Dataset Information

Towards improving tsetse fly paratransgenesis: stable colonization of Glossina morsitans morsitans with genetically modified Sodalis.

ABSTRACT: BACKGROUND:Tsetse flies (Glossina sp.) refractory to trypanosome infection are currently being explored as potential tools to contribute in the control of human and animal African trypanosomiasis. One approach to disrupt trypanosome transmission by the tsetse fly vector involves the use of paratransgenesis, a technique that aims to reduce vector competence of disease vectors via genetic modification of their microbiota. An important prerequisite for developing paratransgenic tsetse flies is the stable repopulation of tsetse flies and their progeny with its genetically modified Sodalis symbiont without interfering with host fitness. RESULTS:In this study, we assessed by qPCR analysis the ability of a chromosomally GFP-tagged Sodalis (recSodalis) strain to efficiently colonize various tsetse tissues and its transmission to the next generation of offspring using different introduction approaches. When introduced in the adult stage of the fly via thoracic microinjection, recSodalis is maintained at high densities for at least 21 days. However, no vertical transmission to the offspring was observed. Oral administration of recSodalis did not lead to the colonization of either adult flies or their offspring. Finally, introduction of recSodalis via microinjection of third-instar larvae resulted in stably colonized adult tsetse flies. Moreover, the subsequent generations of offspring were also efficiently colonized with recSodalis. We show that proper colonization of the female reproductive tissues by recSodalis is an important determinant for vertical transmission. CONCLUSIONS:Intralarval microinjection of recSodalis proves to be essential to achieve optimal colonization of flies with genetically modified Sodalis and its subsequent dissemination into the following generations of progeny. This study provides the proof-of-concept that Sodalis can be used to drive expression of exogenous transgenes in Glossina morsitans morsitans colonies representing a valuable contribution to the development of a paratransgenic tsetse fly based control strategy.

SUBMITTER: De Vooght L

PROVIDER: S-EPMC6251102 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Towards improving tsetse fly paratransgenesis: stable colonization of Glossina morsitans morsitans with genetically modified Sodalis.

De Vooght Linda L Van Keer Severien S Van Den Abbeele Jan J

BMC microbiology 20181123 Suppl 1

<h4>Background</h4>Tsetse flies (Glossina sp.) refractory to trypanosome infection are currently being explored as potential tools to contribute in the control of human and animal African trypanosomiasis. One approach to disrupt trypanosome transmission by the tsetse fly vector involves the use of paratransgenesis, a technique that aims to reduce vector competence of disease vectors via genetic modification of their microbiota. An important prerequisite for developing paratransgenic tsetse flies ...[more]

PMID: 30470181

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Project description:Tsetse flies (Glossina spp.) feed exclusively on vertebrate blood. After a blood meal, the enteric endosymbiont Sodalis glossinidius is exposed to various environmental stressors including high levels of heme. To investigate how S. glossinidius morsitans (Sgm), the Sodalis subspecies that resides within the gut of G. morsitans, tolerates the heme-induced oxidative environment of tsetse's midgut, we used RNAseq to identify bacterial genes that are differentially expressed in cells cultured in high versus lower heme environments. Our analysis identified 436 genes that were significantly differentially expressed (> or < 2-fold) in the presence of high heme [219 heme-induced genes (HIGs) and 217 heme-repressed genes (HRGs)]. HIGs were enriched in Gene Ontology (GO) terms related to regulation of a variety of biological functions, including gene expression and metabolic processes. We observed that 11 out of 13 Sgm genes that were heme regulated in vitro were similarly regulated in bacteria that resided within tsetse's midgut 24 hr (high heme environment) and 96 hr (low heme environment) after the flies had consumed a blood meal. We used intron mutagenesis to make insertion mutations in 12 Sgm HIGs and observed no significant change in growth in vitro in any of the mutant strains in high versus low heme conditions. However, Sgm strains that carried mutations in genes encoding a putative undefined phosphotransferase sugar (PTS) system component (SG2427), fucose transporter (SG0182), bacterioferritin (SG2280), and a DNA-binding protein (SGP1-0002), presented growth and/or survival defects in tsetse midguts as compared to normal Sgm. These findings suggest that the uptake up of sugars and storage of iron represent strategies that Sgm employs to successfully reside within the high heme environment of its tsetse host's midgut. Our results are of epidemiological relevance, as many hematophagous arthropods house gut-associated bacteria that mediate their host's competency as a vector of disease-causing pathogens.

Project description:Tsetse fly exhibit species-specific olfactory uniqueness potentially underpinned by differences in their chemosensory protein repertoire. We assessed 1) expansions of chemosensory protein orthologs in Glossina morsitans morsitans, Glossina pallidipes, Glossina austeni, Glossina palpalis gambiensis, Glossina fuscipes fuscipes and Glossina brevipalpis tsetse fly species using Café analysis (to identify species-specific expansions) and 2) differential expressions of the orthologs and associated proteins in male G. m. morsitans antennae and head tissues using RNA-Seq approaches (to establish associated functional molecular pathways). We established accelerated and significant (P<0.05, λ = 2.60452e-7) expansions of gene families in G. m. morsitans Odorant receptor (Or)71a, Or46a, Ir75a,d, Ionotropic receptor (Ir) 31a, Ir84a, Ir64a and Odorant binding protein (Obp) 83a-b), G. pallidipes Or67a,c, Or49a, Or92a, Or85b-c,f and Obp73a, G. f. fuscipes Ir21a, Gustatory receptor (Gr) 21a and Gr63a), G. p. gambiensis clumsy, Ir25a and Ir8a, and G. brevipalpis Ir68a and missing orthologs in each tsetse fly species. Most abundantly expressed transcripts in male G. m. morsitans included specific Or (Orco, Or56a, 65a-c, Or47b, Or67b, GMOY012254, GMOY009475, and GMOY006265), Gr (Gr21a, Gr63a, GMOY013297 and GMOY013298), Ir (Ir8a, Ir25a and Ir41a) and Obp (Obp19a, lush, Obp28a, Obp83a-b Obp44a, GMOY012275 and GMOY013254) orthologs. Most enriched biological processes in the head were associated with vision, muscle activity and neuropeptide regulations, amino acid/nucleotide metabolism and circulatory system processes. Antennal enrichments (>90% of chemosensory transcripts) included cilium-associated mechanoreceptors, chemo-sensation, neuronal controlled growth/differentiation and regeneration/responses to stress. The expanded and tsetse fly species specific orthologs includes those associated with known tsetse fly responsive ligands (4-methyl phenol, 4-propyl phenol, acetic acid, butanol and carbon dioxide) and potential tsetse fly species-specific responsive ligands (2-oxopentanoic acid, phenylacetaldehyde, hydroxycinnamic acid, 2-heptanone, caffeine, geosmin, DEET and (cVA) pheromone). Some of the orthologs can potentially modulate several tsetse fly species-specific behavioral (male-male courtship, hunger/host seeking, cool avoidance, hygrosensory and feeding) phenotypes. The putative tsetse fly specific chemosensory gene orthologs and their respective ligands provide candidate gene targets and kairomones for respective downstream functional genomic and field evaluations that can effectively expand toolbox of species-specific tsetse fly attractants, repellents and other tsetse fly behavioral modulators.

Project description:Female tsetse flies undergo viviparous reproduction, generating one larva each gonotrophic cycle. Larval nourishment is provided by the mother in the form of milk secretions. The milk consists mostly of lipids during early larval development and shifts to a balanced combination of protein and lipids in the late larval instars. Provisioning of adequate lipids to the accessory gland is an indispensable process for tsetse fecundity. This work investigates the roles of Brummer lipase (Bmm) and the adipokinetic hormone (AKH)/adipokinetic hormone receptor (AKHR) systems on lipid metabolism and mobilization during lactation in tsetse. The contributions of each system were investigated by a knockdown approach utilizing siRNA injections. Starvation experiments revealed that silencing of either system results in prolonged female lifespan. Simultaneous suppression of bmm and akhr prolonged survival further than either individual knockdown. Knockdown of akhr and bmm transcript levels resulted in high levels of whole body lipids at death, indicating an inability to utilize lipid reserves during starvation. Silencing of bmm resulted in delayed oocyte development. Respective reductions in fecundity of 20 and 50% were observed upon knockdown of akhr and bmm, while simultaneous knockdown of both genes resulted in 80% reduction of larval production. Omission of one bloodmeal during larvigenesis (nutritional stress) after simultaneous knockdown led to almost complete suppression of larval production. This phenotype likely results from tsetse's inability to utilize lipid reserves as loss of both lipolysis systems leads to accumulation and retention of stored lipids during pregnancy. This shows that both Bmm lipolysis and AKH/AKHR signaling are critical for lipolysis required for milk production during tsetse pregnancy, and identifies the underlying mechanisms of lipid metabolism critical to tsetse lactation. The similarities in the lipid metabolic pathways and other aspects of milk production between tsetse and mammals indicate that this fly could be used as a novel model for lactation research.

Project description:Tsetse flies are the principal insect vectors of African trypanosomes-sleeping sickness in humans and Nagana in cattle. One of the tsetse fly species, Glossina morsitans morsitans, is host to the parasite, Trypanosoma brucei, a major cause of African trypanosomiasis. Precise details of the life cycle have yet to be established, but the parasite life cycle involves crossing the insect peritrophic matrix (PM). The PM consists of the polysaccharide chitin, several hundred proteins, and both glycosamino- and galactosaminoglycan (GAG) polysaccharides. Owing to the technical challenges of detecting small amounts of GAG polysaccharides, their conclusive identification and composition have not been possible until now. Following removal of PMs from the insects and the application of heparinases (bacterial lyase enzymes that are specific for heparan sulphate (HS) GAG polysaccharides), dot blots with a HS-specific antibody showed heparan sulphate proteoglycans (HSPGs) to be present, consistent with Glossina morsitans morsitans genome analysis, as well as the likely expression of the HSPGs syndecan and perlecan. Exhaustive HS digestion with heparinases, fluorescent labeling of the resulting disaccharides with BODIPY fluorophore, and separation by strong anion exchange chromatography then demonstrated the presence of HS for the first time and provided the disaccharide composition. There were no significant differences in the type of disaccharide species present between genders or between ages (24 vs. 48 h post emergence), although the HS from female flies was more heavily sulphated overall. Significant differences, which may relate to differences in infection between genders or ages, were evident, however, in overall levels of 2-O-sulphation between sexes and, for females, between 24 and 48 h post-emergence, implying a change in expression or activity for the 2-O-sulphotransferase enzyme. The presence of significant quantities of disaccharides containing the monosaccharide GlcNAc6S contrasts with previous findings in Drosophila melanogaster and suggests subtle differences in HS fine structure between species of the Diptera.

Project description:BackgroundThe tsetse transmitted parasitic flagellate Trypanosoma congolense causes animal African trypanosomosis (AAT) across sub-Saharan Africa. AAT negatively impacts agricultural, economic, nutritional and subsequently, health status of the affected populace. The molecular mechanisms that underlie T. congolense's developmental program within tsetse are largely unknown due to considerable challenges with obtaining sufficient parasite cells to perform molecular studies.MethodsIn this study, we used RNA-seq to profile T. congolense gene expression during development in two distinct tsetse tissues, the cardia and proboscis. Indirect immunofluorescent antibody test (IFA) and confocal laser scanning microscope was used to localize the expression of a putative protein encoded by the hypothetical protein (TcIL3000_0_02370).ResultsConsistent with current knowledge, genes coding several variant surface glycoproteins (including metacyclic specific VSGs), and the surface coat protein, congolense epimastigote specific protein, were upregulated in parasites in the proboscis (PB-parasites). Additionally, our results indicate that parasites in tsetse's cardia (C-parasites) and PB employ oxidative phosphorylation and amino acid metabolism for energy. Several genes upregulated in C-parasites encoded receptor-type adenylate cyclases, surface carboxylate transporter family proteins (or PADs), transport proteins, RNA-binding proteins and procyclin isoforms. Gene ontology analysis of products of genes upregulated in C-parasites showed enrichment of terms broadly associated with nucleotides, microtubules, cell membrane and its components, cell signaling, quorum sensing and several transport activities, suggesting that the parasites colonizing the cardia may monitor their environment and regulate their density and movement in this tissue. Additionally, cell surface protein (CSP) encoding genes associated with the Fam50 'GARP', 'iii' and 'i' subfamilies were also significantly upregulated in C-parasites, suggesting that they are important for the long non-dividing trypomastigotes to colonize tsetse's cardia. The putative products of genes that were upregulated in PB-parasites were linked to nucleosomes, cytoplasm and membrane-bound organelles, which suggest that parasites in this niche undergo cell division in line with prior findings. Most of the CSPs upregulated in PB-parasites were hypothetical, thus requiring further functional characterization. Expression of one such hypothetical protein (TcIL3000_0_02370) was analyzed using immunofluorescence and confocal laser scanning microscopy, which together revealed preferential expression of this protein on the entire surface coat of T. congolense parasite stages that colonize G. m. morsitans' proboscis.ConclusionCollectively, our results provide insight into T. congolense gene expression profiles in distinct niches within the tsetse vector. Our results show that the hypothetical protein TcIL3000_0_02370, is expressed on the entire surface of the trypanosomes inhabiting tsetse's proboscis. We discuss our results in terms of their relevance to disease transmission processes.

Project description:Transcription initiation regulation is mediated by sequence-specific interactions between DNA-binding proteins (transcription factors) and cis-elements, where BRE, TATA, INR, DPE and MTE motifs constitute canonical core motifs for basal transcription initiation of genes. Accurate identification of transcription start site (TSS) and their corresponding promoter regions is critical for delineation of these motifs. To this end, the genome scale analysis of core promoter architecture in insects has been confined to Drosophila. The recently sequenced Tsetse fly genome provides a unique opportunity to analyze transcription initiation regulation machinery in blood-feeding insects.A computational method for identification of TSS in newly sequenced Tsetse fly genome was evaluated, using TSS seq tags sampled from two developmental stages namely; larvae and pupae. There were 3134 tag clusters among which 45.4% (1424) of the tag clusters mapped to first coding exons or their proximal predicted 5'UTR regions and 1.0% (31) tag clusters mapping to transposons, within a threshold of 100 tags per cluster. These 1393 non transposon-derived core promoters had propensity for AT nucleotides. The -1/+1 and 1/+1 positions in D. melanogaster, and G. m. morsitans had propensity for CA and AA dinucleotides respectively. The 1393 tag clusters comprised narrow promoters (5%), broad with peak promoters (23%) and broad without peak promoters (72%). Two-way motif co-occurrence analysis showed that the MTE-DPE pair is over-represented in broad core promoters. The frequently occurring triplet motifs in all promoter classes are the INR-MTE-DPE, TATA-MTE-DPE and TATA-INR-DPE. Promoters without the TATA motif had higher frequency of the MTE and INR motifs than those observed in Drosophila, where the DPE motif occur more frequently in promoters without TATA motif. Gene ontology terms associated with developmental processes were overrepresented in the narrow and broad with peak promoters.The study has identified different motif combinations associated with broad promoters in a blood-feeding insect. In the case of TATA-less core promoters, G.m. morsitans uses the MTE to compensate for the lack of a TATA motif. The increasing availability of TSS seq data allows for revision of existing gene annotation datasets with the potential of identifying new transcriptional units.

Dataset Information

Towards improving tsetse fly paratransgenesis: stable colonization of Glossina morsitans morsitans with genetically modified Sodalis.

Publications

Towards improving tsetse fly paratransgenesis: stable colonization of Glossina morsitans morsitans with genetically modified Sodalis.

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