Investigation of pectin deficiency in modulating the bioflavonoid profile of orange processing waste: A sustainable valorization of industrial waste.

Orange processing waste (OPW) generated by the processing of oranges, as well as other citrus fruits, is a major source of pectin in the market nowadays. The residues generated during the pectin extraction process may contain many phytochemicals, including flavonoids. We use state-of-the-art techniques such as liquid chromatography high-resolution mass spectrometry (LC-HRMS/MS) and feature-based molecular network (FBMN) to annotate the flavonoids in OPWs. In particular, four flavonoids, hesperidin, naringin, diosmin, and hesperetin were quantified in the samples by LC-TDQ-MS. In total, 32 flavonoids from different classes were annotated, of which 16 were polymethoxylated flavonoids, 13 were flavonoid glycosides and 3 were flavanone aglycones. The results showed that flavonoid glycosides remain in high concentrations in OPWs from pectin factories even after pectin extraction by harsh conditions. The results show an exciting opportunity to harness the untapped potential of pectin factory waste as a renewable source for the extraction of glycoside flavonoids.

Corrected and republished from: "Extracellular Vesicle Formation in Cryptococcus deuterogattii Impacts Fungal Virulence".

Small molecules are components of fungal extracellular vesicles (EVs), but their biological roles are only superficially known. NOP16 is a eukaryotic gene that is required for the activity of benzimidazoles against Cryptococcus deuterogattii. In this study, during the phenotypic characterization of C. deuterogattii mutants expected to lack NOP16 expression, we observed a reduced EV production. Whole-genome sequencing, RNA-Seq, and cellular proteomics revealed that, contrary to our initial findings, these mutants expressed Nop16 but exhibited altered expression of 14 genes potentially involved in sugar transport. Based on this observation, we designated these mutant strains as Past1 and Past2, representing potentially altered sugar transport. Analysis of the small molecule composition of EVs produced by wild-type cells and the Past1 and Past2 mutant strains revealed not only a reduced number of EVs but also an altered small molecule composition. In a Galleria mellonella model of infection, the Past1 and Past2 mutant strains were hypovirulent. The hypovirulent phenotype was reverted when EVs produced by wild-type cells, but not mutant EVs, were co-injected with the mutant cells in G. mellonella. These results connect EV biogenesis, cargo, and cryptococcal virulence.

Aspergillus fumigatus mitogen-activated protein kinase MpkA is involved in 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. The Mitogen-Activated Protein 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. The sensor histidine kinase SlnASln1 is important for modulation of MpkA phosphorylation. Our work emphasizes the importance of MpkA and compartmentalization of cellular events for GT production and self-defense.

Metabolomics approach to understand molecular mechanisms involved in fungal pathogen-citrus pathosystems.

Citrus is a crucial crop with a significant economic impact globally. However, postharvest decay caused by fungal pathogens poses a considerable threat, leading to substantial financial losses. Penicillium digitatum, Penicillium italicum, Geotrichum citri-aurantii and Phyllosticta citricarpa are the main fungal pathogens, causing green mold, blue mold, sour rot and citrus black spot diseases, respectively. The use of chemical fungicides as a control strategy in citrus raises concerns about food and environmental safety. Therefore, understanding the molecular basis of host-pathogen interactions is essential to find safer alternatives. This review highlights the potential of the metabolomics approach in the search for bioactive compounds involved in the pathogen-citrus interaction, and how the integration of metabolomics and genomics contributes to the understanding of secondary metabolites associated with fungal virulence and the fungal infection mechanisms. Our goal is to provide a pipeline combining metabolomics and genomics that can effectively guide researchers to perform studies aiming to contribute to the understanding of the fundamental chemical and biochemical aspects of pathogen-host interactions, in order to effectively develop new alternatives for fungal diseases in citrus cultivation. We intend to inspire the scientific community to question unexplored biological systems, and to employ diverse analytical approaches and metabolomics techniques to address outstanding questions about the non-studied pathosystems from a chemical biology perspective.

Analytical determination of tryptoquialanines A and B: Ensuring the quality and safety of orange juices.

Although orange juice is a popular beverage worldwide, fruit distribution, storage, and processing can facilitate fungal infection by Penicillium digitatum; leading to the production of tremorgenic alkaloids, specifically tryptoquialanines A (TA) and B (TB). An Analytical method was developed and validated based on QuEChERS and LC-MS/MS analysis to determine the levels of TA and TB in fresh, industrial, and homemade orange juices. Excellent linearity was observed in the method over a high range of 1-1000 µg/kg and low range of 1-75 µg/kg with R2 ≥ 0.998. The LOD and LOQ were 1 and 3 µg/kg, respectively. Recoveries showed values between 57 and 83 %, with RSD ≤ 13 %. Our data indicated a higher prevalence of mycotoxin TA in fresh and industrial orange juices. Reduction in TA and TB content after thermal and HPP treatments were ≤ 32 %. However, thermal treatment was more effective in reducing TA and TB contents.

Traversing the Cell Wall: The Chitinolytic Activity of Histoplasma capsulatum Extracellular Vesicles Facilitates Their Release.

Histoplasma capsulatum is the causative agent of histoplasmosis. Treating this fungal infection conventionally has significant limitations, prompting the search for alternative therapies. In this context, fungal extracellular vesicles (EVs) hold relevant potential as both therapeutic agents and targets for the treatment of fungal infections. To explore this further, we conducted a study using pharmacological inhibitors of chitinase (methylxanthines) to investigate their potential to reduce EV release and its subsequent impact on fungal virulence in an in vivo invertebrate model. Our findings revealed that a subinhibitory concentration of the methylxanthine, caffeine, effectively reduces EV release, leading to a modulation of H. capsulatum virulence. To the best of our knowledge, this is the first reported instance of a pharmacological inhibitor that reduces fungal EV release without any observed fungicidal effects.

Transcriptional profiling of a fungal granuloma reveals a low metabolic activity of Paracoccidioides brasiliensis yeasts and an actively regulated host immune response.

Granulomas are important immunological structures in the host defense against the fungus Paracoccidioides brasiliensis, the main etiologic agent of Paracoccidioidomycosis (PCM), a granulomatous systemic mycosis endemic in Latin America. We have performed transcriptional and proteomic studies of yeasts present in the pulmonary granulomas of PCM aiming to identify relevant genes and proteins that act under stressing conditions. C57BL/6 mice were infected with 1x106 yeasts and after 8- and 12-weeks of infection, granulomatous lesions were obtained for extraction of fungal and murine RNAs and fungal proteins. Dual transcriptional profiling was done comparing lung cells and P. brasiliensis yeasts from granulomas with uninfected lung cells and the original yeast suspension used in the infection, respectively. Mouse transcripts indicated a lung malfunction, with low expression of genes related to muscle contraction and organization. In addition, an increased expression of transcripts related to the activity of neutrophils, eosinophils, macrophages, lymphocytes as well as an elevated expression of IL-1ß, TNF-α, IFN-γ, IL-17 transcripts were observed. The increased expression of transcripts for CTLA-4, PD-1 and arginase-1, provided evidence of immune regulatory mechanisms within the granulomatous lesions. Also, our results indicate iron as a key element for the granuloma to function, where a high number of transcripts related to fungal siderophores for iron uptake was observed, a mechanism of fungal virulence not previously described in granulomas. Furthermore, transcriptomics and proteomics analyzes indicated a low fungal activity within the granuloma, as demonstrated by the decreased expression of genes and proteins related to energy metabolism and cell cycle.

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.

Unveiling Chemical Interactions Between Plants and Fungi Using Metabolomics Approaches.

Metabolomics has been extensively used in clinical studies in the search for new biomarkers of human diseases. However, this approach has also been highlighted in agriculture and biological sciences, once metabolomics studies have been assisting researchers to deduce new chemical mechanisms involved in biological interactions that occur between microorganisms and plants. In this sense, the knowledge of the biological role of each metabolite (virulence factors, signaling compounds, antimicrobial metabolites, among others) and the affected biochemical pathways during the interaction contribute to a better understand of different ecological relationships established in nature. The current chapter addresses five different applications of the metabolomics approach in fungal-plant interactions research: (1) Discovery of biomarkers in pathogen-host interactions, (2) plant diseases diagnosis, (3) chemotaxonomy, (4) plant defense, and (5) plant resistance; using mass spectrometry and/or nuclear magnetic resonance spectroscopy, which are the techniques most used in metabolomics.

Chemical-Biology and Metabolomics Studies in Phage-Host Interactions.

For many years, several studies have explored the molecular mechanisms involved in the infection of bacteria by their specific phages to understand the main infection strategies and the host defense strategies. The modulation of the mechanisms involved in the infection, as well as the expression of key substances in the development of the different life cycles of phages, function as a natural source of strategies capable of promoting the control of different pathogens that are harmful to human and animal health. Therefore, this chapter aims to provide an overview of the mechanisms involved in virus-bacteria interaction to explore the main compounds produced or altered as a chemical survival strategy and the metabolism modulation when occurring a host-phage interaction. In this context, emphasis will be given to the chemistry of peptides/proteins and enzymes encoded by bacteriophages in the control of pathogenic bacteria and the use of secondary metabolites recently reported as active participants in the mechanisms of phage-bacteria interaction. Finally, metabolomics strategies developed to gain new insights into the metabolism involved in the phage-host interaction and the metabolomics workflow in host-phage interaction will be presented.

Genome mining reveals secondary metabolites of Antarctic bacterium Streptomyces albidoflavus related to antimicrobial and antiproliferative activities.

The urgent need for new antimicrobials arises from antimicrobial resistance. Actinobacteria, especially Streptomyces genus, are responsible for production of numerous clinical antibiotics and anticancer agents. Genome mining reveals the biosynthetic gene clusters (BGCs) related to secondary metabolites and the genetic potential of a strain to produce natural products. However, this potential may not be expressed under laboratory conditions. In the present study, the Antarctic bacterium was taxonomically affiliated as Streptomyces albidoflavus ANT_B131 (CBMAI 1855). The crude extracts showed antimicrobial activity against both fungi, Gram-positive and Gram-negative bacteria and antiproliferative activity against five human tumor cell lines. Whole-genome sequencing reveals a genome size of 6.96 Mb, and the genome mining identified 24 BGCs, representing 13.3% of the genome. The use of three culture media and three extraction methods reveals the expression and recovery of 20.8% of the BGCs. The natural products identified included compounds, such as surugamide A, surugamide D, desferrioxamine B + Al, desferrioxamine E, and ectoine. This study reveals the potential of S. albidoflavus ANT_B131 as a natural product producer. Yet, the diversity of culture media and extraction methods could enhance the BGCs expression and recovery of natural products, and could be a strategy to intensify the BGC expression of natural products.

Metabolomic analysis reveals stress tolerance mechanisms in common bean (Phaseolus vulgaris L.) related to treatment with a biostimulant obtained from Corynebacterium glutamicum.

Microbial biostimulants have emerged as a sustainable alternative to increase the productivity and quality of important crops. Despite this, the effects of the treatment on plant metabolism are poorly understood. Thus, this study investigated the metabolic response of common bean (Phaseolus vulgaris) related to the treatment with a biostimulant obtained from the extract of Corynebacterium glutamicum that showed positive effects on the development, growth, and yield of crops previously. By untargeted metabolomic analysis using UHPLC-MS/MS, plants and seeds were subjected to treatment with the biostimulant. Under ideal growth conditions, the plants treated exhibited higher concentration levels of glutamic acid, nicotiflorin and glycosylated lipids derived from linolenic acid. The foliar application of the biostimulant under water stress conditions increased the chlorophyll content by 17% and induced the accumulation of flavonols, mainly quercetin derivatives. Also, germination seed assays exhibited longer radicle lengths for seeds treated compared to the untreated control even in the absence of light (13-18% increase, p-value <0.05). Metabolomic analysis of the seeds indicated changes in concentration levels of amino acids (tryptophan, phenylalanine, tyrosine, glutamine, and arginine) and their derivatives. The results point out the enhancement of abiotic stress tolerance and the metabolic processes triggered in this crop associated with the treatment with the biostimulant, giving the first insights into stress tolerance mechanisms in P. vulgaris.

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.

Metabolite Profiling of Pleurotus ostreatus Grown on Sisal Agro-Industrial Waste Supplemented with Cocoa Almond Tegument and Wheat Bran.

Pleurotus ostreatus is an edible fungus with high nutritional value that uses industrial and agricultural lignocellulosic residues as substrates for growth and reproduction. Understanding their growth metabolic dynamics on agro-industrial wastes would help to develop economically viable and eco-friendly biotechnological strategies for food production. Thus, we used UHPLC/MS/MS and GNPS as an innovative approach to investigate the chemical composition of two strains of P. ostreatus, coded as BH (Black Hirataki) and WH (White Hirataki), grown on sisal waste mixture (SW) supplemented with 20 % cocoa almond tegument (CAT) or 20 % of wheat bran (WB). Metabolite dereplication allowed the identification of 53 metabolites, which included glycerophospholipids, fatty acids, monoacylglycerols, steroids, carbohydrates, amino acids, and flavonoids. This is the first report of the identification of these compounds in P. ostreatus, except for the steroid ergosterol. Most of the metabolites described in this work possess potential biological activities, which support the nutraceutical properties of P. ostreatus. Thus, the results of this study provide essential leads to the understanding of white-rot fungi chemical plasticity aiming at developing alternative biotechnologies strategies for waste recycling.

Molecular networking as a tool to annotate the metabolites of Bacillus sp. and Serratia marcescens isolates and evaluate their fungicidal effects against Magnapothe oryzae and Bipolaris oryzae.

Rhizobacteria are valuable sources of compounds that can be used for the integrated management of diseases in rice. Here, we aimed to explore the metabolism and organize and annotate the metabolites of Bacillus sp. and Serratia marcescens isolates using molecular networking and evaluate their fungicidal effects against Magnaporthe oryzae and Bipolaris oryzae. We obtained bacterial extracts after 6 and 16-h incubation via liquid-liquid extraction using ethyl acetate as solvent. We performed UHPLC-MS analysis and data processing using molecular networking and conducted biological assays in rice plants. Using the Global Natural Product Social spectral libraries, we annotated the following compounds: austinoneol, Phe-Pro, N-acetyl-l-leucine, Leu-Gly, Ile-Leu, Phe-Pro, 2,5-piperazinedione, 3-(1H-indol-3-methyl)-6-methyl-cyclo(d-Trp-l-Pro), and cholic acid. Results of the biological assays showed that the bacterial extracts reduced the mycelial growth of both pathogens in all treatments compared to the control. In the greenhouse setup, 8 days after the challenge for leaf gray spot and leaf blast, all treatments affected up to 4.4% of the leaf area, with an area under disease progress curve of 13.24, showing significant difference compared to the control, which affected 23% of the leaf area, with an AUDPC of 44.65. Our study provides potential new sources of natural products to be applied in the integrated management of rice. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03547-6.

Biotransformation of white and black mustard grains through germination and enzymatic hydrolysis revealed important metabolites for antioxidant properties and cytotoxic activity against Caco-2 cells.

Germination and enzymatic hydrolysis are biological processes with well-recognized positive effects on phenolic composition and antioxidant potential. This study aimed to apply those processes to white (Sinapsis alba) and black (Brassica nigra) mustard grains and to analyze the influences on the total phenolic content (TPC); phenolic and peptide profile determined by ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS); antioxidant potential (DPPH, ABTS, and FRAP assays); and cytotoxicity against Caco-2, a human colorectal adenocarcinoma cell line. Enzyme combinations for hydrolysis were different for each mustard grain, but for both species, enzymatic hydrolysis and germination showed a positive effect on antioxidant properties. From UPLC-HRMS analysis and molecular network studies, 14 peptides and 17 phenolic compounds were identified as metabolites released from mustard after processes application, which were strongly correlated with increased antioxidant activity. In addition, enzymatic hydrolysis applied in germinated mustard grains for both mustards increased the cytotoxic activity against Caco-2 human colorectal adenocarcinoma cell line.

Musa spp. cultivars as a neutralising source against some toxic activities of Bothrops and Crotalus genus snake venoms.

Accidents involving snakes from Bothrops spp. and Crotalus spp. constitute the most important cause of envenomation in Brazil and Argentina. Musa spp. (banana) have been reported to be used in popular medicine against snakebite by the members of the Canudos Settlement, located in Goiás. In this way, the aim of this work was to evaluate the antivenom effect of the Ouro (AA), Prata (AAB), Prata-anã (AAB) and Figo (ABB) cultivars against in vitro (phospholipase, coagulation and proteolytic) and in vivo (lethality and toxicity) activities caused by the venoms and toxicity (Artemia salina nauplii and Danio rerio embryos) of Musa spp. as well as the annotation of chemical compounds possibly related to these activities. From the in vitro antiophidic tests with the sap, we observed 100% inhibition of the phospholipase and coagulant activities with the cultivars Prata-anã and Figo against the venoms of B. alternatus and C. d. collineatus, B. diporus and B. pauloensis, respectively, and neutralisation of the lethality against the B. diporus venom. It was observed that the cultivars of Musa spp. did not show toxicity against Artemia salina nauplii and Danio rerio embryos. The sap analysis via HPLC-MS/MS allowed the annotation of the 13 compounds: abscisic acid, shikimic acid, citric acid, quinic acid, afzelechin, Glp-hexose, glucose, sucrose, isorhamnetin-3-O-galactoside-6-raminoside, kaempferol-3-glucoside-3-raminoside, myricetin-3-O-rutinoside, procyanidin B1 and rutin. Therefore, it can be seen that Musa spp. is a potential therapeutic agent that can act to neutralise the effects caused by snakebites.

A Close Look into the Composition and Functions of Fungal Extracellular Vesicles Produced by Phytopathogens.

Fungal extracellular vesicles (EVs) were first described in human pathogens. In a few years, the field of fungal EVs evolved to include several studies with plant pathogens, in which extracellularly released vesicles play fundamental biological roles. In recent years, solid progress has been made in the determination of the composition of EVs produced by phytopathogens. In addition, EV biomarkers are now known in fungal plant pathogens, and the production of EVs during plant infection has been demonstrated. In this manuscript, we review the recent progress in the field of fungal EVs, with a focus on plant pathogens. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023.