TcTASV-C, a protein family in Trypanosoma cruzi that is predominantly trypomastigote-stage specific and secreted to the medium

Abstract

Among the several multigene families codified by the genome of T. cruzi, the TcTASV family was the latest discovered. The TcTASV (Trypomastigote, Alanine, Serine, Valine) family is composed of ∼40 members, with conserved carboxi- and amino-termini but with a variable central core. According to the length and sequence of the central region the family is split into 3 subfamilies. The TcTASV family is conserved in the genomes of - at least - lineages TcI and TcVI and has no orthologues in other trypanosomatids. In the present work we focus on the study of the TcTASV-C subfamily, composed by 16 genes in the CL Brener strain. We determined that TcTASV-C is preferentially expressed in trypomastigotes, but it is not a major component of the parasite. Both immunoflourescence and flow cytometry experiments indicated that TcTASV-C has a clonal expression, i.e. it is not expressed by all the parasites of a certain population at the same time. We also determined that TcTASV-C is phosphorylated and glycosylated. TASV-C is attached to the parasite surface by a GPI anchor and is shed spontaneously into the medium. About 30% of sera from infected hosts reacted with TcTASV-C, confirming its exposition to the immune system. Its superficial localization and secretory nature suggest a possible role in host-parasite interactions.

Figure 1. Sequence of the protein product of Tcruzi_1863-4-1211-93, a representative member of TcTASV-C subfamily.

The internal region that was cloned to produce the recombinant protein TcTASV-C_GST is underlined (amino acids 65 to 330). Amino acids that are predicted to have post-translational modifications are highlighted. The signal peptide that is present in the N-terminal region and the consensus sequence for the addition of a GPI anchor in the C-terminal region are both highlighted in blue (black letters). The first amino acids (white letters, highlighted in blue) correspond to a wrong-predicted amino-terminal region.

Figure 2. TcTASV-C subfamily is expressed mainly in trypomastigotes, and in different T.cruzi strains.

A. Western blot of total protein extracts from CL Brener trypomastigotes (T), epimastigotes (E), amastigotes (A) and metacyclic trypomastigotes (M) using affinity-purified anti-TcTASV-C antibodies (upper panel). The stripped membrane was tested again with anti-GDH serum to verify comparable loading between stages (lower panel). B. A similar western as in (A) but over exposed to evidence the expression of TcTASV-C in other T. cruzi life stages. C. TcTASV-C expression in trypomastigotes and amastigotes from CL Brener strain and in trypomastigotes of RA and Sylvio strains.

Figure 3. TcTASV-C is attached to the parasite membrane through a GPI anchor and spontaneously shed to the medium.

Live, cell-derived CL Brener trypomastigotes were treated with 2 U of PI-PLC at 37°C, mock-treated at 37°C or left untreated at 0°C for 1 h. Parasites were centrifuged and both pellets (pe) and supernatants (sn) were analyzed by western blot using purified anti-TcTASV-C antibodies (upper panel). Trypomastigote proteins (Tryp) were also included in the western. The membrane was stripped and re-probed with anti-TcSR62 serum to verify whether there had been spontaneous lysis of the parasites (middle panel). The total protein transferred in each line is shown by Poinceau S staining (lower panel).

Figure 4. TcTASV-C is phosphorylated and glycosylated.

Lysates of T. cruzi trypomastigotes were treated with CIAP (A) or glycosidases (B), electrophoresed on a 12% SDS-PAGE gel, transferred to nitrocellulose membrane and TcTASV-C detected using anti-TcTASV-C antibodies.

Figure 5. TcTASV-c is expressed at the trypomastigote surface.

A–C: Indirect immunofluorescence was performed on unpermeabilized trypomastigotes using anti-TcTASV-C antibodies. Asterisks in A denote parasites that do not express TcTASV-C, whose DNA content was labeled with DAPI. B and C: Magnification showing the surface pattern of the TcTASV-C distribution. DNA labeling: A and B: DAPI; C: propidium iodide (PI). D–E: Live trypomastigotes (1×10⁶/assay) from CL Brener (D) or RA (E) strains were reacted with affinity-purified anti-TcTASV-C antibodies (blue) for 1 hour at 4°C and processed for analysis by flow cytometry. The specificity of the binding to TcTASV-C proteins was confirmed by pre-adsorption of the antibodies with the recombinant protein TcTASV-C before incubation with the parasites (pre-adsorbed, green line). Negative (IgG from normal mice; black line) and positive (sera from T. cruzi-infected mice; purple line) controls were included.

Figure 6. TcTASV-C is recognized by sera from infected hosts.

The reactivity of sera from rabbits (A, B) and humans (C, D) against TcTASV-C (and GST) was evaluated by ELISA. Results are presented as absorbance at 450 nm (A, C) or as the ratio between the ODs obtained for each serum against TcTASV-C and GST (B, D). Asterisks in A and C denote differences in the mean values (p<0.005). Dotted lines in B and D represent the cut-off, calculated as the mean + 2SD of control (uninfected) sera. Human's sera: infected: N = 30; uninfected: N = 12. Rabbit's sera: infected: 28; uninfected: 12.