Fungal Transformation and Schistosomicidal Effects of Pimaradienoic Acid

The schistosomicidal effects of pimaradienoic acid (PA) and two derivatives, obtained by fungal transformation in the presence of Aspergillus ochraceus, were investigated. PA was the only compound with antischistosomal activity among the three diterpenes studied, with the ability to significantly reduce the viability of the parasites at concentrations ranging from 25 to 100 mu M. PA also promoted morphological alterations of the tegument of Schistosoma mansoni, separated all the worm couples, and affected the production and development of eggs. Moreover, this compound was devoid of toxicity toward human fibroblasts. In a preliminary in vivo experiment, PA at a dose of 100 mg/kg significantly diminished the number of parasites in infected Balb/c mice. Taken together, these results show that PA may be potentially employed in the discovery of novel schistosomicidal agents, and that diterpenes are an important class of natural compounds for the investigation of agents capable of fighting the parasite responsible for human schistosomiasis.

5-Lipoxygenase Deficiency Impairs Innate and Adaptive Immune Responses during Fungal Infection

5-lipoxygenase-derived products have been implicated in both the inhibition and promotion of chronic infection. Here, we sought to investigate the roles of endogenous 5-lipoxygenase products and exogenous leukotrienes during Histoplasma capsulatum infection in vivo and in vitro. 5-LO deficiency led to increased lung CFU, decreased nitric oxide production and a deficient primary immune response during active fungal infection. Moreover, H. capsulatum-infected 5-LO-/- mice showed an intense influx of neutrophils and an impaired ability to generate and recruit effector T cells to the lung. The fungal susceptibility of 5-LO-/- mice correlated with a lower rate of macrophage ingestion of IgG-H. capsulatum relative to WT macrophages. Conversely, exogenous LTB4 and LTC4 restored macrophage phagocytosis in 5-LO deficient mice. Our results demonstrate that leukotrienes are required to control chronic fungal infection by amplifying both the innate and adaptive immune response during histoplasmosis.

The fungal metabolite eugenitin as additive for Aspergillus niveus glucoamylase activation

Endophytic microorganisms live inside tissues of host plants apparently do not causing warning to them, and area promising source of bioactive molecules as antimicrobial and antitumoral drugs. In this work, we report the isolation of eugenitin from cultures of the endophyte Mycoleptodiscus indicus and its potential as additive for Aspergillus niveus glucoamylase activation. The glucoamylase hydrolytic activity increased twofold using 5 mM of eugenitin and this activation could be explained by the binding mode of eugenitin with the three-dimensional structure of glucoamylase. The in silica prediction of ligand binding sites revealed at least 9 possible interaction sites able to accommodate eugenitin on glucoamylase from Hypocrea jecorina. Besides, we evaluated the effect of pH and temperature on activity and stability, as well as in the hydrolysis of different substrates and kinetic parameters either in presence or absence of eugenitin. The results displayed by eugenitin as additive to glucoamylase activation are promising and provide novel perspectives for applications of fungal metabolites. (C) 2011 Elsevier B.V. All rights reserved.

Prophylaxis of fungal infections in transplant patients

We thank the Clinical Directors from the Hospital das Clínicas da Faculdade de Medicina da USP for their support: Prof. Jose Otávio Costa Auler Junior, Prof. Tarcísio Eloi Pessoa de Barros Filho and Prof. Eloísa Bonfá

Aspergillus fumigatus mitochondrial electron transport chain mediates oxidative stress homeostasis, hypoxia responses and fungal pathogenesis

We previously observed that hypoxia is an important component of host microenvironments during pulmonary fungal infections. However, mechanisms of fungal growth in these in vivo hypoxic conditions are poorly understood. Here, we report that mitochondrial respiration is active in hypoxia (1% oxygen) and critical for fungal pathogenesis. We generated Aspergillus fumigatus alternative oxidase (aoxA) and cytochrome C (cycA) null mutants and assessed their ability to tolerate hypoxia, macrophage killing and virulence. In contrast to ?aoxA, ?cycA was found to be significantly impaired in conidia germination, growth in normoxia and hypoxia, and displayed attenuated virulence. Intriguingly, loss of cycA results in increased levels of AoxA activity, which results in increased resistance to oxidative stress, macrophage killing and long-term persistence in murine lungs. Thus, our results demonstrate a previously unidentified role for fungal mitochondrial respiration in the pathogenesis of aspergillosis, and lay the foundation for future research into its role in hypoxia signalling and adaptation.

Endo-xylanase GH11 activation by the fungal metabolite eugenitin

Eugenitin, a chromone derivative and a metabolite of the endophyte Mycoleptodiscus indicus, at 5 mM activated a recombinant GH11 endo-xylanase by 40 %. The in silico prediction of ligand-binding sites on the three-dimensional structure of the endo-xylanase revealed that eugenitin interacts mainly by a hydrogen bond with a serine residue and a stacking interaction of the heterocyclic aromatic ring system with a tryptophan residue. Eugenitin improved the GH11 endo-xylanase activity on different substrates, modified the optimal pH and temperature activities and slightly affected the kinetic parameters of the enzyme.

The COP9 signalosome counteracts the accumulation of cullin SCF ubiquitin E3 RING ligases during fungal development

Defects in the COP9 signalosome (CSN) impair multicellular development, including embryonic plant or animal death or a block in sexual development of the fungus Aspergillus nidulans. CSN deneddylates cullin-RING ligases (CRLs), which are activated by covalent linkage to ubiquitin-like NEDD8. Deneddylation allows CRL disassembly for subsequent reassembly. An attractive hypothesis is a consecutive order of CRLs for development, which demands repeated cycles of neddylation and deneddylation for reassembling CRLs. Interruption of these cycles could explain developmental blocks caused by csn mutations. This predicts an accumulation of neddylated CRLs exhibiting developmental functions when CSN is dysfunctional. We tested this hypothesis in A. nidulans, which tolerates reduced levels of neddylation for growth. We show that only genes for CRL subunits or neddylation are essential, whereas CSN is primarily required for development. We used functional tagged NEDD8, recruiting all three fungal cullins. Cullins are associated with the CSN1/CsnA subunit when deneddylation is defective. Two CRLs were identified which are specifically involved in differentiation and accumulate during the developmental block. This suggests that an active CSN complex is required to counteract the accumulation of specific CRLs during development.

Production of xylanase and beta-xylosidase from autohydrolysis liquor of corncob using two fungal strains

Agroindustrial residues are materials often rich in cellulose and hemicellulose. The use of these substrates for the microbial production of enzymes of industrial interest is mainly due to their high availability associated with their low cost. In this work, corncob (CCs) particles decomposed to soluble compounds (liquor) were incorporated in the microbial growth medium through autohydrolysis, as a strategy to increase and undervalue xylanase and beta-xylosidase production by Aspergillus terricola and Aspergillus ochraceus. The CCs autohydrolysis liquor produced at 200 A degrees C for 5, 15, 30 or 50 min was used as the sole carbon source or associated with untreated CC. The best condition for enzyme synthesis was observed with CCs submitted to 30 min of autohydrolysis. The enzymatic production with untreated CCs plus CC liquor was higher than with birchwood xylan for both microorganisms. A. terricola produced 750 total U of xylanase (144 h cultivation) and 30 total U of beta-xylosidase (96-168 h) with 0.75% untreated CCs and 6% CCs liquor, against 650 total U of xylanase and 2 total U of beta-xylosidase in xylan; A. ochraceus produced 605 total U of xylanase and 56 total U of beta-xylosidase (168 h cultivation) with 1% untreated CCs and 10% CCs liquor against 400 total U of xylanase and 38 total U of beta-xylosidase in xylan. These results indicate that the treatment of agroindustrial wastes through autohydrolysis can be a viable strategy in the production of high levels of xylanolytic enzymes.

Murine Dendritic Cells Transcriptional Modulation upon Paracoccidioides brasiliensis Infection

Limited information is available regarding the modulation of genes involved in the innate host response to Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis. Therefore, we sought to characterize, for the first time, the transcriptional profile of murine bone marrow-derived dendritic cells (DCs) at an early stage following their initial interaction with P. brasiliensis. DCs connect innate and adaptive immunity by recognizing invading pathogens and determining the type of effector T-cell that mediates an immune response. Gene expression profiles were analyzed using microarray and validated using real-time RT-PCR and protein secretion studies. A total of 299 genes were differentially expressed, many of which are involved in immunity, signal transduction, transcription and apoptosis. Genes encoding the cytokines IL-12 and TNF-alpha, along with the chemokines CCL22, CCL27 and CXCL10, were up-regulated, suggesting that P. brasiliensis induces a potent proinflammatory response in DCs. In contrast, pattern recognition receptor (PRR)-encoding genes, particularly those related to Toll-like receptors, were down-regulated or unchanged. This result prompted us to evaluate the expression profiles of dectin-1 and mannose receptor, two other important fungal PRRs that were not included in the microarray target cDNA sequences. Unlike the mannose receptor, the dectin-1 receptor gene was significantly induced, suggesting that this beta-glucan receptor participates in the recognition of P. brasiliensis. We also used a receptor inhibition assay to evaluate the roles of these receptors in coordinating the expression of several immune-related genes in DCs upon fungal exposure. Altogether, our results provide an initial characterization of early host responses to P. brasiliensis and a basis for better understanding the infectious process of this important neglected pathogen.

Pathogen-Induced Proapoptotic Phenotype and High CD95 (Fas) Expression Accompany a Suboptimal CD8(+) T-Cell Response: Reversal by Adenoviral Vaccine

MHC class la-restricted CD8(+) T cells are important mediators of the adaptive immune response against infections caused by intracellular microorganisms. Whereas antigen-specific effector CD8(+) T cells can clear infection caused by intracellular pathogens, in some circumstances, the immune response is suboptimal and the microorganisms survive, causing host death or chronic infection. Here, we explored the cellular and molecular mechanisms that could explain why CD8(+) T-cell-mediated immunity during infection with the human protozoan parasite Trypanosoma cruzi is not optimal. For that purpose, we compared the CD8(+) T-cell mediated immune responses in mice infected with T. cruzi or vaccinated with a recombinant adenovirus expressing an immunodominant parasite antigen. Several functional and phenotypic characteristics of specific CD8(+) T cells overlapped. Among few exceptions was an accelerated expansion of the immune response in adenoviral vaccinated mice when compared to infected ones. Also, there was an upregulated expression of the apoptotic-signaling receptor CD95 on the surface of specific T cells from infected mice, which was not observed in the case of adenoviral-vaccinated mice. Most importantly, adenoviral vaccine provided at the time of infection significantly reduced the upregulation of CD95 expression and the proapoptotic phenotype of pathogen-specific CD8(+) cells expanded during infection. In parallel, infected adenovirus-vaccinated mice had a stronger CD8(+) T-cell mediated immune response and survived an otherwise lethal infection. We concluded that a suboptimal CD8(+) T-cell response is associated with an upregulation of CD95 expression and a proapoptotic phenotype. Both can be blocked by adenoviral vaccination.

Biochemical and metabolic profiles of Trichoderma strains isolated from common bean crops in the Brazilian Cerrado, and potential antagonism against Sclerotinia sclerotiorum

Some species of Trichoderma have successfully been used in the commercial biological control of fungal pathogens, e.g., Sclerotinia sclerotiorum, an economically important pathogen of common beans (Phaseolus vulgaris L.). The objectives of the present study were (1) to provide molecular characterization of Trichoderma strains isolated from the Brazilian Cerrado; (2) to assess the metabolic profile of each strain by means of Biolog FF Microplates; and (3) to evaluate the ability of each strain to antagonize S. sclerotiorum via the production of cell wall-degrading enzymes (CWDEs), volatile antibiotics, and dual-culture tests. Among 21 isolates, we identified 42.86 % as Trichoderma asperellum, 33.33 % as Trichoderma harzianum, 14.29 % as Trichoderma tomentosum, 4.76 % as Trichoderma koningiopsis, and 4.76 % as Trichoderma erinaceum. Trichoderma asperellum showed the highest CWDE activity. However, no species secreted a specific group of CWDEs. Trichoderma asperellum 364/01, T. asperellum 483/02, and T. asperellum 356/02 exhibited high and medium specific activities for key enzymes in the mycoparasitic process, but a low capacity for antagonism. We observed no significant correlation between CWDE and antagonism, or between metabolic profile and antagonism. The diversity of Trichoderma species, and in particular of T. harzianum, was clearly reflected in their metabolic profiles. Our findings indicate that the selection of Trichoderma candidates for biological control should be based primarily on the environmental fitness of competitive isolates and the target pathogen. (C) 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Chiral HPLC analysis of donepezil, 5-O-desmethyl donepezil and 6-O-desmethyl donepezil in culture medium: application to fungal biotransformation studies

An high performance liquid chromatography (HPLC) method for the enantioselective determination of donepezil (DPZ), 5-O-desmethyl donepezil (5-ODD), and 6-O-desmethyl donepezil (6-ODD) in Czapek culture medium to be applied to biotransformation studies with fungi is described for the first time. The HPLC analysis was carried out using a Chiralpak AD-H column with hexane/ethanol/methanol (75:20:5, v/v/v) plus 0.3 % triethylamine as mobile phase and UV detection at 270 nm. Sample preparation was carried out by liquid-liquid extraction using ethyl acetate as extractor solvent. The method was linear over the concentration range of 100-10,000 ng mL(-1) for each enantiomer of DPZ (r a parts per thousand yenaEuro parts per thousand 0.9985) and of 100-5,000 ng mL(-1) for each enantiomer of 5-ODD (r a parts per thousand yenaEuro parts per thousand 0.9977) and 6-ODD (r a parts per thousand yenaEuro parts per thousand 0.9951). Within-day and between-day precision and accuracy evaluated by relative standard deviations and relative errors, respectively, were lower than 15 % for all analytes. The validated method was used to assess DPZ biotransformation by the fungi Beauveria bassiana American Type Culture Collection (ATCC) 7159 and Cunninghamella elegans ATCC 10028B. Using the fungus B. bassiana ATCC 7159, a predominant formation of (R)-5-ODD was observed while for the fungus C. elegans ATCC 10028B, DPZ was biotransformed to (R)-6-ODD with an enantiomeric excess of 100 %.

Endophytic fungi from Vitis labrusca L. ('Niagara Rosada') and its potential for the biological control of Fusarium oxysporum

We investigated the diversity of endophytic fungi found on grape (Vitis labrusca cv. Niagara Rosada) leaves collected from Salesopolis, SP, Brazil. The fungi were isolated and characterized by amplified ribosomal DNA restriction analysis, followed by sequencing of the ITS1-5.8S-ITS2 rDNA. In addition, the ability of these endophytic fungi to inhibit the grapevine pathogen Fusarium oxysporum f. sp herbemontis was determined in vitro. We also observed that the climatic factors, such as temperature and rainfall, have no effect on the frequency of infection by endophytic fungi. The endophytic fungal community that was identified included Aporospora terricola, Aureobasidium pullulans, Bjerkandera adusta, Colletotrichum boninense, C. gloeosporioides, Diaporthe helianthi, D. phaseolorum, Epicoccum nigrum, Flavodon flavus, Fusarium subglutinans, F. sacchari, Guignardia mangiferae, Lenzites elegans, Paraphaeosphaeria pilleata, Phanerochaete sordida, Phyllosticta sp, Pleurotus nebrodensis, Preussia africana, Tinctoporellus epiniltinus, and Xylaria berteri. Among these isolates, two, C. gloeosporioides and F. flavus, showed potential antagonistic activity against F. oxysporum f. sp herbemontis. We suggest the involvement of the fungal endophyte community of V. labrusca in protecting the host plant against pathogenic Fusarium species. Possibly, some endophytic isolates could be selected for the development of biological control agents for grape fungal disease; alternatively, management strategies could be tailored to increase these beneficial fungi.

“Features of two proteins of Leptospira interrogans with potential role in host-pathogen interactions”

Abstract Background Leptospirosis is considered a re-emerging infectious disease caused by pathogenic spirochaetes of the genus Leptospira. Pathogenic leptospires have the ability to survive and disseminate to multiple organs after penetrating the host. Leptospires were shown to express surface proteins that interact with the extracellular matrix (ECM) and to plasminogen (PLG). This study examined the interaction of two putative leptospiral proteins with laminin, collagen Type I, collagen Type IV, cellular fibronectin, plasma fibronectin, PLG, factor H and C4bp. Results We show that two leptospiral proteins encoded by LIC11834 and LIC12253 genes interact with laminin in a dose - dependent and saturable mode, with dissociation equilibrium constants (KD) of 367.5 and 415.4 nM, respectively. These proteins were named Lsa33 and Lsa25 (Leptospiral surface adhesin) for LIC11834 and LIC12253, respectively. Metaperiodate - treated laminin reduced Lsa25 - laminin interaction, suggesting that sugar moieties of this ligand participate in this interaction. The Lsa33 is also PLG - binding receptor, with a KD of 23.53 nM, capable of generating plasmin in the presence of an activator. Although in a weak manner, both proteins interact with C4bp, a regulator of complement classical route. In silico analysis together with proteinase K and immunoflorescence data suggest that these proteins might be surface exposed. Moreover, the recombinant proteins partially inhibited leptospiral adherence to immobilized laminin and PLG. Conclusions We believe that these multifunctional proteins have the potential to participate in the interaction of leptospires to hosts by mediating adhesion and by helping the bacteria to escape the immune system and to overcome tissue barriers. To our knowledge, Lsa33 is the first leptospiral protein described to date with the capability of binding laminin, PLG and C4bp in vitro.

Evaluating methods for the isolation of marine-derived fungal strains and production of bioactive secondary metabolites

In the present investigation we evaluate methods for the isolation and growth of marine-derived fungal strains in artificial media for the production of secondary metabolites. Inoculation of marine macroorganisms fragments in Petri dishes proved to be the most convenient procedure for the isolation of the largest number of strains. Among the growth media used, 3% malt extract showed the best result for strains isolation and growth, and yielded the largest number of strains from marine macroorganisms. The percentage of strains isolated using each of the growth media which yielded cytotoxic and/or antibiotic extracts was in the range of 23-35%, regardless of the growth media used. Further investigation of extracts obtained from different marine-derived fungal strains yielded several bioactive secondary metabolites, among which (E)-4-methoxy-5-(3-methoxybut-1-enyl)-6-methyl-2H-pyran-2-one is a new metabolite isolated from the Penicillium paxilli strain Ma(G)K.