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

Identificação de Candida dubliniensis isoladas no Brasil, através do método comercial ID 32C

The purpose of the present study was to evaluate the identification of 19 Brazilian C. dubliniensis based on the biochemical profile exhibited when tested by the commercial identification kit ID 32C (bioMerieux). Thirteen of the isolates were rigorously identified as C. dubliniensis and the remaining isolates (six) were considered as having a doubtful profile but the software also suggested that there was 83.6% of chances for them to be C. dubliniensis. As well as pointed by the literature the identification obtained by phenotypic tests should be considered presumptive for C. dubliniensis due to variability of this new species.Dezenove culturas de C. dubliniensis isoladas no Brasil, previamente identificadas através de métodos genotípicos, foram avaliadas pelo kit comercial ID 32C (bioMerieux). Treze culturas foram identificadas como C. dubliniensis, mas as demais (seis) evidenciaram perfil duvidoso, embora o software do sistema sugerisse 83,6% de chances das mesmas pertencerem à espécie C. dubliniensis. A literatura tem registrado grande variabilidade fenotípica com esta espécie e, por isto, as identificações obtidas com este sistema deverão ser consideradas como presuntivas.Universidade Federal de Santa Maria Centro de Ciências da Saúde Departamento de Microbiologia e ParasitologiaUniversidade Federal do Rio Grande do SulUniversidade Federal do Rio Grande do NorteUniversidade Federal de São Paulo (UNIFESP)UNIFESPSciEL

Carbohydrate assimilation profiles of Brazilian Candida dubliniensis isolates based on ID 32C system

The purpose of the present study was to evaluate the identification of 19 Brazilian C. dubliniensis based on the biochemical profile exhibited when tested by the commercial identification kit ID 32C (bioMerieux). Thirteen of the isolates were rigorously identified as C. dubliniensis and the remaining isolates (six) were considered as having a doubtful profile but the software also suggested that there was 83.6% of chances for them to be C. dubliniensis. As well as pointed by the literature the identification obtained by phenotypic tests should be considered presumptive for C. dubliniensis due to variability of this new species.Dezenove culturas de C. dubliniensis isoladas no Brasil, previamente identificadas através de métodos genotípicos, foram avaliadas pelo kit comercial ID 32C (bioMerieux). Treze culturas foram identificadas como C. dubliniensis, mas as demais (seis) evidenciaram perfil duvidoso, embora o software do sistema sugerisse 83,6% de chances das mesmas pertencerem à espécie C. dubliniensis. A literatura tem registrado grande variabilidade fenotípica com esta espécie e, por isto, as identificações obtidas com este sistema deverão ser consideradas como presuntivas

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

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

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

A highly active triterpene derivative capable of biofilm damage to control cryptococcus spp.

Cryptococcus neoformans is an encapsulated yeast responsible for more than 180,000 deaths per year. The standard therapeutic approach against cryptococcosis is a combination of amphotericin B with flucytosine. In countries where cryptococcosis is most prevalent, 5-fluorocytosine is rarely available, and amphotericin B requires intravenous administration. C. neoformans biofilm formation is related to increased drug resistance, which is an important outcome for hospitalized patients. Here, we describe new molecules with anti-cryptococcal activity. A collection of 66 semisynthetic derivatives of ursolic acid and betulinic acid was tested against mature biofilms of C. neoformans at 25 mu M. Out of these, eight derivatives including terpenes, benzazoles, flavonoids, and quinolines were able to cause damage and eradicate mature biofilms. Four terpene compounds demonstrated significative growth inhibition of C. neoformans. Our study identified a pentacyclic triterpenoid derived from betulinic acid (LAFIS13) as a potential drug for anti-cryptococcal treatment. This compound appears to be highly active with low toxicity at minimal inhibitory concentration and capable of biofilm eradication912CAPES - Coordenação de Aperfeiçoamento de Pessoal e Nível SuperiorFAPESP – Fundação de Amparo à Pesquisa Do Estado De São PauloSem informação420276/2016-5; 302637/2017-6; 307191/2016-