Human Fungal Pathogen Research Articles

Hidden allies: how extracellular vesicles drive biofilm formation, stress adaptation, and host–immune interactions in human fungal pathogens

ABSTRACT Pathogenic fungi pose a significant threat to human health, especially given the rising incidence of invasive fungal infections and the emergence of drug-resistant strains. This requires the development of vaccines and the advancement of antifungal strategies. Recent studies have focused on the roles of fungal extracellular vesicles (EVs) in intercellular communication and host-pathogen interactions. EVs are nanosized, lipid membrane-bound particles that facilitate the transfer of proteins, lipids, and nucleic acids. Here, we review the multifaceted functions of EVs produced by different human fungal pathogens, highlighting their importance in the response of fungal cells to different environmental cues and their interactions with host immune cells. We summarize the current state of research on EVs and how leveraging this knowledge can lead to innovative approaches in vaccine development and antifungal treatment.

Nomenclature for human and animal fungal pathogens and diseases: a proposal for standardized terminology.

Medically important pathogenic fungi invade vertebrate tissue and are considered primary when part of their nature life cycle is associated with an animal host and are usually able to infect immunocompetent hosts. Opportunistic fungal pathogens complete their life cycle in environmental habitats or occur as commensals within or on the vertebrate body, but under certain conditions can thrive upon infecting humans. The extent of host damage in opportunistic infections largely depends on the portal and modality of entry as well as on the host's immune and metabolic status. Diseases caused by primary pathogens and common opportunists, causing the top approximately 80% of fungal diseases [D. W. Denning, Lancet Infect Dis, 24:e428-e438, 2024, https://doi.org/10.1016/S1473-3099(23)00692-8], tend to follow a predictive pattern, while those by occasional opportunists are more variable. For this reason, it is recommended that diseases caused by primary pathogens and the common opportunists are named after the etiologic agent, for example, histoplasmosis and aspergillosis, while this should not be done for occasional opportunists that should be named as [causative fungus] [clinical syndrome], for example, Alternaria alternata cutaneous infection. The addition of a descriptor that identifies the location or clinical type of infection is required, as the general name alone may cover widely different clinical syndromes, for example, "rhinocerebral mucormycosis." A list of major recommended human and animal disease entities (nomenclature) is provided in alignment with their causative agents. Fungal disease names may encompass several genera of etiologic agents, consequently being less susceptible to taxonomic changes of the causative species, for example, mucormycosis covers numerous mucormycetous molds.

Structure-guided design and synthesis of C22- and C32-modified FK520 analogs with enhanced activity against human pathogenic fungi.

Invasive fungal infections cause significant mortality worldwide, and current antifungal treatments are often ineffective, toxic, or face growing resistance. This research identifies calcineurin (CaN), a critical protein for fungal survival, as a potential target for developing new antifungal drugs. Although existing CaN inhibitors such as FK506 (tacrolimus) and FK520 (ascomycin) possess antifungal properties, their immunosuppressive effects limit their clinical utility. By studying the structure of human and fungal FKBP12-FK506 or FK520 complexes with CaN, we have designed and synthesized modified FK520 derivatives with strong antifungal activity and reduced immunosuppressive effects. These new derivatives are expected to have significantly improved therapeutic profiles, offering hope for more effective and safer antifungal treatments.

Enhanced antifungal activity of siRNA-loaded anionic liposomes against the human pathogenic fungus Aspergillus fumigatus.

We developed siRNA-loaded anionic liposomes, co-encapsulating low-dose amphotericin B, to enhance siRNA penetration through the fungal cell wall of Aspergillus fumigatus. Targeting mRNAs of three key genes, these liposomes visibly inhibited fungal growth, demonstrating for the first time the antifungal potential of siRNA against human fungal pathogens.

Tracking the uptake of labelled host-derived extracellular vesicles by the human fungal pathogen Aspergillus fumigatus

Abstract Extracellular vesicles (EVs) have gained attention as facilitators of intercellular as well as interkingdom communication during host-microbe interactions. Recently we showed that upon infection, host polymorphonuclear leukocytes produce antifungal EVs targeting the clinically important fungal pathogen Aspergillus fumigatus; however, the small size of EVs (< 1 µm) complicates their functional analysis. Here, we employed a more tractable, reporter-based system to label host alveolar epithelial cell-derived EVs and enabled their visualisation during in vitro A. fumigatus interaction. Fusion of EV marker proteins (CD63, CD9, and CD81) with a Nanoluciferase (NLuc) and a green fluorescent protein (GFP) facilitated their relative quantification by luminescence and visualization by a fluorescence signal. The use of an NLuc fused with a GFP is advantageous as it allows for quantification and visualisation of EVs simultaneously without additional external manipulation and to distinguish subpopulations of EVs. Using this system, visualisation and tracking of EVs was possible using confocal laser scanning microscopy and advanced imaging analysis. These experiments revealed the propensity of host cell-derived EVs to associate with the fungal cell wall and ultimately colocalize with the cell membrane of A. fumigatus hyphae in large numbers. In conclusion, we have created a series of tools to better define the complex interplay of host-derived EVs with microbial pathogens.

Candida glabrata maintains two HAP1 ohnologs, HAP1A and HAP1B, for distinct roles in ergosterol gene regulation to mediate sterol homeostasis under azole and hypoxic conditions.

Invasive and drug-resistant fungal infections pose a significant public health concern. Candida glabrata, a human fungal pathogen, is often difficult to treat due to its intrinsic resistance to azole antifungal drugs and its capacity to develop clinical drug resistance. Therefore, understanding the pathways that facilitate fungal growth and environmental adaptation may lead to novel drug targets and/or more efficacious antifungal therapies. While the mechanisms of azole resistance in Candida species have been extensively studied, the roles of zinc cluster transcription factors, such as Hap1A and Hap1B, in C. glabrata have remained largely unexplored until now. Our research shows that these factors play distinct yet crucial roles in regulating ergosterol homeostasis under azole drug treatment and oxygen-limiting growth conditions. These findings offer new insights into how this pathogen adapts to different environmental conditions and enhances our understanding of factors that alter drug susceptibility and/or resistance.

Candida albicans isolates contain frequent heterozygous structural variants and transposable elements within genes and centromeres.

The human fungal pathogen Candida albicans poses a significant burden on global health, causing high rates of mortality and antifungal drug resistance. C. albicans is a heterozygous diploid organism that reproduces asexually. Structural variants (SVs) are an important source of genomic rearrangement, particularly in species that lack sexual recombination. To comprehensively investigate SVs across clinical isolates of C. albicans, we conducted long read sequencing and genome-wide SV analysis in three distantly related clinical isolates. Our work included a new, comprehensive analysis of transposable element (TE) composition, location and diversity. SVs and TEs are frequently close to coding sequences and many SVs are heterozygous, suggesting that SVs might impact gene and allele-specific expression. Most SVs are uniquely present in only one clinical isolate, indicating that SVs represent a significant source of intra-species genetic variation. We identified multiple, distinct SVs at the centromeres of Chromosome 4 and Chromosome 5, including inversions and transposon polymorphisms. These two chromosomes are often aneuploid in drug resistant clinical isolates, and can form isochromosome structures with breakpoints near the centromere. Further screening of 100 clinical isolates confirmed the widespread presence of centromeric SVs in C. albicans, often appearing in a heterozygous state, indicating that SVs are contributing to centromere evolution in C. albicans Together, these findings highlight that SVs and TEs are common across diverse clinical isolates of C. albicans and that the centromeres of this organism are important sites of genome rearrangement.

Candida albicans pathways that protect against organic peroxides and lipid peroxidation.

Human fungal pathogens must survive diverse reactive oxygen species (ROS) produced by host immune cells that can oxidize a range of cellular molecules including proteins, lipids, and DNA. Formation of lipid radicals can be especially damaging, as it leads to a chain reaction of lipid peroxidation that causes widespread damage to the plasma membrane. Most previous studies on antioxidant pathways in fungal pathogens have been conducted with hydrogen peroxide, so the pathways used to combat organic peroxides and lipid peroxidation are not well understood. The most well-known peroxidase in Candida albicans, catalase, can only act on hydrogen peroxide. We therefore characterized a family of four glutathione peroxidases (GPxs) that were predicted to play an important role in reducing organic peroxides. One of the GPxs, Gpx3 is also known to activate the Cap1 transcription factor that plays the major role in inducing antioxidant genes in response to ROS. Surprisingly, we found that the only measurable role of the GPxs is activation of Cap1 and did not find a significant role for GPxs in the direct detoxification of peroxides. Furthermore, a CAP1 deletion mutant strain was highly sensitive to organic peroxides and oxidized lipids, indicating an important role for antioxidant genes upregulated by Cap1 in protecting cells from organic peroxides. We identified GLR1 (Glutathione reductase), a gene upregulated by Cap1, as important for protecting cells from oxidized lipids, implicating glutathione utilizing enzymes in the protection against lipid peroxidation. Furthermore, an RNA-sequencing study in C. albicans showed upregulation of a diverse set of antioxidant genes and protein damage pathways in response to organic peroxides. Overall, our results identify novel mechanisms by which C. albicans responds to oxidative stress resistance which open new avenues for understanding how fungal pathogens resist ROS in the host.

Gladiolin produced by pathogenic Burkholderia synergizes with amphotericin B through membrane lipid rearrangements.

Amphotericin B (AmpB) is one of the oldest antifungal drugs in clinical use. It is an effective therapeutic, but it comes with toxicity issues due to the similarities between its fungal target (the membrane lipid ergosterol) and its mammalian counterpart (cholesterol). One strategy to improve its activity/toxicity relationship is by combinatorial therapy with potentiators, which would enable a lower therapeutic dose of AmpB. Here, we report on the discovery of the antibiotic gladiolin as a potentiator of AmpB against several priority human fungal pathogens and fungal biofilms, with no increased toxicity against mammalian cells. We show that gladiolin potentiates AmpB by increasing and accelerating membrane damage. Our findings also provide insights into the on-going debate about the mechanism of action of AmpB by indicating that both proposed mechanisms, extraction of ergosterol from membranes and pore formation, are potentiated by gladiolin.

Shining a light on Candida-induced epithelial damage with a luciferase reporter.

Host cell damage is a key parameter for research in infection biology, drug testing, and substance safety screening. In this study, we introduce a luciferase reporter system as a new and reliable assay to measure cell damage and validate it with the pathogenic yeast, Candida albicans, as a test case. We transduced human epithelial cell lines with a lentiviral vector to stably express an optimized luciferase enzyme, Nanoluc. Upon cell damage, the release of cytoplasmic luciferase into the extracellular space can be easily detected by a luminometer. We used the luciferase reporter system to investigate the damage caused by C. albicans to different newly generated epithelial reporter cell lines. We found that fungus-induced cell damage, as determined by established methods, correlated tightly with the release of the luciferase. The new luciferase reporter system is a simple, sensitive, robust, and inexpensive method for measuring host cell damage and has a sensitivity comparable to the standard assay, release of lactate dehydrogenase. It is suitable for high-throughput studies of pathogenesis mechanisms of any microbe, for antimicrobial drug screening, and many other applications.IMPORTANCEWe present a quick, easy, inexpensive, and reliable assay to measure damage to mammalian cells. To this end, we created reporter cell lines which artificially express luciferase, an enzyme that can be easily detected in the supernatant when these cells are damaged. We used infections with the well-investigated fungal pathogen of humans, Candida albicans, as a test case of our system. Using our reporter, we were able to recapitulate the known effects of strain variability, gene deletions, and antifungal treatments on host cell damage. This easily adaptable reporter system can be used to screen for damage in infection models with different microbial species, assay cell-damaging potential of substances, discover new non-toxic antibiotics, and many other damage-based applications.

BLNK negatively regulates innate antifungal immunity through inhibiting c-Cbl-mediated macrophage migration

B cell linker protein (BLNK) is crucial for orchestrating B cell receptor-associated spleen tyrosine kinase (Syk) signaling. However, the role of BLNK in Syk-coupled C-type lectin receptor (CLR) signaling in macrophages remains unclear. Here, we delineate that CLRs govern the Syk-mediated activation of BLNK, thereby impeding macrophage migration by disrupting podosome ring formation upon stimulation with fungal β-glucans or α-mannans. Mechanistically, BLNK instigates its association with casitas B-lineage lymphoma (c-Cbl), competitively impeding the interaction between c-Cbl and Src-family kinase Fyn. This interference disrupts Fyn-mediated phosphorylation of c-Cbl and subsequent c-Cbl-associated F-actin assembly. Consequently, BLNK deficiency intensifies CLR-mediated recruitment of the c-Cbl/phosphatidylinositol 3-kinase complex to the F-actin cytoskeleton, thereby enhancing macrophage migration. Notably, mice with monocyte-specific BLNK deficiency exhibit heightened resistance to infection with Candida albicans, a prominent human fungal pathogen. This resistance is attributed to the increased infiltration of Ly6C+ macrophages into renal tissue. These findings unveil a previously unrecognized role of BLNK for the negative regulation of macrophage migration through inhibiting CLR-mediated podosome ring formation during fungal infections.

RNAi epimutations conferring antifungal drug resistance are inheritable.

Epimutations modify gene expression and lead to phenotypic variation while the encoding DNA sequence remains unchanged. Epimutations mediated by RNA interference (RNAi) and/or chromatin modifications can confer antifungal drug resistance and may impact virulence traits in fungi (1-5). However, whether these epigenetic modifications can be transmitted across generations following sexual reproduction was unclear. This study demonstrates that RNAi epimutations conferring antifungal drug resistance are transgenerationally inherited in the human fungal pathogen Mucor circinelloides . Our research revealed that RNAi-based antifungal resistance follows a DNA sequence-independent, non-Mendelian inheritance pattern. Small RNAs (sRNAs) are the exclusive determinants of inheritance, transmitting drug resistance independently of other known repressive epigenetic modifications. Unique sRNA signature patterns can be traced through inheritance from parent to progeny, further supporting RNA as an alternative molecule for transmitting information across generations. This study marks a significant advance in understanding epigenetic inheritance, highlighting RNAi-exclusive epimutations as a widespread phenomenon in the pathogenic M. circinelloides species complex. Understanding how epimutations occur, propagate, and confer resistance may enable their detection in other eukaryotic pathogens, provide solutions for challenges posed by rising antimicrobial drug resistance (AMR), and also advance research on phenotypic adaptability and its evolutionary implications.

Do morphogenetic switching and intraspecies variation enhance virulence of Candida auris?

Intraspecies variations that affect pathogenicity and antifungal resistance traits pose a serious obstacle to efficient therapy of Candida auris infections. Recent reports indicate that mutations determine drug susceptibility and virulence. However, mutations alone cannot fully explain a bewildering variety of phenotypes in clinical isolates from known C. auris clades, suggesting an unprecedented complexity underlying virulence traits and antifungal resistance. Hence, we wish to discuss how phenotypic plasticity promotes morphogenetic switching and how that contributes to intraspecies variations in the human fungal pathogen C. auris. Further, we will also discuss how intraspecies variations and morphogenetic events can impact the progress in molecular mycology research that aims to find better treatments for C. auris infections. Finally, we will present our opinion as to the most relevant questions to be addressed when trying to better understand the pathophysiology of C. auris.

Biological and genomic analyses of Clavispora sputumsp. nov., a novel potential fungal pathogen closely related to Clavispora lusitaniae (syn. Candida lusitaniae) and Candida auris

Several human fungal pathogens, including drug-resistant Candida auris and species of the Candida haemulonii complex, have emerged over the past two decades, posing new threats to human health. In this study, we report the isolation and identification of a novel species belonging to the genus Clavispora, herein named as Clavisporasputum, from a clinical sputum sample of a COVID-19 patient. Cl. sputum is phylogenetically closely related to fungal pathogens Clavispora lusitaniae (syn. Candida lusitaniae) and C. auris. When grown on CHROMagar Candida Plus medium, Cl. sputum exhibited a similar coloration to C. auris strain CBS12372. Cl. sputum was able to develop weak filaments on CM medium. Although Cl. sputum and Cl. lusitaniae are phylogenetically closely related, comparative genomic and synteny analyses indicated significant chromosomal rearrangements between the two species. Although Cl. sputum could not grow at 37 °C under regular culture condition, an increased fungal burden in the lung tissue of a mouse systemic infection model implies that it could be a potential opportunistic pathogenic yeast in humans.

Genome-Scale Metabolic Models in Fungal Pathogens: Past, Present, and Future.

Fungi are diverse organisms with various characteristics and functions. Some play a role in recycling essential elements, such as nitrogen and carbon, while others are utilized in the food and drink production industry. Some others are known to cause diseases in various organisms, including humans. Fungal pathogens cause superficial, subcutaneous, and systemic infections. Consequently, many scientists have focused on studying the factors contributing to the development of human diseases. Therefore, multiple approaches have been assessed to examine the biology of these intriguing organisms. The genome-scale metabolic models (GEMs) have demonstrated many advantages to microbial metabolism studies and the ability to propose novel therapeutic alternatives. Despite significant advancements, much remains to be elucidated regarding the use of this tool for investigating fungal metabolism. This review aims to compile the data provided by the published GEMs of human fungal pathogens. It gives specific examples of the most significant contributions made by these models, examines the advantages and difficulties associated with using such models, and explores the novel approaches suggested to enhance and refine their development.

Giant transposons promote strain heterogeneity in a major fungal pathogen.

No "one size fits all" option exists for treating fungal infections in large part due to genetic and phenotypic variation among strains. Accounting for strain heterogeneity is thus fundamental for developing efficacious treatments and strategies for safeguarding human health. Here, we report significant progress towards achieving this goal by uncovering a previously hidden mechanism generating heterogeneity in the major human fungal pathogen Aspergillus fumigatus : giant transposons called Starships that span dozens of kilobases and mobilize fungal genes as cargo. By conducting the first systematic investigation of these unusual transposons in a single fungal species, we demonstrate their contributions to population-level variation at the genome, pangenome and transcriptome levels. The Starship atlas we developed will not only help account for variation introduced by these elements in laboratory experiments but will serve as a foundational resource for determining how Starships shape clinically-relevant phenotypes, such as antifungal resistance and pathogenicity.

Auranofin is active against Histoplasma capsulatum and reduces the expression of virulence-related genes.

Auranofin is an approved anti-rheumatic drug that has a broad-range inhibitory action against several microorganisms, including human pathogenic fungi. The auranofin activity against Histoplasma capsulatum, the dimorphic fungus that causes histoplasmosis, has not been properly addressed. Since there are few therapeutic options for this life-threatening systemic mycosis, this study evaluated the effects of auranofin on H. capsulatum growth and expression of virulence factors. Minimal inhibitory and fungicidal concentrations (MIC and MFC, respectively) of auranofin against 15 H. capsulatum strains with distinct genetic backgrounds were determined using the yeast form of the fungus and a microdilution protocol. Auranofin activity was also assessed on a macrophage model of infection and on a Tenebrio molitor invertebrate animal model. Expression of virulence-related genes was compared between auranofin treated and untreated H. capsulatum yeast cells using a quantitative PCR assay. Auranofin affected the growth of different strains of H. capsulatum, with MIC and MFC values ranging from 1.25 to 5.0 μM and from 2.5 to >10 μM, respectively. Auranofin was able to kill intracellular H. capsulatum yeast cells and conferred protection against the fungus in the experimental animal model of infection. Moreover, the expression of catalase A, HSP70, superoxide dismutase, thioredoxin reductase, serine proteinase, cytochrome C peroxidase, histone 2B, formamidase, metallopeptidase, Y20 and YPS3 proteins were reduced after six hours of auranofin treatment. CONCLUSIONS/SIGNIFICANCE: Auranofin is fungicidal against H. capsulatum and reduces the expression of several virulence-related genes, which makes this anti-rheumatic drug a good candidate for new medicines against histoplasmosis.

The development of single-domain VHH nanobodies that target the Candida albicans cell surface.

Candida albicans causes life-threatening invasive infections that are hard to diagnose and treat, with drug resistance leading to treatment failure. The goal of this study was to develop VHH (single variable domain on a heavy chain) nanobodies to detect drug-resistant infections. Llamas were immunized with a mixture of heat killed and fixed C. albicans cells of different morphologies. Llama lymphocyte RNA was used to generate phage display libraries that were tested for binding to C. albicans cells or cell wall fractions, and single antibody domains were isolated. The libraries were panned against echinocandin-resistant C. albicans isolates and counter-selected against echinocandin-susceptible isolates with the aim of isolating binding domains specific for antigens on drug-resistant cells. Thirty diverse VHH nanobodies were selected, and binding characteristics were assessed via dose-response ELISA. Binding was tested against a variety of C. albicans isolates and other Candida species, indicating that the VHHs were specific for C. albicans. The VHH nanobodies were sorted into four distinct groups based on their binding patterns. Two of the groups bound preferentially to the yeast cell poles and hyphae, respectively. Nanobody binding to C. albicans deletion mutants was tested by fluorescence microscopy and ELISA to identify the antigen targets. VHH19 nanobody, belonging to the largest group, recognized the Als4 adhesin. VHH14 antibody in the hyphae-specific group recognized Als3. None of the isolated VHH nanobodies was selective for drug-resistant clinical isolates. Our data indicate that this approach can generate valuable single-domain antibodies specific to C. albicans proteins.IMPORTANCEThe human fungal pathogen Candida albicans causes a range of diseases from superficial mucosal infections such as oral and vaginal thrush to life-threatening, systemic infections. Accurate and rapid diagnosis of these infections remains challenging, and currently, there are no rapid ways to diagnose drug-resistant infections without performing drug susceptibility testing from blood culture, which can take several days. In this proof-of-concept study, we have generated a diverse set of single domain VHH antibodies (nanobodies) from llamas that recognize and bind specifically to C. albicans cell surface. The nanobodies were classified into four groups based on their binding patterns, for example, cell poles or hyphae. Specific nanobodies were verified as recognizing the important adhesin Als4 or the hyphae associated invasin Als3, respectively. The data validate the approach that small VHH antibody domains hold future promise for diagnostic applications and as probes to study the fungal cell surface.

Antibiofilm Activity of PDMS/TiO2 against Candida glabrata through Inhibited Hydrophobic Recovery.

Coatings with antibiofilm properties are desirable for biomedical applications. Titanium dioxide (TiO2) has been explored as an antimicrobial agent in materials development primarily due to it being an excellent photocatalyst. Candida glabrata (C. glabrata) is an emerging human fungal pathogen with known high resistance to oxidative stress. Here, we fabricated a polydimethylsiloxane/titanium dioxide (PDMS/TiO2) nanocomposite coating and tested its antibiofilm activities against C. glabrata. The resulting nanocomposite exhibited >50% reduction in C. glabrata biofilm formation with 2.5 wt % TiO2 loading, even in the dark. Through ROS detection and surface characterization, the antibiofilm activity was attributed to the synergistic interaction of TiO2 nanoparticles with the PDMS matrix, which resulted in the impediment of hydrophobic recovery. This work provides a design strategy to develop antibiofilm coatings against C. glabrata.

Identification, characterization, and structure of a tRNA splicing enzyme RNA 5'-OH kinase from the pathogenic fungi Mucorales.

Fungal Trl1 is an essential tRNA splicing enzyme composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase end-healing domains that convert the 2',3'-cyclic-PO4 and 5'-OH ends of tRNA exons into the 3'-OH,2'-PO4 and 5'-PO4 termini required for sealing by an N-terminal ATP-dependent ligase domain. Trifunctional Trl1 enzymes are present in most human fungal pathogens and are untapped targets for anti-fungal drug discovery. Mucorales species, deemed high priority human pathogens by WHO, elaborate a noncanonical tRNA splicing apparatus in which a stand-alone monofunctional RNA ligase enzyme joins 3'-OH,2'-PO4 and 5'-PO4 termini. Here we identify a stand-alone Mucor circinelloides polynucleotide kinase (MciKIN) and affirm its biological activity in tRNA splicing by genetic complementation in yeast. Recombinant MciKIN catalyzes magnesium-dependent phosphorylation of 5'-OH RNA and DNA ends in vitro. MciKIN displays a strong preference for GTP as the phosphate donor in the kinase reaction, a trait shared with the stand-alone RNA kinase homologs from Mucorales species Rhizopus azygosporus (RazKIN) and Lichtheimia corymbifera (LcoKIN) and with the kinase domains of fungal Trl1 enzymes. We report a 1.65 Å crystal structure of RazKIN in complex with GDP•Mg2+ that illuminates the basis for guanosine nucleotide specificity.