Predicting the Proteins of Angomonas deanei, Strigomonas culicis and Their Respective Endosymbionts Reveals New Aspects of the Trypanosomatidae Family

Endosymbiont-bearing trypanosomatids have been considered excellent models for the study of cell evolution because the host protozoan co-evolves with an intracellular bacterium in a mutualistic relationship. Such protozoa inhabit a single invertebrate host during their entire life cycle and exhibit special characteristics that group them in a particular phylogenetic cluster of the Trypanosomatidae family, thus classified as monoxenics. in an effort to better understand such symbiotic association, we used DNA pyrosequencing and a reference-guided assembly to generate reads that predicted 16,960 and 12,162 open reading frames (ORFs) in two symbiont-bearing trypanosomatids, Angomonas deanei (previously named as Crithidia deanei) and Strigomonas culicis (first known as Blastocrithidia culicis), respectively. Identification of each ORF was based primarily on TriTrypDB using tblastn, and each ORF was confirmed by employing getorf from EMBOSS and Newbler 2.6 when necessary. the monoxenic organisms revealed conserved housekeeping functions when compared to other trypanosomatids, especially compared with Leishmania major. However, major differences were found in ORFs corresponding to the cytoskeleton, the kinetoplast, and the paraflagellar structure. the monoxenic organisms also contain a large number of genes for cytosolic calpain-like and surface gp63 metalloproteases and a reduced number of compartmentalized cysteine proteases in comparison to other TriTryp organisms, reflecting adaptations to the presence of the symbiont. the assembled bacterial endosymbiont sequences exhibit a high A+T content with a total of 787 and 769 ORFs for the Angomonas deanei and Strigomonas culicis endosymbionts, respectively, and indicate that these organisms hold a common ancestor related to the Alcaligenaceae family. Importantly, both symbionts contain enzymes that complement essential host cell biosynthetic pathways, such as those for amino acid, lipid and purine/pyrimidine metabolism. These findings increase our understanding of the intricate symbiotic relationship between the bacterium and the trypanosomatid host and provide clues to better understand eukaryotic cell evolution.Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)ERC AdG SISYPHEUniv Fed Rio de Janeiro, Inst Biofis Carlos Chagas Filho, Lab Ultraestrutura Celular Hertha Meyer, BR-21941 Rio de Janeiro, BrazilUniv Fed Rio de Janeiro, Inst Biofis Carlos Chagas Filho, Lab Metab Macromol Firmino Torres de Castro, BR-21941 Rio de Janeiro, BrazilLab Bioinformat, Lab Nacl Computacao Cient, Rio de Janeiro, BrazilINRIA Grenoble Rhone Alpes, BAMBOO Team, Villeurbanne, FranceUniv Lyon 1, CNRS, UMR5558, Lab Biometrie & Biol Evolut, F-69622 Villeurbanne, FranceUniv Estadual Campinas, Inst Biol, Dept Genet Evolucao & Bioagentes, São Paulo, BrazilUniv São Paulo, Fac Ciencias Farmaceut Ribeirao Preto, Dept Ciencias Farmaceut, São Paulo, BrazilLab Nacl Ciencia & Tecnol Bioetano, São Paulo, BrazilUniv Fed Minas Gerais, Inst Ciencias Biol, Dept Bioquim & Imunol, Belo Horizonte, MG, BrazilUniv Fed Goias, Inst Ciencias Biol, Mol Biol Lab, Goiania, Go, BrazilFundacao Oswaldo Cruz, Inst Carlos Chagas, Lab Biol Mol Tripanossomatideos, Curitiba, Parana, BrazilFundacao Oswaldo Cruz, Inst Carlos Chagas, Lab Genom Func, Curitiba, Parana, BrazilUniv Estadual Campinas, Ctr Pluridisciplinar Pesquisas Quim Biol & Agr, São Paulo, BrazilUniv Fed Minas Gerais, Inst Ciencias Biol, Dept Parasitol, Belo Horizonte, MG, BrazilUniv Fed Santa Catarina, Dept Microbiol Imunol & Parasitol, Ctr Ciencias Biol, Lab Protozool & Bioinformat, Florianopolis, SC, BrazilUniv Fed Vicosa, Dept Bioquim & Biol Mol, Ctr Ciencias Biol & Saude, Vicosa, MG, BrazilInst Butantan, Lab Especial Ciclo Celular, São Paulo, BrazilUniv São Paulo, Dept Biol, Fac Filosofia Ciencias & Letras Ribeirao Preto, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilWeb of Scienc

Capsules from Pathogenic and Non-Pathogenic Cryptococcus spp. Manifest Significant Differences in Structure and Ability to Protect against Phagocytic Cells

Capsule production is common among bacterial species, but relatively rare in eukaryotic microorganisms. Members of the fungal Cryptococcus genus are known to produce capsules, which are major determinants of virulence in the highly pathogenic species Cryptococcus neoformans and Cryptococcus gattii. Although the lack of virulence of many species of the Cryptococcus genus can be explained solely by the lack of mammalian thermotolerance, it is uncertain whether the capsules from these organisms are comparable to those of the pathogenic cryptococci. In this study, we compared the characteristic of the capsule from the non-pathogenic environmental yeast Cryptococcus liquefaciens with that of C. neoformans. Microscopic observations revealed that C. liquefaciens has a capsule visible in India ink preparations that was also efficiently labeled by three antibodies generated to specific C. neoformans capsular antigens. Capsular polysaccharides of C. liquefaciens were incorporated onto the cell surface of acapsular C. neoformans mutant cells. Polysaccharide composition determinations in combination with confocal microscopy revealed that C. liquefaciens capsule consisted of mannose, xylose, glucose, glucuronic acid, galactose and N-acetylglucosamine. Physical chemical analysis of the C. liquefaciens polysaccharides in comparison with C. neoformans samples revealed significant differences in viscosity, elastic properties and macromolecular structure parameters of polysaccharide solutions such as rigidity, effective diameter, zeta potential and molecular mass, which nevertheless appeared to be characteristics of linear polysaccharides that also comprise capsular polysaccharide of C. neoformans. The environmental yeast, however, showed enhanced susceptibility to the antimicrobial activity of the environmental phagocytes, suggesting that the C. liquefaciens capsular components are insufficient in protecting yeast cells against killing by amoeba. These results suggest that capsular structures in pathogenic Cryptococcus species and environmental species share similar features, but also manifest significant difference that could influence their potential to virulence

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

Binding of the wheat germ lectin to Cryptococcus neoformans chitooligomers affects multiple mechanisms required for fungal pathogenesis

The principal capsular component of Cryptococcus neoformans, glucuronoxylomannan (GXM), interacts with surface glycans, including chitin-like oligomers. Although the role of GXM in cryptococcal infection has been well explored, there is no information on how chitooligomers affect fungal pathogenesis. In this study, surface chitooligomers of C. neoformans were blocked through the use of the wheat germ lectin (WGA) and the effects on animal pathogenesis, interaction with host cells, fungal growth and capsule formation were analyzed. Treatment of C. neoformans cells with WGA followed by infection of mice delayed mortality relative to animals infected with untreated fungal cells. This observation was associated with reduced brain colonization by lectin-treated cryptococci. Blocking chitooligomers also rendered yeast cells less efficient in their ability to associate with phagocytes. WGA did not affect fungal viability, but inhibited GXM release to the extracellular space and capsule formation. In WGA-treated yeast cells, genes that are involved in capsule formation and GXM traffic had their transcription levels decreased in comparison with untreated cells. Our results suggest that cellular pathways required for capsule formation and pathogenic mechanisms are affected by blocking chitin-derived structures at the cell surface of C. neoformans. Targeting chitooligomers with specific ligands may reveal new therapeutic alternatives to control cryptococcosis.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil)Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP, Brazil)Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ, Brazil)NIH, AI033142NIH, AI033774NIH, AI052733NIH, HL059842Center for AIDS Research at Einstei

Physical properties of capsular and exo-PS from <i>C. liquefaciens</i> obtained by DLS.

<p>Panel A and B show size distribution of PS fibers capsular and exo-PS samples, respectively.</p

Surface architecture of <i>C. liquefaciens</i>.

<p>Panel A) Uvitex staining (blue) of <i>C. liquefaciens</i> strain showing chitin distribution at the cell wall. Panel B) Staining of <i>C. liquefaciens</i> cell with mAb 18B7 showing that the antibody raised to <i>C. neoformans</i> GXM cross reacts with <i>C. liquefaciens</i> components. Panel C) Merge of panels A and B. Panel D) Light microscopy of acapsular <i>C. neoformans</i> cap67 cells. Panel E) Exo-PS from <i>C. liquefaciens</i> attaching to an acapsular <i>C. neoformans</i> mutant with the PS labeled with mAb 18B7-FITC. Panel F) Incorporation of <i>C. neoformans</i> (control) exo-PS by acapsular <i>C. neoformans</i> cells with labeling of PS by mAb 18B7-FITC. Panel G) <i>C. liquefaciens</i> labeled with 1 mAb 8B7-FITC Panel H) <i>C. liquefaciens</i> stained with WGA-rhodamine. Panel I). Merge of G and H with <i>C. neoformans</i> labeled with mAb 18B7 (green fluorescence) and WGA (red fluorescence).</p

Zeta Potential of exo- and capsular-PS from <i>C. liquefaciens</i>.

<p>The charges of the different <i>C. liquefaciens</i> PS forms were significantly different (P Value<0.01 calculated by <i>Student's T test (two-tailed)</i>).</p

Traversing the Cell Wall: The Chitinolytic Activity of <i>Histoplasma capsulatum</i> Extracellular Vesicles Facilitates Their Release

Histoplasma capsulatum is the causative agent of histoplasmosis. Treating this fungal infection conventionally has significant limitations, prompting the search for alternative therapies. In this context, fungal extracellular vesicles (EVs) hold relevant potential as both therapeutic agents and targets for the treatment of fungal infections. To explore this further, we conducted a study using pharmacological inhibitors of chitinase (methylxanthines) to investigate their potential to reduce EV release and its subsequent impact on fungal virulence in an in vivo invertebrate model. Our findings revealed that a subinhibitory concentration of the methylxanthine, caffeine, effectively reduces EV release, leading to a modulation of H. capsulatum virulence. To the best of our knowledge, this is the first reported instance of a pharmacological inhibitor that reduces fungal EV release without any observed fungicidal effects

Microscopic visualization of <i>C. liquefaciens and C. neoformans capsules and growth profile</i>.

<p>Panel A) India ink stain of <i>C. neoformans</i> cells. Panel B) Counterstaining of <i>C. liquefaciens</i> with India ink. Panel C) Growth curves of <i>C. neoformans</i> and <i>C. liquefaciens</i> at 30°C in minimal media.</p