Deciphering the role of mitochondria in human fungal drug resistance

ABSTRACT

As pathogenic fungi increasingly threaten public health, particularly in immunocompromised populations, understanding the mechanisms behind fungal drug resistance has become critical. This review focuses on the pivotal role of mitochondria in this process. Evidence indicates that mitochondria are essential not only for cellular energy metabolism and responses to oxidative stress but also for significantly influencing the expression and activity of drug efflux pumps, which facilitate the expulsion of antifungal agents. Moreover, the intricate roles of mitochondria in iron homoeostasis and calcium signalling are closely linked to the development of drug resistance in fungi. By elucidating these mechanisms, we can identify potential therapeutic targets and pave the way for more effective strategies to combat resistant fungal infections.

GRAPHICAL ABSTRACT

1. Introduction

Fungal infections have increasingly been recognised as a major global health concern, especially among individuals with compromised immune systems (Rhodes and Fisher 2019; Lionakis and Drummond 2023; Denning 2024). Pathogenic fungi, such as species of Candida and Aspergillus, are responsible for a wide array of infections ranging from superficial skin conditions to severe, life-threatening systemic diseases (Mukaremera et al. 2017; Talapko et al. 2021; Earle et al. 2023; Lass-Flörl and Kanj 2024). The World Health Organization (WHO) published its first-ever list of priority fungal pathogens in October 2022, organising 19 fungal species into three tiers of urgency (critical, high, and medium) to direct research initiatives. The critical category includes pathogens Cryptococcus neoformans, Candida auris, Aspergillus fumigatus, and Candida albicans; the high-priority category contains Nakaseomyces glabrata (Candida glabrata), Histoplasma spp., Eumycetoma causative agents, Mucorales, Fusarium spp., Candida tropicalis, and Candida parapsilosis; while the medium-priority category comprises Scedosporium spp., Lomentospora prolificans, Coccidioides spp., Pichia kudriavzeveii (Candida krusei), Cryptococcus gattii, Talaromyces marneffei, Pneumocystis jirovecii, and Paracoccidioides spp. (WHO 2022). Human fungal infections, despite their significant impact, remain under-researched. Recent estimates indicate that approximately 6.5 million people are affected each year, resulting in 3.7 million deaths – a significant increase from the previous estimate of 1.5 to 2.0 million deaths annually (Brown et al. 2024; Denning 2024). The increasing rates of antifungal resistance further complicate the management of these infections, leading to heightened morbidity and mortality (Fisher and Alastruey-Izquierdo 2022; Kaki 2023; Lee et al. 2023; Hoenigl et al. 2024; van Rhijn et al. 2024; Zheng et al. 2024). Frequently utilised antifungal agents, such as azoles, echinocandins, and polyenes, are facing significant challenges due to the rise of resistant strains (Kano et al. 2020; Jamiu et al. 2021; Fisher and Alastruey-Izquierdo 2022; Mahendrarajan and Bari 2022; Rivelli Zea and Toyotome 2022; Czajka et al. 2023; Daneshnia et al. 2023; Xu et al. 2024).

Mitochondria, often referred to as the powerhouses of the cell, are essential for energy production, primarily through a process known as oxidative phosphorylation, which generates adenosine triphosphate (ATP), the energy currency of the cell. However, in fungi, mitochondria transcend mere ATP generation by regulating various metabolic pathways, influencing cellular signalling, and mediating responses to environmental stresses (Li et al. 2020; Black et al. 2021; Xue et al. 2023a, 2024). Research highlights the intricate relationship between mitochondrial activity and the virulence of fungal pathogens, drawing significant interest within the field of mycology (Shingu-Vazquez and Traven 2011; Black et al. 2021; Xue et al. 2023a, 2024; Hu et al. 2024). Understanding how these organisms adapt to hostile conditions and elude host immune responses is vital for developing new therapeutic strategies. Recent advancements have highlighted the crucial role of mitochondria in fungal drug resistance (Koch and Traven 2020; Song et al. 2020). Mitochondria contribute to metabolic flexibility, reactive oxygen species (ROS) management, and iron homoeostasis, all of which are vital for fungal survival under therapeutic pressure (Black et al. 2021). The complex interplay between mitochondrial function and drug resistance mechanisms is influenced by factors such as metabolic states, environmental stressors, and genetic variations, which can significantly impact the overall fitness of fungal pathogens. Changes in mitochondrial morphology, including processes like fusion and fission, are tightly regulated and can influence a pathogen’s ability to respond to stress and adapt to new environments. For example, when confronted with oxidative stress or nutrient scarcity, fungi can modify their mitochondrial architecture to enhance survival and pathogenic capabilities (Chang and Doering 2018; Koch and Traven 2020; Song et al. 2020). Additionally, mitochondria play a central role in iron metabolism, vital for the survival and virulence of numerous fungal pathogens (Black et al. 2021; Xue et al. 2023a). Iron acts as a cofactor for various enzymes involved in respiration and the synthesis of essential biomolecules. As fungal pathogens compete with their hosts for this critical nutrient, the regulation of iron uptake and homoeostasis becomes a key element in determining their pathogenicity (Kronstad et al. 2013). Research has shown that alterations in mitochondrial function can significantly affect iron metabolism (Xue et al. 2023b, 2024). This connection links mitochondria to mechanisms of virulence and resistance against antifungal agents (Black et al. 2021). Moreover, mitochondria are essential for responding to stressors encountered in the host environment. Fungal pathogens frequently face challenges such as oxidative stress, heat shock, and nutrient limitation upon entering the host. Mitochondria help alleviate these stresses by modulating metabolic pathways, enhancing antioxidant defences, and regulating cellular signalling cascades (Weerasinghe and Stölting 2024). The capacity of fungi to endure oxidative stress is often associated with mitochondrial function, as ROS production can trigger adaptive responses that enhance survival and virulence (Chang and Doering 2018; Black et al. 2021, 2024). The ability of fungi to adapt to elevated temperatures – common in mammalian hosts – is significantly influenced by mitochondrial functions. The interaction between temperature-sensitive mitochondrial proteins and metabolic pathways is crucial for the survival of these pathogens in the host environment. Maintaining mitochondrial integrity is vital for cellular processes, as any disruption can impair adaptability to thermal challenges and affect virulence (Caza et al. 2016; Xie et al. 2017; Horianopoulos et al. 2020; Gao et al. 2022). The multifaceted roles of mitochondria in fungal pathogens suggest that they are not merely passive contributors to energy metabolism but active participants in signalling and regulatory networks that determine virulence and drug resistance. Understanding the pathogenic potential of fungi related to mitochondrial function is essential for designing innovative antifungal strategies to combat the growing challenge of drug resistance. As antifungal resistance continues to rise, grasping these connections becomes increasingly crucial (Li and Calderone 2017; Duvenage et al. 2019; Song et al. 2020). This review aims to elucidate the complex relationship between mitochondrial function and fungal drug resistance, focusing on how mitochondrial processes, iron homoeostasis, and cellular signalling pathways influence this resistance.

2. Connection between mitochondrial function and drug resistance

The intricate relationship between mitochondrial function and drug resistance in fungi highlights the critical role of mitochondria in various cellular processes. This connection can be organised into several key aspects that contribute to our understanding of how mitochondrial function impacts the efficacy of antifungal treatments.

2.1. Mitochondrial function and energy metabolism

Mitochondria are often referred to as the powerhouses of the cell due to their essential role in energy metabolism. They primarily generate ATP through oxidative phosphorylation, relying on the electron transport chain located in the inner mitochondrial membrane. ATP production supports various cellular functions across multiple species including fungi. For instance, the respiratory capacity of fungi significantly influences their ability to resist various stressors, including antifungal drugs (Tanida et al. 2006; Black et al. 2021). Consequently, fungi that cannot efficiently produce ATP may struggle to activate stress response pathways, diminishing their resistance to drug treatment (Li et al. 2021). In addition to ATP synthesis, mitochondria are actively involved in several biosynthetic pathways that contribute to cellular homoeostasis. The tricarboxylic acid (TCA) cycle, occurring within the mitochondrial matrix, is central to energy production and provides important intermediates for the synthesis of amino acids and lipids (Ene et al. 2014; Garbe and Vylkova 2019; Ryu et al. 2024). Lipid synthesis in mitochondria is crucial for maintaining membrane integrity and fluidity (Horvath and Daum 2013; Schuster and Steinberg 2020). A well-maintained membrane is vital for cell survival, as it serves as a barrier against environmental stressors and plays a significant role in drug resistance. Disruption of membrane integrity can enhance the susceptibility of fungal cells to antifungal agents, as compromised membranes may allow for increased drug influx or decreased drug efflux. Moreover, mitochondria play a crucial role in amino acid metabolism, which is vital for protein synthesis and cellular signalling. They are involved in specific pathways that regulate the availability of essential amino acids and their derivatives. For instance, mitochondria contribute to the synthesis of glutamate, a key amino acid that plays a significant role in energy metabolism (Ene et al. 2014; Garbe and Vylkova 2019; Paudel et al. 2021). Amino acids not only serve as building blocks for proteins but also act as signalling molecules that help fungi respond to stress. The metabolic pathways that produce these amino acids are intricately linked to the TCA cycle, demonstrating a direct correlation between energy metabolism and the availability of crucial cellular components. Additionally, mitochondria are pivotal in haem biosynthesis, which is intricately linked to fungal resistance mutations in the ERG11 gene affecting the haem-binding site, as well as to the regulation of iron homoeostasis (Feng et al. 2010; Black et al. 2021).

2.2. Role of mitochondrial proteins in drug resistance

The role of mitochondrial proteins in drug resistance has attracted significant attention, particularly regarding azole resistance in various Candida species. Investigations using mitochondrial proteomics have focused on several strains, including C. albicans, N. glabrata, P. kudriavzevii, and C. auris. These studies have revealed significant differences in mitochondrial protein expression between azole-sensitive and azole-resistant strains. Specifically, researchers identified 417, 165, and 25 differentially expressed mitochondrial proteins (DEMPs) in the resistant strains of C. albicans, N. glabrata, and C. auris, respectively. The profiles of these DEMPs are primarily associated with crucial cellular functions, including ribosomal biogenesis, glycolysis, the TCA cycle, transport mechanisms, ergosterol biosynthesis, and mannan synthesis for the cell wall. Notably, C. albicans and C. auris exhibited heightened activity in these processes, whereas N. glabrata and P. kudriavzevii displayed significantly lower metabolic activity due to their fermentative nature. Additionally, the azole-resistant strain of C. albicans produced elevated levels of various transcription factors, including Rtg3, which correlates with the activation of complex I within the mitochondrial electron transport chain (ETC). In contrast, N. glabrata and P. kudriavzevii showed reduced mitochondrial function, particularly in complexes IV and V of the ETC, along with decreased expression of critical proteins such as Hog1, Tor1, and Snf1/Snf4 (Zhou et al. 2024). This research is significant as Candida species frequently cause various invasive infections, and their resistance to azole antifungals poses substantial treatment challenges (Zhou et al. 2024). Understanding the mechanisms contributing to drug resistance in pathogenic Candida species is vital for developing improved prevention strategies and discovering new therapeutic targets.

2.3. Mitochondrial respiration and drug efflux pumps

Mitochondrial respiration is closely associated with the expression and activity of drug efflux pumps in fungal pathogens (Shingu-Vazquez and Traven 2011). Drug efflux pumps, such as Cdr1 and Mdr1 in Candida (Puri et al. 1999; Dunkel et al. 2008) and similar pumps in Aspergillus (Tobin et al. 1997), are crucial for expelling antifungal agents from the cell, thereby reducing drug efficacy. Enhanced mitochondrial respiration correlates with increased activity of these efflux pumps in both Candida and Aspergillus species. Mitochondria also play a significant role in maintaining cellular redox balance and generating ROS, which influence drug resistance through signalling pathways. Specifically, ROS produced during mitochondrial respiration can modulate the expression of genes involved in drug efflux and stress responses (Black et al. 2021, 2024; Xue et al. 2024). This interplay suggests that mitochondrial respiration is not merely a metabolic process but also a critical regulator of cellular defence mechanisms against antifungal treatments. A key mitochondrial protein, Bcs1A, is essential for the assembly of respiratory chain complexes in A. fumigatus, which is critical for mitochondrial respiration and ATP generation. Mutations or dysregulation of Bcs1A can disrupt mitochondrial function, creating a feedback loop that enhances the expression of drug efflux pumps (Yang et al. 2024).

2.4. Calcium signaling and mitochondrial dysfunction

In fungi, various external stressors trigger an increase in cytosolic Ca²⁺ levels. This elevation activates the calcium sensor protein calmodulin, which in turn activates the phosphatase calcineurin. Calcineurin dephosphorylates the transcription factor CrzA/Crz1, facilitating its translocation into the nucleus (Juvvadi et al. 2014, 2017). Studies have shown that calcium-signalling inhibitors, such as EGTA and FK506, enhance the efficacy of conventional antifungal agents like ITC and VRC against human fungal pathogens such as C. albicans and A. fumigatus, as demonstrated by both in vitro plate assays and in vivo animal models (Raad et al. 2008; Liu et al. 2015). Mitochondria function as dynamic reservoirs of Ca²⁺, regulating intracellular calcium concentrations by sequestering and releasing calcium ions (Ganitkevich 2003). They are integral to calcium signalling, which is essential for numerous cellular processes, including drug resistance. Research has indicated that mitochondrial dysfunction in A. fumigatus leads to the upregulation of multidrug transporters, which play a critical role in antifungal resistance (Li et al. 2020). Additionally, disruptions in calcium signalling pathways, particularly those related to mitochondrial calcium uptake, significantly contribute to increased antifungal resistance (Li et al. 2020). Mitochondria can influence intracellular calcium levels through their respiratory functions. Alterations in calcium signalling can activate stress response pathways, resulting in the expression of resistance-related genes. This mechanism highlights the crucial role of mitochondrial function in maintaining calcium homoeostasis and its subsequent influence on drug resistance (Li et al. 2020).

2.5. Iron homeostasis and drug susceptibility

The role of mitochondria in regulating iron homoeostasis is intricately linked to the efficacy of specific antifungal agents, such as fluconazole (Song et al. 2020). Iron is essential for cellular metabolism and mitochondrial function, facilitating various iron-dependent processes, including haem and iron-sulphur cluster synthesis, which are vital for the electron transport chain and energy production (Xue et al. 2023a, 2024). Evidence suggests that low iron levels can enhance fungal sensitivity to azole drugs, as iron is crucial for ergosterol biosynthesis, a key component of fungal cell membranes. Conversely, excessive iron can lead to mitochondrial dysfunction and increased ROS production, potentially altering drug metabolism and contributing to resistance. Recent findings by Van Genechten et al. (2024) emphasise the importance of iron homoeostasis in determining azole susceptibility. Fungal pathogens have evolved sophisticated strategies to sequester iron from their surroundings, despite its limited availability in host tissues. These strategies include the production of siderophores and the utilisation of haemoglobin and transferrin as sources of iron (Jung et al. 2008, 2021; Kronstad et al. 2013; Bairwa et al. 2017; Xue et al. 2023a, 2023b). The efficiency of these iron acquisition mechanisms significantly impacts the efficacy of antifungal drugs, as enhanced iron uptake can facilitate increased ergosterol synthesis, thereby reinforcing the integrity of fungal cell membranes and conferring resilience against azole treatments (Van Genechten et al. 2024).

2.6. Mitochondrial function and stress response pathways

The regulatory networks governing mitochondrial function and drug resistance are complex and involve multiple signalling pathways. ROS generated during mitochondrial respiration can act as signalling molecules that modulate the expression of stress response genes and drug efflux pumps. One prominent example is A. fumigatus Mba1, associated with the mitochondrial membrane. Mba1 has been linked to antifungal resistance through its ability to modulate the production of ROS. Elevated ROS levels can trigger stress responses that enhance the expression of resistance-related genes, thereby contributing to the survival of fungal pathogens in the presence of antifungal drugs (Zhu et al. 2023). This indicates that mitochondrial ROS not only contributes to oxidative stress but also plays a fundamental role in the adaptive responses of fungi under antifungal pressure. Moreover, mitochondrial dysfunction could lead to altered expression of genes involved in the biosynthesis of cell wall components. The integrity of the fungal cell surface is vital for the effectiveness of antifungal treatments; any modifications in cell wall composition can diminish the efficacy of these drugs. A key component of cell wall integrity is β-glucans, which are essential for maintaining the structural stability of the cell wall. Research has demonstrated that inhibiting β-glucan synthesis can enhance the effectiveness of antifungal therapies. For instance, the echinocandin class of antifungals, which target the enzyme responsible for β-glucan synthesis (1,3-β-D-glucan synthase), shows increased efficacy in fungi with compromised cell walls (Douglas et al. 1997; Onishi et al. 2000; Hu et al. 2023). In addition, C. albicans adapts to hypoxic conditions by activating a mitochondrial cAMP-PKA signalling pathway, which leads to the induction of β-glucan masking (Pradhan et al. 2018). This highlights the need for further research to explore the interplay between mitochondrial function and antifungal susceptibility in greater detail.

3. Advancements in mitochondrion-targeted antifungal agents

Recent advancements in drug discovery have led to significant progress in the development of mitochondrion-targeted antifungal agents, highlighting the potential of various novel compounds in combating fungal infections (Qin et al. 2023). One noteworthy agent, F901318 (olorofim), selectively inhibits dihydroorotate dehydrogenase (DHODH), which is localised in the inner mitochondrial membrane and serves as a key enzyme in the de novo pyrimidine synthesis pathway. This compound has shown substantial efficacy against Aspergillus species, demonstrating a notable selectivity for fungal DHODH compared to its mammalian counterpart (Oliver et al. 2016). In vivo studies have suggested that F901318 enhances survival rates and reduces fungal loads in murine models of invasive aspergillosis (Wiederhold et al. 2018; Seyedmousavi et al. 2019). Another significant compound, T-2307, an arylamide compound, has demonstrated broad-spectrum antifungal activity, showing potent effects against resistant strains of Candida, Cryptococcus, and Aspergillus species (Mitsuyama et al. 2008; Nishikawa et al. 2017; Wiederhold et al. 2020; Gerlach et al. 2021). It achieves this by targeting mitochondrial respiratory chain complexes III and IV, resulting in mitochondrial dysfunction and collapse of the mitochondrial membrane potential. Importantly, T-2307 exhibits remarkable selectivity for fungal mitochondria, providing a substantial advantage over conventional antifungal agents (Shibata et al. 2012; Wiederhold 2021). Ilicicolin H, a natural polyketide, has also emerged as a significant player in the field of mitochondrion-targeted antifungals. It inhibits cytochrome bc1 reductase within the mitochondrial respiratory chain, exhibiting broad-spectrum antifungal activity against various fungi, including those resistant to existing therapies. Its ability to selectively bind to fungal proteins underscores its potential as a therapeutic agent (Singh et al. 2012, 2013). Moreover, the indazole derivatives Inz-1 and Inz-5 have shown promise in inhibiting fungal cytochrome bc1 activity. These compounds demonstrate significant antifungal potency while maintaining high selectivity over mammalian cells. Inz-5, in particular, has been highlighted for its enhanced potency and stability, making it a strong candidate for further development against azole-resistant fungal strains (Vincent et al. 2016). Efforts to target mitochondrial functions extend beyond synthetic compounds, as natural products have also provided a rich source of antifungal agents with mitochondrial implications. Compounds like berberine (da Silva et al. 2016; Tong et al. 2021) and resveratrol (Jung et al. 2005; Singh and Singh 2019; Vestergaard and Ingmer 2019) have demonstrated antifungal properties by impairing mitochondrial function, enhancing their therapeutic potential against resistant fungal strains. Despite the progress made in developing mitochondria-targeted antifungals, the majority of these agents remain in preclinical or early clinical stages. The ongoing challenge is to identify additional selective targets and develop compounds that can effectively exploit mitochondrial pathways in fungi while minimising adverse effects on human cells.

4. Challenges

Despite the growing understanding of mitochondrial function in fungal drug resistance (Koch and Traven 2020; Alves and Gourlay 2024), several challenges hinder the effective translation of these insights into therapeutic strategies. These challenges can be categorised into several key areas:

4.1. Complexity of mitochondrial functions

While targeting mitochondrial functions in fungi may provide a pathway to combat drug resistance, there is a significant risk that these interventions could also affect human mitochondrial functions. Mitochondria in human cells share similarities with those in fungi, which raises concerns about potential off-target effects that could lead to toxicity. Such adverse effects could manifest as metabolic disorders, increased oxidative stress, or impaired cellular functions in host cells, ultimately resulting in severe side effects for patients undergoing treatment. Therefore, a careful balance must be maintained when designing mitochondrion-targeted therapies to minimise collateral damage to human cells. Fungi are highly adaptable organisms, and targeted disruption of mitochondrial functions may prompt the emergence of compensatory mechanisms. For instance, if a specific mitochondrial pathway is inhibited, fungi may activate alternative pathways or enhance the expression of efflux pumps to maintain their metabolic and energetic balance (Verma et al. 2018). This adaptability not only undermines the effectiveness of antifungal agents but can also lead to the rapid development of resistant strains. Understanding these compensatory responses is crucial for predicting treatment outcomes and designing more resilient therapeutic strategies.

4.2. Diversity and heterogeneity among fungal species

The diversity among fungal species presents significant challenges in the development of antifungal agents: Fungi comprise a vast and diverse kingdom with varying mitochondrial genomics and physiological characteristics (Wilson and Xu 2012; Tang et al. 2024). Different fungi may possess unique mitochondrial structures, gene sequences, and metabolic pathways. This variability influences their mechanisms of drug resistance and complicates the development of broad-spectrum antifungal agents. For instance, resistance mechanisms in C. auris may differ from those observed in A. fumigatus (Lee et al. 2023), requiring tailored approaches for each pathogen. Therefore, a comprehensive understanding of the specific mitochondrial characteristics and resistance mechanisms of various fungal species is essential for effective drug development. Fungal pathogens exhibit rapid evolutionary capabilities, which may be driven by the selective pressure imposed by antifungal treatments (Revie et al. 2018). This adaptability can lead to the emergence of resistant strains that possess mitochondrial adaptations conferring competitive advantages. For example, changes in mitochondrial morphology, such as alterations in fission and fusion function, may enhance the survival of fungi under stressful conditions, including the presence of antifungal agents (Neubauer et al. 2015). Understanding these adaptive mechanisms is crucial for anticipating potential resistance pathways and designing counteractive strategies to stay ahead of evolving fungal pathogens.

4.3. Limitations in research tools and methodologies

The tools and methodologies available for studying mitochondrial function in fungi are still evolving, presenting challenges in research: To elucidate the precise roles of mitochondrial proteins and function in drug resistance, there is a pressing need for more advanced imaging techniques and genetic manipulation tools. Current methodologies may lack the resolution or specificity required to assess mitochondrial functions accurately, making it challenging to correlate specific mitochondrial alterations with drug resistance phenotypes. This technological gap hinders the ability to investigate how mitochondrial function influences the susceptibility of different fungal species to antifungal agents. The absence of standardised methodologies for assessing mitochondrial function across different fungal species can lead to inconsistent findings in the literature. Variability in experimental conditions, such as growth media, temperature, or stressors applied, can influence mitochondrial behaviour and consequently impact the interpretation of results. Inconsistent methodologies make it difficult to compare findings across studies, complicating efforts to build a cohesive understanding of the role of mitochondrial function in antifungal resistance.

4.4. Translation from laboratory to clinical practice

Translating laboratory findings into clinical practice for mitochondrion-targeted therapies against fungal infections presents notable challenges. One major concern is the potential off-target effects on human mitochondria due to the evolutionary conservation of mitochondrial functions between fungi and humans. This necessitates thorough preclinical testing to ensure safety and efficacy. Additionally, the robust cell walls of fungi can impede the effective penetration of therapeutic agents, highlighting the need for novel delivery systems to enhance bioavailability. Identifying unique therapeutic targets is also complicated by the close evolutionary relationship between pathogenic and non-pathogenic fungi, making it essential to find molecular targets with differential expression or function in pathogenic strains. Addressing these challenges requires an integrated approach that combines insights from laboratory research with a deep understanding of fungal biology and human physiology, facilitating the development of effective antifungal therapies.

5. Conclusions and future directions

The rising incidence of fungal infections, coupled with the emergence of drug-resistant strains, underscores the urgent need for a comprehensive understanding of the mechanisms of antifungal drug resistance. This review has highlighted the pivotal role of mitochondrial function in modulating antifungal sensitivity (Figure 1). Mitochondria, traditionally recognised as the powerhouses of the cell, are now understood to play multifaceted roles beyond energy production. They are integral to various metabolic processes, oxidative stress responses, and calcium signalling pathways, all of which significantly influence the efficacy of antifungal agents. Thus, a deeper exploration of the interplay between mitochondrial function and drug resistance is essential for the development of innovative therapeutic strategies.

Figure 1.

Mechanisms underlying mitochondrial regulation of antifungal resistance. (A) Calcium signaling and transcriptional regulation: mitochondrial dysfunction leads to abnormal cytoplasmic Ca²⁺ transients, resulting in the persistent nuclear localization of the zinc finger transcription factor CrzA. This triggers the overexpression of multidrug transporter genes (mdr1, abcC, atrA, atrB, abcE, and atrF), contributing to azole resistance in Aspergillus fumigatus (Li et al. 2020). (B–C) ROS production: deletion of the mitochondrial protein MBA1 in A. fumigatus (Zhu et al. 2023) or the ERMES complex component Gem1 in Candida glabrata (Okamoto et al. 2023) results in increased ROS production. (D) Enhanced efficacy of amphotericin B: lansoprazole enhances the effectiveness of amphotericin B against multidrug-resistant Candida auris by disrupting fungal respiration. (E) Iron metabolism and ergosterol biosynthesis: mitochondrial dysfunction deregulates iron metabolism and disrupts ergosterol biosynthesis, increasing the susceptibility of Candida albicans to azoles (Thomas et al. 2013; Song et al. 2020). (F) Mutational effects: the Δbcs1A mutant exhibits increased resistance to azoles, terbinafine, and simvastatin, indicating a role for Bcs1A in the antifungal susceptibility of A. fumigatus (Yang et al. 2024). These mechanisms highlight the critical role of mitochondrial function in regulating antifungal resistance.

Recent studies have provided valuable insights into the complex function of mitochondrial proteins that are crucial for understanding resistance mechanisms. For instance, the research conducted by Vögtle et al. (2017) utilised stable isotope labelling coupled with quantitative mass spectrometry to elucidate the intricate distribution of submitochondrial proteins in Saccharomyces cerevisiae. Similarly, Morgenstern et al. (2021) focused on the quantitative function of the human mitochondrial proteome within a cellular context, aiming to understand how these functions contribute to cellular function and response to stress. Such methodologies represent a promising avenue for further research into the mitochondrial aspects of fungal resistance mechanisms. Future investigations should focus on deciphering the specific molecular mechanisms by which mitochondrial function influences the expression of drug efflux pumps and other resistance-related factors. Advanced imaging techniques and genetic manipulation tools will play a critical role in dissecting the functions of key mitochondrial proteins across various fungal species. For instance, employing CRISPR/Cas9 technology allows researchers to selectively knock out or activate genes associated with mitochondrial function, thereby elucidating their roles in the development of resistance. The development of novel antifungal agents that target mitochondrial functions must be accompanied by rigorous preclinical and clinical evaluations to ascertain their safety and efficacy. Understanding the pharmacokinetics and pharmacofunction of these agents in the context of mitochondrion-targeted therapies is crucial for their successful translation into clinical practice. As the landscape of antifungal treatments continues to evolve, these factors will be pivotal in ensuring that new therapies are both effective and safe for patients. A particularly promising direction for future studies lies in the exploration of combination therapies that integrate mitochondrion-targeted agents with existing antifungal medications. By harnessing the potential synergies between these treatments, it may be possible to enhance the overall efficacy of antifungal regimens while minimising the risk of resistance development. For example, combining a novel mitochondrion-targeted drug with an established antifungal could increase the effective concentration of the latter, thereby improving treatment outcomes. Moreover, the integration of nanotechnology into antifungal treatment strategies presents an exciting opportunity (Zaioncz et al. 2017; Sharifi et al. 2024; Zhu et al. 2024). By developing nanoparticle-based delivery systems that specifically target fungal mitochondria, researchers can improve the therapeutic outcomes of antifungal agents while concurrently reducing side effects. This targeted approach not only enhances the concentration of drugs at the site of action but also minimises systemic exposure, which is often associated with adverse effects.

In addition to these pharmacological strategies, technological advancements such as single-cell sequencing are revolutionising our understanding of drug resistance at a granular level. Single-cell sequencing technology facilitates the analysis of gene expression and variability at the single-cell level, thereby revealing the heterogeneity of drug resistance among different fungal cells. Such insights can help identify specific mitochondrial genes or regulatory pathways that play critical roles in resistance, ultimately guiding more targeted therapeutic interventions. Real-time imaging technologies, such as fluorescence microscopy, enable researchers to observe changes in mitochondrial function and their interactions with other cellular structures in real-time. This technology allows for dynamic monitoring of mitochondrial morphological alterations and functional states in response to antifungal drug stress. By capturing these changes as they occur, scientists can gain invaluable insights into the immediate responses of fungi to antifungal treatment and the subsequent adaptations that may confer resistance. Mass spectrometry techniques are also invaluable in this context. By analysing alterations in the mitochondrial proteome, researchers can identify specific mitochondrial proteins associated with drug resistance. These proteins may serve as novel drug targets or help in elucidating how mitochondria influence cellular responses to antifungal agents. Furthermore, integrating multi-omics approaches – combining genomics, transcriptomics, and metabolomics – can provide a comprehensive analysis of the relationship between mitochondrial function and fungal resistance. This holistic view may reveal potential regulatory mechanisms and adaptive strategies employed by fungi in the face of antifungal pressures.

In conclusion, an integrated approach that combines mitochondrial biology with antifungal drug development holds great promise for yielding more effective treatment options against resistant fungal infections. The complexities of mitochondrial function in fungi provide critical insights not only into their pathogenic mechanisms but also into innovative therapeutic strategies to combat these resilient organisms. As we advance our understanding of these intricate biological processes, we pave the way for tailored and more effective antifungal treatments that could significantly improve patient outcomes in the face of an escalating global health challenge. Ultimately, addressing the multifaceted nature of fungal drug resistance will require a collaborative effort across various fields of research, including molecular biology, pharmacology, and clinical medicine. By fostering interdisciplinary partnerships and leveraging cutting-edge technologies, the scientific community can develop more robust strategies aimed at mitigating the threat posed by drug-resistant fungal infections. The future of antifungal therapy lies in our ability to adapt and innovate, ensuring that we remain one step ahead of these increasingly formidable pathogens.

Funding Statement

This work was supported by the Natural Science Research of Jiangsu Higher Education Institutions [24KJD430010], the Nantong Jiangsu Scientific Research Project [JC2023043], HaiYa Young Scientist Foundation of Shenzhen University General Hospital [2024-HY013], and the Natural Science Foundation of Jiangsu Province [BK20240948].

Disclosure statement

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

Author contributions

Yuanyuan Ma and Yachun Zhou were the primary contributors to the writing of the manuscript. Liang Yang and Peng Xue conducted a thorough review and provided critical feedback. Tianyuan Jia and Zilin Zhuang contributed to the corrections of the manuscript. All authors have reviewed and approved the final version of the manuscript.