RESUMO
African sleeping sickness is caused by Trypanosoma brucei, a parasite transmitted by the bite of a tsetse fly. Trypanosome infection induces a severe transcriptional downregulation of tsetse genes encoding for salivary proteins, which reduces its anti-hemostatic and anti-clotting properties. To better understand trypanosome transmission and the possible role of glycans in insect bloodfeeding, we characterized the N-glycome of tsetse saliva glycoproteins. Tsetse salivary N-glycans were enzymatically released, tagged with either 2-aminobenzamide (2-AB) or procainamide, and analyzed by HILIC-UHPLC-FLR coupled online with positive-ion ESI-LC-MS/MS. We found that the N-glycan profiles of T. brucei-infected and naïve tsetse salivary glycoproteins are almost identical, consisting mainly (>50%) of highly processed Man3GlcNAc2 in addition to several other paucimannose, high mannose, and few hybrid-type N-glycans. In overlay assays, these sugars were differentially recognized by the mannose receptor and DC-SIGN C-type lectins. We also show that salivary glycoproteins bind strongly to the surface of transmissible metacyclic trypanosomes. We suggest that although the repertoire of tsetse salivary N-glycans does not change during a trypanosome infection, the interactions with mannosylated glycoproteins may influence parasite transmission into the vertebrate host.
Assuntos
Glicoproteínas/metabolismo , Lectinas Tipo C/metabolismo , Polissacarídeos/metabolismo , Proteínas e Peptídeos Salivares/metabolismo , Trypanosoma/metabolismo , Moscas Tsé-Tsé/parasitologia , Animais , Cromatografia Líquida , Concanavalina A , Glicosídeo Hidrolases , Insetos Vetores/parasitologia , Saliva , Glândulas Salivares/parasitologia , Espectrometria de Massas em Tandem , Trypanosoma brucei brucei/genética , Tripanossomíase Africana/parasitologiaRESUMO
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.
Assuntos
Biologia Computacional , Biblioteca Gênica , Proteínas de Insetos/genética , Saliva/química , Glândulas Salivares/química , Triatoma/metabolismo , Sequência de Aminoácidos , Animais , Apirase/química , Sangue , Brasil , Clonagem Molecular , Ingestão de Alimentos , Hemeproteínas , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Insetos Vetores , Filogenia , Glândulas Salivares/fisiologia , Proteínas e Peptídeos Salivares , Alinhamento de Sequência , Análise de Sequência de DNA , Serina Endopeptidases/isolamento & purificação , Serina Endopeptidases/metabolismo , Triatoma/classificação , Triatoma/genética , Trypanosoma cruzi/fisiologiaRESUMO
To assist haematophagy, Rhodnius prolixus produces several bioactive molecules in its saliva which it injects into the host skin. The most abundant of these molecules are the nitrophorins (NPs). In this work, we reduced the expression of NP1-4 in the saliva of R. prolixus by RNAi and evaluated the subsequent feeding performance of the bugs using the cibarial pump electromyogram either on the dorsal skin or on the tail vein of the mice. NPs salivary mRNA was reduced by >99% in comparison to controls. Saliva from knockdown nymphs also presented 82% less haemproteins while the total protein was not reduced. Knockdown nymphs feeding on the skin had lower ingestion rates mainly due to the longer cumulative probing time and lower cibarial pump frequency. Another difference was that knockdown insects bit approximately 5 times more. No differences were observed between groups fed on the tail vein. When the feeding sites were compared, nymphs fed on the tail vein had higher effective ingestion rates. These findings endorse the importance of the NPs for the ability of bugs to complete the meal in a short total contact time with a low number of bites, decreasing the perception of the insect by the host.