Viability Assay of Trichoderma stromaticum Conidia Inside Human Peripheral Blood Mononuclear-Derived Macrophages.

Macrophages represent a crucial line of defense and are responsible for preventing the growth and colonization of pathogens in different tissues. Conidial phagocytosis is a key process that allows for the investigation of the cytoplasmic and molecular events involved in macrophage-pathogen interactions, as well as for the determination of the time of death of internalized conidia. The technique involving the phagocytosis of fungal conidia by macrophages is widely used for studies evaluating the modulation of the immune responses against fungi. The evasion of phagocytosis and escape of phagosomes are mechanisms of fungal virulence. Here, we report the methods that can be used for the analysis of the phagocytosis, clearance, and viability of T. stromaticum conidia, a fungus which is used as a biocontrol and biofertilizer agent and is capable of inducing human infections. The protocol consists of 1) Trichoderma culture, 2) washing to obtain conidia, 3) the isolation of peripheral blood mononuclear cells (PBMCs) using the polysucrose solution method and the differentiation of the PBMCs into macrophages, 4) an in vitro phagocytosis method using round glass coverslips and coloration, and 5) a clearance assay to assess the conidia viability after conidia phagocytosis. In summary, these techniques can be used to measure the fungal clearance efficiency of macrophages.

Protease inhibitors from Theobroma cacao impair SARS-CoV-2 replication in vitro.

SARS-CoV-2 is a newly emerging virus from the Coronaviridae family that has already infected over 700 million people worldwide and killed over 6 million. This virus uses protease molecules to replicate and infect the host, which makes these molecules targets for therapeutic substances to eliminate the virus and treat infected people. Through the protein-protein molecular docking approach, we detected two cystatins from Theobroma cacao, TcCYS3 and TcCYS4, described as papain-like protease inhibitors. These inhibitors decreased SARS-CoV-2 genomic copies without toxicity to Vero cells. There is a need to perform comprehensive studies in relevant animal models and to investigate the action mechanisms of protease inhibitors from Theobroma cacao that control the replication of SARS-CoV-2 in human cells.

Trichoderma after crossing kingdoms: infections in human populations.

Trichoderma is a saprophytic fungus that is used worldwide as a biocontrol and biofertilizer agent. Although considered nonpathogenic until recently, reports of human infections produced by members of the Trichoderma genus are increasing. Numerous sources of infection were proposed based upon patient data and phylogenetic analysis, including air, agriculture, and healthcare facilities, but the deficit of knowledge concerning Trichoderma infections makes patient treatment difficult. These issues are compounded by isolates that present profiles which exhibit high minimum inhibitory concentration values to available antifungal drugs. The aim of this review is to present the global distribution and sources of infections that affect both immunocompetent and immunocompromised hosts, clinical features, therapeutic strategies that are used to treat patients, as well as highlighting treatments with the best responses. In addition, the antifungal susceptibility profiles of Trichoderma isolates that have emerged in recent decades were examined and which antifungal drugs need to be further evaluated as potential candidates to treat Trichoderma infections are also indicated.

The ethanolic extract of the fungus Trichoderma stromaticum decreases the Toxoplasma gondii replication in vitro and ameliorates the experimental toxoplasmosis in vivo.

Trichoderma are fungi that are well-known to inhibit the growth of a variety of plant pathogens. Currently, there is an increasing search for new drugs to treat toxoplasmosis. The aims of this study were to investigate the effect of ExtTs in the control of Toxoplasma gondii proliferation in vitro and the course of toxoplasmosis in a mouse model. Firstly, the cytotoxicity of the ExtTs was evaluated by cultivating macrophages with different concentrations of the extract and cell viability was assessed by the MTT assay. Next, the infectivity of the T. gondii treated with extract was analyzed by infecting J774 macrophages. To evaluate the effect of the ExtTs in vivo, C57BL/6 mice were infected orally with T. gondii, ME-49, treated daily with ExtTs, and clinical, biochemical and histological changes were monitored. It was demonstrated that the extract did not affect the host cellular viability and, the treatment of parasites with ExtTs altered their morphology and decreased their ability to proliferate inside macrophages. Additionally, the treatment of mice with ExtTs decreased the parasitism and inflammation in the small intestine and liver of infected mice in parallel with increased IL-10/TNF ratio systemically and prevented alterations to serum VLDL and triglyceride levels. Thus, ExtTs could be considered an alternative/complementary therapy to control toxoplasmosis.

Trichoderma stromaticum spores induce autophagy and downregulate inflammatory mediators in human peripheral blood-derived macrophages.

Trichoderma spp. are usually considered safe and normally used as biocontrol and biofertilization. Safety for human health is evaluated by several tests that detect various effects such as allergenicity, toxicity, infectivity, and pathogenicity. However, they do not evaluate the effects of the agent upon the immune system. The aim of this study was to investigate the interaction between T. stromaticum spores and mammalian cells to assess the immunomodulatory potential of the spores of this fungus. First, mouse macrophage cell line J774 and human macrophages were exposed to fungal spores and analyzed for structural features, through scanning and transmission electron microscopy. Then, various analysis were performed in human macrophages as to their effect in some functional and molecular aspects of the immune system through immunocytochemistry, flow cytometry and gene expression assays. We demonstrated that T. stromaticum spores induces autophagy and autophagy-related genes (ATGs) and downmodulate inflammatory mediators, including ROS, NLRP3, the cytokines IL-1ß, IL-18, IL-12 and IL-10, as well as TLR2, TLR4, miR-146b and miR-155, which may lead to an augmented susceptibility to pathogens. Our study shows the extension of damages the biofungicide Tricovab® can cause in the innate immune response. Further studies are necessary to elucidate other innate and adaptive immune responses and, consequently, the safety of this fungus when in contact with humans.

Antimicrobial activity and partial chemical structure of acylpolyamines isolated from the venom of the spider Acanthoscurria natalensis.

Background: Acylpolyamines are one of the main non-peptide compounds present in spider venom and represent a promising alternative in the search for new molecules with antimicrobial action. Methods: The venom of Acanthoscurria natalensis spider was fractionated by reverse-phase liquid chromatography (RP-HPLC) and the antimicrobial activity of the fractions was tested using a liquid growth inhibition assay. The main antimicrobial fraction containing acylpolyamines (ApAn) was submitted to two additional chromatographic steps and analyzed by MALDI-TOF. Fractions of interest were accumulated for ultraviolet (UV) spectroscopy and ESI-MS/MS analysis and for minimum inhibitory concentration (MIC) and hemolytic activity determination. Results: Five acylpolyamines were isolated from the venom with molecular masses between 614 Da and 756 Da, being named ApAn728, ApAn614a, ApAn614b, ApAn742 and ApAn756. The analysis of UV absorption profile of each ApAn and the fragmentation pattern obtained by ESI-MS/MS suggested the presence of a tyrosyl unit as chromophore and a terminal polyamine chain consistent with structural units PA43 or PA53. ApAn presented MIC between 128 µM and 256 µM against Escherichia coli and Staphylococcus aureus, without causing hemolysis against mouse erythrocytes. Conclusion: The antimicrobial and non-hemolytic properties of the analyzed ApAn may be relevant for their application as possible therapeutic agents and the identification of an unconventional chromophore for spider acylpolyamines suggests an even greater chemical diversity.

Antimicrobial activity and partial chemical structure of acylpolyamines isolated from the venom of the spider Acanthoscurria natalensis

Background: Acylpolyamines are one of the main non-peptide compounds present in spider venom and represent a promising alternative in the search for new molecules with antimicrobial action. Methods: The venom of Acanthoscurria natalensis spider was fractionated by reverse-phase liquid chromatography (RP-HPLC) and the antimicrobial activity of the fractions was tested using a liquid growth inhibition assay. The main antimicrobial fraction containing acylpolyamines (ApAn) was submitted to two additional chromatographic steps and analyzed by MALDI-TOF. Fractions of interest were accumulated for ultraviolet (UV) spectroscopy and ESI-MS/MS analysis and for minimum inhibitory concentration (MIC) and hemolytic activity determination. Results: Five acylpolyamines were isolated from the venom with molecular masses between 614 Da and 756 Da, being named ApAn728, ApAn614a, ApAn614b, ApAn742 and ApAn756. The analysis of UV absorption profile of each ApAn and the fragmentation pattern obtained by ESI-MS/MS suggested the presence of a tyrosyl unit as chromophore and a terminal polyamine chain consistent with structural units PA43 or PA53. ApAn presented MIC between 128 µM and 256 µM against Escherichia coli and Staphylococcus aureus, without causing hemolysis against mouse erythrocytes. Conclusion: The antimicrobial and non-hemolytic properties of the analyzed ApAn may be relevant for their application as possible therapeutic agents and the identification of an unconventional chromophore for spider acylpolyamines suggests an even greater chemical diversity.(AU)

Antimicrobial activity and partial chemical structure of acylpolyamines isolated from the venom of the spider Acanthoscurria natalensis

Abstract Background: Acylpolyamines are one of the main non-peptide compounds present in spider venom and represent a promising alternative in the search for new molecules with antimicrobial action. Methods: The venom of Acanthoscurria natalensis spider was fractionated by reverse-phase liquid chromatography (RP-HPLC) and the antimicrobial activity of the fractions was tested using a liquid growth inhibition assay. The main antimicrobial fraction containing acylpolyamines (ApAn) was submitted to two additional chromatographic steps and analyzed by MALDI-TOF. Fractions of interest were accumulated for ultraviolet (UV) spectroscopy and ESI-MS/MS analysis and for minimum inhibitory concentration (MIC) and hemolytic activity determination. Results: Five acylpolyamines were isolated from the venom with molecular masses between 614 Da and 756 Da, being named ApAn728, ApAn614a, ApAn614b, ApAn742 and ApAn756. The analysis of UV absorption profile of each ApAn and the fragmentation pattern obtained by ESI-MS/MS suggested the presence of a tyrosyl unit as chromophore and a terminal polyamine chain consistent with structural units PA43 or PA53. ApAn presented MIC between 128 µM and 256 µM against Escherichia coli and Staphylococcus aureus, without causing hemolysis against mouse erythrocytes. Conclusion: The antimicrobial and non-hemolytic properties of the analyzed ApAn may be relevant for their application as possible therapeutic agents and the identification of an unconventional chromophore for spider acylpolyamines suggests an even greater chemical diversity.

Toxoplasma gondii induces extracellular traps release in cat neutrophils.

Neutrophils respond differently to violations of the body's physiological barriers during infections. Extracellular traps comprise one of the mechanisms used by these cells to reduce the spread of pathogens to neighboring tissues, as well as ensure a high concentration of antimicrobial agents at the site of infection. To date, this innate defense mechanism has not been previously demonstrated in neutrophils of cats exposed to Toxoplasma gondii. The aim of this study was to characterize the in vitro release of neutrophil extracellular traps (NETs) when neutrophils isolated from cats were exposed to T. gondii. First, cellular viability was tested at different time points after parasite exposure. The production of reactive oxygen species (ROS) and lactate dehydrogenase and the amount of extracellular DNA were quantified. In addition, the number of parasites associated with neutrophils was determined, and the observed NETs formed were microscopically characterized. Results showed that (i) in culture, neutrophils isolated from cats presented diminished cellular viability after 4 h of incubation, and when neutrophils were incubated with T. gondii, they displayed cytotoxic effects after 3 h of interaction; (ii) neutrophils were able to release structures composed of DNA and histones, characterized as NETs under optical, immunofluorescence, and electron scanning microscopy, when stimulated with T. gondii; (iii) only 11.4% of neutrophils were able to discharge NETs during 3 h of incubation; however, it was observed through extracellular quantification of DNA that this small number of cells were able to display different behavior compared to a negative control (no parasite) group; (iv) significant differences in ROS production were observed in neutrophils exposed to T. gondii. In conclusion, our results showed that neutrophils isolated from cats exposed to T. gondii release structures composed of DNA and histones, similar to what has already been described in other neutrophil species infected with the parasite.

Ethanolic extract of the fungus Trichoderma stromaticum decreases inflammation and ameliorates experimental cerebral malaria in C57BL/6 mice.

Increased resistance to the first-line treatment against P. falciparum malaria, artemisinin-based combination therapies, has been reported. Here, we tested the effect of crude ethanolic extract of the fungus Trichoderma stromaticum (Ext-Ts) on the growth of P. falciparum NF54 in infected human red blood cells (ihRBCs) and its anti-malarial and anti-inflammatory properties in a mouse model of experimental cerebral malaria. For this purpose, ihRBCs were treated with Ext-Ts and analysed for parasitaemia; C57BL/6 mice were infected with P. berghei ANKA (PbA), treated daily with Ext-Ts, and clinical, biochemical, histological and immunological features of the disease were monitored. It was observed that Ext-Ts presented a dose-dependent ability to control P. falciparum in ihRBCs. In addition, it was demonstrated that Ext-Ts treatment of PbA-infected mice was able to increase survival, prevent neurological signs and decrease parasitaemia at the beginning of infection. These effects were associated with systemically decreased levels of lipids and IFN-γ, ICAM-1, VCAM-1 and CCR5 cerebral expression, preserving blood brain barrier integrity and attenuating the inflammatory lesions in the brain, liver and lungs. These results suggest that Ext-Ts could be a source of immunomodulatory and antimalarial compounds that could improve the treatment of cerebral malaria.

Toxoplasma gondii antigen SAG2A differentially modulates IL-1ß expression in resistant and susceptible murine peritoneal cells.

The cell surface of Toxoplasma gondii is covered by antigens (SAGs) from the SRS family anchored by glycosylphosphatidylinositol (GPI) and includes antigens from the SAG2 family. Among these, the SAG2A surface antigen shows great potential in activating humoral responses and has been used in characterizing the acute phase of infection and in the serological diagnosis of toxoplasmosis. In this study, we aimed to evaluate rSAG2A-induced proteins in BALB/c and C57BL/c mice macrophages and to evaluate the phenotypic polarization induced in the process. We treated the peritoneal macrophages from mouse strains that were resistant or susceptible to T. gondii with rSAG2A to analyze their proteomic profile by mass spectrometry and systems biology. We also examined the gene expression of these cells by RT-qPCR using the phenotypic markers of M1 and M2 macrophages. Differences were observed in the expression of proteins involved in the inflammatory process in both resistant and susceptible cells, and macrophages were preferentially induced to obtain a pro-inflammatory immune response (M1) via the overexpression of IL-1ß in mice susceptible to this parasite. These data suggest that the SAG2A antigen induces phenotypic and classical activation of macrophages in both resistant and susceptible strains of mice during the acute phase of the disease.

18-Des-hydroxy Cytochalasin: an antiparasitic compound of Diaporthe phaseolorum-92C, an endophytic fungus isolated from Combretum lanceolatum Pohl ex Eichler.

Chemical investigation of the ethyl acetate extract from the endophytic fungus Diaporthe phaseolorum-92C (92C) isolated from the roots of Combretum lanceolatum led to the isolation of 18-des-hydroxy Cytochalasin H (compound 1). The trypanocidal and schistosomicidal activity and cytotoxicity of the extract from 92C were evaluated. The schistosomicidal, leishmanicidal, antimicrobial, and antioxidant actions, as well as the antitumor activity against the breast cancer cells MDA-MB-231 and MCF-7, and the cytotoxicity towards normal human lung fibroblasts GM07492A of compound 1 was tested. The extract from 92C (20 µg/mL) exerted potent trypanocidal activity, reducing 82% of the number of amastigotes and trypomastigotes of Trypanosoma cruzi. Compound 1 at 50 µg/mL killed 50% of Schistosoma mansoni adult worms. Compound 1 reduced the viability of Leishmania amazonenses promastigotes (IC50 = 9.2 µg/mL) and of the cancer cells MDA-MB-231 and MCF-7 (IC50 = 17.5 and 8.88 µg/mL, respectively), presented moderate antioxidant activity, and gave IC50 of 2049.7 ± 39.9 µg/mL for the cytotoxicity towards normal cells GM07492A. This knowledge is highly relevant to the search for new promising compounds for therapeutic purposes.

Gene expression profiling of bone marrow mesenchymal stem cells from Osteogenesis Imperfecta patients during osteoblast differentiation.

Mesenchymal stem cells (MSCs) are precursors present in adult bone marrow that are able to differentiate into osteoblasts, adipocytes and chondroblasts that have gained great importance as a source for cell therapy. Recently, a number of studies involving the analysis of gene expression of undifferentiated MSCs and of MSCs in the differentiation into multiple lineage processes were observed but there is no information concerning the gene expression of MSCs from Osteogenesis Imperfecta (OI) patients. Osteogenesis Imperfecta is characterized as a genetic disorder in which a generalized osteopenia leads to excessive bone fragility and severe bone deformities. The aim of this study was to analyze gene expression profile during osteogenic differentiation from BMMSCs (Bone Marrow Mesenchymal Stem Cells) obtained from patients with Osteogenesis Imperfecta and from control subjects. Bone marrow samples were collected from three normal subjects and five patients with OI. Mononuclear cells were isolated for obtaining mesenchymal cells that had been expanded until osteogenic differentiation was induced. RNA was harvested at seven time points during the osteogenic differentiation period (D0, D+1, D+2, D+7, D+12, D+17 and D+21). Gene expression analysis was performed by the microarray technique and identified several differentially expressed genes. Some important genes for osteoblast differentiation had lower expression in OI patients, suggesting a smaller commitment of these patient's MSCs with the osteogenic lineage. Other genes also had their differential expression confirmed by RT-qPCR. An increase in the expression of genes related to adipocytes was observed, suggesting an increase of adipogenic differentiation at the expense osteogenic differentiation.

Osteoblastic differentiation of bone marrow mesenchymal stromal cells in Bruck Syndrome.

BACKGROUND: Osteogenesis Imperfecta (OI) (OMIM %259450) is a heterogeneous group of inherited disorders characterized by increased bone fragility, with clinical severity ranging from mild to lethal. The majority of OI cases are caused by mutations in COL1A1 or COL1A2. Bruck Syndrome (BS) is a further recessively-inherited OI-like phenotype in which bone fragility is associated with the unusual finding of pterygia and contractures of the large joints. Notably, several studies have failed to show any abnormalities in the biosynthesis of collagen 1 in BS patientes. Evidence was obtained for a specific defect of the procollagen telopeptide lysine hydroxylation in BS, whereas mutations in the gene PLOD2 have been identified. Recently, several studies described FKBP10 mutations in OI-like and BS patients, suggesting that FKBP10 is a bonafide BS locus. METHODS: We analyzed the coding region and intron/exon boundaries of COL1A1, COL1A2, PLOD2 and FKBP10 genes by sequence analysis using an ABI PRISM 3130 automated sequencer and Big Dye Terminator Sequencing protocol. Mononuclear cells obtained from the bone marrow of BS, OI patients and healthy donors were cultured and osteogenic differentiation was induced. The gene expression of osteoblast specific markers were also evaluated during the osteoblastic differentiation of mesenchymal stem cell (MSC) by qRT-PCR using an ABI7500 Sequence Detection System. RESULTS: No mutations in COL1A1, COL1A2 or PLOD2 were found in BS patient. We found a homozygous 1-base-pair duplication (c.831dupC) that is predicted to produce a translational frameshift mutation and a premature protein truncation 17 aminoacids downstream (p.Gly278ArgfsX95). The gene expression of osteoblast specific markers BGLAP, COL1A1, MSX2, SPARC and VDR was evaluated by Real Time RT-PCR during differentiation into osteoblasts and results showed similar patterns of osteoblast markers expression in BS and healthy controls. On the other hand, when compared with OI patients, the expression pattern of these genes was found to be different. CONCLUSIONS: Our work suggests that the gene expression profiles observed during mesenchymal stromal cell differentiation into osteoblast are distinct in BS patients as compared to OI patients. The present study shows for the first time that genes involved in osteogenesis are differentially expressed in BS and OI patients.

Evaluation of the allergenicity potential of TcPR-10 protein from Theobroma cacao.

BACKGROUND: The pathogenesis related protein PR10 (TcPR-10), obtained from the Theobroma cacao-Moniliophthora perniciosa interaction library, presents antifungal activity against M. perniciosa and acts in vitro as a ribonuclease. However, despite its biotechnological potential, the TcPR-10 has the P-loop motif similar to those of some allergenic proteins such as Bet v 1 (Betula verrucosa) and Pru av 1 (Prunus avium). The insertion of mutations in this motif can produce proteins with reduced allergenic power. The objective of the present work was to evaluate the allergenic potential of the wild type and mutant recombinant TcPR-10 using bioinformatics tools and immunological assays. METHODOLOGY/PRINCIPAL FINDINGS: Mutant substitutions (T10P, I30V, H45S) were inserted in the TcPR-10 gene by site-directed mutagenesis, cloned into pET28a and expressed in Escherichia coli BL21(DE3) cells. Changes in molecular surface caused by the mutant substitutions was evaluated by comparative protein modeling using the three-dimensional structure of the major cherry allergen, Pru av 1 as a template. The immunological assays were carried out in 8-12 week old female BALB/c mice. The mice were sensitized with the proteins (wild type and mutants) via subcutaneous and challenged intranasal for induction of allergic airway inflammation. CONCLUSIONS/SIGNIFICANCE: We showed that the wild TcPR-10 protein has allergenic potential, whereas the insertion of mutations produced proteins with reduced capacity of IgE production and cellular infiltration in the lungs. On the other hand, in vitro assays show that the TcPR-10 mutants still present antifungal and ribonuclease activity against M. perniciosa RNA. In conclusion, the mutant proteins present less allergenic potential than the wild TcPR-10, without the loss of interesting biotechnological properties.