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The Trypanosoma brucei MISP family of invariant proteins are co-expressed with BARP as triple helical bundle structures on the surface of salivary gland forms, but is dispensable for parasite development within the tsetse vector

ABSTRACT: Trypanosoma brucei spp. develop into mammalian-infectious metacyclic trypomastigotes inside tsetse salivary glands. Besides acquiring a variant surface glycoprotein (VSG) coat, little is known about the metacyclic expression of invariant surface antigens. Proteomics analyses of saliva from T. brucei-infected flies identified, in addition to VSG and Brucei Alanine-Rich Protein (BARP) peptides, a family of GPIanchored surface proteins herein named Metacyclic Invariant Surface Proteins (MISP). The MISP family is encoded by five paralog genes with >80% protein identity, which are exclusively expressed by salivary gland stages of the parasite and peak in metacyclic stage, as shown by confocal microscopy and immuno-high resolution scanning electron microscopy. Crystallographic analysis of a MISP isoform (MISP360) and a high confidence model of BARP revealed a triple helical bundle architecture commonly found in other trypanosome surface proteins. Molecular modelling combined with live fluorescent microscopy suggests that MISP N-termini are extended above the VSG coat. However, vaccination with recombinant MISP360 isoform did not protect mice against a T. brucei infectious tsetse bite. Lastly, both RNAi knock down and CRISPR-Cas9-driven knock out of all MISP paralogues suggest they are not essential for parasite development in the tsetse vector. We speculate that MISP may be relevant during trypanosome inoculation or establishment in the vertebrate’s skin.

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ORGANISM(S): Trypanosoma Brucei

SUBMITTER: Douglas Lamont

LAB HEAD: Dr Alvaro Acosta Serrano

PROVIDER: PXD039684 | Pride | 2023-05-10

REPOSITORIES: Pride

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The Trypanosoma brucei MISP family of invariant proteins is co-expressed with BARP as triple helical bundle structures on the surface of salivary gland forms, but is dispensable for parasite development within the tsetse vector.

Casas-Sanchez Aitor A Ramaswamy Raghavendran R Perally Samïrah S Haines Lee R LR Rose Clair C Aguilera-Flores Marcela M Portillo Susana S Verbeelen Margot M Hussain Shahid S Smithson Laura L Yunta Cristina C Lehane Michael J MJ Vaughan Sue S van den Abbeele Jan J Almeida Igor C IC Boulanger Martin J MJ Acosta-Serrano Álvaro Á

PLoS pathogens 20230330 3

Trypanosoma brucei spp. develop into mammalian-infectious metacyclic trypomastigotes inside tsetse salivary glands. Besides acquiring a variant surface glycoprotein (VSG) coat, little is known about the metacyclic expression of invariant surface antigens. Proteomic analyses of saliva from T. brucei-infected tsetse flies identified, in addition to VSG and Brucei Alanine-Rich Protein (BARP) peptides, a family of glycosylphosphatidylinositol (GPI)-anchored surface proteins herein named as Metacycli ...[more]

PMID: 36996244

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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.

Project description:The host range of African trypanosomes is influenced by innate protective molecules in the blood of primates. A subfraction of human high-density lipoprotein (HDL) containing apolipoprotein A-I, apolipoprotein L-I, and haptoglobin-related protein is toxic to Trypanosoma brucei brucei but not the human sleeping sickness parasite Trypanosoma brucei rhodesiense. It is thought that T. b. rhodesiense evolved from a T. b. brucei-like ancestor and expresses a defense protein that ablates the antitrypanosomal activity of human HDL. To directly investigate this possibility, we developed an in vitro selection to generate human HDL-resistant T. b. brucei. Here we show that conversion of T. b. brucei from human HDL sensitive to resistant correlates with changes in the expression of the variant surface glycoprotein (VSG) and abolished uptake of the cytotoxic human HDLs. Complete transcriptome analysis of the HDL-susceptible and -resistant trypanosomes confirmed that VSG switching had occurred but failed to reveal the expression of other genes specifically associated with human HDL resistance, including the serum resistance-associated gene (SRA) of T. b. rhodesiense. In addition, we found that while the original active expression site was still utilized, expression of three expression site-associated genes (ESAG) was altered in the HDL-resistant trypanosomes. These findings demonstrate that resistance to human HDLs can be acquired by T. b. brucei. Keywords: Trypanosoma, VSG, antigenic switching, HDL-resistance Bloodstream stages of the Lister strain 427 T. b. brucei (MiTat 1.2), expressing VSG221, were used in these studies. Cells were cultured in HMI-9 medium with the addition of heat inactivated fetal bovine serum (FBS) (10%) and Serum Plus (10%). T. b. brucei 427-221 is an antigenically stable line and contains a single copy of the vsg221 gene within the 221 expression site (221ES). At a cell density of approximately 1,000,000 cells/ml, T. b. brucei 427-221 were exposed to various amounts of human HDLs for 24 h in a 6 well plate. Surviving trypanosomes were counted using a hemocytometer then diluted into fresh HMI-9 medium and allowed to recover for 5-14 days. Once the cells had grown to a density of approximately 1,000,000 cells/ml, they were once again incubated with human HDLs. Each round of selection was performed with increasing concentrations of human HDLs and freezer stocks were prepared for each surviving population. Over nine months we conducted eight rounds of human HDL selection, resulting in a population of T. b. brucei that survived incubation with 800 μl of human HDLs (160 lytic U).

Project description:The infectious metacyclic forms of Trypanosoma brucei result from a complex development in the tsetse fly vThe infectious metacyclic forms of Trypanosoma brucei result from a complex development in the tsetse fly vector. When they infect mammals, they cause African sleeping sickness in humans. Due to scarcity of biological material and difficulties of the tsetse fly as an experimental system, very limited information is available concerning the gene expression profile of metacyclic Trapanosoma forms. We used an in vitro system based on expressing the RNA binding protein 6 (RBP6) to obtain infectious metacyclics and determined their protein and mRNA repertoires by mass-spectrometry (MS) based proteomics and mRNA sequencing (RNAseq) in comparison to non-infectious procyclic trypanosomes. This comparison showed that metacyclics are quiescent cells, and we propose this influences the choice of a monocistronic variant surface glycoprotein expression site. Metacyclics have a largely bloodstream-form type transcriptome, and thus are programmed to translate a bloodstream-form type proteome upon entry into the mammalian host and resumption of cell division. Genes encoding cell surface components showed the largest changes between procyclics and metacyclics, observed at both the transcript and protein levels. Genes encoding metabolic enzymes exhibited expression in metacyclics with features of both procyclic and bloodstream forms, suggesting that this intermediate-type metabolism is dictated by the availability of nutrients in the tsetse fly vector. ector. When they infect mammals, they cause African sleeping sickness in humans. Due to scarcity of biological material and difficulties of the tsetse fly as an experimental system, very limited information is available concerning the gene expression profile of metacyclic Trapanosoma forms. We used an in vitro system based on expressing the RNA binding protein 6 (RBP6) to obtain infectious metacyclics and determined their protein and mRNA repertoires by mass-spectrometry (MS) based proteomics and mRNA sequencing (RNAseq) in comparison to non-infectious procyclic trypanosomes. This comparison showed that metacyclics are quiescent cells, and we propose this influences the choice of a monocistronic variant surface glycoprotein expression site. Metacyclics have a largely bloodstream-form type transcriptome, and thus are programmed to translate a bloodstream-form type proteome upon entry into the mammalian host and resumption of cell division. Genes encoding cell surface components showed the largest changes between procyclics and metacyclics, observed at both the transcript and protein levels. Genes encoding metabolic enzymes exhibited expression in metacyclics with features of both procyclic and bloodstream forms, suggesting that this intermediate-type metabolism is dictated by the availability of nutrients in the tsetse fly vector.

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