Geometrical distribution of Cryptococcus neoformans mediates flower-like biofilm development

Microbial biofilms are highly structured and dynamic communities in which phenotypic diversification allows microorganisms to adapt to different environments under distinct conditions. The environmentally ubiquitous pathogen Cryptococcus neoformans colonizes many niches of the human body and implanted medical devices in the form of biofilms, an important virulence factor. A new approach was used to characterize the underlying geometrical distribution of C. neoformans cells during the adhesion stage of biofilm formation. Geometrical aspects of adhered cells were calculated from the Delaunay triangulation and Voronoi diagramobtained fromscanning electronmicroscopy images (SEM). A correlation between increased biofilm formation and higher ordering of the underlying cell distribution was found. Mature biofilm aggregates were analyzed by applying an adapted protocol developed for ultrastructure visualization of cryptococcal cells by SEM. Flower-like clusters consisting of cells embedded in a dense layer of extracellular matrix were observed as well as distinct levels of spatial organization: adhered cells, clusters of cells and community of clusters. The results add insights into yeast motility during the dispersion stage of biofilm formation. This study highlights the importance of cellular organization for biofilm growth and presents a novel application of the geometrical method of analysis

Genome-Wide Analysis of Secondary Metabolite Gene Clusters in Ophiostoma ulmi and Ophiostoma novo-ulmi Reveals a Fujikurin-Like Gene Cluster with a Putative Role in Infection

The emergence of new microbial pathogens can result in destructive outbreaks, since their hosts have limited resistance and pathogens may be excessively aggressive. Described as the major ecological incident of the twentieth century, Dutch elm disease, caused by ascomycete fungi from the Ophiostoma genus, has caused a significant decline in elm tree populations (Ulmus sp.) in North America and Europe. Genome sequencing of the two main causative agents of Dutch elm disease (Ophiostoma ulmi and Ophiostoma novo-ulmi), along with closely related species with different lifestyles, allows for unique comparisons to be made to identify how pathogens and virulence determinants have emerged. Among several established virulence determinants, secondary metabolites (SMs) have been suggested to play significant roles during phytopathogen infection. Interestingly, the secondary metabolism of Dutch elm pathogens remains almost unexplored, and little is known about how SM biosynthetic genes are organized in these species. To better understand the metabolic potential of O. ulmi and O. novo-ulmi, we performed a deep survey and description of SM biosynthetic gene clusters (BGCs) in these species and assessed their conservation among eight species from the Ophiostomataceae family. Among 19 identified BGCs, a fujikurin-like gene cluster (OpPKS8) was unique to Dutch elm pathogens. Phylogenetic analysis revealed that orthologs for this gene cluster are widespread among phytopathogens and plant-associated fungi, suggesting that OpPKS8 may have been horizontally acquired by the Ophiostoma genus. Moreover, the detailed identification of several BGCs paves the way for future in-depth research and supports the potential impact of secondary metabolism on Ophiostoma genus’ lifestyle

Pathogenic diversity amongst serotype C VGIII and VGIV Cryptococcus gattii isolates

Cryptococcus gattii is one of the causative agents of human cryptococcosis. Highly virulent strains of serotype B C. gattii have been studied in detail, but little information is available on the pathogenic properties of serotype C isolates. In this study, we analyzed pathogenic determinants in three serotype C C. gattii isolates (106.97, ATCC 24066 and WM 779). Isolate ATCC 24066 (molecular type VGIII) differed from isolates WM 779 and 106.97 (both VGIV) in capsule dimensions, expression of CAP genes, chitooligomer distribution, and induction of host chitinase activity. Isolate WM 779 was more efficient than the others in producing pigments and all three isolates had distinct patterns of reactivity with antibodies to glucuronoxylomannan. This great phenotypic diversity reflected in differential pathogenicity. VGIV isolates WM 779 and 106.97 were similar in their ability to cause lethality and produced higher pulmonary fungal burden in a murine model of cryptococcosis, while isolate ATCC 24066 (VGIII) was unable to reach the brain and caused reduced lethality in intranasally infected mice. These results demonstrate a high diversity in the pathogenic potential of isolates of C. gattii belonging to the molecular types VGIII and VGIV

The calcium transporter Pmc1 provides Ca2+ tolerance and influences the progression of murine cryptococcal infection

Submitted by Fabricia Pimenta ([email protected]) on 2019-01-31T18:17:20Z No. of bitstreams: 1 ve_Marcio_Rodrigues_etal_CDTS_2013c.pdf: 654095 bytes, checksum: 2c4916d4e295d8e8485b645fd88d7f58 (MD5)Approved for entry into archive by Fabricia Pimenta ([email protected]) on 2019-03-07T20:00:12Z (GMT) No. of bitstreams: 1 ve_Marcio_Rodrigues_etal_CDTS_2013c.pdf: 654095 bytes, checksum: 2c4916d4e295d8e8485b645fd88d7f58 (MD5)Made available in DSpace on 2019-03-07T20:00:12Z (GMT). No. of bitstreams: 1 ve_Marcio_Rodrigues_etal_CDTS_2013c.pdf: 654095 bytes, checksum: 2c4916d4e295d8e8485b645fd88d7f58 (MD5) Previous issue date: 2013-10Universidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Porto Alegre, RS, Brasil.Universidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Porto Alegre, RS, Brasil.Universidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Porto Alegre, RS, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Professor Paulo de G oes. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Centro de Desenvolvimento Tecnológico em Saúde. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Professor Paulo de G oes. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Porto Alegre, RS, Brasil.Universidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Porto Alegre, RS, Brasil.The Ca(2+)-calcineurin signaling pathway in the human fungal pathogen Cryptococcus neoformans is essential for adaptation to the host environment during infection. Calcium transporters regulate cytosolic calcium concentrations, providing Ca(2+) loading into storage organelles. The three calcium transporters that have been characterized in C. neoformans, Cch1, Eca1 and Vcx1, are required for fungal virulence, supporting a role for calcium-mediated signaling in cryptococcal pathogenesis. In the present study, we report the functional characterization of the putative vacuolar calcium ATPase Pmc1 in C. neoformans. Our results demonstrate that Pmc1 provides tolerance to high Ca(2+) concentrations. The double knockout of C. neoformans PMC1 and VCX1 genes impaired the intracellular calcium transport, resulting in a significant increase in cytosolic calcium levels. Furthermore, Pmc1 was essential for both the progression of pulmonary infection and brain colonization in mice, emphasizing the crucial role of calcium signaling and transport for cryptococcal pathogenesis

Genotypic and Phenotypic Diversity of Cryptococcus gattii VGII Clinical Isolates and Its Impact on Virulence

The Cryptococcus gattii species complex harbors the main etiological agents of cryptococcosis in immunocompetent patients. C. gattii molecular type VGII predominates in the north and northeastern regions of Brazil, leading to high morbidity and mortality rates. C. gattii VGII isolates have a strong clinical relevance and phenotypic variations. These phenotypic variations among C. gattii species complex isolates suggest that some strains are more virulent than others, but little information is available related to the pathogenic properties of those strains. In this study, we analyzed some virulence determinants of C. gattii VGII strains (CG01, CG02, and CG03) isolated from patients in the state of Piauí, Brazil. The C. gattii R265 VGIIa strain, which was isolated from the Vancouver outbreak, differed from C. gattii CG01, CG02 and CG03 isolates (also classified as VGII) when analyzed the capsular dimensions, melanin production, urease activity, as well as the glucuronoxylomannan (GXM) secretion. Those differences directly reflected in their virulence potential. In addition, CG02 displayed higher virulence compared to R265 (VGIIa) strain in a cryptococcal murine model of infection. Lastly, we examined the genotypic diversity of these strains through Multilocus Sequence Type (MLST) and one new subtype was described for the CG02 isolate. This study confirms the presence and the phenotypic and genotypic diversity of highly virulent strains in the Northeast region of Brazil

Modulation of Zinc Homeostasis in Acanthamoeba castellanii as a Possible Antifungal Strategy against Cryptococcus gattii

Cryptococcus gattii is a basidiomycetous yeast that can be found in the environment and is one of the agents of cryptococcosis, a life-threatening disease. During its life cycle, cryptococcal cells take hold inside environmental predators such as amoebae. Despite their evolutionary distance, macrophages and amoebae share conserved similar steps of phagocytosis and microbial killing. To evaluate whether amoebae also share other antifungal strategies developed by macrophages, we investigated nutritional immunity against cryptococcal cells. We focused on zinc homeostasis modulation in Acanthamoeba castellanii infected with C. gattii. The intracellular proliferation rate (IPR) in amoebae was determined using C. gattii R265 and mutants for the ZIP1 gene, which displays defects of growth in zinc-limiting conditions. We detected a reduced IPR in cells lacking the ZIP1 gene compared to wild-type strains, suggesting that amoebae produce a low zinc environment to engulfed cells. Furthermore, flow cytometry analysis employing the zinc probe Zinpyr-1 confirmed the reduced concentration of zinc in cryptococcal-infected amoebae. qRT-PCR analysis of zinc transporter-coding genes suggests that zinc export by members of the ZnT family would be involved in the reduced intracellular zinc concentration. These results indicate that amoebae may use nutritional immunity to reduce fungal cell proliferation by reducing zinc availability for the pathogen

The heat shock protein (Hsp) 70 of Cryptococcus neoformans is associated with the fungal cell surface and influences the interaction between yeast and host cells

Submitted by Fabricia Pimenta ([email protected]) on 2019-01-31T17:40:15Z No. of bitstreams: 1 ve_Marcio_Rodrigues_etal_CDTS_2013a.pdf: 2964965 bytes, checksum: d8cee31a594193a7d05450efbc188c29 (MD5)Approved for entry into archive by Claudete Fernandes ([email protected]) on 2019-12-13T19:03:14Z (GMT) No. of bitstreams: 1 ve_Marcio_Rodrigues_etal_CDTS_2013a.pdf: 2964965 bytes, checksum: d8cee31a594193a7d05450efbc188c29 (MD5)Made available in DSpace on 2019-12-13T19:03:14Z (GMT). No. of bitstreams: 1 ve_Marcio_Rodrigues_etal_CDTS_2013a.pdf: 2964965 bytes, checksum: d8cee31a594193a7d05450efbc188c29 (MD5) Previous issue date: 2013Universidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Porto Alegre, RS, Brasil.Universidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Porto Alegre, RS, Brasil.Universidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Porto Alegre, RS, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Paulo de Góes. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Porto Alegre, RS, Brasil.Universidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Porto Alegre, RS, Brasil / Universidade Federal do Rio Grande do Sul. Departamento de Biologia Molecular e Biotecnologia. Porto Alegre, RS, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Paulo de Góes. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Centro de Desenvolvimento Tecnológico em Saúde. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Porto Alegre, RS, Brasil / Universidade Federal do Rio Grande do Sul. Departamento de Biologia Molecular e Biotecnologia. Porto Alegre, RS, Brasil.Universidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Porto Alegre, RS, Brasil / Universidade Federal do Rio Grande do Sul. Departamento de Biologia Molecular e Biotecnologia. Porto Alegre, RS, Brasil.The pathogenic yeast Cryptococcus neoformans secretes numerous proteins, such as heat shock proteins, by unconventional mechanisms during its interaction with host cells. Hsp70 is a conserved chaperone that plays important roles in various cellular processes, including the interaction of fungi with host immune cells. Here, we report that sera from individuals with cryptococcosis infection recognize a recombinant C. neoformans Hsp70 (Cn_rHsp70). Moreover, immunofluorescence assays using antibodies against Cn_rHsp70 revealed the localization of this protein at the cell surface mainly in association with the capsular network. We found that the addition of Cn_rHsp70 positively modulated the interaction of C. neoformans with human alveolar epithelial cells and decreased fungal killing by mouse macrophages, without affecting phagocytosis rates. Immunofluorescence analysis showed that there was a competitive association among the receptor, GXM and Cn_rHsp70, indicating that the Hsp70-binding sites in host cells appear to be shared by glucuronoxylomannan (GXM), the major capsular antigen in C. neoformans. Our observations suggest additional mechanisms by which Hsp70 influences the interaction of C. neoformans with host cells

Análise química e biológica de espécies do gênero Hypericum nativas da região sul do Brasil

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The vacuolar-sorting protein Snf7 is required for export of virulence determinants in members of the Cryptococcus neoformans complex

Submitted by Fabricia Pimenta ([email protected]) on 2019-02-01T15:54:49Z No. of bitstreams: 1 ve_Marcio_Rodrigues_etal_CDTS_2014b.pdf: 1516608 bytes, checksum: 73e7476e70644ff13558d511695598ca (MD5)Approved for entry into archive by Fabricia Pimenta ([email protected]) on 2019-03-07T19:09:52Z (GMT) No. of bitstreams: 1 ve_Marcio_Rodrigues_etal_CDTS_2014b.pdf: 1516608 bytes, checksum: 73e7476e70644ff13558d511695598ca (MD5)Made available in DSpace on 2019-03-07T19:09:52Z (GMT). No. of bitstreams: 1 ve_Marcio_Rodrigues_etal_CDTS_2014b.pdf: 1516608 bytes, checksum: 73e7476e70644ff13558d511695598ca (MD5) Previous issue date: 2014-09-02Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Professor Paulo de Góes. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Porto Alegre, RS, Brasil.Universidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Porto Alegre, RS, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biofisica Carlos Chagas Filho. Laboratório de Ultraestrutura Celular Hertha Meyer. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Metrologia, Normalização e Qualidade Industrial. Laboratório de Biologia. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biofisica Carlos Chagas Filho. Laboratório de Ultraestrutura Celular Hertha Meyer. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Metrologia, Normalização e Qualidade Industrial. Laboratório de Biologia. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Porto Alegre, RS, Brasil.Universidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Porto Alegre, RS, Brasil / Universidade Federal do Rio Grande do Sul. Departamento de Biologia Molecular e Biotecnologia. Porto Alegre, RS, Brasil.Universidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Porto Alegre, RS, Brasil / Universidade Federal do Rio Grande do Sul. Departamento de Biologia Molecular e Biotecnologia. Porto Alegre, RS, BrasilFundação Oswaldo Cruz. Centro de Desenvolvimento Tecnológico em Saúde. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Professor Paulo de Góes. Rio de Janeiro, RJ, Brasil.Fungal pathogenesis requires a number of extracellularly released virulence factors. Recent studies demonstrating that most fungal extracellular molecules lack secretory tags suggest that unconventional secretion mechanisms and fungal virulence are strictly connected. Proteins of the endosomal sorting complex required for transport (ESCRT) have been recently associated with polysaccharide export in the yeast-like human pathogen Cryptococcus neoformans. Snf7 is a key ESCRT operator required for unconventional secretion in Eukaryotes. In this study we generated snf7Δ mutant strains of C. neoformans and its sibling species C. gattii. Lack of Snf7 resulted in important alterations in polysaccharide secretion, capsular formation and pigmentation. This phenotype culminated with loss of virulence in an intranasal model of murine infection in both species. Our data support the notion that Snf7 expression regulates virulence in C. neoformans and C. gattii by ablating polysaccharide and melanin traffic. These results are in agreement with the observation that unconventional secretion is essential for cryptococcal pathogenesis and strongly suggest the occurrence of still obscure mechanisms of exportation of non-protein molecules in Eukaryotes

The Anti-helminthic Compound Mebendazole Has Multiple Antifungal Effects against Cryptococcus neoformans

Submitted by Fabricia Pimenta ([email protected]) on 2018-06-29T19:03:08Z No. of bitstreams: 1 ve_Marcio_Rodrigues_etal_CDTS_2017a.pdf: 3873194 bytes, checksum: 99621cb2d1cf68a20e492eae8b6b1757 (MD5)Approved for entry into archive by Fabricia Pimenta ([email protected]) on 2018-07-26T16:28:40Z (GMT) No. of bitstreams: 1 ve_Marcio_Rodrigues_etal_CDTS_2017a.pdf: 3873194 bytes, checksum: 99621cb2d1cf68a20e492eae8b6b1757 (MD5)Made available in DSpace on 2018-07-26T16:28:40Z (GMT). No. of bitstreams: 1 ve_Marcio_Rodrigues_etal_CDTS_2017a.pdf: 3873194 bytes, checksum: 99621cb2d1cf68a20e492eae8b6b1757 (MD5) Previous issue date: 2017-03-28Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Paulo de Góes. Laboratório de Biologia Celular de Leveduras Patogênicas Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Porto Alegre, RS, Brasil.Universidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Porto Alegre, RS, Brasil.Fundação Oswaldo Cruz. Centro de Desenvolvimento Tecnológico em Saúde. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Departamento de Biologia Molecular e Biotecnologia. Porto Alegre, RS, Brasil.Universidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Departamento de Biologia Molecular e Biotecnologia. Porto Alegre, RS, Brasil.Universidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Departamento de Biologia Molecular e Biotecnologia. Porto Alegre, RS, Brasil.Veterans Administration Medical Center. Northport, NY, USA / Stony Brook University. Department of Molecular Genetics and Microbiology. Stony Brook, NY, USA.Universidade Federal do Rio Grande do Sul. Centro de Biotecnologia. Departamento de Biologia Molecular e Biotecnologia. Porto Alegre, RS, Brasil.Fundação Oswaldo Cruz. Centro de Desenvolvimento Tecnológico em Saúde. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Paulo de Góes. Laboratório de Biologia Celular de Leveduras Patogênicas Rio de Janeiro, RJ, Brasil.Cryptococcus neoformans is the most lethal pathogen of the central nervous system. The gold standard treatment of cryptococcosis, a combination of amphotericin B with 5-fluorocytosine, involves broad toxicity, high costs, low efficacy, and limited worldwide availability. Although the need for new antifungals is clear, drug research and development (R&D) is costly and time-consuming. Thus, drug repurposing is an alternative to R&D and to the currently available tools for treating fungal diseases. Here we screened a collection of compounds approved for use in humans seeking for those with anti-cryptococcal activity. We found that benzimidazoles consist of a broad class of chemicals inhibiting C. neoformans growth. Mebendazole and fenbendazole were the most efficient antifungals showing in vitro fungicidal activity. Since previous studies showed that mebendazole reaches the brain in biologically active concentrations, this compound was selected for further studies. Mebendazole showed antifungal activity against phagocytized C. neoformans, affected cryptococcal biofilms profoundly and caused marked morphological alterations in C. neoformans, including reduction of capsular dimensions. Amphotericin B and mebendazole had additive anti-cryptococcal effects. Mebendazole was also active against the C. neoformans sibling species, C. gattii. To further characterize the effects of the drug a random C. gattii mutant library was screened and indicated that the antifungal activity of mebendazole requires previously unknown cryptococcal targets. Our results indicate that mebendazole is as a promising prototype for the future development of anti-cryptococcal drugs