Triatomines : trypanosomatids, bacteria, and viruses potential vectors?

Triatominae bugs are the vectors of Chagas disease, a major concern to public health especially in Latin America, where vector-borne Chagas disease has undergone resurgence due mainly to diminished triatomine control in many endemic municipalities. Although the majority of Triatominae species occurs in the Americas, species belonging to the genus Linshcosteus occur in India, and species belonging to the Triatoma rubrofasciata complex have been also identified in Africa, the Middle East, South-East Asia, and in the Western Pacific. Not all of Triatominae species have been found to be infected with Trypanosoma cruzi, but the possibility of establishing vector transmission to areas where Chagas disease was previously non-endemic has increased with global population mobility. Additionally, the worldwide distribution of triatomines is concerning, as they are able to enter in contact and harbor other pathogens, leading us to wonder if they would have competence and capacity to transmit them to humans during the bite or after successful blood feeding, spreading other infectious diseases. In this review, we searched the literature for infectious agents transmitted to humans by Triatominae. There are reports suggesting that triatominesmay be competent vectors for pathogens such as Serratiamarcescens, Bartonella, andMycobacteriumleprae, and that triatomine infection with other microrganisms may interfere with triatomine-T. cruzi interactions, altering their competence and possibly their capacity to transmit Chagas disease

Revealing a novel Otubain-Like Enzyme from Leishmania infantum with deubiquitinating activity toward K48-linked substrate

Deubiquitinating enzymes (DUBs) play an important role in regulating a variety of eukaryotic processes. In this context, exploring the role of deubiquitination in Leishmania infantum could be a promising alternative to search new therapeutic targets for leishmaniasis. Here we present the first characterization of a DUB from L. infantum, otubain (OtuLi), and its localization within parasite. The recombinant OtuLi (rOtuLi) showed improved activity on lysine 48 (K48)-linked over K63-linked tetra-ubiquitin (Ub) and site-directed mutations on amino acids close to the catalytic site (F82) or involved in Ub interaction (L265 and F182) caused structural changes as shown by molecular dynamics, resulting in a reduction or loss of enzyme activity, respectively. Furthermore, rOtuLi stimulates lipid droplet biogenesis (an inflammatory marker) and induces IL-6 and TNF-a secretion in peritoneal macrophages, both proinflammatory cytokines. Our findings suggest that OtuLi is a cytoplasmic enzyme with K48-linked substrate specificity that could play a part in proinflammatory response in stimulated murine macrophages

Triatomines: Trypanosomatids, Bacteria, and Viruses Potential Vectors? [Review]

Triatominae bugs are the vectors of Chagas disease, a major concern to public health especially in Latin America, where vector-borne Chagas disease has undergone resurgence due mainly to diminished triatomine control in many endemic municipalities. Although the majority of Triatominae species occurs in the Americas, species belonging to the genus Linshcosteus occur in India, and species belonging to the Triatoma rubrofasciata complex have been also identified in Africa, the Middle East, South-East Asia, and in the Western Pacific. Not all of Triatominae species have been found to be infected with Trypanosoma cruzi, but the possibility of establishing vector transmission to areas where Chagas disease was previously non-endemic has increased with global population mobility. Additionally, the worldwide distribution of triatomines is concerning, as they are able to enter in contact and harbor other pathogens, leading us to wonder if they would have competence and capacity to transmit them to humans during the bite or after successful blood feeding, spreading other infectious diseases. In this review, we searched the literature for infectious agents transmitted to humans by Triatominae. There are reports suggesting that triatomines may be competent vectors for pathogens such as Serratia marcescens, Bartonella, and Mycobacterium leprae, and that triatomine infection with other microrganisms may interfere with triatomine-T. cruzi interactions, altering their competence and possibly their capacity to transmit Chagas disease

The Trypanosoma cruzi Virulence Factor Oligopeptidase B (OPBTc) Assembles into an Active and Stable Dimer

Oligopeptidase B, a processing enzyme of the prolyl oligopeptidase family, is considered as an important virulence factor in trypanosomiasis. Trypanosoma cruzi oligopeptidase B (OPBTc) is involved in host cell invasion by generating a Ca2+-agonist necessary for recruitment and fusion of host lysosomes at the site of parasite attachment. The underlying mechanism remains unknown and further structural and functional characterization of OPBTc may help clarify its physiological function and lead to the development of new therapeutic molecules to treat Chagas disease. In the present work, size exclusion chromatography and analytical ultracentrifugation experiments demonstrate that OPBTc is a dimer in solution, an association salt and pH-resistant and independent of intermolecular disulfide bonds. The enzyme retains its dimeric structure and is fully active up to 42°C. OPBTc is inactivated and its tertiary, but not secondary, structure is disrupted at higher temperatures, as monitored by circular dichroism and fluorescence spectroscopy. It has a highly stable secondary structure over a broad range of pH, undergoes subtle tertiary structure changes at low pH and is less stable under moderate ionic strength conditions. These results bring new insights into the structural properties of OPBTc, contributing to future studies on the rational design of OPBTc inhibitors as a promising strategy for Chagas disease chemotherapy

Proteases of haematophagous arthropod vectors are involved in blood-feeding, yolk formation and immunity : a review

Ticks, triatomines, mosquitoes and sand flies comprise a large number of haematophagous arthropods considered vectors of human infectious diseases. While consuming blood to obtain the nutrients necessary to carry on life functions, these insects can transmit pathogenic microorganisms to the vertebrate host. Among the molecules related to the blood-feeding habit, proteases play an essential role. In this review, we provide a panorama of proteases from arthropod vectors involved in haematophagy, in digestion, in egg development and in immunity. As these molecules act in central biological processes, proteases from haematophagous vectors of infectious diseases may influence vector competence to transmit pathogens to their prey, and thus could be valuable targets for vectorial control

Prolil oligopeptidases de tripanossomos : aspectos estruturais e funcionais

Tese (doutorado)—Universidade de Brasília, Faculdade de Medicina, 2010.A linha de pesquisa do nosso grupo visa à identificação e caracterização de alvos potenciais para a quimioterapia da doença de Chagas tendo as proteases como principal enfoque, uma vez que estão envolvidas em processos biológicos fundamentais para a viabilidade e virulência de patógenos. Neste contexto, essa tese aborda alguns aspectos estruturais e funcionais de serino-proteases pertencente à família Prolil Oligopeptidase: a oligopeptidase B (OPBTc) e prolil oligopeptidase (POPTc 80) de Trypanosoma cruzi e a prolil oligopeptidase (POP Tb) de Trypanosoma brucei. A OPBTc participa da invasão celular por meio do recrutamento e fusão de lisossomos da célula hospedeira até o local de ligação do parasito por uma via dependente de Ca2+. Por meio deste trabalho, a OPBTc tornou-se a primeira oligopeptidase B a ter sua estrutura dimérica confirmada por ultracentrifugação analítica. Experimentos de dicroísmo circular mostraram que em altas temperaturas, a enzima conserva sua estrutura secundária, mas não a terciária. A perda da estrutura terciária por tratamento térmico acompanha a diminuição da atividade da enzima e a perda da dimerização, que ocorrem a partir de 50 °C. No entanto, a associação do dímero é resistente a altas concentrações de sal e a tratamento com reagentes redutores, indicando que esta interação não ocorre devido a pontes dissulfeto intermoleculares. Este trabalho também mostra a caracterização funcional da POP de T. brucei, protozoário causador da doença do sono. Esta protease foi capaz de hidrolisar colágeno tipo I semi-purificado in vitro e colágeno nativo em mesentério de rato, além de peptídeos hormonais. Foi observado ainda, que POP Tb é liberada na corrente sanguínea de camundongos infectados pelo T. brucei, onde permanece ativa, sugerindo sua contribuição para a patogênese da doença do sono. Como já proposto em estudos prévios, a POP Tc80 está envolvida na entrada do T. cruzi em célula hospedeiras não fagocíticas e alguns de seus substratos naturais foram caracterizados mostrando que a mesmo hidrolisa colágenos do tipo I e IV e fibronectina. O mecanismo desta catálise não é completamente entendido, porém cálculos de interação entre o modelo estrutural teórico da POP Tc80 com o colágeno mostraram que ocorre uma abertura dos domínios da enzima para entrada do substrato. Para verificar essa hipótese, mutações sítios dirigidas na POP Tc80 acarretaram na perda da sua atividade devido à rigidez da enzima, impedindo o acesso do substrato à fenda catalítica. Finalmente, ainda na tentativa de esclarecer o papel da POP Tc80 na infecção do T. cruzi, foi montada uma estratégia para silenciar seu gene. Os resultados preliminares indicam que um alelo do gene poptc80 sofreu recombinação homóloga sendo trocado pelo marcador de seleção, o gene de resistência a neomicina.The research activities of our group aim the identication and characterization of potential targets for chemotherapy oT Chagas:s disease, and proteases are the main focus since they are involved in biological processes essential for the viability and virulence of pathogens. In this context, this thesis addresses some structural and functional features of seine-proteases that belong to the Prolil Oligopeptidase family: the oligopeptidase B (OPBTc) and prolyl oligopeptidase (POP Tc80) of Trypanosoma cruzi and the prolyl oligopeptidase (POPTb) of Trypanosoma brucei. The OPBTc participates in cell invasion through the recruitment and fusion of the host cell lysosomes to the binding site of the parasite by a Ca2+ dependent pathway. In this work, the OPBTc had a structural approach, being the first oligopeptidase B to have its dimeric structure confirmed by analytical ultracentrifugation. Circular dichroism experiments showed that at high temperatures, the enzyme retains its secondary structure but not the tertiary. The loss of tertiary structure by heat treatment follows the decline of enzyme catalytic ability, which occurs from 50 "C. However, the association of the dimer is resistant to salt high concentration and treatment with reducing reagents, indicating that this interaction is not dueto intermolecular disulfide bonds. This work also shows the functional characterization of the enzyme POP of T. brucei {protozoan that causes sleeping sickness), the POPTb. This protease was able to hydrolyze native and semi-purified collagen type I and peptide hormones. In addition, POPTb is released into the bloodstream of T. brucei infected mice, where it remains active, suggesting their contribution to the pathogenesis of sleeping sickness. As shown in previous studies, the POP Tc80 is involved in the entry of the parasite in the non-phagocytic host cell and some of its natural substrates were characterized showing that the enzyme hydrolyzes collagen type I and IV and fibronectin. The mechanism of this catalysis is not completely understood, but docking analysis of the theoretical structural model of POP Tc80 with collagen, showed that the enzyme domains open to substrate entry. To investigate this hypothesis, site directed mutations in POP Tc80 resulted in loss of its activity due to the rigidity of the enzyme, preventing the substrate access to the catalytic pocket. In a attempt to clarify the role of POP Tc80 in T. cruzi infection, it was set up a strategy to silence its gene. Preliminary results indicate that one allele of the poptc80 gene has been replaced by the selection marquee, the gene for resistance to neomycin, through homologous recombination

Identification of novel Trypanosoma cruzi prolyl oligopeptidase inhibitors by structure-based virtual screening

International audienceWe have previously demonstrated that the secreted prolyl oligopeptidase of Trypanosoma cruzi (POPTc80) is involved in the infection process by facilitating parasite migration through the extracellular matrix. We have built a 3D structural model where POPTc80 is formed by a catalytic α/β-hydrolase domain and a β-propeller domain, and in which the substrate docks at the inter-domain interface, suggesting a "jaw opening" gating access mechanism. This preliminary model was refined by molecular dynamics simulations and next used for a virtual screening campaign, whose predictions were tested by standard binding assays. This strategy was successful as all 13 tested molecules suggested from the in silico calculations were found out to be active POPTc80 inhibitors in the micromolar range (lowest K i at 667 nM). This work paves the way for future development of innovative drugs against Chagas disease