilitates the expression of stress response genes, enabling fungi to withstand adverse conditions. In the human pathogenic fungus C. neoformans, the calcineurin pathway is vital for survival in hostile host environments, where the immune system regulates calcium concentrations and other stressors [73,74]. Mutants lacking key components of this pathway exhibit heightened sensitivity to stress, such as high temperature and osmotic imbalance, and, more importantly, reduced virulence, highlighting the role of calcium signaling not only in environmental adaptation but also in the pathogenic potential of fungi, emphasizing calcineurin and CRZ1 as promising targets for antifungal strategies [69,75].

Research on calcium-mediated signalling and fungal virulence

Recent studies have highlighted the importance of calcium-mediated signaling in the virulence of fungal pathogens. The calcineurin signaling pathway, activated by calcium, is crucial for various aspects of fungal development and pathogenicity, including stress responses, cell wall integrity, and the production of virulence factors. For example, in Aspergillus fumigatus, calcium signaling regulates the expression of genes involved in drug resistance and virulence, demonstrating the multifaceted role of this pathway in fungal biology (Table 1). Additionally, in Candida albicans, the calcium signaling pathway plays a crucial role in conferring resistance and tolerance to azole drugs [37,76]. Some studies have also shown that blocking calcium signals with calcium inhibitors, in combination with drugs such as azoles, can inhibit fungal growth more effectively [77,78]. Furthermore, research on the interactions between calcium signaling and other pathways, such as the PHO pathway, reveals a complex network of regulatory mechanisms that fungi utilize to adapt to their environments and increase their virulence. Understanding these signaling pathways offers insights into potential therapeutic targets for antifungal drug development, as disrupting calcium signaling could impair fungal virulence and enhance treatment efficacy [69,] [79,80]. In addition, the existence of calcineurin-targeting drugs and their binding structures could provide critical insights into potential therapeutic targets, aiding the development of new compounds that block fungal virulence without compromising immune defense [81].

Relationships between calcium transport proteins and fungal drug resistance

Mechanisms of antifungal drugs

Antifungal drugs primarily function by targeting the cell walls or cell membranes of fungal pathogens. The most common classes include azoles, polyenes, and echinocandins, each with distinct mechanisms of action. Azoles inhibit the activity of lanosterol 14α-demethylase, a key enzyme involved in ergosterol synthesis and essential for maintaining cell membrane integrity [82–84]. Examples of azole drugs include fluconazole and itraconazole [78–80]. In contrast, polyenes bind to ergosterol, creating pores that lead to cell lysis, and in Candida albicans, they stimulate the CWI pathway [85]. An example of a polyene drug is amphotericin B [86–88]. Lastly, echinocandins disrupt the integrity of the fungal cell wall by inhibiting the synthesis of the cell wall component (1,3)-β-d-glucan [83–86]; examples of echinocandin drugs include caspofungin, micafungin, and anidulafungin [86,87,89]. The synthesis of chitin and glucan plays a crucial role in maintaining fungal cell integrity during growth and adaptation to stress [90]. Despite these mechanisms, the emergence of drug-resistant strains poses significant challenges in clinical settings. Resistance often arises from mutations in drug-target proteins or the upregulation of efflux pumps that expel drugs from the cell, thereby reducing their efficacy. Recent studies have highlighted the role of calcium transport proteins in modulating responses to antifungal agents, suggesting that alterations in calcium homeostasis can affect drug susceptibility (Table 2). For example, the metal chaperone protein MtmA in Aspergillus fumigatus has been shown to negatively regulate resistance by influencing calcium signaling pathways, indicating a complex interplay between calcium transport and antifungal drug action [91]. Another metal chaperone protein in Candida albicans is Hsp90, which plays a critical role in resistance to echinocandins by regulating the calcineurin pathway. Inhibiting Hsp90 reduces resistance and enhances the efficacy of echinocandin [92–94]. Further disruption of mitochondrial function and altered calcium levels activate the CrzA pathway, leading to the upregulation of drug efflux pumps, which contributes to azole resistance in Aspergillus fumigatus [95]. Another limitation is the fungistatic nature of azole drugs rather than their fungicidal nature, which likely contributes to resistance in immunocompromised patients [96,97]. Finally, another major challenge is the side effects associated with antifungal drugs. For example, amphotericin B is known to cause nephrotoxicity [90], whereas azoles can lead to hepatotoxicity [95] and interact with cytochrome P450 enzymes either as substrates or inhibitors, causing further toxicity [96]. These limitations underscore the urgent need for the development of safer and more effective antifungal agents.

Table 2.

Calcium transport proteins and their resistance to antifungals.

Antifungal dug class	Mode of action	calcium transporter involvement.	Effect on resistance
Azoles	Inhibits the activity of lanosterol 14-a- Demethylase [82]	Cch1/Mid1 mediate Ca2+ entry [29] Vcx1 removes Excess Ca2+ [54]	supports membrane stress stability and tolerance through Crz1 activation
Polyenes	Binds to ergosterol, creating pores leading to cell lysis [86]	Vcx1 regulates Ca2+ homeostasis [53] NCLX regulates. excess Ca2+ [51]	limits of toxic Ca2+ levels to maintain Intracellular stability
Echinocandins	Inhibit the 1,3 betta-d-glucan synthesis [126] [22],	Cch1/Mid1 activates calcineurin during stress Vcx1 maintains Ca2+ Balance [50]	Activates cell wall repair genes (e.g.; chitin synthesis).

The impact of calcium transport on drug accumulation

Calcium transport proteins play crucial roles in maintaining intracellular calcium levels, which are essential for various cellular functions, including drug accumulation. Fungal cells utilize calcium channels and pumps to regulate calcium homeostasis, which directly impacts the efficacy of antifungal treatments. Studies have demonstrated that disruptions in calcium signaling can lead to increased drug resistance. For example, mutations that affect calcium transport mechanisms can result in altered cytosolic calcium levels, which in turn influence the expression of multidrug resistance-associated transport genes (Figure 2). This was observed in A. fumigatus, where calcium signaling was linked to the upregulation of these transporters, leading to increased resistance to azole antifungals. Similarly, in C. neoformans, calcium transporters are essential for withstanding antifungal pressure, and strains lacking these transporters exhibit increased susceptibility to fluconazole [98]. In C. albicans, combining calcineurin inhibitors such as cyclosporine A with azole antifungals counteracts resistance by disrupting the calcium-dependent signaling pathway [99]. Additionally, the use of calcium chelators has been shown to restore drug susceptibility, emphasizing the importance of calcium transport in modulating drug accumulation within fungal cells [100].

Figure 2.

Calcium signaling pathway in pathogenic fungi. the diagram shows how external stress signals trigger calcium influx through the Cch1-mid1 channel complex. Elevated levels of cytosolic Ca2+ activate calmodulin and the phosphate calcineurin, which dephosphorylates the transcription factor Crz1. Crz1 then enters the nucleus to activate stress-responsive genes [61]. These signals regulate fungal virulence, drug resistance, cell wall integrity and adaptation to environmental stress. Organelles such as the vacuole, ER, mitochondria, and Golgi maintain calcium balance via pumps and exchangers [69].

Regulation of antifungal resistance gene expression

The expression of antifungal resistance genes is intricately linked to calcium signaling pathways. Calcium ions act as secondary messengers in various signaling cascades, influencing the transcription of genes associated with drug resistance. For example, the transcription factor CrzA, which is activated by elevated calcium levels, plays a pivotal role in regulating the expression of multidrug resistance genes in response to antifungal stress. In C. neoformans, deletion of flavin carrier protein 1 (FLC1) led to increased cytosolic calcium levels and increased expression of Crz1-dependent genes, which are crucial for virulence and drug resistance. In addition, CRZ1-deficient mutants exhibit hypersensitivity to chemicals such as chloride and chitosan, a dysregulated transcriptional response to alkaline stress and defects in cellular morphology and mating [101]. In C. neoformans, this hypersensitivity suggests that CRZ1 mutants struggle with ionic stress and cell wall integrity, which may weaken their survival and virulence. These findings suggest that calcium transport proteins not only maintain homeostasis but also modulate the expression of resistance genes in response to antifungal treatment, thereby contributing to the overall resistance phenotype of fungal pathogens [102] (Figure 2).

Calcium-mediated signal transduction and resistance research

Research into calcium-mediated signal transduction pathways has revealed their significant role in the development of antifungal resistance. Calcium signaling is crucial for various cellular processes, including the stress response and adaptation to antifungal agents. In Aspergillus fumigatus, for example, calcium signaling pathways have been shown to activate stress response mechanisms that confer resistance to azole treatment. The interplay between calcium signaling and mitochondrial function has also been highlighted, where mitochondrial dysfunction can trigger calcium-dependent signaling pathways, leading to the upregulation of resistance-associated genes. These findings indicate that targeting calcium signaling pathways may represent a novel therapeutic strategy to combat antifungal resistance. Disruption of these pathways has shown promise in reversing resistance and enhancing the efficacy of existing antifungal treatments, suggesting that further exploration of calcium transport proteins could yield valuable insights into overcoming drug resistance in fungal pathogens [103]. In addition, blocking multiple pathways, such as PKC, HOG, and calcineurin, in Candida albicans can prevent paradoxical effects, which may also be a potential antifungal strategy for Aspergillus fumigatus [104]. Furthermore, calcineurin is essential for the virulence of C. neoformans and may identify key signal transduction components involved in fungal pathogenesis that could serve as potential therapeutic targets [74,105].

The interplay between calcium-dependent signaling and multistress response networks in fungi

Fungal pathogens are constantly challenged by a variety of environmental stresses (e.g. oxidative stress, osmotic stress, membrane disruption, and antifungal therapy), and calcium-dependent signaling is central to triggering the adaptive response to a variety of stressful environments. When exposed to stress, intracellular calcium influx occurs through channels such as Cch1–Mid1, which activate downstream effectors, including the calcineurin – Crz1 signaling pathway, and regulate the expression of gene targets involved in ion homeostasis, cell wall rearrangement, and oxidative stress defenses [106]. Calcium signaling does not work alone; it involves a variety of stress response networks, including the high-osmolarity glycerol (HOG) pathway, the protein kinase C (PKC) pathway, and MAP kinase cascades [104]. Those connections of concurrent and cross-regulating networks enable the fungus to accept multiple stress signals and coordinate a single adaptive response. For example, in Candida albicans and Aspergillus fumigatus, calcineurin signaling is necessary for survival under antifungal therapy, endoplasmic reticulum stress, and thermal stress, indicating that calcium is a central regulator of stress adaptation in fungi. The connections between calcium signaling and other stress response systems could provide potential for the development of synergistic antifungal approaches that target multiple response pathways at once [106].

Calcium transport proteins in therapy potential

Inhibitory effects of calcium transport protein inhibitors on fungal growth

Calcium transport proteins play crucial roles in various biological processes, including the regulation of fungal growth and development. Recent studies have highlighted the potential of calcium transport protein inhibitors as antifungal agents. For example, the inhibition of calcium signaling pathways in fungi has been shown to significantly suppress growth and pathogenicity, particularly in species such as C. neoformans. The Rab GTPase Ypt7 in C. neoformans has been identified as essential for vacuole biogenesis and iron acquisition, indicating that targeting calcium transport processes could impair fungal survival and virulence [107]. Deletion of the Cch1-Mid1 calcium channel impairs calcium uptake, reduces pathogenicity and enhances susceptibility to oxidative stress [11,108]. Additionally, natural compounds such as tryptanthrin have demonstrated antifungal activity against Cryptococcus species via mechanisms involving the regulation of calcium transport pathways and cell cycle arrest [109]. These findings suggest that calcium transport protein inhibitors could serve as effective therapeutic agents for combating fungal infections, particularly in immunocompromised patients. Despite these findings, there are significant structural similarities between fungal calcium transport proteins and their mammalian counterparts, particularly in the Cch1–Mid1 and calcineurin pathway components, which raises the possibility of off-target effects [110]. Moreover, phylogenetic analysis of the fungal calcium transport system revealed both functional conservation and evolutionary divergence at the species level, which is likely indicative of the potential for selective targeting [24]. Although some drugs, including BAPTA and verapamil, can disrupt calcium regulation in fungi in vitro, they are not suitable for clinical use in human patients due to safety or specificity concerns. In addition, different fungal species may utilize calcium channels and signaling pathways that depend on calcium, indicating that treatments may need to be tailored to individual fungi. Furthermore, by targeting calcium transport proteins as antifungals, researchers will need to develop antifungal drugs that specifically target fungal proteins without significantly impacting similar human proteins.

Structural and pharmacological advances in targeting fungal calcium channels

Recent advances in structural biology provide more clarity regarding the structure and regulatory aspects of fungal calcium channels, specifically the members of the high-affinity calcium uptake system HACS (for example, components (such as Cch1 and Mid1 from C. albicans and A. fumigatus) that are important for fungal virulence). Calcium channels are essential for maintaining calcium homeostasis, facilitating hyphal development, resistance to oxidative stress, and cell wall integrity [110]. Importantly, these features of fungal calcium channels, which contribute to virulence and host adaptation, can be exploited in the future by recognizing certain fungal-specific structural and regulatory features, unlike their mammalian counterparts. Functionally, certain calcium channels have been validated as potential antifungal targets, as well as in pharmacological studies where they have been structurally identified to exhibit a certain level of antifungal activity, despite the rise in antifungal resistance relative to current antifungal treatments [81].

Synergistic effects of calcium transport protein inhibitors with other therapeutic approaches

The combination of calcium transport protein inhibitors with other therapeutic modalities has shown promise in enhancing antifungal efficacy. For example, the use of tryptanthrin in conjunction with calcineurin inhibitors, such as FK506 and cyclosporine A, has resulted in a synergistic effect against C. neoformans, thereby enhancing the overall antifungal response [109]. Experimental studies have demonstrated that calcineurin inhibitors (FK506 and cyclosporine) synergize with caspofungin to disrupt A. fumigatus hyphal development [111], show similar synergy against echinocandin-resistant Candida lusitaniae strains [112], and exhibit growth inhibition against C. neoformans [113], whereas combinations of azoles of azoles (fluconazole, terbinafine, fenpropimorph, voriconazole, and posaconazole) with these inhibitors enhance antifungal efficacy against C. albicans [114–120], with the cyclosporine-fluconazole combination proving more effective than amphotericin B or fluconazole alone [121]. Calcium channel blockers (verapamil and flunarizine) further synergize with fluconazole against resistant C. albicans by disrupting Cch1-mid1-mediated calcium homeostasis [122]. This synergism may be attributed to the modulation of calcium signaling pathways, which are critical for fungal growth and survival. Furthermore, recent advancements in nanomedicine have opened up new avenues for delivering calcium transport inhibitors in conjunction with conventional antifungal therapies, potentially enhancing their bioavailability and therapeutic effectiveness [123]. The integration of these strategies could lead to more effective treatment regimens against resistant fungal infections, highlighting the need for further research into combination therapies involving calcium transport protein inhibitors.

Challenges and opportunities in clinical translation

Despite the promising potential of calcium transport protein inhibitors in antifungal therapy, several challenges hinder their clinical translation. One significant obstacle is the complexity of calcium signaling pathways, which are often intertwined with various cellular processes, making it difficult to selectively target specific calcium transport proteins without affecting normal cellular functions [124]. Furthermore, the ability of fungi to switch between their morphological states is a key factor in causing diseases in humans, since it allows them to survive in different environments and even invade host tissues, such as the opportunistic human pathogen C. albicans [125]. Moreover, the variability in patient responses to calcium modulation therapies complicates the development of standardized treatment protocols. However, recent advancements in drug delivery systems, such as nanoparticles and targeted therapies, present new opportunities to increase the specificity and efficacy of these inhibitors [123]. Additionally, the fungal cell wall is composed of molecules not found in human cells, making it an ideal target for the development of clinical antifungal compounds that do not harm human cells [3]. Furthermore, A. fumigatus calcineurin has a unique serine-proline-rich region that is conserved in filamentous fungi but absent in humans, presenting an opportunity to develop selective antifungal drugs [57]. Additionally, ongoing research into the molecular mechanisms of calcium transport proteins in fungi may provide insights into the optimization of therapeutic strategies. Addressing these challenges while leveraging emerging technologies could pave the way for effective clinical applications of calcium transport protein inhibitors in the treatment of fungal infections.

Conclusion

The regulation of pathogenicity and drug resistance in human fungal pathogens is a complex process that is significantly influenced by calcium transport proteins. This review highlights the critical role these proteins play in the virulence of fungi, emphasizing their potential as key targets for novel antifungal therapies. The intricate interplay between calcium signaling and fungal pathogenicity suggests that a deeper understanding of the molecular mechanisms underlying calcium transport could open new avenues for treatment strategies that effectively combat fungal infections. As we delve into the molecular pathways involving calcium transport proteins, it is essential to recognize the diverse findings across different studies. While some studies have established a clear connection between calcium homeostasis and fungal virulence, other studies present contrasting findings or emphasize alternative pathways. This variance underscores the need for a balanced interpretation of the existing data, promoting an integrative approach that reconciles different perspectives and findings. A collaborative effort among researchers is crucial to harmonize these disparate results, ultimately leading to a comprehensive understanding of the role of calcium transport proteins in fungal biology. Moreover, despite the promising insights gained thus far, a significant gap remains in translating these findings into practical clinical applications. Future experimental and clinical studies are imperative to validate the proposed mechanisms and assess the therapeutic potential of targeting calcium transport proteins. By prioritizing research investigating the clinical implications of these proteins in various fungal species, we can develop more effective antifungal agents and address the growing challenge of antifungal resistance. In conclusion, while the current body of research provides a solid foundation for understanding the importance of calcium transport proteins in fungal virulence and drug resistance, further investigations are necessary. By fostering a multifaceted research agenda that encompasses both basic and applied studies, we can pave the way for innovative antifungal therapies that enhance our ability to combat fungal infections in an era of increasing resistance.

Funding Statement

This work was supported by grants from:. National Natural Science Foundation of China [32300020] (W.W.). Natural Science Foundation of Anhui Province [2023AH050735] (W.W.).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

There is no new data produced in this manuscript.