The Flagellar Arginine Kinase in Trypanosoma brucei Is Important for Infection in Tsetse Flies

Cher-Pheng Ooi, Brice Rotureau, Simonetta Gribaldo, Christina Georgikou, Daria Julkowska, Thierry Blisnick, Sylvie Perrot, Ines Subota, Philippe Bastin
Source: PLoS ONE
Publication Date: (2015)
Issue: 10(7): e0133676
Research Area:
Basic Research
Cells used in publication:
Trypanosoma brucei
Species: unicellular
Tissue Origin: blood
Nucleofectorâ„¢ I/II/2b
For the purpose of generating a Trypanosoma brucei AnTat 1.1 cell line deprived of AK3, the authors used a double knockout approach. Nucleofections were carried out using the Amaxa Nucleofector (Lonza)(Burkard et al MBP2007) and clonal populations were obtained with sub-cloning by limiting dilution. Generation of the ?ak3 cell line was accomplished by 2 successive rounds of Nucleofection to replace both alleles of endogenous AK3 in WT with BLA and PURO genes. For the purpose of generating an add-back cell line (AB), the AK3::Ty1 gene was synthesized and integrated into the pHD430 plasmid by Genecust Europe to generate the pHD430AK3::Ty1 plasmid. This plasmid was linearized with the NotI restriction enzyme prior to Nucleofection for integration into the ribosomal intergenic spacer in the ?ak3 cell line. An additional round of nucleofection was carried out on clonal AB cell line with the pHD360 vector that was targeted to the tubulin locus to ensure expression of tet-repressor, hence rendering the production of the AK3::Ty1 protein tetracycline-inducible. As the generated cell lines were to be used for tsetse infection experiments, only one clonal cell line from each was chosen for further analyses. Lonza summary: The authors managed to knockdown the AK3, a gene encoding the flagellar arginine kinase, in Trypanosoma brucei. They used a successive double Nucleofection approach to stably impair both AK3 alleles through DNA recombination and different selection markers. The motility and infectivity of the modified parasites was impaired in its tsetse fly vector, and this phenotype was revertable by overexpression of AK3. The authors conclude that AK3 might play an important role in the parasite Trypanosoma brucei infection of tsetse fly.
African trypanosomes are flagellated parasites that cause sleeping sickness. Parasites are transmitted from one mammalian host to another by the bite of a tsetse fly. Trypanosoma brucei possesses three different genes for arginine kinase (AK) including one (AK3) that encodes a protein localised to the flagellum. AK3 is characterised by the presence of a unique amino-terminal insertion that specifies flagellar targeting. We show here a phylogenetic analysis revealing that flagellar AK arose in two independent duplication events in T. brucei and T. congolense, the two species of African trypanosomes that infect the tsetse midgut. In T. brucei, AK3 is detected in all stages of parasite development in the fly (in the midgut and in the salivary glands) as well as in bloodstream cells, but with predominance at insect stages. Genetic knockout leads to a slight reduction in motility and impairs parasite infectivity towards tsetse flies in single and competition experiments, both phenotypes being reverted upon expression of an epitope-tagged version of AK3. We speculate that this flagellar arginine kinase is important for T. brucei infection of tsetse, especially in the context of mixed infections and that its flagellar targeting relies on a system equivalent to that discovered for calflagins, a family of trypanosome flagellum calcium binding proteins.