La noción de familia en la mente infantil: un estudio evolutivo en dos contextos familiares

Tesis doctoral inédita de la Universidad Autónoma de Madrid, Facultad de Psicología. Fecha de lectura: 199

TcTASV: a novel protein family in trypanosoma cruzi identified from a subtractive trypomastigote cDNA library.

BACKGROUND: The identification and characterization of antigens expressed in Trypanosoma cruzi stages that parasitize mammals are essential steps for the development of new vaccines and diagnostics. Genes that are preferentially expressed in trypomastigotes may be involved in key processes that define the biology of trypomastigotes, like cell invasion and immune system evasion. METHODOLOGY/PRINCIPAL FINDINGS: With the initial aim of identifying trypomastigote-specific expressed tags, we constructed and sequenced an epimastigote-subtracted trypomastigote cDNA library (library TcT-E). More than 45% of the sequenced clones of the library could not be mapped to previously annotated mRNAs or proteins. We validated the presence of these transcripts by reverse northern blot and northern blot experiments, therefore providing novel information about the mRNA expression of these genes in trypomastigotes. A 280-bp consensus element (TcT-E element, TcT-Eelem) located at the 3' untranslated region (3' UTR) of many different open reading frames (ORFs) was identified after clustering the TcT-E dataset. Using an RT-PCR approach, we were able to amplify different mature mRNAs containing the same TcT-Eelem in the 3' UTR. The proteins encoded by these ORFs are members of a novel surface protein family in T. cruzi, (which we named TcTASV for T. cruzi Trypomastigote, Alanine, Serine and Valine rich proteins). All members of the TcTASV family have conserved coding amino- and carboxy-termini, and a central variable core that allows partitioning of TcTASV proteins into three subfamilies. Analysis of the T. cruzi genome database resulted in the identification of 38 genes/ORFs for the whole TcTASV family in the reference CL-Brener strain (lineage II). Because this protein family was not found in other trypanosomatids, we also looked for the presence of TcTASV genes in other evolutionary lineages of T. cruzi, sequencing 48 and 28 TcTASVs members from the RA (lineage II) and Dm28 (lineage I) T. cruzi strains respectively. Detailed phylogenetic analyses of TcTASV gene products show that this gene family is different from previously characterized mucin (TcMUCII), mucin-like, and MASP protein families. CONCLUSIONS/SIGNIFICANCE: We identified TcTASV, a new gene family of surface proteins in T. cruzi

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

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

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

<p>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).</p

TcTASV-C is phosphorylated and glycosylated.

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

TcTASV-c is expressed at the trypomastigote surface.

<p><b>A–C:</b> 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). <b>D–E:</b> 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 <i>T. cruzi</i>-infected mice; purple line) controls were included.</p

TcTASV-C subfamily is expressed mainly in trypomastigotes, and in different <i>T.cruzi</i> strains.

<p><b>A.</b> 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>B.</b> A similar western as in (A) but over exposed to evidence the expression of TcTASV-C in other <i>T. cruzi</i> life stages<b>. C.</b> TcTASV-C expression in trypomastigotes and amastigotes from CL Brener strain and in trypomastigotes of RA and Sylvio strains.</p

TcTASV-C is recognized by sera from infected hosts.

<p>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.</p

Sequence of the protein product of <i>Tcruzi_1863-4-1211-93</i>, a representative member of TcTASV-C subfamily.

<p>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.</p