Project description:Background: Tsetse flies serve as biological vectors for several species of African trypanosomes. In order to survive, proliferate and establish a midgut infection, trypanosomes must cross the tsetse fly peritrophic matrix (PM), an acellular gut lining surrounding the blood meal. Crossing of this multi layered structure occurs at least twice during parasite migration and development, but the mechanism of how they do so is poorly understood. In order to better comprehend the molecular events surrounding trypanosome crossing of the tsetse PM, a mass spectrometry-based approach was applied to investigate the PM protein composition using Glossina morsitans morsitans as a model organism. Methods: Urea-SDS extracts of tsetse PM proteins were either subject to an in solution tryptic digestion or fractionated on 1D SDS-PAGE and the resulting bands digested with trypsin. The tryptic fragments from both preparations were purified and analysed by 2D-LC-MS/MS. Tandem MS data were searched against the Glossina-morsitans-Yale_PEPTIDES_GmorY1.1 database downloaded from VectorBase (https://www.vectorbase.org/proteomes) using the Mascot (version 2.3.02, Matrix Science) search engine. Search parameters were a precursor mass tolerance of 10 ppm for the in-solution digest using the LTQ-Orbitrap Velos and 0.6 Da for the lower resolution LTQ instrument. Fragment mass tolerance was 0.6 Da for both instruments. One missed cleavage was permitted, carbamidomethylation was set as a fixed modification and oxidation (M) was included as a variable modification. For in-solution data, the false discovery rate was <1%, and individual ion scores >30 were considered to indicate identity or extensive homology (p <0.05 ). Results: Overall, over 200 proteins were identified, several of those containing Chitin Binding Domains (CBD), a signature of insect PM proteins, including novel peritrophins and peritrophin-like glycoproteins, which are essential in maintaining PM architecture and may act as trypanosome adhesins. Furthermore, a minimum of 27 proteins were also identified from the tsetse secondary endosymbiont, Sodalis glossinidius, suggesting this bacterium is probably in close association with the tsetse PM. Conclusion: To our knowledge this is the first report on the protein composition of G. m. morsitans, an important vector of African trypanosomes. Further functional analyses of these proteins will lead to a better understanding of the tsetse physiology as well as to identification of potential targets to block trypanosome development within the tsetse.

Project description:Tsetse flies (Glossina spp.) are major vectors of African trypanosomes, causing either Human or Animal African Trypanosomiasis (HAT or AAT). Several approaches are developed to control the disease among which the anti-vector Sterile Insect Technique. Another approach in the frame of anti-vector strategies could consist in controlling the fly’s vector competence which needs identifying factors (genes, proteins, biological pathways, …) involved in this process. The present work aims to verify whether protein candidates identified under experimental controlled conditions on insectary-reared tsetse flies have their counterpart in field-collected flies. Glossina palpalis palpalis flies naturally infected with Trypanosoma congolense were sampled in two HAT/AAT foci in Southern Cameroon. After dissection, the proteome from guts of parasite-infected flies were compared to that from uninfected flies in order to identify quantitative and/or qualitative changes associated to infection. A total of 3291 proteins were identified of which 1818 could be quantified. The comparative analysis allowed identifying 175 proteins with significant decreased abundance in infected as compared to uninfected flies, while 61 proteins displayed increased abundance. Among the former are RNA binding proteins, kinases, actin, ribosomal proteins, endocytosis proteins, oxido-reductases, as well as proteins that are unusually found such as tsetse salivary proteins (Tsal) or Yolk proteins. Among the proteins with increased abundance are fructose-1,6-biphosphatase, serine proteases, membrane trafficking proteins, death proteins (or apoptosis proteins), and SERPINs (inhibitor of serine proteases, enzymes considered as trypanosome virulence factors) that displayed highest increased abundance. Sodalis, Wiggleswothia and Wolbachia proteins are strongly under-represented, particularly when compared to data from similar experimentation conducted under controlled conditions on T. brucei gambiense infected (or uninfected) G. palpalis gambiensis insectary reared flies. Comparing the overall recorded data, 364 proteins identified in gut extracts from field flies were shown to have a homologue in insectary flies. Discrepancies between the two studies may arise from differences in the species of studied flies and trypanosomes as well as in differences in environmental conditions in which the two experiments were carried out. Finally, the present study together with former proteomic and transcriptomic studies on the secretome of trypanosomes, on the gut extracts from insectary reared and on field collected tsetse flies, provide a pool of data and information on which to draw in order to perform further investigations on, for example, mammal host immunization or on fly vector competence modification via para-transgenic approaches.

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Wigglesworthia glossinidia endosymbiont of Glossina brevipalpis

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