Análise proteômica comparativa da saliva entre diferentes períodos após a alimentação sanguínea de Triatoma dimidiata, triatomíneo vetor da doença de Chagas

Dissertação (mestrado) — Universidade de Brasília, Faculdade de Medicina, Programa de Pós-Graduação em Ciências Médicas, 2016.Os triatomíneos são artrópodes hematófagos durante todo o seu ciclo de vida e para garantir o sucesso de seu repasto, desenvolveram estratégias adaptativas que contrapõem o sistema hemostático e imune do hospedeiro, injetando moléculas farmacologicamente ativas durante esse processo. Por possuirem diversas moléculas com interesse farmacológico, a saliva desses artrópodes hematófagos passou a ser alvo de pesquisas. Para ampliar o conhecimento sobre essas moléculas, esse trabalho teve como objetivos: 1) investigar a expressão protéica na saliva de Triatoma dimidiata em diferentes tempos após a alimentação sanguínea, por meio da análise proteômica comparativa por LC-MS/MS, eletroforese bidimensional e análises in silico, e 2) caracterizar in silico a sequência aminoacídica do antígeno 5 salivar de Rhodnius neglectus (RNAV), além da expressão dessa molécula em Escherichia coli. A análise proteômica comparativa da saliva de T. dimidiata levou à identificação de 362 proteínas, das quais 65 eram específicas da amostra de cinco dias após a alimentação sanguínea (dpa) e 79 específicas da amostra de 10 dpa, além de contribuir para validar dados do transcritoma dessa espécie disponível na literatura. A análise in silico do RNAV sugere que a molécula é secretada pela via clássica, que modificações pós-traducionais como fosforilação e glicosilação devem ocorrer na proteína, e que existem aproximadamente 25 resíduos de aminoácidos que podem estar presentes em epítopos lineares para células B. Não foi possível obter o RNAV recombinante. O conhecimento das proteínas salivares do T. dimidiata e sua comparação com as de outras espécies de artrópodes hematófagos pode ser útil para entender processos biológicos fundamentais como sobrevivência, adaptação e as interações desses vetores com seus hospedeiros e com os agentes infecciosos que eles são capazes de transmitir.Triatomine are bloodsucking arthropods throughout their life cycle and to ensure the success of their meal, they have developed adaptive strategies that counteract the hemostatic and immune system of the host, injecting pharmacologically active molecules during this process. By owning several molecules with pharmacological interest, bloodsucking arthropods saliva has become the subject of various research. To enhance our knowledge about these molecules, this study aimed 1) to investigate the protein expression in Triatoma dimidiata saliva at different times after the blood feeding through comparative proteomic analysis using LC-MS/MS, 2-DE electrophoresis and bioinformatics, and 2) to characterize in silico the salivary antigen 5 of Rhodnius neglectus (RNAV), beyond its expressin in Escherichia coli. The comparative proteomic analysis of T. dimidiata saliva revealed 362 proteins, 65 specific of the sample collected 5 days after feeding and 79 specific of the sample collected 10 days after feeding, and aided to validate available data from T. dimidiata transcriptome. RNAV in silico analysis revealed this protein may be secreted by the classical pathway, phosphorilation and glycosilation sites, and the existence of 25 amino acid residues that may be present in linear epitopes to B cells. Our attempt failed to produce recombinant RNAV. Knowing T. dimidiata salivary proteins and comparison with those from other species of hematophagous arthropods may help to understand fundamental biological processes such as survival, adaptation, vector-host and vector-pathogens interactions

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 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

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

Sialoma do Triatoma sordida, triatomíneo peridomiciliar vetor da Doença de Chagas

Tese (doutorado) — Universidade de Brasília, Faculdade de Ciências Médicas, Programa de Pós-Graduação em Ciências Médicas, 2021.Os triatomíneos são os vetores da Doença de Chagas (DC), por serem hematófagos, desenvolveram adaptações morfológicas e moleculares para conseguirem se alimentar de sangue e assim adquirir os nutrientes necessários para a sua sobrevivência e reprodução. A saliva destes artrópodes, é constituída de moléculas que interagem com os sistemas hemostático e imune dos hospedeiros vertebrados, a fim de contrapô-los, manter o sangue fluido e garantir o sucesso no repasto. Com o intuito de compreender a complexidade das moléculas que estão envolvidas neste processo, o objetivo do presente estudo foi investigar o perfil de expressão dos genes das glândulas salivares do Triatoma sordida, triatomíneo peridomiciliar e importante vetor da DC no Brasil. Foi realizado o sequenciamento de alto rendimento do tipo Illumina com o RNA total extraído das glândulas salivares e espectrometria de massas do tipo LC-MS/MS com amostra da saliva, obtivemos 57.645.372 reads que foram montadas em 26.670 contigs (CDS). Destes, 16.683 (62,6% CDS) foram anotados e separados em 7 categorias, destas, 3 se destacaram: Secretadas (FPKM= 79,5%), Proteínas constitutivas (FPKM= 12,1%) e Desconhecidas (FPKM= 7,6%). Entre os transcritos com maior expressão podemos destacar as Lipocalinas, Trialisina curta/Trialisina, Inibidor de serino protease do tipo Kazal, Antígeno 5, Tripsina Salivar, Inositol fosfato fosfatase. No proteoma, 132 proteínas foram identificadas, dentre os grupos com maior quantidade de espectros (Ce), temos: Lipocalinas (Ce= 42%), Enzimas (Ce= 22,5%), Famílias específicas de Hemípteras (Ce= 10,1%), Famílias de Proteínas Ubíquas (Ce= 5,9%), Outras proteínas salivares secretadas (Ce= 4,9%) e Inibidores de Protease (Ce= 1,4%). No proteoma também foi relatada pela primeira vez a Anexina, que pode estar envolvida coma imunidade do inseto. Neste trabalho, apresentamos o primeiro sialoma do T. sordida, demonstrando o padrão de expressão das proteínas salivares. Os resultados obtidos corroboram com alguns já descritos em outros sialomas de triatomíneos. O estudo dos compostos salivares auxilia a compreensão acerca da biologia dos vetores, da evolução da hematofagia, além de demonstrar o potencial biotecnológico existente nestes animais.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) e Fundação de Apoio a Pesquisa do Distrito Federal (FAP/DF).Triatomines are the vectors of Chagas disease (CD), as they are hematophagous, they have developed morphological and molecular adaptations in order to be able to feed on blood and thus acquire the nutrients necessary for their reproduction and survival. The saliva of these arthropods is composed of molecules that interact with the hemostatic and immune systems of vertebrate hosts, so as to neutralize them, maintain blood fluid and ensure a successful meal. Intending to understand the complexity of the molecules involved in this process, the aim of this study was to investigate the expression profile of genes in the salivary glands of Triatoma sordida, peridomiciliary triatomine and an important vector of CD in Brazil. Using high-throughput Illumina – type sequencing performed with total RNA extracted from salivary glands and LCMS/MS type mass spectrometry with saliva sample, obtained 57,645,372 reads that were mounted in 26,670 contigs (CDS). Of these, 16,683 (62.6% CDS) were annoted and separated into 7 categories which 3 stood out: Secreted (79.5% FPKM), Housekeeping (12,1% FPKM) and Unknowns (7.6%FPKM). Among the transcripts with greater expression, we can highlight Lipocalins, Short triallysin/triallysin, Kazal type, Antigen 5, Salivary trypsin, Inositol phosphate phosphatase. In the proteome, 132 proteins were identified , among the groups with the greatest amount of spectra, we have : Lipocalins (42%Sc), Enzymes (22,5%Sc), Hemiptera specific families (10,1% Sc),Ubiquitous protein families (5,9%Sc), Other salivary secreted protein (4,9%Sc) and Protease inhibitors (1,4% Sc). Annexin was also reported for the first time in the proteome, which may be involved in the insect’s immunity. The results obtained corroborate those already described in other triatomine sialomes. The study of salivary compounds helps to understand the biology of vectors, the evolution of the hematophagy, in addition to demonstrating the biotechnological potential existing in these animals

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

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