Dataset Information

Generation of a nanobody targeting the paraflagellar rod protein of trypanosomes.

ABSTRACT: Trypanosomes are protozoan parasites that cause diseases in humans and livestock for which no vaccines are available. Disease eradication requires sensitive diagnostic tools and efficient treatment strategies. Immunodiagnostics based on antigen detection are preferable to antibody detection because the latter cannot differentiate between active infection and cure. Classical monoclonal antibodies are inaccessible to cryptic epitopes (based on their size-150 kDa), costly to produce and require cold chain maintenance, a condition that is difficult to achieve in trypanosomiasis endemic regions, which are mostly rural. Nanobodies are recombinant, heat-stable, small-sized (15 kDa), antigen-specific, single-domain, variable fragments derived from heavy chain-only antibodies in camelids. Because of numerous advantages over classical antibodies, we investigated the use of nanobodies for the targeting of trypanosome-specific antigens and diagnostic potential. An alpaca was immunized using lysates of Trypanosoma evansi. Using phage display and bio-panning techniques, a cross-reactive nanobody (Nb392) targeting all trypanosome species and isolates tested was selected. Imunoblotting, immunofluorescence microscopy, immunoprecipitation and mass spectrometry assays were combined to identify the target recognized. Nb392 targets paraflagellar rod protein (PFR1) of T. evansi, T. brucei, T. congolense and T. vivax. Two different RNAi mutants with defective PFR assembly (PFR2RNAi and KIF9BRNAi) were used to confirm its specificity. In conclusion, using a complex protein mixture for alpaca immunization, we generated a highly specific nanobody (Nb392) that targets a conserved trypanosome protein, i.e., PFR1 in the flagella of trypanosomes. Nb392 is an excellent marker for the PFR and can be useful in the diagnosis of trypanosomiasis. In addition, as demonstrated, Nb392 can be a useful research or PFR protein isolation tool.

SUBMITTER: Obishakin E

PROVIDER: S-EPMC4281110 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Generation of a nanobody targeting the paraflagellar rod protein of trypanosomes.

Obishakin Emmanuel E Stijlemans Benoit B Santi-Rocca Julien J Vandenberghe Isabel I Devreese Bart B Muldermans Serge S Bastin Philippe P Magez Stefan S

PloS one 20141231 12

Trypanosomes are protozoan parasites that cause diseases in humans and livestock for which no vaccines are available. Disease eradication requires sensitive diagnostic tools and efficient treatment strategies. Immunodiagnostics based on antigen detection are preferable to antibody detection because the latter cannot differentiate between active infection and cure. Classical monoclonal antibodies are inaccessible to cryptic epitopes (based on their size-150 kDa), costly to produce and require col ...[more]

PMID: 25551637

Similar Datasets

Project description:UNLABELLED:Trypanosoma cruzi is the etiologic agent of Chagas disease, and current methods for its genetic manipulation have been highly inefficient. We report here the use of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system for disrupting genes in the parasite by three different strategies. The utility of the method was established by silencing genes encoding the GP72 protein, which is required for flagellar attachment, and paraflagellar rod proteins 1 and 2 (PFR1, PFR2), key components of the parasite flagellum. We used either vectors containing single guide RNA (sgRNA) and Cas9, separately or together, or one vector containing sgRNA and Cas9 plus donor DNA for homologous recombination to rapidly generate mutant cell lines in which the PFR1, PFR2, and GP72 genes have been disrupted. We demonstrate that genome editing of these endogenous genes in T. cruzi is successful without detectable toxicity of Cas9. Our results indicate that PFR1, PFR2, and GP72 contribute to flagellar attachment to the cell body and motility of the parasites. Therefore, CRISPR/Cas9 allows efficient gene disruption in an almost genetically intractable parasite and suggest that this method will improve the functional analyses of its genome. IMPORTANCE:Trypanosoma cruzi is the agent of Chagas disease, which affects millions of people worldwide. Vaccines to prevent this disease are not available, and drug treatments are not completely effective. The study of the biology of this parasite through genetic approaches will make possible the development of new preventive or treatment options. Previous attempts to use the CRISPR/Cas9 in T. cruzi found a detectable but low frequency of Cas9-facilitated homologous recombination and fluorescent marker swap between exogenous genes, while Cas9 was toxic to the cells. In this report, we describe new approaches that generate complete disruption of an endogenous gene without toxicity to the parasites and establish the relevance of several proteins for flagellar attachment and motility.

Dataset Information

Generation of a nanobody targeting the paraflagellar rod protein of trypanosomes.

Publications

Generation of a nanobody targeting the paraflagellar rod protein of trypanosomes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets