Characterization of recombinant Trypanosoma brucei gambiense Translationally Controlled Tumor Protein (rTbgTCTP) and its interaction with Glossina midgut bacteria

Abstract

In humans, sleeping sickness (i.e. Human African Trypanosomiasis) is caused by the protozoan parasites Trypanosoma brucei gambiense (Tbg) in West and Central Africa, and T. b. rhodesiense in East Africa. We previously showed in vitro that Tbg is able to excrete/secrete a large number of proteins, including Translationally Controlled Tumor Protein (TCTP). Moreover, the tctp gene was described previously to be expressed in Tbg-infected flies. Aside from its involvement in diverse cellular processes, we have investigated a possible alternative role within the interactions occurring between the trypanosome parasite, its tsetse fly vector, and the associated midgut bacteria. In this context, the Tbg tctp gene was synthesized and cloned into the baculovirus vector pAcGHLT-A, and the corresponding protein was produced using the baculovirus Spodoptera frugicola (strain 9) / insect cell system. The purified recombinant protein rTbgTCTP was incubated together with bacteria isolated from the gut of tsetse flies, and was shown to bind to 24 out of the 39 tested bacteria strains belonging to several genera. Furthermore, it was shown to affect the growth of the majority of these bacteria, especially when cultivated under microaerobiosis and anaerobiosis. Finally, we discuss the potential for TCTP to modulate the fly microbiome composition toward favoring trypanosome survival.

Keywords: Glossina; Translationally Controlled Tumor Protein (TCTP); Trypanosoma brucei gambiense; bacteria midgut flora.

Figure 1.

(A) Protein sequence of rTbgTCTP with linearized epitope (pink); amino acids involved in the epitope conformation are presented in bold type. (B) Purification profile for rTbgTCTP on a nickel-charged affinity resin (Ni-NTA) column. (C) sodium dodecyl sulfate - PAGE analysis of rTbgTCTP. Lane 1: protein molecular marker (Novex Sharp Pre-Stained Protein Standard, Thermo Fisher Scientific); lane 2: electrophoresis of rTbgTCTP (10 µg) purified on a Ni-NTA column stained with Coomassie Blue; lane 3: western blot of rTbgTCTP (10 µg) on nitrocellulose membrane using anti-His monoclonal antibody (mAb).

Figure 2.

(A) The Tbg 927.8.6750 tertiary structure. The part corresponding to the epitope is in pink. (B) Linear epitope (pink) of Tbg 927.8.6750 from Trypanosoma brucei gambiense (Bepiprep 1.0 server) and the conformational epitope 3P3K (green; DiscoTope 2.0 server). (C) Lysine localization of 3P3K (red).

Figure 3.

Phylogenetic tree for TCTP among trypanosomatids and their corresponding host species (PhyML). The PhyML website was used to construct the phylogenetic tree based on the amino acid sequences of the corresponding TCTP and default parameters of the server. Numbers above branches indicate bootstrap values.

Figure 4.

Binding between bacteria and rTbgTCTP as revealed by western blot using anti-His mAb. Lane 1: molecular weight markers (Novex Sharp Pre-Stained Protein Standard, Thermo Fisher Scientific); lane 2: purified rTbgTCTP; lane 3: bacteria (Providencia spp. from Glossina palpalis palpalis) incubated without rTbgTCTP as a control; lane 4: bacteria incubated with rTbgTCTP and subsequently treated with β-mercaptoethanol.

Figure 5.

Growth kinetics of the bacteria strains grown in the absence of rTbgTCTP. The bacterial concentration was measured using the OD at 600 nm. Bacteria were cultivated in Mitsuhashi medium at 25°C (A-C) or 37°C (D-F), in either aerobiosis (A, D), microaerobiosis (B, E) or anaerobiosis (C, F).

Figure 6.

Effect of different concentrations of rTbgTCTP on bacteria growth. The effect was tested at 25°C (A) and 37°C (B-E), under 3 different atmosphere compositions: aerobiosis (white column), microaerobiosis (gray column), and anaerobiosis (black column). Histograms compare, for a given experimental condition (and for each time), the OD of the tested culture (with TCTP at a given concentration) with the OD of the corresponding control culture without TCTP (assigned to the value 100%). The results are the mean of triplicate experiments.

Figure 6.

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Figure 6.

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