Interação entre o papaya meleira virus (PMeV) e o mamoeiro (Carica papaya L.)

A meleira do mamoeiro é causada pelo Papaya meleira virus (PMeV), de genoma de dsRNA, que induz uma exsudação espontânea de látex fluido à partir de frutos e folhas. Inicialmente, objetivou-se estabelecer uma metodologia molecular simplificada para o diagnóstico precoce do vírus. A metodologia baseou-se na extração e análise do dsRNA viral, a partir de látex e tecidos da planta. A transmissão do vírus pela mosca-branca Trialeurodes variabilis foi avaliada, observando-se que o inseto não transmitiu o vírus, embora tenha sido capaz de adquirí-lo a partir de mamoeiros infectados. Foi confirmado que a infecção ocorre quando látex infectado é injetado no ápíce do caule do mamoeiro. A avaliação da distribuição do PMeV em mamoeiros em diferentes fases da infecção revelou a ocorrência do vírus, principalmente, nos órgãos ricos em laticíferos. Neste tipo de célula, o vírus reduziu a quantidade e alterou a morfologia sueprficial dos polímeros presentes no látex, ligando-se fortemente à estes polímeros. O PMeV induziu um acúmulo dos elementos potássio e fósforo no látex, que também apresentou teores mais elevados de água. Observou-se também no látex infectado a síntese de alcalóides e um acúmulo de cristais de oxalato de cálcio. Este acúmulo foi positivamente correlacionado com a síntese de H2O2 nos laticíferos de plantas doentes, que também foi observada nas células companheiras do floema. Os resultados obtidos sugerem a existência de um mecanismo de defesa do mamoeiro contra o PMeV que envolve a síntese de H2O2 pelos laticíferos e células do floema.Papaya sticky disease' is caused by Papaya meleira virus (PMeV) which has a dsRNA molecule as genome. It induces an exudation of fluid latex from the fruits and leaves. Initially, this work aimed to establish an easy molecular diagnostic method to early virus detection. The stablished method was based on virus de dsRNA extraction from papaya latex and tissues. Afterwards, the PMeV transmission by Trialeurodes variabilis whitefly was evaluated. Our observation was that as far as the insect caried the virus, it did not transmit it to the papaya plant. We also established that PMev infection occurs only when latex from diseased plants was injected in healthy ones. The evaluation of PMeV allocation within papaya at different infecton stages demonstrated that the virus was present mainly in laticifers richly tissues. In this cell type, the virus strongly reduced the amount of latex polymers, also altering their superficial morphology. Using transmission elecron microscopy, PMeV was observed strongly linked to the polymers. The virus induced an ncrease in water and potassium and phosphorus elements levels in the latex. Also, it was observed an increase in alkaloids and calcium oxalate crystal accumation. This accumulation was positively correlated with H2O2 synthesis, which was also observed at phloem companion cells. Taken together, these results suggest the existence of papaya defense mechanism against PMeV involving the H2O2 synthesis by laticifers and phloem cells

Extracellular Vesicles from Bothrops jararaca Venom Are Diverse in Structure and Protein Composition and Interact with Mammalian Cells

Snake venoms are complex cocktails of non-toxic and toxic molecules that work synergistically for the envenoming outcome. Alongside the immediate consequences, chronic manifestations and long-term sequelae can occur. Recently, extracellular vesicles (EVs) were found in snake venom. EVs mediate cellular communication through long distances, delivering proteins and nucleic acids that modulate the recipient cell’s function. However, the biological roles of snake venom EVs, including possible cross-organism communication, are still unknown. This knowledge may expand the understanding of envenoming mechanisms. In the present study, we isolated and characterized the EVs from Bothrops jararaca venom (Bj-EVs), giving insights into their biological roles. Fresh venom was submitted to differential centrifugation, resulting in two EV populations with typical morphology and size range. Several conserved EV markers and a subset of venom related EV markers, represented mainly by processing enzymes, were identified by proteomic analysis. The most abundant protein family observed in Bj-EVs was 5’-nucleotidase, known to be immunosuppressive and a low abundant and ubiquitous toxin in snake venoms. Additionally, we demonstrated that mammalian cells efficiently internalize Bj-EVs. The commercial antibothropic antivenom partially recognizes Bj-EVs and inhibits cellular EV uptake. Based on the proteomic results and the in vitro interaction assays using macrophages and muscle cells, we propose that Bj-EVs may be involved not only in venom production and processing but also in host immune modulation and long-term effects of envenoming

Proteomic Analysis of <i>Trypanosoma cruzi</i> Secretome: Characterization of Two Populations of Extracellular Vesicles and Soluble Proteins

Microorganisms use specialized systems to export virulence factors into host cells. Secretion of effector proteins into the extracellular environment has been described in <i>Trypanosoma cruzi</i>; however, a comprehensive proteomic analysis of the secretome and the secretion mechanisms involved remain elusive. Here, we present evidence that <i>T. cruzi</i> releases proteins associated with vesicles that are formed by at least two different mechanisms. Transmission electron microscopy showed larger vesicles budding from the plasma membrane of noninfective epimastigotes and infective metacyclic trypomastigotes, as well as smaller vesicles within the flagellar pocket of both forms. Parasite conditioned culture supernatant was fractionated and characterized by morphological, immunochemical, and proteomic analyses. Three fractions were obtained by differential ultracentrifugation: the first enriched in larger vesicles resembling ectosomes, the second enriched in smaller vesicles resembling exosomes, and a third fraction enriched in soluble proteins not associated with extracellular vesicles. Label-free quantitative proteomic analysis revealed a rich collection of proteins involved in metabolism, signaling, nucleic acid binding, and parasite survival and virulence. These findings support the notion that <i>T. cruzi</i> uses different secretion pathways to excrete/secrete proteins. Moreover, our results suggest that metacyclic forms may use extracellular vesicles to deliver cargo into host cells

Proteomic Analysis of <i>Trypanosoma cruzi</i> Secretome: Characterization of Two Populations of Extracellular Vesicles and Soluble Proteins

Microorganisms use specialized systems to export virulence factors into host cells. Secretion of effector proteins into the extracellular environment has been described in <i>Trypanosoma cruzi</i>; however, a comprehensive proteomic analysis of the secretome and the secretion mechanisms involved remain elusive. Here, we present evidence that <i>T. cruzi</i> releases proteins associated with vesicles that are formed by at least two different mechanisms. Transmission electron microscopy showed larger vesicles budding from the plasma membrane of noninfective epimastigotes and infective metacyclic trypomastigotes, as well as smaller vesicles within the flagellar pocket of both forms. Parasite conditioned culture supernatant was fractionated and characterized by morphological, immunochemical, and proteomic analyses. Three fractions were obtained by differential ultracentrifugation: the first enriched in larger vesicles resembling ectosomes, the second enriched in smaller vesicles resembling exosomes, and a third fraction enriched in soluble proteins not associated with extracellular vesicles. Label-free quantitative proteomic analysis revealed a rich collection of proteins involved in metabolism, signaling, nucleic acid binding, and parasite survival and virulence. These findings support the notion that <i>T. cruzi</i> uses different secretion pathways to excrete/secrete proteins. Moreover, our results suggest that metacyclic forms may use extracellular vesicles to deliver cargo into host cells

Proteomic Analysis of <i>Trypanosoma cruzi</i> Secretome: Characterization of Two Populations of Extracellular Vesicles and Soluble Proteins

Microorganisms use specialized systems to export virulence factors into host cells. Secretion of effector proteins into the extracellular environment has been described in <i>Trypanosoma cruzi</i>; however, a comprehensive proteomic analysis of the secretome and the secretion mechanisms involved remain elusive. Here, we present evidence that <i>T. cruzi</i> releases proteins associated with vesicles that are formed by at least two different mechanisms. Transmission electron microscopy showed larger vesicles budding from the plasma membrane of noninfective epimastigotes and infective metacyclic trypomastigotes, as well as smaller vesicles within the flagellar pocket of both forms. Parasite conditioned culture supernatant was fractionated and characterized by morphological, immunochemical, and proteomic analyses. Three fractions were obtained by differential ultracentrifugation: the first enriched in larger vesicles resembling ectosomes, the second enriched in smaller vesicles resembling exosomes, and a third fraction enriched in soluble proteins not associated with extracellular vesicles. Label-free quantitative proteomic analysis revealed a rich collection of proteins involved in metabolism, signaling, nucleic acid binding, and parasite survival and virulence. These findings support the notion that <i>T. cruzi</i> uses different secretion pathways to excrete/secrete proteins. Moreover, our results suggest that metacyclic forms may use extracellular vesicles to deliver cargo into host cells