Atividade proteolítica salivar de triatomíneos vetores da Doença de Chagas

Trabalho de Conclusão de Curso (graduação)—Universidade de Brasília, Faculdade de Ceilândia, 2017.Os triatomíneos hematófagos, vetores da Doença de Chagas, são insetos cujo crescimento, mudas e reprodução são regulados pelo repasto sanguíneo. Alguns RNAs mensageiros de proteases foram descritos em sialotranscriptomas de triatomíneos hematófagos, sugerindo que essas proteínas sejam expressas na saliva desses animais. O objetivo desse estudo foi identificar e caracterizar atividades proteolíticas na saliva de Rhodnius prolixus e R. neglectus em diferentes dias pós-repasto sanguíneo (dprs). Os triatomíneos foram mantidos em colônia no Biotério do IB, UnB. Cinco, 12 e 20 dprs, as glândulas salivares foram dissecadas para a obtenção da saliva. O perfil proteico foi analisado por eletroforese em SDS-PAGE; a atividade gelatinolítica por zimografia em gel; e a atividade proteolítica sobre o substrato fluorogênico Z-Phe-Arg-AMC em solução, na ausência ou presença de inibidores de proteases. O perfil proteico das salivas nos diferentes períodos após o repasto sanguíneo é semelhante. As proteínas salivares que apresentaram atividade proteolítica no ensaio zimográfico possuem massas moleculares entre 20 e 70 kDa. As salivas apresentaram atividade enzimática sobre o peptídeo fluorogênico Z-Phe-Arg-AMC, inibidas apenas nas amostras de 20 dias após o repasto sanguíneo por fenantrolina (R. prolixus, 48,86%; e R. neglectus, 38,43% de inibição). Esses resultados sugerem a presença, principalmente, de metaloproteases na saliva de R. prolixus e R. neglectus. No entanto, uma investigação mais profunda é necessária e poderá ajudar a elucidar o papel dessas proteases salivares no processamento de peptídeos bioativos presentes na saliva e/ou no hospedeiro vertebrado e suas implicações para o repasto sanguíneo e a transmissão do T. cruzi.Hematophagous triatomines, vectors of Chagas Disease, are insects which growth, molts and reproduction are regulated by blood meal. Some protease mRNAs have been described in sialotranscriptomes of hematophagous triatomines, suggesting these proteins are expressed in their saliva. This study aimed to identify and to characterize proteolytic activities in Rhodnius prolixus and R. neglectus saliva in different days after the blood meal has been taken (dabm). The triatomines were reared in the Insectarium of the Biological Sciences Institute, UnB. Five, 12 and 20 dabm, salivary glands were dissected to obtain saliva. Protein profile was analyzed by electrophoresis in SDS-PAGE; gelatinolytic activity by gel-zymography; and proteolytic activity on fluorogenic substrate Z-Phe-Arg-AMC in solution, in protease inhibitors absence or presence. Saliva protein profile in different dabm is similar. Saliva proteins that showed proteolytic activity in gel-zymography have molecular masses ranging from 20-70 kDa. Salivas showed enzymatic activity in fluorogenic peptide Z-Phe-Arg-AMC, inhibited only in 20 dabm by phenanthroline (R. prolixus, 48.86 %; R. neglectus, 38.43 % of inhibition). These results suggest expression, mainly, of metalloproteases in R. prolixus and R. neglectus saliva. However, a deep investigation is necessary and may help to elucidate the salivary proteases roles in the generation of bioactive peptides in saliva and/or in the vertebrate host and their implications to blood meal and T. cruzi transmission

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

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

Insights into the microRNA landscape of Rhodnius prolixus, a vector of Chagas disease

Abstract The growing interest in microRNAs (miRNAs) over recent years has led to their characterization in numerous organisms. However, there is currently a lack of data available on miRNAs from triatomine bugs (Reduviidae: Triatominae), which are the vectors of the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease. A comprehensive understanding of the molecular biology of vectors provides new insights into insect-host interactions and insect control approaches, which are key methods to prevent disease incidence in endemic areas. In this work, we describe the miRNome profiles from gut, hemolymph, and salivary gland tissues of the Rhodnius prolixus triatomine. Small RNA sequencing data revealed abundant expression of miRNAs, along with tRNA- and rRNA-derived fragments. Fifty-two mature miRNAs, previously reported in Ecdysozoa, were identified, including 39 ubiquitously expressed in the three tissues. Additionally, 112, 73, and 78 novel miRNAs were predicted in the gut, hemolymph, and salivary glands, respectively. In silico prediction showed that the top eight most highly expressed miRNAs from salivary glands potentially target human blood-expressed genes, suggesting that R. prolixus may modulate the host’s gene expression at the bite site. This study provides the first characterization of miRNAs in a Triatominae species, shedding light on the role of these crucial regulatory molecules

Proteomic mapping of multifunctional complexes within Triatomine saliva

Triatomines are hematophagous insects that transmit Trypanosoma cruzi, the etiological agent of Chagas disease. This neglected tropical disease represents a global health issue as it is spreading worldwide. The saliva of Triatominae contains miscellaneous proteins crucial for blood feeding acquisition, counteracting host's hemostasis while performing vasodilatory, anti-platelet and anti-coagulant activities, besides modulating inflammation and immune responses. Since a set of biological processes are mediated by protein complexes, here, the sialocomplexomes (salivary protein complexes) of five species of Triatominae were studied to explore the protein-protein interaction networks. Salivary multiprotein complexes from Triatoma infestans, Triatoma dimidiata, Dipetalogaster maxima, Rhodnius prolixus, and Rhodnius neglectus were investigated by Blue-Native- polyacrylamide gel electrophoresis coupled with liquid chromatography tandem mass spectrometry. More than 70 protein groups, uncovering the landscape of the Triatominae salivary interactome, were revealed. Triabin, actin, thioredoxin peroxidase and an uncharacterized protein were identified in sialocomplexes of the five species, while hexamerin, heat shock protein and histone were identified in sialocomplexes of four species. Salivary proteins related to triatomine immunity as well as those required during blood feeding process such as apyrases, antigen 5, procalins, and nitrophorins compose different complexes. Furthermore, unique proteins for each triatomine species were revealed. This study represents the first Triatominae sialocomplexome reference to date and shows that the approach used is a reliable tool for the analysis of Triatominae salivary proteins assembled into complexes

An integrative sialomic analysis reveals molecules from Triatoma sordida (Hemiptera: Reduviidae)

Triatomines have evolved salivary glands that produce versatile molecules with various biological functions, including those leading their interactions with vertebrate hosts’ hemostatic and immunological systems. Here, using high-throughput transcriptomics and proteomics, we report the first sialome study on the synanthropic triatomine Triatoma sordida. As a result, 57,645,372 reads were assembled into 26,670 coding sequences (CDS). From these, a total of 16,683 were successfully annotated. The sialotranscriptomic profile shows Lipocalin as the most abundant protein family within putative secreted transcripts. Trialysins and Kazal-type protease inhibitors have high transcript levels followed by ubiquitous protein families and enzyme classes. Interestingly, abundant trialysin and Kazal-type members are highlighted in this triatomine sialotranscriptome. Furthermore, we identified 132 proteins in T. sordida salivary gland soluble extract through LC-MS/MS spectrometry. Lipocalins, Hemiptera specific families, CRISP/Antigen-5 and Kazal-type protein inhibitors proteins were identified. Our study provides a comprehensive description of the transcript and protein compositions of the salivary glands of T. sordida. It significantly enhances the information in the Triatominae sialome databanks reported so far, improving the understanding of the vector’s biology, the hematophagous behaviour, and the Triatominae subfamily’s evolution