Members of the Fusarium oxysporum species complex are globally distributed soil fungi and plant pathogens that increasingly cause severe and often treatment-refractory human infections. Because clinical and non-clinical isolates arise from overlapping ecological reservoirs, the biological features that contribute to enhanced virulence in mammalian hosts remain incompletely defined. Here, we identify remodeling of ergosterol homeostasis as a distinguishing characteristic of clinically derived isolates, which displayed increased ergosterol biosynthetic activity, elevated extracellular ergosterol release, reduced azole accumulation, and increased pathogenicity in a murine infection model. Functionally, heightened sterol flux promoted caspase-1–dependent macrophage pyroptosis while selectively suppressing chemokine production through IL-10/TGF-β–associated pathways, resulting in impaired neutrophil recruitment despite robust proinflammatory cytokine induction. We propose that enhanced sterol biosynthesis, potentially favored under azole exposure, amplifies pathogenic potential in mammalian hosts. These findings redefine ergosterol as an immunomodulatory virulence determinant linking sterol metabolism, host adaptation, and disease severity.

Invasive fungal infections are associated with high mortality and are increasingly difficult to treat due to a limited antifungal arsenal and the rapid emergence of drug resistance. Novel therapeutic strategies that combine potent antifungal activity, low host toxicity, in vivo stability, and a reduced propensity for resistance development are urgently needed. Antimicrobial peptides (AMPs) stand out as a promising class of compounds to combat antimicrobial resistance. Leveraging the unique properties of AMPs, we previously developed a novel approach to synthesize random peptide mixtures (RPMs) with robust bactericidal activity against drug-resistant bacteria. Here, we evaluate the antifungal potential of RPMs and demonstrate species-dependent, broad-spectrum activity of FK20 (L-phenylalanine-L-lysine, 20-mer) against major human fungal pathogens, including Candida spp., Cryptococcus neoformans, and Aspergillus fumigatus, with particularly high potency against the multidrug-resistant pathogen Candida auris. Mechanistic analyses revealed rapid membrane and cell wall disruption accompanied by intracellular penetration, consistent with membrane-active antifungal activity. Importantly, experimental evolution assays demonstrated a markedly reduced capacity for resistance development in C. auris. FK20 inhibited biofilm formation and displayed substantial activity against mature, pre-formed biofilms, both alone and synergistically in combination with caspofungin. Finally, FK20 showed significant therapeutic efficacy in a murine model of systemic candidiasis. Collectively, these findings establish RPMs as a versatile antifungal platform with broad-spectrum activity, biofilm efficacy, and a low resistance footprint, highlighting their promise as a novel therapeutic strategy against drug-resistant fungal infections. IMPORTANCE:The rising prevalence of invasive fungal infections, particularly among immunocompromised individuals, has become a critical public health concern. However, antifungal drug development has not kept pace with this growing need, and treatment options remain limited to a small number of drug classes. The emergence of multidrug-resistant fungal pathogens, such as Candida auris, further exacerbates this crisis by reducing the efficacy of existing therapeutics and increasing the risk of treatment failure. In this study, we evaluate the antifungal potential of FK20, a random peptide mixture (RPM) composed of L-phenylalanine and L-lysine. FK20 displays potent activity against C. auris and other clinically relevant Candida species, impairs biofilm formation, and exhibits synergy with caspofungin. Importantly, FK20 limits the emergence of resistance and demonstrates therapeutic efficacy in a murine model of systemic candidiasis. These findings establish RPMs as a promising new class of antifungals with broad-spectrum activity and clinical potential against drug-resistant fungal infections.

Aspergillus fumigatus accounts for approximately 65% of all invasive fungal infections in humans, with mortality rates from aspergillosis approaching 50%. Fungal virulence in plant pathogenic fungi can be modulated by viruses that infect fungi, also known as mycoviruses. However, their impact on fungal pathogenesis in mammals has remained largely unexplored. Here, utilizing an A. fumigatus strain naturally infected with the double-stranded RNA virus, A. fumigatus polymycovirus-1M (AfuPmV-1M), we found that the mycovirus confers a substantial survival advantage to the fungus under oxidative stress, heat stress and within the murine lung. Virus-cured fungal strains exhibited reduced conidiation, melanin production and levels of proteins involved in RNA metabolism and stress response, resulting in diminished fitness and virulence in mice. Finally, antiviral treatment during infection reduced AfuPmV-1M viral load, leading to improved survival in infected mice. Taken together, these data suggest that mycoviruses play an important and often underappreciated role as molecular 'backseat drivers' in fungal fitness, stress tolerance and disease progression.

Regulated cell death (RCD) is a highly conserved and coordinated cellular demise process. In animals, the fate of cells depends on the precise regulation of RCD, with inhibitors of apoptosis proteins (IAPs) playing a major role as negative regulators of cellular death. In fungi, RCD regulates crucial processes, including growth, development, stress response, host-pathogen interactions and more. However, the biochemical details of this process are not well understood in this kingdom. IAPs are, remarkably, one of the few RCD/Apoptosis-regulatory proteins that are conserved in fungi. Here, we performed large-scale bioinformatic analyses of IAPs in sequenced fungal genomes. While most fungal organisms have a single IAP gene (81.65 % of the genomes analyzed here), some fungi lack IAPs altogether and others have multiple IAP-like genes. Using the Aspergillus nidulans IAP, AnBir1, we show that this protein is required for survival and regulates cell death by inhibiting caspase-like activity. Moreover, disrupting RCD by constitutively expressing AnBir1 impacted fundamental processes, including development, stress response and secondary metabolism. We also show that fungal RCD can be hijacked for therapeutic purposes. Using virus-induced gene silencing (VIGS), we targeted the AnBir1 homolog of the plant pathogenic fungus Sclerotinia sclerotiorum (SsBir1) during plant infection. Targeting SsBir1 resulted in enhanced resistance to S. sclerotiorum infection. We propose that IAPs play a critical role in regulating caspase activities and other RCD-related processes in fungi and may constitute a novel therapeutic target for fungal infections.

Fusarium oxysporum is a cross-kingdom pathogen. While some strains cause disseminated fusariosis and blinding corneal infections in humans, others are responsible for devastating vascular wilt diseases in plants. To better understand the distinct adaptations of F. oxysporum to animal or plant hosts, we conducted a comparative phenotypic and genetic analysis of two strains: MRL8996 (isolated from a keratitis patient) and Fol4287 (isolated from a wilted tomato [Solanum lycopersicum]). Infection of mouse corneas and tomato plants revealed that, while both strains cause symptoms in both hosts, MRL8996 caused more severe corneal disease in mice, whereas Fol4287 induced more pronounced wilting symptoms in tomato plants. In vitro assays using abiotic stress treatments revealed that the human pathogen MRL8996 was better adapted to elevated temperatures, whereas the plant pathogen Fol4287 was more tolerant to osmotic and cell wall stresses. Both strains displayed broad resistance to antifungal treatment, with MRL8996 exhibiting the paradoxical effect of increased tolerance to higher concentrations of the antifungal caspofungin. We identified a set of accessory chromosomes (ACs) that encode genes with different functions and have distinct transposon profiles between MRL8996 and Fol4287. Interestingly, ACs from both genomes also encode proteins with shared functions, such as chromatin remodeling and post-translational protein modifications. Our phenotypic assays and comparative genomics analyses lay the foundation for future studies correlating genotypes with phenotype and for developing targeted antifungals for agricultural and clinical uses.IMPORTANCEFusarium oxysporum is a cross-kingdom fungal pathogen that infects both plants and animals. In addition to causing many devastating wilt diseases, this group of organisms was recently recognized by the World Health Organization as a high-priority threat to human health. Climate change has increased the risk of Fusarium infections, as Fusarium strains are highly adaptable to changing environments. Deciphering fungal adaptation mechanisms is crucial to developing appropriate control strategies. We performed a comparative analysis of Fusarium strains using an animal (mouse) and plant (tomato) host and in vitro conditions that mimic abiotic stress. We also performed comparative genomics analyses to highlight the genetic differences between human and plant pathogens and correlate their phenotypic and genotypic variations. We uncovered important functional hubs shared by plant and human pathogens, such as chromatin modification, transcriptional regulation, and signal transduction, which could be used to identify novel antifungal targets.

Fusarium oxysporum is a cross-kingdom pathogen. While some strains cause disseminated fusariosis and blinding corneal infections in humans, others are responsible for devastating vascular wilt diseases in plants. To better understand the distinct adaptations of F. oxysporum to animal or plant hosts, we conducted a comparative phenotypic and genetic analysis of two strains: MRL8996 (isolated from a keratitis patient) and Fol4287 (isolated from a wilted tomato [Solanum lycopersicum]). Infection of mouse corneas and tomato plants revealed that, while both strains cause symptoms in both hosts, MRL8996 caused more severe corneal disease in mice, whereas Fol4287 induced more pronounced wilting symptoms in tomato plants. In vitro assays using abiotic stress treatments revealed that the human pathogen MRL8996 was better adapted to elevated temperatures, whereas the plant pathogen Fol4287 was more tolerant to osmotic and cell wall stresses. Both strains displayed broad resistance to antifungal treatment, with MRL8996 exhibiting the paradoxical effect of increased tolerance to higher concentrations of the antifungal caspofungin. We identified a set of accessory chromosomes (ACs) that encode genes with different functions and have distinct transposon profiles between MRL8996 and Fol4287. Interestingly, ACs from both genomes also encode proteins with shared functions, such as chromatin remodeling and post-translational protein modifications. Our phenotypic assays and comparative genomics analyses lay the foundation for future studies correlating genotype with phenotype and for developing targeted antifungals for agricultural and clinical uses.

The anticonvulsant drug carbamazepine is ubiquitous in the environment and has even even detected in human urine after consuming produce irrigated with reclaimed wastewater. Whether unintentional carbamazepine exposure through food and water affects public health is unknown. Its potential adverse effects are particularly concerning during pregnancy, as carbamazepine increases the risk of intrauterine growth restriction and congenital malformations in fetuses of carbamazepine-prescribed mothers. While environmental carbamazepine levels are much lower than clinical doses, its impact on early embryonic development, a period highly susceptible to malformations, requires investigation. This study used mice to examine the effect of exposing female mice to environmentally relevant carbamazepine concentrations (200/500/2000 ng/L in their drinking water) on embryos at gestation day 9.5. While no obvious malformations or compromised survival rates were observed, embryonic growth was delayed in a dose-dependent manner; developmental stages were younger than expected, fewer somites had formed, and heart maturation was delayed. Molecular analysis revealed a reduced expression of key developmental genes and decreased proliferation, linking growth delay to perturbed mechanisms. This study is the first to link maternal exposure to environmentally relevant carbamazepine concentrations with growth delay in mammalian embryos. Given that prenatal growth restriction contributes to human morbidity, this finding calls for further risk analyses of environmental pharmaceuticals on fetal health.

Over the past billion years, the fungal kingdom has diversified to more than two million species, with over 95% still undescribed. Beyond the well-known macroscopic mushrooms and microscopic yeast, fungi are heterotrophs that feed on almost any organic carbon, recycling nutrients through the decay of dead plants and animals and sequestering carbon into Earth's ecosystems. Human-directed applications of fungi extend from leavened bread, alcoholic beverages and biofuels to pharmaceuticals, including antibiotics and psychoactive compounds. Conversely, fungal infections pose risks to ecosystems ranging from crops to wildlife to humans; these risks are driven, in part, by human and animal movement, and might be accelerating with climate change. Genomic surveys are expanding our knowledge of the true biodiversity of the fungal kingdom, and genome-editing tools make it possible to imagine harnessing these organisms to fuel the bioeconomy. Here, we examine the fungal threats facing civilization and investigate opportunities to use fungi to combat these threats.

Invasive fungal infections, characterized by high mortality rates, present a growing health concern due to the limited spectrum of effective drugs, escalating numbers of immunocompromised patients, and the rapid emergence of drug resistance. Addressing this challenge requires alternative therapeutic strategies. Candidate drugs should fulfill key requirements to qualify for clinical use, including negligible host toxicity, high in vitro and in vivo antifungal activity, in vivo stability, and modes of actions that reduce the potential for the emergence of resistant strains. Antimicrobial peptides (AMPs) stand out as a promising class of compounds to combat antimicrobial resistance. Capitalizing on the unique characteristics of AMPs, we have developed a novel approach, to synthesize random peptide mixtures (RPMs) with robust bactericidal properties against drug-resistant bacteria. Here, we evaluate the antifungal potential of RPMs. Our findings demonstrate high potency of RPMs against Candida auris, a newly emerged multidrug-resistant human fungal pathogen. In vitro studies demonstrated the effective eradication of C. auris by FK20 RPMs, with scanning electron microscopy revealing substantial damage to the fungal outer membrane, while confocal microscopy confirms their ability to penetrate C. auris cells. Importantly, our study elucidates that FK20 RPMs effectively hinder the development of resistance in C. auris, as confirmed by experimental evolution assays. Moreover, a synergistic effect is observed when combining FK20 RPMs with the antifungal drug caspofungin, effectively inhibiting biofilm formation. Lastly, we demonstrate the significant therapeutic potential of FK20 RPM in a murine model of systemic candidiasis. Our findings underscore the promise of RPMs as novel, efficacious, and safe treatment for drug-resistant fungal infections. ### Competing Interest Statement The authors have declared no competing interest.

The mycobiota are a critical part of the gut microbiome, but host–fungal interactions and specific functional contributions of commensal fungi to host fitness remain incompletely understood. Here, we report the identification of a new fungal commensal, Kazachstania heterogenica var. weizmannii, isolated from murine intestines. K. weizmannii exposure prevented Candida albicans colonization and significantly reduced the commensal C. albicans burden in colonized animals. Following immunosuppression of C. albicans colonized mice, competitive fungal commensalism thereby mitigated fatal candidiasis. Metagenome analysis revealed K. heterogenica or K. weizmannii presence among human commensals. Our results reveal competitive fungal commensalism within the intestinal microbiota, independent of bacteria and immune responses, that could bear potential therapeutic value for the management of C. albicans–mediated diseases.

We describe a previously unappreciated role for Bruton’s tyrosine kinase (BTK) in fungal immune surveillance against aspergillosis, an unforeseen complication of BTK inhibitors (BTKi) used for treating B cell lymphoid malignancies. We studied BTK-dependent fungal responses in neutrophils from diverse populations, including healthy donors, patients who were treated with BTKi, and X-linked agammaglobulinemia patients. Upon fungal exposure, BTK was activated in human neutrophils in a TLR2-, Dectin-1-, and FcγR-dependent manner, triggering the oxidative burst. BTK inhibition selectively impeded neutrophil-mediated damage to Aspergillus hyphae, primary granule release, and the fungus-induced oxidative burst by abrogating NADPH oxidase subunit p40phox and GTPase RAC2 activation. Moreover, neutrophil-specific Btk deletion in mice enhanced aspergillosis susceptibility by impairing neutrophil function, not recruitment or lifespan. Conversely, GM-CSF partially mitigated these deficits by enhancing p47phox activation. Our findings underline the crucial role of BTK signaling in neutrophils for antifungal immunity and provide a rationale for GM-CSF use to offset these deficits in patients who are susceptible.

Fungal pathogens pose a significant threat to global health. Aspergillus fumigatus accounts for approximately 65% of all invasive fungal infections in humans, with mortality rates from aspergillosis reaching nearly 50%. Fungal virulence in plant pathogenic fungi can be modified by mycoviruses, viruses that infect fungi. However, their impact on fungal pathogenesis in mammals has remained largely unexplored. Here, utilizing an A. fumigatus strain naturally infected with Aspergillus fumigatus polymycovirus-1M (AfuPmV-1M), we found that the mycovirus confers a significant survival advantage to the fungus under conditions of oxidative stress, heat stress, and within the murine lung. Thus, AfuPmV-1M modulates fungal fitness, resulting in increased virulence and the progression of exacerbated fungal disease. Moreover, antiviral treatment reverses the exacerbated virus-mediated virulence, representing a promising "antipathogenicity" therapy against virus-bearing pathogenic fungi. Taken together, these data suggest that mycoviruses play a significant role as "backseat drivers" in human fungal diseases, presenting critical clinical implications.

ABSTRACT As chemical pollution is constantly increasing, the impact on the environment and public health must be investigated. This study focuses on the anticonvulsant drug carbamazepine (CBZ), which is ubiquitously present in the environment. Due to its physicochemical properties and stability during wastewater treatment, CBZ is detected in reclaimed wastewater, surface water and groundwater. In water-scarce regions heavily relying on treated wastewater for crop irrigation, CBZ is detected in arable land, produce and even in humans consuming crops irrigated with recealimed wastewater. Aalthough environmental levels of CBZ are very low, risks associated with unintentional exposure to CBZ are essential to be revealed. In perinatal medicine, CBZ is a teratogen; its prescription to pregnant women increases the risk for fetal malformations. This raises the concern of whether environmental exposure to CBZ may also impact embryogenesis. Studies in zebrafish and chick embryos or in cell culture have indicated negative outcomes upon exposure to low CBZ levels. Yet, these systems do not recapitulate the manner by which human fetuses are exposed to pharmaceuticals via maternal uptake. Here, we employed the mouse model to determine whether maternal exposure to environmental-relevant doses of CBZ will impact embryonic development. No effects on fertility, number of gestation sacs, gross embryonic malformations or fetal survival were detected. Yet, embryos were growth-delayed compared to controls ( p =0.0011), as manifested in lower embryonic stage and somite number, earlier morphological features and reduction in mitotically-active cells. This study provides the first evidence for the effect of environmental concentration of CBZ on the developmental kinetics of maternally-exposed mammalian embryos. While the developmental delay was relatively modest, its consistency in high number of biological replicates, together with the known implication of developmental delay on post-natal health, calls for further in-depth risk analyses to reveal the effects of pharmaceuticals released to the environment on public health.

Myeloid phagocytes of the respiratory immune system, such as neutrophils, monocytes, and alveolar macrophages, are essential for immunity to Aspergillus fumigatus , the most common etiologic agent of mold pneumonia worldwide. Following engulfment of A. fumigatus conidia, fusion of the phagosome with the lysosome, is a critical process for killing conidia. TFEB and TFE3 are transcription factors that regulate lysosomal biogenesis under stress and are activated by inflammatory stimuli in macrophages, but it is unknown whether TFEB and TFE3 contribute to anti- Aspergillus immunity during infection. We found that lung neutrophils express TFEB and TFE3, and their target genes were upregulated during A. fumigatus lung infection. Additionally, A. fumigatus infection induced nuclear accumulation of TFEB and TFE3 in macrophages in a process regulated by Dectin-1 and CARD9 signaling. Genetic deletion of Tfeb and Tfe3 impaired macrophage killing of A. fumigatus conidia. However, in a murine immune competent Aspergillus infection model with genetic deficiency of Tfeb and Tfe3 in hematopoietic cells, we surprisingly found that lung myeloid phagocytes had no defects in conidial phagocytosis or killing. Loss of TFEB and TFE3 did not impact murine survival or clearance of A. fumigatus from the lungs. Our findings indicate that myeloid phagocytes activate TFEB and TFE3 in response to A. fumigatus , and while this pathway promotes macrophage fungicidal activity in vitro , genetic loss can be functionally compensated at the portal of infection in the lung, resulting in no measurable defect in fungal control and host survival.

Subverting the host immune system is a major task for any given pathogen to assure its survival and proliferation. For the opportunistic human pathogen Bacillus cereus (Bc), immune evasion enables the establishment of potent infections. In various species of the Bc group, the pleiotropic regulator PlcR and its cognate cell-cell signaling peptide PapR7 regulate virulence gene expression in response to fluctuations in population density, i.e., a quorum-sensing (QS) system. However, how QS exerts its effects during infections and whether PlcR confers the immune evading ability remain unclear. Herein, we report how interception of the QS communication in Bc obliterates the ability to affect the host immune system. Here, we designed a peptide- based QS inhibitor that suppresses PlcR-dependent virulence factor expression and attenuates Bc infectivity in mouse models. We demonstrate that the QS peptidic inhibitor blocks host immune system-mediated eradication by reducing the expression of PlcR-regulated major tox-ins similarly to the profile that was observed for isogenic strains. Our findings provide evidence that Bc infectivity is regulated by QS circuit-mediated destruction of host immunity, thus reveal a interesting strategy to limit Bc virulence and enhance host defense. This peptidic quorum-quenching agent constitutes a readily accessible chemical tool for studying how other pathogen QS systems modulate host immunity and forms a basis for development of anti-infective therapeutics.

OPINION article Front. Cell. Infect. Microbiol., 13 October 2022Sec. Fungal Pathogenesis Volume 12 - 2022 | https://doi.org/10.3389/fcimb.2022.1020608

Fluorescence-based techniques enable researchers to monitor physiologic processes, specifically fungal cell viability and death, during cellular encounters with the mammalian immune system with single event resolution. By incorporating two independent fluorescent probes in fungal organisms either prior to, or ensuing experimental infection in mice or in cultured leukocytes, it is possible to distinguish and quantify live and killed fungal cells to interrogate genetic, pharmacologic, and cellular determinants that shape host-fungal cell outcomes. This chapter reviews the techniques and applications of fluorescent fungal reporters of viability, with emphasis on the filamentous mold Aspergillus fumigatus.