RESUMEN
Neutrophil extracellular traps (NETs) evolved as a unique effector mechanism contributing to resistance against infection that can also promote tissue damage in inflammatory conditions. Malaria infection can trigger NET release, but the mechanisms and consequences of NET formation in this context remain poorly characterized. Here we show that patients suffering from severe malaria had increased amounts of circulating DNA and increased neutrophil elastase (NE) levels in plasma. We used cultured erythrocytes and isolated human neutrophils to show that Plasmodium-infected red blood cells release macrophage migration inhibitory factor (MIF), which in turn caused NET formation by neutrophils in a mechanism dependent on the C-X-C chemokine receptor type 4 (CXCR4). NET production was dependent on histone citrullination by peptidyl arginine deiminase-4 (PAD4) and independent of reactive oxygen species (ROS), myeloperoxidase (MPO) or NE. In vitro, NETs functioned to restrain parasite dissemination in a mechanism dependent on MPO and NE activities. Finally, C57/B6 mice infected with P. berghei ANKA, a well-established model of cerebral malaria, presented high amounts of circulating DNA, while treatment with DNAse increased parasitemia and accelerated mortality, indicating a role for NETs in resistance against Plasmodium infection.
Asunto(s)
Eritrocitos/inmunología , Trampas Extracelulares/inmunología , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Malaria/inmunología , Neutrófilos/inmunología , Plasmodium/inmunología , Receptores CXCR4/metabolismo , Animales , Eritrocitos/metabolismo , Eritrocitos/parasitología , Trampas Extracelulares/metabolismo , Trampas Extracelulares/parasitología , Humanos , Malaria/metabolismo , Malaria/parasitología , Malaria/patología , Ratones , Ratones Endogámicos C57BL , Neutrófilos/metabolismo , Neutrófilos/parasitología , Parasitemia/inmunología , Parasitemia/metabolismo , Parasitemia/parasitología , Parasitemia/patologíaRESUMEN
The synganglion is the central nervous system of ticks and, as such, controls tick physiology. It does so through the production and release of signaling molecules, many of which are neuropeptides. These peptides can function as neurotransmitters, neuromodulators and/or neurohormones, although in most cases their functions remain to be established. We identified and performed in silico characterization of neuropeptides present in different life stages and organs of Rhipicephalus microplus, generating transcriptomes from ovary, salivary glands, fat body, midgut and embryo. Annotation of synganglion transcripts led to the identification of 32 functional categories of proteins, of which the most abundant were: secreted, energetic metabolism and oxidant metabolism/detoxification. Neuropeptide precursors are among the sequences over-represented in R. microplus synganglion, with at least 5-fold higher transcription compared with other stages/organs. A total of 52 neuropeptide precursors were identified: ACP, achatin, allatostatins A, CC and CCC, allatotropin, bursicon A/B, calcitonin A and B, CCAP, CCHamide, CCRFamide, CCH/ITP, corazonin, DH31, DH44, eclosion hormone, EFLamide, EFLGGPamide, elevenin, ETH, FMRFamide myosuppressin-like, glycoprotein A2/B5, gonadulin, IGF, inotocin, insulin-like peptides, iPTH, leucokinin, myoinhibitory peptide, NPF 1 and 2, orcokinin, proctolin, pyrokinin/periviscerokinin, relaxin, RYamide, SIFamide, sNPF, sulfakinin, tachykinin and trissin. Several of these neuropeptides have not been previously reported in ticks, as the presence of ETH that was first clearly identified in Parasitiformes, which include ticks and mites. Prediction of the mature neuropeptides from precursor sequences was performed using available information about these peptides from other species, conserved domains and motifs. Almost all neuropeptides identified are also present in other tick species. Characterizing the role of neuropeptides and their respective receptors in tick physiology can aid the evaluation of their potential as drug targets.
Asunto(s)
Ixodidae , Neuropéptidos , Rhipicephalus , Animales , Femenino , Ixodidae/metabolismo , Neuropéptidos/química , Neuropéptidos/genética , Neuropéptidos/metabolismo , Péptidos , Rhipicephalus/genética , Rhipicephalus/metabolismo , TranscriptomaRESUMEN
Triatoma dimidiata, a Chagas disease vector widely distributed along Central America, has great capability for domestic adaptation as the majority of specimens caught inside human dwellings or in peridomestic areas fed human blood. Exploring the salivary compounds that overcome host haemostatic and immune responses is of great scientific interest. Here, we provide a deeper insight into its salivary gland molecules. We used high-throughput RNA sequencing to examine in depth the T. dimidiata salivary gland transcriptome. From >51 million reads assembled, 92.21% are related to putative secreted proteins. Lipocalin is the most abundant gene family, confirming it is an expanded family in Triatoma genus salivary repertoire. Other putatively secreted members include phosphatases, odorant binding protein, hemolysin, proteases, protease inhibitors, antigen-5 and antimicrobial peptides. This work expands the previous set of functionally annotated sequences from T. dimidiata salivary glands available in NCBI from 388 to 3815. Additionally, we complemented the salivary analysis through proteomics (available data via ProteomeXchange with identifier PXD008510), disclosing the set complexity of 119 secreted proteins and validating the transcriptomic results. Our large-scale approach enriches the pharmacologically active molecules database and improves our knowledge about the complexity of salivary compounds from haematophagous vectors and their biological interactions. SIGNIFICANCE: Several haematophagous triatomine species can transmit Trypanosoma cruzi, the etiological agent of Chagas disease. Due to the reemergence of this disease, new drugs for its prevention and treatment are considered priorities. For this reason, the knowledge of vector saliva emerges as relevant biological finding, contributing to the design of different strategies for vector control and disease transmission. Here we report the transcriptomic and proteomic compositions of the salivary glands (sialome) of the reduviid bug Triatoma dimidiata, a relevant Chagas disease vector in Central America. Our results are robust and disclosed unprecedented insights into the notable diversity of its salivary glands content, revealing relevant anti-haemostatic salivary gene families. Our work expands almost ten times the previous set of functionally annotated sequences from T. dimidiata salivary glands available in NCBI. Moreover, using an integrated transcriptomic and proteomic approach, we showed a correlation pattern of transcription and translation processes for the main gene families found, an important contribution to the research of triatomine sialomes. Furthermore, data generated here reinforces the secreted proteins encountered can greatly contribute for haematophagic habit, Trypanosoma cruzi transmission and development of therapeutic agent studies.
Asunto(s)
Glándulas Salivales/química , Triatoma/química , Animales , Enfermedad de Chagas/transmisión , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Insectos Vectores/genética , Transcriptoma/genética , Triatoma/genéticaRESUMEN
Triatoma brasiliensis is the most important autochthon vector of Trypanosoma cruzi in Brazil, where it is widely distributed in the semiarid areas of the Northeast. In order to advance the knowledge of the salivary biomolecules of Triatominae, a salivary gland cDNA library of T. brasiliensis was mass sequenced and analyzed. Polypeptides were sequenced by HPLC/Edman degradation experiments. Then 1712 cDNA sequences were obtained and grouped in 786 clusters. The housekeeping category had 24.4% and 17.8% of the clusters and sequences, respectively. The putatively secreted category contained 47.1% of the clusters and 68.2% of the sequences. Finally, 28.5% of the clusters, containing 14% of all sequences, were classified as unknown. The sialoma of T. brasiliensis showed a high amount and great variety of different lipocalins (93.8% of secreted proteins). Remarkably, a great number of serine proteases that were not observed in previous blood-sucking sialotranscriptomes were found. Nine Kazal peptides were identified, among them one with high homology to the tabanid vasodilator vasotab, suggesting that the Triatoma vasodilator could be a Kazal protein.
Asunto(s)
Biología Computacional , Biblioteca de Genes , Proteínas de Insectos/genética , Saliva/química , Glándulas Salivales/química , Triatoma/metabolismo , Secuencia de Aminoácidos , Animales , Apirasa/química , Sangre , Brasil , Clonación Molecular , Ingestión de Alimentos , Hemoproteínas , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Insectos Vectores , Filogenia , Glándulas Salivales/fisiología , Proteínas y Péptidos Salivales , Alineación de Secuencia , Análisis de Secuencia de ADN , Serina Endopeptidasas/aislamiento & purificación , Serina Endopeptidasas/metabolismo , Triatoma/clasificación , Triatoma/genética , Trypanosoma cruzi/fisiologíaRESUMEN
BACKGROUND: The completed sequence of the Anopheles gambiae genome has enabled genome-wide analyses of gene expression and regulation in this principal vector of human malaria. These investigations have created a demand for efficient methods of cataloguing and analyzing the large quantities of data that have been produced. The organization of genome-wide data into one unified database makes possible the efficient identification of spatial and temporal patterns of gene expression, and by pairing these findings with comparative algorithms, may offer a tool to gain insight into the molecular mechanisms that regulate these expression patterns. DESCRIPTION: We provide a publicly-accessible database and integrated data-mining tool, angaGEDUCI, that unifies 1) stage- and tissue-specific microarray analyses of gene expression in An. gambiae at different developmental stages and temporal separations following a bloodmeal, 2) functional gene annotation, 3) genomic sequence data, and 4) promoter sequence comparison algorithms. The database can be used to study genes expressed in particular stages, tissues, and patterns of interest, and to identify conserved promoter sequence motifs that may play a role in the regulation of such expression. The database is accessible from the address http://www.angaged.bio.uci.edu. CONCLUSION: By combining gene expression, function, and sequence data with integrated sequence comparison algorithms, angaGEDUCI streamlines spatial and temporal pattern-finding and produces a straightforward means of developing predictions and designing experiments to assess how gene expression may be controlled at the molecular level.
Asunto(s)
Algoritmos , Anopheles/genética , Secuencia Conservada/genética , Bases de Datos Genéticas , Perfilación de la Expresión Génica , Regiones Promotoras Genéticas/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Animales , Sitios de Unión/genética , Genes de Insecto/genética , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción/metabolismo , Interfaz Usuario-ComputadorRESUMEN
We have carried out a survey of the genome of Leishmania (Viannia) braziliensis by shotgun sequencing. Approximately 15% of the haploid genome of the parasite (5.15 Mb of genomic sequence) was obtained. A large number of known and putative genes, predicted to be involved in several cellular processes, were identified. Some genomic features were investigated, such as the general G + C content, which was found to be lower than L. major (57% versus 63%). BlastN searches revealed that 60.2% of the clusterized GSS sequences displayed similarity to L. major genomic sequences, while a BlastX search showed that 45.3% of the thus obtained predicted protein sequences showed similarity to annotated proteins of L. major. Further comparison of the degree of conservation between L. major and L. braziliensis revealed that coding regions are much more conserved than non-coding ones. The shotgun sequence analysis of Leishmania braziliensis appears to be an efficient and suitable strategy contributing to the search for vaccines and novel drug targets. The sequence data described in this paper have been submitted to the dbGSS database under the following accession numbers (BX530413 to BX530454; BX530456 to BX530718; BX538354 to BX539305; BX539350 to BX540325; BX541002 to BX544869; BX544893 to BX545685; BX897701 to BX897710; BX905184 to BX907797; BX907798 to BX908381; BX908403 to BX908718). All data including sequences are also available at (www.ebi.ac.uk/embl/).
Asunto(s)
Genoma de Protozoos , Leishmania braziliensis/genética , Análisis de Secuencia de ADN , Animales , Composición de Base , Secuencia Conservada , ADN Protozoario/química , ADN Protozoario/aislamiento & purificación , Genes Protozoarios , Leishmania major/genética , Datos de Secuencia Molecular , Familia de Multigenes , Sistemas de Lectura Abierta , Homología de SecuenciaRESUMEN
The quest for new control strategies for ticks can profit from high throughput genomics. In order to identify genes that are involved in oogenesis and development, in defense, and in hematophagy, the transcriptomes of ovaries, hemocytes, and salivary glands from rapidly ingurgitating females, and of salivary glands from males of Boophilus microplus were PCR amplified, and the expressed sequence tags (EST) of random clones were mass sequenced. So far, more than 1,344 EST have been generated for these tissues, with approximately 30% novelty, depending on the the tissue studied. To date approximately 760 nucleotide sequences from B. microplus are deposited in the NCBI database. Mass sequencing of partial cDNAs of parasite genes can build up this scant database and rapidly generate a large quantity of useful information about potential targets for immunobiological or chemical control.
Asunto(s)
ADN Complementario/análisis , Biblioteca de Genes , Oogénesis/genética , Garrapatas/genética , Garrapatas/patogenicidad , Animales , Bovinos , Enfermedades de los Bovinos/parasitología , Bases de Datos Genéticas , Femenino , Hemocitos , Masculino , Ovario , Reacción en Cadena de la Polimerasa , Glándulas Salivales , Análisis de Secuencia de ADNRESUMEN
The hosts for Antricola delacruzi ticks are insectivorous, cave-dwelling bats on which only larvae are found. The mouthparts of nymphal and adult A. delacruzi are compatible with scavenging feeding because the hypostome is small and toothless. How a single blood meal of a larva provides energy for several molts as well as for oviposition by females is not known. Adults of A. delacruzi possibly feed upon an unknown food source in bat guano, a substrate on which nymphal and adult stages are always found. Guano produced by insectivorous bats contains twice the amount of protein and 60 times the amount of iron as beef. In addition, bacteria and chitin-rich fungi proliferate on guano. Comparative data on the transcriptome of the salivary glands of A. delacruzi is nonexistent and would help to understand the physiological adaptations of salivary glands that accompany different sources of food as well as the steps taken by the Acari toward haematophagy, believed to have evolved from scavenging dead animals. Annotation of the transcriptome of salivary glands from female instars of A. delacruzi collected on guano categorized 5.7% of the clusters of expressed genes as putative secreted proteins. They included abundantly expressed TIL-domain-containing proteins (possible anti-microbials), an abundantly expressed protein similar to a serum amyloid found in the sialotranscriptomes of Ornithodoros spp., a savignygrin, a family of mucin/peritrophin/cuticle-like proteins, anti-microbials and an HIV envelope-like glycoprotein also found in soft ticks. When comparing the transcriptome of A. delacruzi with those of blood-feeding female soft and hard ticks some notable differences were observed; they consisted of the following transcripts over- or under-represented or absent in the sialotranscriptome of A. delacruzi that may reflect its source of food: ferritin, mucins with chitin-binding domains and TIL-domain-containing proteins versus lipocalins, basic tail proteins, metalloproteases, glycine-rich proteins and Kunitz protease inhibitors, respectively.
Asunto(s)
Argasidae/metabolismo , Conducta Alimentaria , Saliva/metabolismo , Transcriptoma , Adaptación Fisiológica , Secuencia de Aminoácidos , Animales , Argasidae/genética , Quirópteros/parasitología , Heces , Femenino , Perfilación de la Expresión Génica , Biblioteca de Genes , Genes Esenciales , Especiación Genética , Datos de Secuencia Molecular , Glándulas Salivales/metabolismoRESUMEN
Aegyptin is a 30 kDa mosquito salivary gland protein that binds to collagen and inhibits platelet aggregation. We have studied the biophysical properties of aegyptin and its mechanism of action. Light-scattering plot showed that aegyptin has an elongated monomeric form, which explains the apparent molecular mass of 110 kDa estimated by gel-filtration chromatography. Surface plasmon resonance identified the sequence RGQOGVMGF (where O is hydroxyproline) that mediates collagen interaction with von Willebrand factor (vWF) as a high-affinity binding site for aegyptin, with a K(D) of approximately 5 nM. Additionally, aegyptin interacts with the linear peptide RGQPGVMGF and heat-denatured collagen, indicating that the triple helix and hydroxyproline are not a prerequisite for binding. However, aegyptin does not interact with scrambled RGQPGVMGF peptide. Aegyptin also recognizes the peptides (GPO)(10) and GFOGER with low affinity (microM range), which respectively represent glycoprotein VI and integrin alpha2beta1 binding sites in collagen. Truncated forms of aegyptin were engineered, and the C-terminus fragment was shown to interact with collagen and to attenuate platelet aggregation. In addition, aegyptin prevents laser-induced carotid thrombus formation in the presence of Rose Bengal in vivo, without significant bleeding in rats. In conclusion, aegyptin interacts with distinct binding sites in collagen, and is useful tool to inhibit platelet-collagen interaction in vitro and in vivo.