Monoclonal antibodies against the 43,000 da glycoprotein from Paracoccidioides brasiliensis modulate laminin-mediated fungal adhesion to epithelial cells and pathogenesis

The surface glycoprotein gp43, a highly immunogenic component of Paracoccidioides brasiliensis, is used in the serodiagnosis of paracoccidioidomycosis (PCM) and has recently been shown to specifically bind the extracellular matrix protein laminin, Binding to laminin induces the increased adhesion of the fungus to epithelial cells; a hamster testicle infection model has shown that the gp43-dependent binding of fungal cells to laminin enhances their pathogenicity in vivo. We report on the production and characterization of 12 monoclonal antibodies against the gp43 that recognize peptide sequences in the molecule detecting at least three different epitopes as well as different isoforms of this antigen. MAbs interfered in the fungal pathogenicity in vivo either by inhibiting or enhancing granuloma formation and tissue destruction, Results suggest that P. brasiliensis propagules may start infection in man by strongly adhering to human lung cells, Thus, laminin-mediated fungal adhesion to human lung carcinoma (A549) cells was much more intense than to Madin-Darby canine kidney cells (MDCK), indicating differences in binding affinity, Subsequent growth of fungi bound to the lung cells could induce the granulomatous inflammatory reaction characteristic of PCM. Both steps are greatly stimulated by laminin binding in infective cells expressing gp43.

Differential gene expression by Paracoccidioides brasiliensis in host interaction conditions: Representational difference analysis identifies candidate genes associated with fungal pathogenesis

Paracoccidioides brasiliensis causes infection by the host inhalation of airborne propagules of the mycelia phase of the fungus. These particles reach the lungs, and disseminate to virtually all organs. Here we describe the identification of differentially expressed genes in studies of host-fungus interaction. We analyzed two cDNA populations of P. brasiliensis, one obtained from infected animals and the other an admixture of fungus and human blood thus mimicking the hematologic events of the fungal dissemination. Our analysis identified transcripts differentially expressed. Genes related to iron acquisition, melanin synthesis and cell defense were specially upregulated in the mouse model of infection. The upregulated transcripts of yeast cells during incubation with human blood were those predominantly related to cell wall remodeling/synthesis. The expression pattern of genes was independently confirmed in host conditions, revealing their potential role in the infection process. This work can facilitate functional studies of novel regulated genes that may be important for the survival and growth strategies of P. brasiliensis in humans. (c) 2006 Elsevier Masson SAS. All rights reserved.

Thermal behaviour of corn starch granules under action of fungal alpha-amylase

Corn starch, partially hydrolyzed by fungal alpha-amylase was investigated by using thermal analysis, microscopy and X-ray diffraction. After enzymatic treatment lower degradation onset temperatures were observed. DSC analysis showed almost similar range of gelatinization temperature, however, the enthalpies of gelatinization increased for the partially hydrolyzed starch granules. According to the X-ray diffraction analysis, stronger cereal pattern peaks were recognized after enzymatic digestion. The results suggested that the hydrolysis was more pronounced in the amorphous part of the starch granules.

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

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

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.

Purificação de lacases PPO-I de Botryosphaeria rhodina

Laccases are glycoprotein polyphenol oxidases which are involved in fungal pathogenicity and they are also useful for biotechnological applications. The ligninolytic ascomycete, Botryosphaeria rhodina, has been studied as producer of exopolysaccharide and PPO-I and PPO-II laccases induced by veratryl alcohol. However, as the induced laccases have not been isolated, the aim of this study was to purify the enzyme and to identify the carbohydrates constituents of the glycosidic moiety. The fungus was cultivated on broth Vogel, 1% glucose and 30.4mM veratryl alcohol during 4.5 days at 28°C/180 rpm. The extracellular fluid showed high carbohydrate concentration and the stability of PPO-I laccase under conditions of refrigeration and freezing at 4°C-18°C over 40 days. The purification was developed by ultrafiltration using a NMWL 100 and 30 kDa membrane, gelfiltration on Sephadex G-100, and ion-exchange chromatography on DEAE-cellulose. The purified laccase was identified as a glycoprotein, weight molecular 113 kDa, consisting of 40% protein and 60% carbohydrate identified by HPAEC-PAD as fucose, galactose, mannose, glucose and glucosamine.

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.

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.

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.