Study of Lomentospora prolificans pathogenicity: Host immune response and fungal virulence factors.

188 p.

Extensive alteration of fungal gene transcript accumulation and elevation of G-protein-regulated cAMP levels by a virulence-attenuating hypovirus.

Persistent infection of the chestnut blight fungus Cryphonectria parasitica with the prototypic hypovirus CHVI-713 results in attenuation of fungal virulence (hypo-virulence) and reduced accumulation of the GTP-binding (G) protein a subunit CPG-1. Transgenic cosuppression of CPG-1 accumulation in the absence of virus infection also confers hypovirulence. We now report the use of mRNA differential display to examine the extent to which virus infection alters fungal gene transcript accumulation and to assess the degree to which modification of CPG-1 signal transduction contributes to this alteration. More than 400 PCR products were identified that either increased (296 products) or decreased (127 products) in abundance as a result of virus infection. Significantly, 65% of these products exhibited similar changes as a result of CPG-1 cosuppression in the absence of virus infection. We also report that both virus infection and CPG-1 cosuppression elevate cAMP levels 3- to 5-fold. Additionally, it was possible to mimic the effect of virus infection and CPG-1 cosuppression on transcript accumulation for representative fungal genes by drug-induced elevation of cAMP levels. These results strengthen and extend previous indications that hypovirus infection causes a significant and persistent alteration of fungal gene expression/transcript accumulation. They further show that this alteration is primarily mediated through modification of the CPG-1 signaling pathway and suggest that, similar to mammalian Gi alpha subunits, CPG-1 functions as a negative modulator of adenylyl cyclase. Finally, these results suggest a role for G-protein-regulated cAMP accumulation in hypovirus-mediated alteration of fungal gene expression.

Virulence profile of ten Paracoccidioides brasiliensis isolates: association with morphologic and genetic patterns

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Produção de Bipolaris euphorbiae em meios de cultura sólidos e líquidos obtidos de grãos e resíduos agroindustriais

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

PATHOGENICITY AND IMMUNOGENICITY OF PARACOCCIDIOIDES-BRASILIENSIS ISOLATES IN THE HUMAN-DISEASE AND IN AN EXPERIMENTAL MURINE MODEL

The pathogenicity and immunogenicity of six recently isolated Paracoccidioides brasiliensis samples derived from patients presenting distinct and well defined clinical forms of paracoccidioidomycosis (PCM) were compared as to their virulence, tropism to different organs and ability to induce specific cellular and humoral immune response in susceptible (B10.A) inbred mice. Isolates Pb44 and Pb47 were obtained from acute cases, Pb50 from a chronic severe form, Pb45 from a chronic moderate case and both Pb56 and Pb57 from chronic mild forms of PCM. Pathogenicity and tropism of each fungal sample were evaluated by LD50% estimation, examination of gross lesions on various organs at 2, 4, 12 and 16 weeks post-infection, and by colony-forming unit (CFU) counts in the lungs at week 16 post-infection of mice. Fungal tropism in human PCM and in B10.A mice was always dissociated. A well defined relationship between virulence of the fungal sample and the clinical findings of the correspondent patient was not evident, although a tendency to higher LD50% and less intense paracoccidioidic lesions was observed in mice infected with Pb56 and Pb57. The specific DTH response patterns varied according to the infectant sample, but positive DTH reactions at the beginning of the infection and a tendency to anergy or low DTH responses at week 12 and/or week 16 post-infection were always observed. A correspondence between the DTH response in humans and in mice was noticeable only when the isolates from the most benign cases (Pb56 and Pb57) were considered. The specific antibody patterns in mice and in the correspondent patients were also not analogous. Collectively, these results indicate that an association between the fungal pathogenicity and immunogenicity in the human disease and in susceptible mice was discernible only when isolates obtained from very mild cases (Pb56 and Pb57) were considered.

Interaction of pathogenic fungi with host cells: Molecular and cellular approaches

This review provides an overview of several molecular and cellular approaches that are likely to supply insights into the host-fungus interaction. Fungi present intra- and/or extracellular host-parasite interfaces, the parasitism phenomenon being dependent on complementary surface molecules. The entry of the pathogen into the host cell is initiated by the fungus adhering to the cell surface, which generates an uptake signal that may induce its cytoplasmatic internalization. Furthermore, microbial pathogens use a variety of their surface molecules to bind to host extracellular matrix (ECM) components to establish an effective infection. on the other hand, integrins mediate the tight adhesion of cells to the ECM at sites referred to as focal adhesions and also play a role in cell signaling. The phosphorylation process is an important mechanism of cell signaling and regulation; it has been implicated recently in defense strategies against a variety of pathogens that alter host-signaling pathways in order to facilitate their invasion and survival within host cells. The study of signal transduction pathways in virulent fungi is especially important in view of their putative role in the regulation of pathogenicity. This review discusses fungal adherence, changes in cytoskeletal organization and signal transduction in relation to host-fungus interaction. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Host organism defense by a peptide-polysaccharide extracted from the fungus Sporothrix schenckii

A peptide-polysaccharide, a peptide-rhamnomannan, was isolated from the pathogenic yeast form of the fungus Sporothrix schenckii. This substance, which may play a role in fungal virulence, was tested in an animal model of systemic disease, and depression of the immune response was observed in the animals between the 4th and 6th week of infection. Concomitantly, this compound showed mitogenic activity when challenged with normal lymphocytes and was also found to be involved in the inflammatory response. These results provide further information for the understanding of fungal implantation in tissues and of the pathogenicity of this systemic mycosis.

Isolation of ergosterol peroxide and its reversion to ergosterol in the pathogenic fungus Sporothrix schenckii

Ergosterol peroxide, a presumed product of the H2O2-dependent enzymatic oxidation of ergosterol, has been isolated from yeast from yeast forms of the pathogenic fungus Sporothrix schenckii. The substance, which may have a role in fungal virulence, has been characterized mainly using spectroscopic methods (1H and 13C nuclear magnetic resonance and high resolution mass spectra). The purified compound showed a molecular formula of C28H44O3, displaying characteristic features of epidioxy sterols and was reverted to ergosterol when submitted to S. schenckii enzymatic extract.

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.

Candida species: Current epidemiology, pathogenicity, biofilm formation, natural antifungal products and new therapeutic options

The incidence of fungal infections has increased significantly, so contributing to morbidity and mortality. This is caused by an increase in antimicrobial resistance and the restricted number of antifungal drugs, which retain many side effects. Candida species are major human fungal pathogens that cause both mucosal and deep tissue infections. Recent evidence suggests that the majority of infections produced by this pathogen are associated with biofilm growth. Biofilms are biological communities with a high degree of organization, in which micro-organisms form structured, coordinated and functional communities. These biological communities are embedded in a self-created extracellular matrix. Biofilm production is also associated with a high level of antimicrobial resistance of the associated organisms. The ability of Candida species to form drugresistant biofilms is an important factor in their contribution to human disease. The study of plants as an alternative to other forms of drug discovery has attracted great attention because, according to the World Health Organization, these would be the best sources for obtaining a wide variety of drugs and could benefit a large population. Furthermore, silver nanoparticles, antibodies and photodynamic inactivation have also been used with good results. This article presents a brief review of the literature regarding the epidemiology of Candida species, as well as their pathogenicity and ability to form biofilms, the antifungal activity of natural products and other therapeutic options. © 2013 SGM.

Antifungal Efficacy during Candida krusei Infection in Non-Conventional Models Correlates with the Yeast In Vitro Susceptibility Profile

The incidence of opportunistic fungal infections has increased in recent decades due to the growing proportion of immunocompromised patients in our society. Candida krusei has been described as a causative agent of disseminated fungal infections in susceptible patients. Although its prevalence remains low among yeast infections (2-5%), its intrinsic resistance to fluconazole makes this yeast important from epidemiologic aspects. Non mammalian organisms are feasible models to study fungal virulence and drug efficacy. In this work we have used the lepidopteran Galleria mellonella and the nematode Caenorhabditis elegans as models to assess antifungal efficacy during infection by C. krusei. This yeast killed G. mellonella at 25, 30 and 37°C and reduced haemocytic density. Infected larvae melanized in a dose-dependent manner. Fluconazole did not protect against C. krusei infection, in contrast to amphotericin B, voriconazole or caspofungin. However, the doses of these antifungals required to obtain larvae protection were always higher during C. krusei infection than during C. albicans infection. Similar results were found in the model host C. elegans. Our work demonstrates that non mammalian models are useful tools to investigate in vivo antifungal efficacy and virulence of C. krusei. © 2013 Scorzoni et al.

Expressão gênica das adesinas e fatores de virulência da fase miceliar e leveduriforme do complexo paracoccidioides e de sua interação com células epiteliais e fagócitos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Signaling via cAMP in Fungi: Interconnections with Mitogen-Activated Protein Kinase Pathways

The cAMP signal transduction pathway controls a wide variety of processes in fungi. For example, considerable progress has been made in describing the involvement of cAMP pathway components in the control of morphogenesis in Saccharomyces cerevisiae, Ustilago maydis, and Magnaporthe grisea. These morphological processes include the establishment of filamentous growth in S. cerevisiae and U. maydis, and the differentiation of an appressorial infection structure in M. grisea. The discovery that appressorium formation requires cAMP signaling provides an immediate connection to fungal virulence. This connection may have broader implications among fungal pathogens because recent work indicates that cAMP signaling controls the expression of virulence traits in the human pathogen Cryptococcus neoformans. In this fungus, cAMP also influences mating, as has been found for Schizosaccharomyces pombe and as may occur in U. maydis. Finally, cAMP and mitogen- activated protein kinase pathways appear to function coordinately to control the response of certain fungi, e.g., Saccharomyces cerevisiae and Schizosaccharomyces pombe, to environmental stress. There are clues that interconnections between these pathways may be common in the control of many fungal processes.

A Multifaceted Study of Scedosporium boydii Cell Wall Changes during Germination and Identification of GPI-Anchored Proteins

International audience

Biochemical characterization of potential virulence markers in the human fungal pathogen Pseudallescheria boydii

The ubiquitous Pseudallescheria boydii (anamorph Scedosporium apiospermum) is a saprophytic filamentous fungus recognized as a potent etiologic agent of a wide variety of infections in immunocompromised as well as in immunocompetent patients. Very little is known about the virulence factors expressed by this fungal pathogen. The present review provides an overview of recent discoveries related to the identification and biochemical characterization of potential virulence attributes produced by P. boydii, with special emphasis on surface and released molecules. These structures include polysaccharides (glucans), glycopeptides (peptidorhamnomannans), glycolipids (glucosylceramides) and hydrolytic enzymes (proteases, phosphatases and superoxide dismutase), which have been implicated in some fundamental cellular processes in P. boydii including growth, differentiation and interaction with host molecules. Elucidation of the structure of cell surface components as well as the secreted molecules, especially those that function as virulence determinants, is of great relevance to understand the pathogenic mechanisms of P. boydii.