Sirtuin E deacetylase is required for full virulence of Aspergillus fumigatus.

Aspergillus fumigatus represents a public health problem due to the high mortality rate in immunosuppressed patients and the emergence of antifungal-resistant isolates. Protein acetylation is a crucial post-translational modification that controls gene expression and biological processes. The strategic manipulation of enzymes involved in protein acetylation has emerged as a promising therapeutic approach for addressing fungal infections. Sirtuins, NAD+-dependent lysine deacetylases, regulate protein acetylation and gene expression in eukaryotes. However, their role in the human pathogenic fungus A. fumigatus remains unclear. This study constructs six single knockout strains of A. fumigatus and a strain lacking all predicted sirtuins (SIRTKO). The mutant strains are viable under laboratory conditions, indicating that sirtuins are not essential genes. Phenotypic assays suggest sirtuins' involvement in cell wall integrity, secondary metabolite production, thermotolerance, and virulence. Deletion of sirE attenuates virulence in murine and Galleria mellonella infection models. The absence of SirE alters the acetylation status of proteins, including histones and non-histones, and triggers significant changes in the expression of genes associated with secondary metabolism, cell wall biosynthesis, and virulence factors. These findings encourage testing sirtuin inhibitors as potential therapeutic strategies to combat A. fumigatus infections or in combination therapy with available antifungals.

The StuA Transcription Factor and Alternative Splicing Mechanisms Drive the Levels of MAPK Hog1 Transcripts in the Dermatophyte Trichophyton rubrum.

Trichophyton rubrum is a human fungal pathogen that causes dermatophytosis, an infection that affects keratinized tissues. Integrated molecular signals coordinate mechanisms that control pathogenicity. Transcriptional regulation is a core regulation of relevant fungal processes. Previous RNA sequencing data revealed that the absence of the transcription factor StuA resulted in the differential expression of the MAPK-related high glycerol osmolarity gene (hog1) in T. rubrum. Here we validated the role of StuA in regulating the transcript levels of hog1. We showed through RT-qPCR that transcriptional regulation controls hog1 levels in response to glucose, keratin, and co-culture with human keratinocytes. In addition, we also detected hog1 pre-mRNA transcripts that underwent alternative splicing, presenting intron retention in a StuA-dependent mechanism. Our findings suggest that StuA and alternative splicing simultaneously, but not dependently, coordinate hog1 transcript levels in T. rubrum. As a means of preventing and treating dermatophytosis, our results contribute to the search for new potential drug therapies based on the molecular aspects of signaling pathways in T. rubrum.

Extracellular vesicles from the mycoparasitic fungus Trichoderma harzianum.

Trichoderma harzianum is a filamentous fungus that can act as a mycoparasite, saprophyte, or a plant symbiotic. It is widely used as a biological control agent against phytopathogenic fungi and can also be used for plant growth promotion and biofortification. Interaction between T. harzianum and phytopathogenic fungi involves mycoparasitism, competition, and antibiosis. Extracellular vesicles (EVs) have been described as presenting a central role in mechanisms of communication and interaction among fungus and their hosts. In this study, we characterized extracellular vesicles of T. harzianum produced during growth in the presence of glucose or S. sclerotiorum mycelia. A set of vesicular proteins was identified using proteomic approach, mainly presenting predicted signal peptides.

Extracellular vesicles from virulent P. brasiliensis induce TLR4 and dectin-1 expression in innate cells and promote enhanced Th1/Th17 response.

Extracellular vesicles (EVs) are membrane-enclosed nanoparticles that transport several biomolecules and are involved in important mechanisms and functions related to the pathophysiology of fungal diseases. EVs from Paracoccidioides brasiliensis, the main causative agent of Paracoccidioidomycosis (PCM), modulate the immune response of macrophages. In this study, we assessed the EVs proteome from a virulent P. brasiliensis isolated from granulomatous lesions and compared their immunomodulatory ability with EVs isolated from the fungus before the animal passage (control EVs) when challenging macrophages and dendritic cells (DCs). Proteome showed that virulent EVs have a higher abundance of virulence factors such as GP43, protein 14-3-3, GAPDH, as well as virulence factors never described in PCM, such as aspartyl aminopeptidase and a SidJ analogue compared with control EVs. Virulent extracellular vesicles induced higher expression of TLR4 and Dectin-1 than control EVs in macrophages and dendritic cells (DCs). In opposition, a lower TLR2 expression was induced by virulent EVs. Additionally, virulent EVs induced lower expression of CD80, CD86 and TNF-α, but promoted a higher expression of IL-6 and IL-10, suggesting that EVs isolated from virulent P. brasiliensis-yeast promote a milder DCs and macrophage maturation. Herein, we showed that EVs from virulent fungi stimulated a higher frequency of Th1/Tc1, Th17, and Treg cells, which gives new insights into fungal extracellular vesicles. Taken together, our results suggest that P. brasiliensis utilizes its EVs as virulence bags that manipulate the immune system in its favour, creating a milder immune response and helping with fungal evasion from the immune system.

Deacetylation by sirtuins is important for Aspergillus fumigatus pathogenesis and virulence.

Protein acetylation is a crucial post-translational modification that controls gene expression and a variety of biological processes. Sirtuins, a prominent class of NAD + -dependent lysine deacetylases, serve as key regulators of protein acetylation and gene expression in eukaryotes. In this study, six single knockout strains of fungal pathogen Aspergillus fumigatus were constructed, in addition to a strain lacking all predicted sirtuins (SIRTKO). Phenotypic assays suggest that sirtuins are involved in cell wall integrity, secondary metabolite production, thermotolerance, and virulence. AfsirE deletion resulted in attenuation of virulence, as demonstrated in murine and Galleria infection models. The absence of AfSirE leads to altered acetylation status of proteins, including histones and non-histones, resulting in significant changes in the expression of genes associated with secondary metabolism, cell wall biosynthesis, and virulence factors. These findings encourage testing sirtuin inhibitors as potential therapeutic strategies to combat A. fumigatus infections or in combination therapy with available antifungals.

Vacuoles and peroxisomes are involved in Aspergillus fumigatus gliotoxin production and self-protection.

Aspergillus fumigatus is a saprophytic fungus that can cause a variety of human diseases known as aspergillosis. Mycotoxin gliotoxin (GT) production is important for its virulence and must be tightly regulated to avoid excess production and toxicity to the fungus. GT self-protection by GliT oxidoreductase and GtmA methyltransferase activities is related to the subcellular localization of these enzymes and how GT can be sequestered from the cytoplasm to avoid increased cell damage. Here, we show that GliT:GFP and GtmA:GFP are localized in the cytoplasm and in vacuoles during GT production. Peroxisomes are also required for proper GT production and self-defense. The Mitogen-Activated Protein (MAP) kinase MpkA is essential for GT production and self-protection, interacts physically with GliT and GtmA and it is necessary for their regulation and subsequent presence in the vacuoles. Our work emphasizes the importance of dynamic compartmentalization of cellular events for GT production and self-defense.

Mass Spectrometry Analysis Reveals Lipids Induced by Oxidative Stress in Candida albicans Extracellular Vesicles.

Candida albicans is a commensal fungus in healthy humans that causes infection in immunocompromised individuals through the secretion of several virulence factors. The successful establishment of infection is owing to elaborate strategies to cope with defensive molecules secreted by the host, including responses toward oxidative stress. Extracellular vesicle (EV) release is considered an alternative to the biomolecule secretory mechanism that favors fungal interactions with the host cells. During candidiasis establishment, the host environment becomes oxidative, and it impacts EV release and cargo. To simulate the host oxidative environment, we added menadione (an oxidative stress inducer) to the culture medium, and we explored C. albicans EV metabolites by metabolomics analysis. This study characterized lipidic molecules transported to an extracellular milieu by C. albicans after menadione exposure. Through Liquid Chromatography coupled with Mass Spectrometry (LC-MS) analyses, we identified biomolecules transported by EVs and supernatant. The identified molecules are related to several biological processes, such as glycerophospholipid and sphingolipid pathways, which may act at different levels by tuning compound production in accordance with cell requirements that favor a myriad of adaptive responses. Taken together, our results provide new insights into the role of EVs in fungal biology and host-pathogen interactions.

Aspergillus fumigatus Extracellular Vesicles Display Increased Galleria mellonella Survival but Partial Pro-Inflammatory Response by Macrophages.

Fungal extracellular vesicles (EVs) mediate intra- and interspecies communication and are critical in host-fungus interaction, modulating inflammation and immune responses. In this study, we evaluated the in vitro pro- and anti-inflammatory properties of Aspergillus fumigatus EVs over innate leukocytes. A. fumigatus EVs induced a partial proinflammatory response by macrophages, characterized by increased tumor necrosis factor-alpha production, and increased gene expression of induced nitric oxide synthase and adhesion molecules. EVs induce neither NETosis in human neutrophils nor cytokine secretion by peripheral mononuclear cells. However, prior inoculation of A. fumigatus EVs in Galleria mellonella larvae resulted in increased survival after the fungal challenge. Taken together, these findings show that A. fumigatus EVs play a role in protection against fungal infection, although they induce a partial pro-inflammatory response.

Extracellular Vesicles from Candida haemulonii var. vulnera Modulate Macrophage Oxidative Burst.

Members of the Candida haemulonii species complex are multidrug-resistant emergent yeast pathogens able to cause superficial and invasive infections in risk populations. Fungal extracellular vesicles (EVs) play a critical role in the pathogenicity and virulence of several species and may perform essential functions during infections, such as carrying virulence factors that behave in two-way communications with the host, affecting survival and fungal resistance. Our study aimed to describe EV production from Candida haemulonii var. vulnera and evaluate whether murine macrophage RAW 264.7 cells respond to their stimuli by generating an oxidative response after 24 h. For this purpose, reactive oxygen species detection assays demonstrated that high concentrations of yeast and EVs (1010 particles/mL) of Candida haemulonii did not change macrophage viability. However, the macrophages recognized these EVs and triggered an oxidative response through the classical NOX-2 pathway, increasing O2•- and H2O2 levels. However, this stress did not cause lipid peroxidation in the RAW 264.7 cells and neither lead to the activation of the COX-2-PGE2 pathway. Thus, our data suggest that low concentrations of C. haemulonii EVs are not recognized by the classical pathway of the oxidative burst generated by macrophages, which might be an advantage allowing the transport of virulence factors via EVs, not identified by the host immune system that could work as fine tube regulators during infections caused by C. haemulonii. In contrast, C. haemulonii var. vulnera and high EV concentrations activated microbicidal actions in macrophages. Therefore, we propose that EVs could participate in the virulence of the species and that these particles could be a source of antigens to be exploited as new therapeutic targets.

Addressing Microbial Resistance Worldwide: Challenges over Controlling Life-Threatening Fungal Infections.

Fungal infections are a serious global concern because of their ability to spread and colonize host tissues in immunocompromised individuals. Such infections have been frequently reported worldwide and are currently gaining clinical research relevance owing to their resistant character, representing a bottleneck in treating affected people. Resistant fungi are an emergent public health threat. The upsurge of such pathogens has led to new research toward unraveling the destructive potential evoked by these species. Some fungi-grouped into Candida, Aspergillus, and Cryptococcus-are causative agents of severe and systemic infections. They are associated with high mortality rates and have recently been described as sources of coinfection in COVID-hospitalized patients. Despite the efforts to elucidate the challenges of colonization, dissemination, and infection severity, the immunopathogenesis of fungal diseases remains a pivotal characteristic in fungal burden elimination. The struggle between the host immune system and the physiological strategies of the fungi to maintain cellular viability is complex. In this brief review, we highlight the relevance of drug resistance phenotypes in fungi of clinical significance, taking into consideration their physiopathology and how the scientific community could orchestrate their efforts to avoid fungal infection dissemination and deaths.

Chitin Biosynthesis in Aspergillus Species.

The fungal cell wall (FCW) is a dynamic structure responsible for the maintenance of cellular homeostasis, and is essential for modulating the interaction of the fungus with its environment. It is composed of proteins, lipids, pigments and polysaccharides, including chitin. Chitin synthesis is catalyzed by chitin synthases (CS), and up to eight CS-encoding genes can be found in Aspergillus species. This review discusses in detail the chitin synthesis and regulation in Aspergillus species, and how manipulation of chitin synthesis pathways can modulate fungal growth, enzyme production, virulence and susceptibility to antifungal agents. More specifically, the metabolic steps involved in chitin biosynthesis are described with an emphasis on how the initiation of chitin biosynthesis remains unknown. A description of the classification, localization and transport of CS was also made. Chitin biosynthesis is shown to underlie a complex regulatory network, with extensive cross-talks existing between the different signaling pathways. Furthermore, pathways and recently identified regulators of chitin biosynthesis during the caspofungin paradoxical effect (CPE) are described. The effect of a chitin on the mammalian immune system is also discussed. Lastly, interference with chitin biosynthesis may also be beneficial for biotechnological applications. Even after more than 30 years of research, chitin biosynthesis remains a topic of current interest in mycology.

Caspofungin Affects Extracellular Vesicle Production and Cargo in Candida auris.

Antifungal resistance has become more frequent, either due to the emergence of naturally resistant species or the development of mechanisms that lead to resistance in previously susceptible species. Among these fungal species of global threat, Candida auris stands out for commonly being highly resistant to antifungal drugs, and some isolates are pan-resistant. The rate of mortality linked to C. auris infections varies from 28% to 78%. In this study, we characterized C. auris extracellular vesicles (EVs) in the presence of caspofungin, an echinocandin, which is the recommended first line antifungal for the treatment of infections due to this emerging pathogen. Furthermore, we also analyzed the protein and RNA content of EVs generated by C. auris cultivated with or without treatment with caspofungin. We observed that caspofungin led to the increased production of EVs, and treatment also altered the type and quantity of RNA molecules and proteins enclosed in the EVs. There were distinct classes of RNAs in the EVs with ncRNAs being the most identified molecules, and tRNA-fragments (tRFs) were abundant in each of the strains studied. We also identified anti-sense RNAs, varying from 21 to 55 nt in length. The differentially abundant mRNAs detected in EVs isolated from yeast subjected to caspofungin treatment were related to translation, nucleosome core and cell wall. The differentially regulated proteins identified in the EVs produced during caspofungin treatment were consistent with the results observed with the RNAs, with the enriched terms being related to translation and cell wall. Our study adds new information on how an echinocandin can affect the EV pathway, which is associated with the yeast cell being able to evade treatment and persist in the host. The ability of C. auris to efficiently alter the composition of EVs may represent a mechanism for the fungus to mitigate the effects of antifungal agents.

The Role of Extracellular Vesicles in COVID-19 Pathology.

Extracellular vesicles (EVs) have become a trending topic in recent years; they constitute a new intercellular communication paradigm. Extracellular vesicles are 30-4000 nanometers in diameter particles that are limited by a phospholipid bilayer and contain functional biomolecules, such as proteins, lipids, and nucleic acids. They are released by virtually all types of eukaryotic cells; through their cargoes, EVs are capable of triggering signaling in recipient cells. In addition to their functions in the homeostatic state, EVs have gained attention because of their roles in pathological contexts, eventually contributing to disease progression. In the Coronavirus disease 2019 (COVID-19) pandemic, aside from the scientific race for the development of preventive and therapeutic interventions, it is critical to understand the pathological mechanisms involved in SARS-CoV-2 infection. In this sense, EVs are key players in the main processes of COVID-19. Thus, in this review, we highlight the role of EVs in the establishment of the viral infection and in the procoagulant state, cytokine storm, and immunoregulation of innate and adaptive immune responses.

The RNA Content of Fungal Extracellular Vesicles: At the "Cutting-Edge" of Pathophysiology Regulation.

The role of extracellular vesicles (EVs) in interkingdom communication is widely accepted, and their role in intraspecies communication has been strengthened by recent research. Based on the regulation promoted by EV-associated molecules, the interactions between host and pathogens can reveal different pathways that ultimately affect infection outcomes. As a great part of the regulation is ascribable to RNA contained in EVs, many studies have focused on profiling RNAs in fungal and host EVs, tracking their accumulation during infection, and identifying potential target genes. Herein, we overview the main classes of RNA contained in fungal EVs and the biological processes regulated by these molecules, portraying a state-of-the-art picture of RNAs loaded in fungal EVs, while also raising several questions to drive future investigations. Our compiled data show unambiguously that EVs act as key elements in signaling pathways, and play a crucial role in pathosystems. A complete understanding of the processes that govern RNA content loading and trafficking, and its effect on recipient cells, will lead to improved technologies to ward off infectious agents that threaten human health.

The Expression of Tax and HBZ Genes in Serum-Derived Extracellular Vesicles From HTLV-1 Carriers Correlates to Proviral Load and Inflammatory Markers.

Human T-lymphotropic virus 1 (HTLV-1) is the etiologic agent of adult cell leukemia/lymphoma (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). One of the major questions in HTLV-1 studies is related to the understanding of causes that lead to different clinical manifestations. However, it is well known that the viral genes tax and HTLV-1 basic leucine zipper factor (HBZ) are related to viral infectivity and the development of neurological and hematological diseases. Currently, there is evidence that HTLV-1 infected cells can release small extracellular vesicles (sEVs) involved in the mechanisms of viral particles spreading. Therefore, we evaluated the expression levels of tax and HBZ viral transcripts in serum-derived sEVs from HTLV-1 carriers, as well as the role of these vesicles in the modulation of the immune response. Three HAM/TSP carriers presented detectable levels of tax and HBZ transcripts in sEVs and were positively correlated to the proviral load (PVL) in peripheral blood mononuclear cells (PBMCs). The viral transcripts were only detectable in individuals with a PVL higher than 6,000/105 PBMCs. Additionally, it was observed that HBZ presented a 2-12-folds increase over tax expression units. Gene expression and secretory protein analysis indicated that PBMCs from blood donors and HTLV-1 carriers exposed to increasing doses of tax+ HBZ+ sEVs showed a dose-dependent increase in interferon (IFN)-γ and interleukin (IL)-8 transcripts and proteins. Interestingly, the increase in IL-8 levels was close to those seen in HTLV-1-infected PBMCs with high PVL. Taken together, these findings indicate that the expression of viral transcripts in serum-derived sEVs of HTLV-1 carriers is related to the PVL presented by the infected individual. Additionally, tax+ HBZ+ sEVs can induce the production of inflammatory cytokines in patients with low PVL, which may be related to the development of symptoms in HTLV-1 infection.

Fungal Extracellular Vesicles Are Involved in Intraspecies Intracellular Communication.

Fungal infections are associated with high mortality rates in humans. The risk of fungal diseases creates the urgent need to broaden the knowledge base regarding their pathophysiology. In this sense, the role of extracellular vesicles (EVs) has been described to convey biological information and participate in the fungus-host interaction process. We hypothesized that fungal EVs work as an additional element in the communication routes regulating fungal responses in intraspecies interaction systems. In this respect, the aim of this study was to address the gene regulation profiles prompted by fungal EVs in intraspecies recipient cells. Our data demonstrated the intraspecies uptake of EVs in pathogenic fungi, such as Candida albicans, Aspergillus fumigatus, and Paracoccidioides brasiliensis, and the effects triggered by EVs in fungal cells. In C. albicans, we evaluated the involvement of EVs in the yeast-to-hypha transition, while in P. brasiliensis and A. fumigatus the function of EVs as stress transducers was investigated. P. brasiliensis and A. fumigatus were exposed to an inhibitor of glycosylation or UV light, respectively. The results demonstrated the role of EVs in regulating the expression of target genes and triggering phenotypic changes. The EVs treatment induced cellular proliferation and boosted the yeast to hyphal transition in C. albicans, while they enhanced stress responsiveness in A. fumigatus and P. brasiliensis, establishing a role for EVs in fungal intraspecies communication. Thus, EVs regulate fungal behavior, acting as potent message effectors, and understanding their effects and mechanism(s) of action could be exploited in antifungal therapies. IMPORTANCE Here, we report a study about extracellular vesicles (EVs) as communication mediators in fungi. Our results demonstrated the role of EVs from Candida albicans, Aspergillus fumigatus, and Paracoccidioides brasiliensis regulating the expression of target genes and phenotypic features. We asked whether fungal EVs play a role as message effectors. We show that fungal EVs are involved in fungal interaction systems as potent message effectors, and understanding their effects and mechanisms of action could be exploited in antifungal therapies.

Shaping the gut microbiota by bioactive phytochemicals: An emerging approach for the prevention and treatment of human diseases.

The human digestive tract is the cottage to trillions of live microorganisms, which regulate health and illness. A healthy Gut Microbiota (GM) is necessary for preventing microbial growth, body growth, obesity, cancer, diabetes, and enhancing immunity. The equilibrium in GM's composition and the presence/absence of critical species enable specific responses to be essential for the host's better health condition. Research evidences revealed that the dietary plants and their bioactive phytochemicals (BPs) play an extensive and critical role in shaping the GM to get beneficial health effects. BPs are also known to improve gastrointestinal health and reduce the risk of several diseases by modulating GM-mediated cellular and molecular processes. Regular intake of BPs-rich vegetables, fruits, and herbal preparations promotes probiotic bacteria, including Bifidobacteria and Lactobacillus species, while inhibiting unwanted gut residents' development Escherichia coli, and Salmonella typhimurium etc. Upon consumption, BPs contact the GM that gets transformed before being absorbed from the gastrointestinal tract. Biotransformation of BPs by GM is linked with the enhancement of bioactivity/toxicity diminishment of the BPs compared to parental phytochemicals. Therefore, the current review focuses on the role of BPs in shaping GM for the prevention and treatment of human diseases.

Transcriptional and translational landscape of Candida auris in response to caspofungin.

Candida auris has emerged as a serious worldwide threat by causing opportunistic infections that are frequently resistant to one or more conventional antifungal medications resulting in high mortality rates. Against this backdrop, health warnings around the world have focused efforts on understanding C. auris fungal biology and effective prevention and treatment approaches to combat this fungus. To date, there is little information about the differentially expressed genes when this fungus is treated with conventional antifungals, and caspofungin is a standard echinocandin deployed in the therapy against C. auris. In this work, we treated two distinct strains of C. auris for 24 h with caspofungin, and the cellular responses were evaluated at the morphological, translational and transcriptional levels. We first observed that the echinocandin caused morphological alterations, aggregation of yeast cells, and modifications in the cell wall composition of C. auris. Transcriptomic analysis revealed an upregulation of genes related to the synthesis of the cell wall, ribosome, and cell cycle after exposure to caspofungin. Supporting these findings, the integrated proteomic analysis showed that caspofungin-treated cells were enriched in ribosome-related proteins and cell wall, especially mannoproteins. Altogether, these results provide further insights into the biology of C. auris and expands our understanding regarding the antifungal activity of caspofungin and reveal cellular targets, as the mannose metabolism, that can be further explored for the development of novel antifungals.

Gasdermin D inhibition prevents multiple organ dysfunction during sepsis by blocking NET formation.

Multiple organ dysfunction is the most severe outcome of sepsis progression and is highly correlated with a worse prognosis. Excessive neutrophil extracellular traps (NETs) are critical players in the development of organ failure during sepsis. Therefore, interventions targeting NET release would likely effectively prevent NET-based organ injury associated with this disease. Herein, we demonstrate that the pore-forming protein gasdermin D (GSDMD) is active in neutrophils from septic humans and mice and plays a crucial role in NET release. Inhibition of GSDMD with disulfiram or genic deletion abrogated NET formation, reducing multiple organ dysfunction and sepsis lethality. Mechanistically, we demonstrate that during sepsis, activation of the caspase-11/GSDMD pathway controls NET release by neutrophils during sepsis. In summary, our findings uncover a novel therapeutic use for disulfiram and suggest that GSDMD is a therapeutic target to improve sepsis treatment.