RESUMO
Microbes rarely exist in isolation and instead form complex polymicrobial communities. As a result, microbes have developed intricate offensive and defensive strategies that enhance their fitness in these complex communities. Thus, identifying and understanding the molecular mechanisms controlling polymicrobial interactions is critical for understanding the function of microbial communities. In this study, we show that the gram-negative opportunistic human pathogen Pseudomonas aeruginosa, which frequently causes infection alongside a plethora of other microbes including fungi, encodes a genetic network which can detect and defend against gliotoxin, a potent, disulfide-containing antimicrobial produced by the ubiquitous filamentous fungus Aspergillus fumigatus. We show that gliotoxin exposure disrupts P. aeruginosa zinc homeostasis, leading to transcriptional activation of a gene encoding a previously uncharacterized dithiol oxidase (herein named as DnoP), which detoxifies gliotoxin and structurally related toxins. Despite sharing little homology to the A. fumigatus gliotoxin resistance protein (GliT), the enzymatic mechanism of DnoP from P. aeruginosa appears to be identical that used by A. fumigatus. Thus, DnoP and its transcriptional induction by low zinc represent a rare example of both convergent evolution of toxin defense and environmental cue sensing across kingdoms. Collectively, these data provide compelling evidence that P. aeruginosa has evolved to survive exposure to an A. fumigatus disulfide-containing toxin in the natural environment.
Assuntos
Aspergillus fumigatus , Gliotoxina , Pseudomonas aeruginosa , Gliotoxina/metabolismo , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/genética , Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/genética , Zinco/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Interações Microbianas , Humanos , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genéticaRESUMO
As one of the mycotoxins produced by Aspergillus fumigatus, gliotoxin has a variety of pharmacological effects, such as anti-tumor, antibacterial, immunosuppressive. Antitumor drugs induce tumor cell death in several forms, including apoptosis, autophagy, necrosis and ferroptosis. Ferroptosis is a recently identified unique form of programmed cell death characterized by iron-dependent accumulation of lethal lipid peroxides, which induces cell death. A large amount of preclinical evidence suggests that ferroptosis inducers may enhance the sensitivity of chemotherapy and the induction of ferroptosis may be an effective therapeutic strategy to prevent acquired drug resistance. In our study, gliotoxin was characterized as a ferroptosis inducer and showed strong anti-tumor activity with IC50 of 0.24 µM and 0.45 µM in H1975 and MCF-7 cells at 72 h, respectively. Gliotoxin may provide a new natural template for the designing of ferroptosis inducers.
Assuntos
Produtos Biológicos , Ferroptose , Gliotoxina , Humanos , Gliotoxina/farmacologia , Produtos Biológicos/farmacologia , Ferro/metabolismo , ApoptoseRESUMO
Gliotoxin is a representative compound of the epipolythiodioxopiperazine (ETP) class of fungal metabolites. Histone Lysine Specific Demethylase 1 (LSD1) is highly expressed in a variety of cancers. Herein, a series of 6-heterocyclic carboxylic ester derivatives of gliotoxin was designed and synthesized as new LSD1 inhibitors and their biological evaluations in human gastric MGC-803 and HGC-27 cells were carried out. All of the derivatives effectively suppressed the enzymatic activities of LSD1. In particular, compound 4e exhibited excellent LSD1 inhibition with IC50 = 62.40 nM, as well as anti-proliferation against MGC-803 and HGC-27 cells with IC50 values of 0.31 µM and 0.29 µM, respectively. 4e also had a remarkable capacity to inhibit the colony formation, suppress migration and induce the apoptosis of these two cancer cell lines. In sum, our findings identified and characterized the 6-heterocyclic carboxylic ester derivatives of gliotoxin as potent and cellular active LSD1 inhibitors, which may provide a novel chemotype of LSD1 inhibitors for gastric cancer treatment.
Assuntos
Antineoplásicos , Gliotoxina , Neoplasias Gástricas , Humanos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Neoplasias Gástricas/tratamento farmacológico , Gliotoxina/farmacologia , Gliotoxina/uso terapêutico , Relação Estrutura-Atividade , Linhagem Celular Tumoral , Proliferação de Células , Histona Desmetilases/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêuticoRESUMO
The Mycobacterium tuberculosis (MTB) infection causes tuberculosis (TB) and has been a long-standing public-health threat. It is urgent that we discover novel antitubercular agents to manage the increased incidence of multidrug-resistant (MDR) or extensively drug-resistant (XDR) strains of MTB and tackle the adverse effects of the first- and second-line antitubercular drugs. We previously found that gliotoxin (1), 12, 13-dihydroxy-fumitremorgin C (2), and helvolic acid (3) from the cultures of a deep-sea-derived fungus, Aspergillus sp. SCSIO Ind09F01, showed direct anti-TB effects. As macrophages represent the first line of the host defense system against a mycobacteria infection, here we showed that the gliotoxin exerted potent anti-tuberculosis effects in human THP-1-derived macrophages and mouse-macrophage-leukemia cell line RAW 264.7, using CFU assay and laser confocal scanning microscope analysis. Mechanistically, gliotoxin apparently increased the ratio of LC3-II/LC3-I and Atg5 expression, but did not influence macrophage polarization, IL-1ß, TNF-a, IL-10 production upon MTB infection, or ROS generation. Further study revealed that 3-MA could suppress gliotoxin-promoted autophagy and restore gliotoxin-inhibited MTB infection, indicating that gliotoxin-inhibited MTB infection can be treated through autophagy in macrophages. Therefore, we propose that marine fungi-derived gliotoxin holds the promise for the development of novel drugs for TB therapy.
Assuntos
Gliotoxina , Mycobacterium tuberculosis , Tuberculose , Animais , Camundongos , Humanos , Gliotoxina/farmacologia , Tuberculose/tratamento farmacológico , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Macrófagos , Fungos , AutofagiaRESUMO
The fungal pathogen Aspergillus fumigatus is frequently cultured from the sputum of cystic fibrosis (CF) patients along with the bacterium Pseudomonas aeruginosa. A. fumigatus secretes a range of secondary metabolites, and one of these, gliotoxin, has inhibitory effects on the host immune response. The effect of P. aeruginosa culture filtrate (CuF) on fungal growth and gliotoxin production was investigated. Exposure of A. fumigatus hyphae to P. aeruginosa cells induced increased production of gliotoxin and a decrease in fungal growth. In contrast, exposure of A. fumigatus hyphae to P. aeruginosa CuF led to increased growth and decreased gliotoxin production. Quantitative proteomic analysis was used to characterize the proteomic response of A. fumigatus upon exposure to P. aeruginosa CuF. Changes in the profile of proteins involved in secondary metabolite biosynthesis (e.g. gliotoxin, fumagillin, pseurotin A), and changes to the abundance of proteins involved in oxidative stress (e.g. formate dehydrogenase) and detoxification (e.g. thioredoxin reductase) were observed, indicating that the bacterial secretome had a profound effect on the fungal proteome. Alterations in the abundance of proteins involved in detoxification and oxidative stress highlight the ability of A. fumigatus to differentially regulate protein synthesis in response to environmental stresses imposed by competitors such as P. aeruginosa. Such responses may ultimately have serious detrimental effects on the host.
Assuntos
Aspergillus fumigatus , Pseudomonas aeruginosa , Humanos , Proteoma/metabolismo , Proteômica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , SecretomaRESUMO
Cryptic links between apparently unrelated metabolic systems represent potential new drug targets in fungi. Evidence of such a link between zinc and gliotoxin (GT) biosynthesis in Aspergillus fumigatus is emerging. Expression of some genes of the GT biosynthetic gene cluster gli is influenced by the zinc-dependent transcription activator ZafA, zinc may relieve GT-mediated fungal growth inhibition and, surprisingly, GT biosynthesis is influenced by zinc availability. In A. fumigatus, dithiol gliotoxin (DTG), which has zinc-chelating properties, is converted to either GT or bis-dethiobis(methylthio)gliotoxin (BmGT) by oxidoreductase GliT and methyltransferase GtmA, respectively. A double deletion mutant lacking both GliT and GtmA was previously observed to be hypersensitive to exogenous GT exposure. Here we show that compared to wild-type exposure, exogenous GT and the zinc chelator N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) inhibit A. fumigatus ΔgliTΔgtmA growth, specifically under zinc-limiting conditions, which can be reversed by zinc addition. While GT biosynthesis is evident in zinc-depleted medium, addition of zinc (1 µM) suppressed GT and activated BmGT production. In addition, secretion of the unferrated siderophore, triacetylfusarinine C (TAFC), was evident by A. fumigatus wild-type (at >5 µM zinc) and ΔgtmA (at >1 µM zinc) in a low-iron medium. TAFC secretion suggests that differential zinc-sensing between both strains may influence fungal Fe3+ requirement. Label-free quantitative proteomic analysis of both strains under equivalent differential zinc conditions revealed protein abundance alterations in accordance with altered metabolomic observations, in addition to increased GliT abundance in ΔgtmA at 5 µM zinc, compared to wild-type, supporting a zinc-sensing deficiency in the mutant strain. The relative abundance of a range of oxidoreductase- and secondary metabolism-related enzymes was also evident in a zinc- and strain-dependent manner. Overall, we elaborate new linkages between zinc availability, natural product biosynthesis and oxidative stress homeostasis in A. fumigatus.
Assuntos
Gliotoxina , Aspergillus fumigatus , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Gliotoxina/metabolismo , Gliotoxina/farmacologia , Proteômica , Zinco/metabolismoRESUMO
INTRODUCTION: Antifungal agents are essential in the fight against serious fungal disease, however emerging resistance is threatening an already limited collection of therapeutics. Proteomic analyses of effects of antifungal agents can expand our understanding of multifactorial mechanisms of action and have also proven valuable to elucidate proteomic changes associated with antifungal resistance. AREAS COVERED: This review covers the application of proteomic techniques to examine sensitivity and resistance to antifungals including commonly used therapeutics, amphotericin B, echinocandins and the azoles, based predominantly on studies involving Aspergillus fumigatus, Candida albicans and Candida glabrata from the last 10 years. In addition, non-clinical antimicrobial agents are also discussed, which highlight the potential of proteomics to identify new antifungal targets. EXPERT COMMENTARY: Fungal proteomics has evolved in the last decade with increased genome availability and developments in mass spectrometry. Collectively, these have led to the advancement of proteomic techniques, allowing increased coverage of the proteome. Gel-based proteomics laid the foundation for these types of studies, which has now shifted to the more powerful gel-free proteomics. This has resulted in the identification of key mediators and potential biomarkers of antifungal resistance, as well as elucidating the mechanisms of action of novel and established antifungal agents.
Assuntos
Antifúngicos , Proteoma , Antifúngicos/farmacologia , Equinocandinas , Humanos , Testes de Sensibilidade Microbiana , ProteômicaRESUMO
Sclerotium rolfsii causes destructive soilborne disease in numerous plant species, and biological control may be a promising and sustainable approach for suppressing this widespread pathogen. In this study, the antagonistic effect against S. rolfsii of 10 Trichoderma strains was tested by the dual culture method, and a gliotoxin-producing strain, T. virens T23, was shown to be the most effective, inhibiting growth of S. rolfsii in vitro by 70.2%. To clarify the antagonistic mechanism and gliotoxin biosynthesis regulation of T23, a gliotoxin-deficient mutant was constructed via Agrobacterium tumefaciens-mediated gene knockout in vivo. As expected, disruption of the gene located in the putative gliotoxin biosynthesis gene cluster, gliI-T, resulted in gliotoxin deficiency and attenuation of the antagonistic effect against S. rolfsii, indicating that gliotoxin biosynthesis is regulated by gliI-T and that gliotoxin is an important antifungal metabolite of T23. Transmission electron microscopy revealed that gliotoxin treatment caused marked alterations of the hyphal cells of S. rolfsii depending on the drug concentration, whereby one of the prominent structural alterations was a reduction in the number and length of mitochondrial cristae. When S. rolfsii was exposed to 30 µg/ml of gliotoxin for 12 h, striking plasmolysis and ultrastructural changes were induced. The results demonstrated that gliotoxin is an important secondary metabolite of T. virens T23 in its antagonism against S. rolfsii.
Assuntos
Gliotoxina , Hypocrea , Trichoderma , Basidiomycota , Doenças das PlantasRESUMO
The APSES family proteins are transcription factors (TFs) with a basic helix-loop-helix domain, known to regulate growth, development, secondary metabolism, and other biological processes in Aspergillus species. In the genome of the human opportunistic pathogenic fungus Aspergillus fumigatus, five genes predicted to encode APSES TFs are present. Here, we report the characterization of one of these genes, called mbsA (Afu7g05620). The deletion (Δ) of mbsA resulted in significantly decreased hyphal growth and asexual sporulation (conidiation), and lowered mRNA levels of the key conidiation genes abaA, brlA, and wetA. Moreover, ΔmbsA resulted in reduced spore germination rates, elevated sensitivity toward Nikkomycin Z, and significantly lowered transcripts levels of genes associated with chitin synthesis. The mbsA deletion also resulted in significantly reduced levels of proteins and transcripts of genes associated with the SakA MAP kinase pathway. Importantly, the cell wall hydrophobicity and architecture of the ΔmbsA asexual spores (conidia) were altered, notably lacking the rodlet layer on the surface of the ΔmbsA conidium. Comparative transcriptomic analyses revealed that the ΔmbsA mutant showed higher mRNA levels of gliotoxin (GT) biosynthetic genes, which was corroborated by elevated levels of GT production in the mutant. While the ΔmbsA mutant produced higher amount of GT, ΔmbsA strains showed reduced virulence in the murine model, likely due to the defective spore integrity. In summary, the putative APSES TF MbsA plays a multiple role in governing growth, development, spore wall architecture, GT production, and virulence, which may be associated with the attenuated SakA signaling pathway.
Assuntos
Aspergillus fumigatus/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Esporos Fúngicos/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Aspergillus fumigatus/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Esporos Fúngicos/genética , Fatores de Transcrição/genéticaRESUMO
Gliotoxin is a kind of epipolythiodioxopiperazine derived from different fungi that is characterized by a disulfide bridge. Gliotoxins can be biosynthesized by a gli gene cluster and regulated by a positive GliZ regulator. Gliotoxins show cytotoxic effects via the suppression the function of macrophage immune function, inflammation, antiangiogenesis, DNA damage by ROS production, peroxide damage by the inhibition of various enzymes, and apoptosis through different signal pathways. In the other hand, gliotoxins can also be beneficial with different doses. Low doses of gliotoxin can be used as an antioxidant, in the diagnosis and treatment of HIV, and as an anti-tumor agent in the future. Gliotoxins have also been used in the control of plant pathogens, including Pythium ultimum and Sclerotinia sclerotiorum. Thus, it is important to elucidate the toxic mechanism of gliotoxins. The toxic mechanism of gliotoxins and biosynthetic strategies to reduce the toxicity of gliotoxins and their producing strains are summarized in this review.
Assuntos
Gliotoxina/biossíntese , Gliotoxina/toxicidade , Animais , Ascomicetos/efeitos dos fármacos , Humanos , Família Multigênica/genética , Pythium/efeitos dos fármacosRESUMO
Growing evidence demonstrates that human mesenchymal stromal cells (MSCs) modify their in vivo anti-inflammatory actions depending on the specific inflammatory environment encountered. Understanding this better is crucial to refine MSC-based cell therapies for lung and other diseases. Using acute exacerbations of cystic fibrosis (CF) lung disease as a model, the effects of ex vivo MSC exposure to clinical bronchoalveolar lavage fluid (BALF) samples, as a surrogate for the in vivo clinical lung environment, on MSC viability, gene expression, secreted cytokines, and mitochondrial function were compared with effects of BALF collected from healthy volunteers. CF BALF samples that cultured positive for Aspergillus sp. (Asp) induced rapid MSC death, usually within several hours of exposure. Further analyses suggested the fungal toxin gliotoxin as a potential mediator contributing to CF BALF-induced MSC death. RNA sequencing analyses of MSCs exposed to either Asp+ or Asp- CF BALF samples identified a number of differentially expressed transcripts, including those involved in interferon signaling, antimicrobial gene expression, and cell death. Toxicity did not correlate with bacterial lung infections. These results suggest that the potential use of MSC-based cell therapies for CF or other lung diseases may not be warranted in the presence of Aspergillus.
Assuntos
Anti-Inflamatórios/uso terapêutico , Fibrose Cística/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Líquido da Lavagem Broncoalveolar/microbiologia , Fibrose Cística/metabolismo , Humanos , Pulmão/metabolismo , Pulmão/microbiologia , Transplante de Células-Tronco Mesenquimais/métodosRESUMO
Pyruvate kinase M2 (PKM2) functions as an important rate-limiting enzyme of aerobic glycolysis that is involved in tumor initiation and progression. However, there are few studies on effective PKM2 inhibitors. Gliotoxin is a marine-derived fungal secondary metabolite with multiple biological activities, including immunosuppression, cytotoxicity, and et al. In this study, we found that Gliotoxin directly bound to PKM2 and inhibited its glycolytic activity in a dose-dependent manner accompanied by the decreases in glucose consumption and lactate production in the human glioma cell line U87. Moreover, Gliotoxin suppressed tyrosine kinase activity of PKM2, leading to a dramatic reduction in Stat3 phosphorylation in U87 cells. Furthermore, Gliotoxin suppressed cell viability in U87 cells, and cytotoxicity of Gliotoxin on U87 cells was obviously augmented under hypoxia condition compared to normal condition. Finally, Gliotoxin was demonstrated to induce cell apoptosis of U87 cells and synergize with temozolomide. Our findings identify Gliotoxin as a new PKM2 inhibitor with anti-tumor activity, which lays the foundation for the development of Gliotoxin as a promising anti-tumor drug in the future.
Assuntos
Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Gliotoxina/isolamento & purificação , Gliotoxina/farmacologia , Piruvato Quinase/antagonistas & inibidores , Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Organismos Aquáticos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sistema Livre de Células , Sinergismo Farmacológico , Inibidores Enzimáticos/administração & dosagem , Fungos/química , Gliotoxina/administração & dosagem , Glicólise/efeitos dos fármacos , Humanos , Fosforilação , Temozolomida/administração & dosagemRESUMO
BACKGROUND: Invasive pulmonary aspergillosis (IPA) remains a life-threatening opportunistic infection, but can be difficult to diagnose. New biomarkers are therefore needed. Gliotoxin (GT), a secondary metabolite of Aspergillus fumigatus, and bis(methylthio)gliotoxin (bmGT), a degradation product of GT, have been proposed as potential biomarkers. However, these findings have yet to be confirmed. OBJECTIVES: To identify the diagnostic potential of GT and bmGT in serum and bronchoalveolar lavage fluid (BALf) in haematology patients compared to galactomannan (GM). MATERIALS AND METHODS: We prospectively collected culture supernatant, serum and BALf from patients with culture-positive IPA and measured GT and bmGT concentrations using ultra high-performance liquid chromatography-quadrupole time of flight mass spectrometry. Galactomannan was detected using a commercially available enzyme immunoassay. RESULTS: We included 18 patients with proven (n = 6) and probable (n = 12) IPA, all with positive cultures for Aspergillus fumigatus. BmGT was only detected in serum from one patient (5.6%), whereas GM was positive (optical density ≥ 0.5) in 11/18 patients (61.1%, P = 0.002). We could not find GT in any serum sample. In BALf, bmGT was detected in 8/18 patients (44.4%) and GT in 9/18 patients (50%), compared to GM (optical density ≥ 1.0) in all patients (100%). CONCLUSIONS: Gliotoxin and bis(methylthio)gliotoxin had a very poor performance for diagnosing IPA. As other biomarkers are more sensitive and easier to detect, we would not recommend serum or BALf GT/bmGT to be used in the diagnosis of IPA.
Assuntos
Biomarcadores/sangue , Testes Diagnósticos de Rotina/métodos , Gliotoxina/análogos & derivados , Gliotoxina/sangue , Aspergilose Pulmonar Invasiva/diagnóstico , Líquido da Lavagem Broncoalveolar/química , Galactose/análogos & derivados , Humanos , Mananas/sangue , Estudos Prospectivos , Soro/químicaRESUMO
Death-associated protein kinase 1 (DAPK1) expression induced by diverse death stimuli mediates apoptotic activity in various cancers, including ovarian cancer. In addition, mutual interaction between the tumor suppressor p53 and DAPK1 influences survival and death in several cancer cell lines. However, the exact role and connection of DAPK1 and p53 family proteins (p53, p63, and p73) in drug-resistant ovarian cancer cells have not been studied previously. In this study, we investigated whether DAPK1 induction by gliotoxin derived from marine fungus regulates the level of transcriptionally active p63 (TAp63) to promote apoptosis in an autophagy-dependent manner. Pre-exposure of paclitaxel-resistant ovarian cancer cells to gliotoxin inhibited the expression of multidrug resistant-associated proteins (MDR1 and MRP1-3), disrupted the mitochondrial membrane potential, and induced caspase-dependent apoptosis through autophagy induction after subsequent treatment with paclitaxel. Gene silencing of DAPK1 prevented TAp63-mediated downregulation of MDR1 and MRP1-3 and autophagic cell death after sequential treatment with gliotoxin and then paclitaxel. However, pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, had no effect on the levels of DAPK1 and TAp63 or on the inhibition of MDR1 and MRP1-3. These results suggest that DAPK1-mediated TAp63 upregulation is one of the critical pathways that induce apoptosis in chemoresistant cancer cells.
Assuntos
Morte Celular Autofágica/efeitos dos fármacos , Gliotoxina/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Linhagem Celular Tumoral , Proteínas Quinases Associadas com Morte Celular/genética , Proteínas Quinases Associadas com Morte Celular/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Gliotoxina/uso terapêutico , Humanos , Neoplasias Ovarianas/patologia , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
PURPOSE: The involvement of hepatic stellate cells (HSCs) with ischemia-reperfusion (I/R) injury in rat liver was examined using gliotoxin, which is known to induce HSC apoptosis. METHODS: Male Sprague-Dawley rats were used. HSC was represented by a glial fibrillary acidic protein (GFAP)-positive cell. Liver ischemia was produced by cross-clamping the hepatoduodenal ligament. The degree of I/R injury was evaluated by a release of aminotransferases. Sinusoidal diameter and sinusoidal perfusion rates were examined using intravital fluorescence microscopy. RESULTS: Gliotoxin significantly decreased the number of GFAP-positive cells 48 h after dosing (2.50 ± 0.19% [mean ± SD] in the nontreated group vs. 1.91 ± 0.46% in the gliotoxin-treated group). Liver damage was significantly suppressed by the pretreatment with gliotoxin. Sinusoidal diameters in zone 3 were wider in the gliotoxin group (10.25 ± 0.35 µm) than in the nontreated group (8.21 ± 0.50 µm). The sinusoidal perfusion rate was maintained as well in the gliotoxin group as in normal livers, even after I/R. CONCLUSIONS: Pretreatment with gliotoxin significantly reduced the number of HSCs in the liver and further suppressed liver injury following I/R. It is strongly suggested that HSCs play a functional role in exacerbating the degree of I/R injury of the liver.
Assuntos
Células Estreladas do Fígado/fisiologia , Fígado/irrigação sanguínea , Traumatismo por Reperfusão/etiologia , Animais , Proteína Glial Fibrilar Ácida/análise , Gliotoxina/farmacologia , Células Estreladas do Fígado/efeitos dos fármacos , Células de Kupffer/efeitos dos fármacos , Masculino , Microcirculação/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Zinc performs diverse physiological functions, and virtually all living organisms require zinc as an essential trace element. To identify the detailed function of zinc in fungal pathogenicity, we carried out cDNA microarray analysis using the model system of Aspergillus fumigatus, a fungal pathogen. From microarray analysis, we found that the genes involved in gliotoxin biosynthesis were upregulated when zinc was depleted, and the microarray data were confirmed by northern blot analysis. In particular, zinc deficiency upregulated the expression of GliZ, which encodes a Zn2-Cys6 binuclear transcription factor that regulates the expression of the genes required for gliotoxin biosynthesis. The production of gliotoxin was decreased in a manner inversely proportional to the zinc concentration, and the same result was investigated in the absence of ZafA, which is a zinc-dependent transcription activator. Interestingly, we found two conserved ZafA-binding motifs, 5'-CAAGGT-3', in the upstream region of GliZ on the genome and discovered that deletion of the ZafA-binding motifs resulted in loss of ZafA-binding activity; gliotoxin production was decreased dramatically, as demonstrated with a GliZ deletion mutant. Furthermore, mutation of the ZafA-binding motifs resulted in an increase in the conidial killing activity of human macrophage and neutrophil cells, and virulence was decreased in a murine model. Finally, transcriptomic analysis revealed that the expression of ZafA and GliZ was upregulated during phagocytosis by macrophages. Taken together, these results suggest that zinc plays an important role in the pathogenicity of A. fumigatus by regulating gliotoxin production during the phagocytosis pathway to overcome the host defense system.
Assuntos
Aspergillus fumigatus/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Gliotoxina/biossíntese , Zinco/metabolismo , Animais , Aspergillus fumigatus/genética , Aspergillus fumigatus/patogenicidade , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica , Humanos , Macrófagos , Neutrófilos , VirulênciaRESUMO
Cyclization of linear dipeptidyl precursors derived from nonribosomal peptide synthetases (NRPSs) into 2,5-diketopiperazines (DKPs) is a crucial step in the biosynthesis of a large number of bioactive natural products. However, the mechanism of DKP formation in fungi has remained unclear, despite extensive studies of their biosyntheses. Here we show that DKP formation en route to the fungal virulence factor gliotoxin requires a seemingly extraneous couplet of condensation (C) and thiolation (T) domains in the NRPS GliP. In vivo truncation of GliP to remove the CT couplet or just the T domain abrogated production of gliotoxin and all other gli pathway metabolites. Point mutation of conserved active sites in the C and T domains diminished cyclization activity of GliP in vitro and abolished gliotoxin biosynthesis in vivo. Verified NRPSs of other fungal DKPs terminate with similar CT domain couplets, suggesting a conserved strategy for DKP biosynthesis by fungal NRPSs.
Assuntos
Aspergillus fumigatus/metabolismo , Dicetopiperazinas/metabolismo , Gliotoxina/biossíntese , Dicetopiperazinas/química , Regulação Fúngica da Expressão Gênica , Gliotoxina/química , Estrutura MolecularRESUMO
The destruction of pulmonary epithelium is a major feature of lung diseases caused by the fungal pathogen Aspergillus fumigatus (A. fumigatus). Gliotoxin, a major mycotoxin of A. fumigatus, is widely postulated to be associated with the tissue invasion. However, the mechanism is unclear. In this study, we first discovered that cofilin, a regulator of actin dynamics in the pulmonary epithelial cells, existed mainly in the form of oligomer, which kept it unable to depolymerize actin filaments. Gliotoxin could reduce the formation of cofilin oligomer and promote the release of active cofilin monomer by regulating cofilin phosphorylation balance. Then, the active cofilin induced the dissolution of actin stress fibers to result in the disruption of pulmonary epithelium barrier function. Collectively, our study revealed a novel mechanism of gliotoxin destructing lung epithelium barrier function and for the first time indicated the role of cofilin oligomer in this process.
Assuntos
Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Gliotoxina/toxicidade , Pulmão/efeitos dos fármacos , Fibras de Estresse/metabolismo , Células A549/efeitos dos fármacos , Animais , Aspergillus fumigatus/patogenicidade , Linhagem Celular/efeitos dos fármacos , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Humanos , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , SolubilidadeRESUMO
BACKGROUND: Invasive pulmonary aspergillosis (IPA) has been increasingly reported in patients with underlying respiratory diseases (URD). Early diagnosis of IPA is crucial for mortality reduction and improved prognosis, yet remains difficult. Existing diagnostic tools for IPA largely rely on the detection of biomarkers based on serum or bronchoalveolar lavage fluid (BALF), both of which have their limitations. The use of sputum sample is non-invasive, and Aspergillus detection is feasible; however, the usefulness of sputum biomarkers for the diagnosis of IPA, especially in patients with URD, has not been systematically studied. METHODS: This is a prospective diagnostic trial. At least 118 participants will be recruited from respiratory wards and intensive care units. IPA is defined according to the EORTC/MSG criteria modified for patients with URD. Induced sputum and blood will be collected, and BALF will be obtained by bronchoscopy. Sputum biomarkers, including galactomannan, Aspergillus DNA, triacetylfusarinine and bis(methylthio)gliotoxin will be determined, and the presence of a JF5 antigen will be examined with a lateral fluid device. The sensitivity, specificity, negative predictive value, positive predictive value and diagnostic odds ratio will be computed for different biomarkers and compared using the McNemar χ2 test. Receiver operating characteristic analyses will be performed, and the cut-off values will be established. Participants will receive follow-up evaluations at 3 months and 6 months after recruitment. The difference in hospital stay and survival will be analysed, and the relationships between the levels of biomarkers and hospital stay and survival will be analysed via regression models. DISCUSSION: We have developed and verified the feasibility of Aspergillus-related biomarker assays for sputum. The study findings will contribute to a novel look at the diagnostic performance of sputum biomarkers in IPA and provide important insight into the improvement of the early diagnosis of IPA, particularly in patients with URD. TRIAL REGISTRATION: This study has been registered with the Chinese Clinical Trial Registry ( ChiCTR-DPD-16009070 ) on 24th of August 2016.
Assuntos
Aspergilose Pulmonar Invasiva/diagnóstico , Transtornos Respiratórios/microbiologia , Escarro/microbiologia , Biomarcadores/análise , Líquido da Lavagem Broncoalveolar , Broncoscopia , Protocolos Clínicos , Diagnóstico Precoce , Galactose/análogos & derivados , Humanos , Unidades de Terapia Intensiva , Aspergilose Pulmonar Invasiva/complicações , Tempo de Internação , Mananas/análise , Estudos Prospectivos , Curva ROC , Transtornos Respiratórios/complicações , Sensibilidade e Especificidade , Análise de SobrevidaRESUMO
Acquired drug resistance constitutes an enormous hurdle in cancer treatment, and the search for effective compounds against resistant cancer is still advancing. Marine organisms are a promising natural resource for the discovery and development of anticancer agents. In this study, we examined whether gliotoxin (GTX), a secondary metabolite isolated from marine-derived Aspergillus fumigatus, inhibits the growth of adriamycin (ADR)-resistant non-small-cell lung cancer (NSCLC) cell lines A549/ADR. We investigated the effects of GTX on A549/ADR cell viability with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the induction of apoptosis in A549/ADR cells treated with GTX via fluorescence-activated cell sorting analysis, Hoechst staining, annexin V/propidium iodide staining, tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining, and western blotting. We found that GTX induced apoptosis in A549/ADR cells through the mitochondria-dependent pathway by disrupting mitochondrial membrane potential and activating p53, thereby increasing the expression levels of p21, p53 upregulated modulator of apoptosis (PUMA), Bax, cleaved poly (ADP-ribose) polymerase (PARP), and cleaved caspase-9. More importantly, we discovered that GTX works in conjunction with ADR to exert combinational effects on A549/ADR cells. In conclusion, our results suggest that GTX may have promising effects on ADR-resistant NSCLC cells by inducing mitochondria-dependent apoptosis and through the combined effects of sequential treatment with ADR.