Use of fungal inocula and pumice for decomposing coffee pulp

A study of decomposition of coffee pulp (from Guatemala City, Guatemala) was carried out with several inocula. Fresh and pressed pulps were studied in field and laboratory experiments. Results showed that the best inoculum among the eight examined in the experiments was the fungus Trichoderma viride, the only one not isolated from the coffee pulp itself. This inoculum, together with pumice, accelerated the decomposition of coffee pulp at ambient temperature during a 45-day test period. © 1985.

Laminin-binding epitope on gp43 from Paracoccidioides brasiliensis is recognized by a monoclonal antibody raised against Staphylococcus aureus laminin receptor

Adhesion is regarded as an important step in the pathogenesis of several microorganisms. Thus, the ability to recognize extracellular matrix proteins, such as laminin or fibronectin, has been correlated with invasiveness. Studying the already characterized laminin-binding protein of Paracoccidioides brasiliensis, the 43 kDa glycoprotein (gp43), we evaluated whether MAb 1.H12, raised against the laminin-binding protein from Staphylococcus aureus, cross-reacts with that fungal protein. By immunoblot analysis we show that MAb 1.H12 recognizes gp43. This interaction is able to inhibit the laminin-mediated adhesion to epithelial cells as well as the P. brasiliensis infection in vivo. Moreover, through immunoenzymatic assays, we show that MAb 1.H12 recognizes gp43 in solid phase and that this interaction is partially inhibited by the addition of anti-gp43 MAbs. These results show that MAb 1.H12 recognizes the gp43, suggesting the presence of an epitope similar to those found in the other laminin-binding proteins from phylogenetically very distant cells. These findings reinforce the possibility of evolutionary conservation of such epitopes.

Atherosclerosis morphology and pathogenesis

Atherosclerosis is a very common and important disease being the most important cause of mortality in Brazil. Indeed, in 1995, 23.3% of deaths, all ages, in our country, were the consequence of atherosclerosis. This percentage grows to 26.3% for S. Paulo and 32.7% for Rio Grande do Sul. Morphologically, there are 3 main types of lesions: fatty streaks, fibrous plaques, and complicated lesions. Fatty streaks are inocuous and occur early in life. In some persons, with age, they change into fibrous plaques that may lead to stenosis. They also may become complicated by erosion, calcification, hemorrhage and thrombosis. Atherosclerosis is initiated by endothelial functional alterations responsible for increase in permeability to macromolecules, adhesion, and migration of monocytes-macrophages and lymphocytes plus recruitment of platelets and smooth-muscle medial cells. Adhesion molecules, cytokines, growth factors, and free radicals are locally synthesized, favoring proliferation of extracellular matrix and progression of the lesion. Experimental, clinical, and epidemiological evidence point to the importance of lipids, mainly cholesterol-rich low-density lipoprotein (LDL), as one of the most important molecules involved in the genesis and progression of atherosclerosis. Patients with a genetic disorder of cholesterol metabolism (familial hyperlipidemia), caused by a decrease in the availability of receptors for LDL, develop severe atherosclerosis early in life. A series of other factors, such as age, diabetes melitus, diet, hypertension, lack of exercise, elevated hemocysteinemia, immunological disorders, and coagulation instability, are related to the progression of atherosclerosis. All of them are capable of altering the endothelium or increasing the offer of LDL. All the above-mentioned factors are systemic; but atherosclerosic lesions are focal, located at preferential sites such as the emergence of colaterals, bifurcations, and curvatures of arteries, all areas in which the laminar flow is disturbed. In these areas shear stress is diminished favoring the prolongation of permanence time of lipid particles, cells, cytokines, growth factors, etc., in the vicinity of the endothelium. Moreover, the endothelium has sensors that act as transducers of mechanical forces in biological responses. Experimental data demonstrate that the number and quality of adhesion molecules, cytokines, and growth factors synthetized, as well as the local production of radicals, and pro and anticoagulation factors may change with shear stress favoring or not the local establishment and progression of atherosclerotic lesions.

Specialized fungal parasites and opportunistic fungi in gardens of attine ants

Ants in the tribe Attini (Hymenoptera: Formicidae) comprise about 230 described species that share the same characteristic: all coevolved in an ancient mutualism with basidiomycetous fungi cultivated for food. In this paper we focused on fungi other than the mutualistic cultivar and their roles in the attine ant symbiosis. Specialized fungal parasites in the genus Escovopsis negatively impact the fungus gardens. Many fungal parasites may have small impacts on the ants' fungal colony when the colony is balanced, but then may opportunistically shift to having large impacts if the ants' colony becomes unbalanced. Copyright © 2012 Fernando C. Pagnocca et al.

Role of TLR-2 and fungal surface antigens on innate immune response against Sporothrix schenckii

Sporotrichosis is an infection caused by the dimorphic fungus Sporothrix schenckii. Toll-like receptors (TLRs) play an important role in immunity, since they bind to pathogen surface antigens and initiate the immune response. However, little is known about the role of TLR-2 and fungal surface antigens in the recognition of S. schenckii and in the subsequent immune response. This study aimed to evaluate the involvement of TLR-2 and fungal surface soluble (SolAg) and lipidic (LipAg) antigens in phagocytosis of S. schenckii and production of immune mediators by macrophages obtained from WT and TLR-2 -/- animals. The results showed that TLR-2-/- animals had had statistical lower percentage of macrophages with internalized yeasts compared to WT. SolAg and LipAg impaired phagocytosis and immunological mediator production for both WT and TLR-2-/-. The absence of TLR-2 led to lower production of the cytokines TNF, IL-1β, IL-12 and IL-10 compared to WT animals. These results suggest a new insight in relation to how the immune system, through TLR-2, recognizes and induces the production of mediators in response to the fungus S. schenckii. Copyright © Informa Healthcare USA, Inc.

Photodynamic and Antibiotic Therapy Impair the Pathogenesis of Enterococcus faecium in a Whole Animal Insect Model

Enterococcus faecium has emerged as one of the most important pathogens in healthcare-associated infections worldwide due to its intrinsic and acquired resistance to many antibiotics, including vancomycin. Antimicrobial photodynamic therapy (aPDT) is an alternative therapeutic platform that is currently under investigation for the control and treatment of infections. PDT is based on the use of photoactive dye molecules, widely known as photosensitizer (PS). PS, upon irradiation with visible light, produces reactive oxygen species that can destroy lipids and proteins causing cell death. We employed Galleria mellonella (the greater wax moth) caterpillar fatally infected with E. faecium to develop an invertebrate host model system that can be used to study the antimicrobial PDT (alone or combined with antibiotics). In the establishment of infection by E. faecium in G. mellonella, we found that the G. mellonella death rate was dependent on the number of bacterial cells injected into the insect hemocoel and all E. faecium strains tested were capable of infecting and killing G. mellonella. Antibiotic treatment with ampicillin, gentamicin or the combination of ampicillin and gentamicin prolonged caterpillar survival infected by E. faecium (P = 0.0003, P = 0.0001 and P = 0.0001, respectively). In the study of antimicrobial PDT, we verified that methylene blue (MB) injected into the insect followed by whole body illumination prolonged the caterpillar survival (P = 0.0192). Interestingly, combination therapy of larvae infected with vancomycin-resistant E. faecium, with antimicrobial PDT followed by vancomycin, significantly prolonged the survival of the caterpillars when compared to either antimicrobial PDT (P = 0.0095) or vancomycin treatment alone (P = 0.0025), suggesting that the aPDT made the vancomycin resistant E. faecium strain more susceptible to vancomycin action. In summary, G. mellonella provides an invertebrate model host to study the antimicrobial PDT and to explore combinatorial aPDT-based treatments.

Sulfonation and anticoagulant activity of fungal exocellular β-(1→6)-d-glucan (lasiodiplodan)

An exocellular β-(1→6)-d-glucan (lasiodiplodan) produced by a strain of Lasiodiplodia theobromae (MMLR) grown on sucrose was derivatized by sulfonation to promote anticoagulant activity. The structural features of the sulfonated β-(1→6)-d-glucan were investigated by UV-vis, FT-IR and 13C NMR spectroscopy, and the anticoagulant activity was investigated by the classical coagulation assays APTT, PT and TT using heparin as standard. The content of sulfur and degree of substitution of the sulfonated glucan was 11.73% and 0.95, respectively. UV spectroscopy showed a band at 261 nm due to the unsaturated bond formed in the sulfonation reaction. Results of FT-IR and 13C NMR indicated that sulfonyl groups were inserted on the polysaccharide. The sulfonated β-(1→6)-d-glucan presented anticoagulant activity as demonstrated by the increase in dose dependence of APTT and TT, and these actions most likely occurred because of the inserted sulfonate groups on the polysaccharide. The lasiodiplodan did not inhibit the coagulation tests. © 2012 Elsevier Ltd.

Increase nitric oxide and oxidative stress in dogs experimentally infected by Ehrlichia canis: Effect on the pathogenesis of the disease

The aim of this study was to evaluate nitric oxide levels, lipid peroxidation, protein oxidation and glutathione reductase activity in serum of dogs experimentally infected by Ehrlichia canis. Banked serum samples of dogs divided into two groups were used: negative control (n=5) and infected by E. canis (n=5). The concentration of nitrite/nitrate (NOx), lipid peroxidation (TBARS), advanced oxidation protein products (AOPP), and glutathione reductase (GR) activity in sera were evaluated. Samples were collected on days 0, 3, 6, 18 and 30 post-infection (PI). NOx and TBARS levels were significantly (P<0.05) higher in the infected group at 18 and 30 days PI, as well as AOPP levels at 30 days PI when compared to samples from control group. The GR activity was significant (P<0.05) increased in serum of dogs infected by E. canis on days 18 and 30 PI. Based on the increased levels of NOx, TBARS, AOPP and GR activity we concluded that dogs experimentally infected by E. canis develop a state of redox imbalance and that these changes might be involved in the pathophysiology of the disease. © 2013 Elsevier B.V.

Paracoccidoides brasiliensis 30 kDa Adhesin: Identification as a 14-3-3 Protein, Cloning and Subcellular Localization in Infection Models

Paracoccidoides brasiliensis adhesion to lung epithelial cells is considered an essential event for the establishment of infection and different proteins participate in this process. One of these proteins is a 30 kDa adhesin, pI 4.9 that was described as a laminin ligand in previous studies, and it was more highly expressed in more virulent P. brasiliensis isolates. This protein may contribute to the virulence of this important fungal pathogen. Using Edman degradation and mass spectrometry analysis, this 30 kDa adhesin was identified as a 14-3-3 protein. These proteins are a conserved group of small acidic proteins involved in a variety of processes in eukaryotic organisms. However, the exact function of these proteins in some processes remains unknown. Thus, the goal of the present study was to characterize the role of this protein during the interaction between the fungus and its host. To achieve this goal, we cloned, expressed the 14-3-3 protein in a heterologous system and determined its subcellular localization in in vitro and in vivo infection models. Immunocytochemical analysis revealed the ubiquitous distribution of this protein in the yeast form of P. brasiliensis, with some concentration in the cytoplasm. Additionally, this 14-3-3 protein was also present in P. brasiliensis cells at the sites of infection in C57BL/6 mice intratracheally infected with P. brasiliensis yeast cells for 72 h (acute infections) and 30 days (chronic infection). An apparent increase in the levels of the 14-3-3 protein in the cell wall of the fungus was also noted during the interaction between P. brasiliensis and A549 cells, suggesting that this protein may be involved in host-parasite interactions, since inhibition assays with the protein and this antibody decreased P. brasiliensis adhesion to A549 epithelial cells. Our data may lead to a better understanding of P. brasiliensis interactions with host tissues and paracoccidioidomycosis pathogenesis. © 2013 Silva et al.

Fungal communities in the garden chamber soils of leaf-cutting ants

Leaf-cutting ants modify the properties of the soil adjacent to their nests. Here, we examined whether such an ant-altered environment impacts the belowground fungal communities. Fungal diversity and community structure of soil from the fungus garden chambers of Atta sexdens rubropilosa and Atta bisphaerica, two widespread leaf-cutting ants in Brazil, were determined and compared with non-nest soils. Culture-dependent methods revealed similar species richness but different community compositions between both types of soils. Penicillium janthinellum and Trichoderma spirale were the prevalent isolates in fungus chamber soils and non-nest soils, respectively. In contrast to cultivation methods, analyses of clone libraries based on the internal transcribed spacer (ITS) region indicated that richness of operational taxonomic units significantly differed between soils of the fungus chamber and non-nest soils. FastUnifrac analyses based on ITS sequences further revealed a clear distinction in the community structure between both types of soils. Plectania milleri and an uncultured Clavariaceae fungus were prevalent in fungus chamber soils and non-nest soils, respectively. FastUnifrac analyses also revealed that fungal community structures of soil from the garden chambers markedly differed among ant species. Our findings suggest that leaf-cutting ants affect fungal communities in the soil from the fungus chamber in comparison to non-nest soils. © 2013 WILEY-VCH Verlag GmbH & Co.

Cryptococcosis: epidemiology, fungal resistance, and new alternatives for treatment

Cryptococcosis is an important systemic mycosis and the third most prevalent disease in human immunodeficiency virus (HIV)-positive individuals. The incidence of cryptococcosis is high among the 25 million people with HIV/acquired immunodeficiency syndrome (AIDS), with recent estimates indicating that there are one million cases of cryptococcal meningitis globally per year in AIDS patients. In Cryptococcus neoformans, resistance to azoles may be associated with alterations in the target enzyme encoded by the gene ERG11, lanosterol 14α-demethylase. These alterations are obtained through mutations, or by overexpressing the gene encoding. In addition, C. gattii and C. neoformans present a heteroresistance phenotype, which may be related to increased virulence. Other species beyond C. neoformans and C. gattii, such as C. laurentii, have been diagnosed mainly in patients with immunosuppression. Infections of C. albidus have been isolated in cats and marine mammals. Recent evidence suggests that the majority of infections produced by this pathogen are associated with biofilm growth, which is also related with increased resistance to antifungal agents. Therefore, there is a great need to search for alternative antifungal agents for these fungi. The search for new molecules is currently occurring from nanoparticle drugs of plant peptide origin. This article presents a brief review of the literature regarding the epidemiology of cryptococcosis, as well as fungal resistance and new alternatives for treatment. © 2013 Springer-Verlag Berlin Heidelberg.

Hydroxyurea therapy in sickle cell anemia patients aids to maintain oral fungal colonization balance

Background: The aim of this study was to evaluate the frequency of Candida species and presence of lesions in the oral cavity of patients with sickle cell anemia (SS). Methods: The study included 30 patients diagnosed with sickle cell anemia and taking hydroxyurea for at least 90 days (SS/HU+); and 39 patients with sickle cell anemia and without hydroxyurea therapy (SS/HU-). Two control groups were constituted by healthy individuals matched to the test groups in age, gender, and oral conditions (C/HU+ for SS/HU+ and C/HU- for SS/HU-). Oral clinical examination and anamnesis were performed. Yeasts were collected by oral rinses and identified by API system. Antifungal susceptibility evaluation was performed according to the CLSI methodology. Data obtained for microorganisms counts were compared by Student's t test (SS/HU+ vs. C/HU+ and SS/HU- vs. C/HU-) using MINITAB for Windows 1.4. Significance level was set at 5%. Results: No oral candidosis lesions were detected. Significant differences in yeasts counts were observed between SS/HU- group and the respective control, but there were no differences between SS/HU+ and C/HU+. Candida albicans was the most prevalent species in all groups. Candida famata was observed both in SS and control groups. Candida dubliniensis, Candida glabrata, Candida krusei, Candida tropicalis, Candida pelliculosa, and Candida parapsilosis were observed only in SS groups. Most strains were susceptible to all antifungal agents. Conclusion: Hydroxyurea therapy seems to decrease candidal counts and resistance rate in sickle cell anemia patients. However, further studies should be conducted in the future to confirm this finding. Hydroxyurea therapy in sickle cell anemia patients maintains fungal species balance in oral cavity. © 2013 John Wiley & Sons A/S.

Microbial culture collections as pillars for promoting fungal diversity, conservation and exploitation

Fungi are a diverse group of organisms with an overall global number of 1.5 M up to 3.3 M species on Earth. Besides their ecological roles as decomposers, fungi are important in several aspects of applied research. Here, we review how culture collections may promote the knowledge on diversity, conservation and biotechnological exploitation of fungi. The impact of fungi diversity on biotechnological studies is discussed. We point out the major roles of microbial repositories, including fungal preservation, prospecting, identification, authentication and supply. A survey on the World Data Center for Microorganisms (WDCM) powered by the World Federation for Culture Collections and on the Genetic Heritage Management Council (CGEN) database revealed that 46 Brazilian culture collections registered in these databases are dedicate to preserving fungi. Most of these culture collections are located in the Southeast of Brazil. This scenario also demonstrates that Brazil has many collections focused on fungal strains, but the lack of up-to-date information in WDCM as well as of a solid national platform for culture collections registration do not allow accurate assessment of fungal preservation. © 2013 Elsevier Inc. All rights reserved.

Molecular identification of fungal communities in a soil cultivated with vegetables and soil suppressiveness to Rhizoctonia solani

Fungi constitute an important part of the soil ecosystem, playing key roles in decomposition, cycling processes, and biotic interactions. Molecular methods have been used to assess fungal communities giving a more realistic view of their diversity. For this purpose, total DNA was extracted from bulk soils cultivated with tomato (STC), vegetables (SHC), and native forest (SMS) from three sites of the Taquara Branca river basin in Sumaré County, São Paulo State, Brazil. This metagenomic DNA was used as a template to amplify fungal 18S rDNA sequences, and libraries were constructed in Escherichia coli by cloning PCR products. The plasmid inserts were sequenced and compared to known rDNA sequences in the GenBank database. Of the sequenced clones, 22 were obtained from the SMS sample, 18 from the SHC sample, and 6 from the STC sample. Although most of the clone sequences did not match the sequences present in the database, individual amplified sequences matched with Glomeromycota (SMS), Fungi incertae sedis (SMS), and Neocallimastigomycota (SHC). Most of the sequences from the amplified taxa represent uncultured fungi. The molecular analysis of variance (AMOVA) indicated that fluctuations observed of haplotypes in the composition may be related to herbicide application. © 2013 Silvana Pompéia Val-Moraes et al.

Influence of experimental canine ehrlichiosis on the E-ADA activity and purine levels in serum and possible functional correlations with pathogenesis

The aim of this study was to evaluate adenosine deaminase activity and purines levels in serum of dogs experimentally infected by Ehrlichia canis. Banked serum samples of dogs divided into two groups with five animals each: healthy animals and animals infected by E. canis. The concentration of purines (adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine, inosine, hypoxanthine, xanthine and uric acid), and adenosine deaminase (E-ADA) activity in sera were evaluated. Samples were collected on days 12 and 30 post-infection (PI). The E-ADA activity showed a significant reduction on day 12 PI, and increased on day 30 PI in dogs infected with E. canis. On day 12, an increase in seric concentration of ATP, ADP and adenosine was verified, and different levels of hypoxanthine, xanthine and uric acid had a drastic reduction in infected compared healthy dogs (P< 0.05). However, on day 30 PI, the levels of seric ADP and AMP decreased, unlike the concentration of xanthine and uric acid that increased significantly in infected dogs (P< 0.05). Therefore, the activity of E-ADA and purine levels are altered in experimental canine ehrlichiosis, probably with the purpose of modulating the pathogenesis of the disease related to immune response, oxidative stress and coagulation disorders in acute phase. © 2013 Elsevier B.V.