RESUMEN
Trypanosoma cruzi trypomastigotes invade a great variety of mammalian cells, with several molecules being implicated in this complex event. Herein, the sequence GGIALAG present in prokineticin-2 receptor (PKR2), selected by phage display technology, is described as a new T. cruzi receptor for the Tc85 group of glycoproteins belonging to the gp85/TS superfamily and involved in cellular invasion of mammalian hosts. This finding is confirmed by the inhibitory activity of MCF10-A (human mammary) cell invasion by T. cruzi either by anti-PKR2 antibodies (77%) or GGIALAG-synthetic peptide (42%). Furthermore, interference RNA (iRNA) inhibition of PKR2 expression in MCF10-A cells reduces T. cruzi invasion by 50%. The binding site of Tc85 to PKR2 was localized at the C-terminal end of the molecule, upstream of the conserved FLY sequence, previously implicated in parasite cell invasion. PKR2, a receptor formed by seven membrane-spanning α-helical segments, is mainly present in the central nervous system, peripheral organs, and mature blood cells. Due to its wide distribution, PKR2 could be a suitable receptor for T. cruzi natural infection, contributing to the parasite dissemination throughout the mammalian organism. These findings augment the number and diversity of possible in vivo receptors for T. cruzi and reassure the multiplicity of Tc85 binding sites to mammalian hosts.
Asunto(s)
Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/metabolismo , Trypanosoma cruzi/fisiología , Animales , Bacteriófagos , Sitios de Unión , Línea Celular , Secuencia Conservada , Glicoproteínas/genética , Humanos , Receptores Acoplados a Proteínas G/genética , Receptores de Péptidos/genéticaRESUMEN
Parasite infections are largely dependent on interactions between pathogen and different host cell populations to guarantee a successful infectious process. This is particularly true for obligatory intracellular parasites as Plasmodium, Toxoplasma, and Leishmania, to name a few. Adhesion to and entry into the cell are essential steps requiring specific parasite and host cell molecules. The large amount of possible involved molecules poses additional difficulties for their identification by the classical biochemical approaches. In this respect, the search for alternative techniques should be pursued. Among them two powerful methodologies can be employed, both relying upon the construction of highly diverse combinatorial libraries of peptides or oligonucleotides that randomly bind with high affinity to targets on the cell surface and are selectively displaced by putative ligands. These are, respectively, the peptide-based phage display and the oligonucleotide-based aptamer techniques. The phage display technique has been extensively employed for the identification of novel ligands in vitro and in vivo in different areas such as cancer, vaccine development, and epitope mapping. Particularly, phage display has been employed in the investigation of pathogen-host interactions. Although this methodology has been used for some parasites with encouraging results, in trypanosomatids its use is, as yet, scanty. RNA and DNA aptamers, developed by the SELEX process (Systematic Evolution of Ligands by Exponential Enrichment), were described over two decades ago and since then contributed to a large number of structured nucleic acids for diagnostic or therapeutic purposes or for the understanding of the cell biology. Similarly to the phage display technique scarce use of the SELEX process has been used in the probing of parasite-host interaction. In this review, an overall survey on the use of both phage display and aptamer technologies in different pathogenic organisms will be discussed. Using these techniques, recent results on the interaction of Trypanosoma cruzi with the host will be highlighted focusing on members of the 85 kDa protein family, a subset of the gp85/TS superfamily.
RESUMEN
Trypanosoma cruzi is a protozoan parasite that infects vertebrates, causing in humans a pathological condition known as Chagas' disease. The infection of host cells by T. cruzi involves a vast collection of molecules, including a family of 85 kDa GPI-anchored glycoproteins belonging to the gp85/trans-sialidase superfamily, which contains a conserved cell-binding sequence (VTVXNVFLYNR) known as FLY, for short. Herein, it is shown that BALB/c mice administered with a single dose (1 µg/animal, intraperitoneally) of FLY-synthetic peptide are more susceptible to infection by T. cruzi, with increased systemic parasitaemia (2-fold) and mortality. Higher tissue parasitism was observed in bladder (7·6-fold), heart (3-fold) and small intestine (3·6-fold). Moreover, an intense inflammatory response and increment of CD4+ T cells (1·7-fold) were detected in the heart of FLY-primed and infected animals, with a 5-fold relative increase of CD4+CD25+FoxP3+ T (Treg) cells. Mice treated with anti-CD25 antibodies prior to infection, showed a decrease in parasitaemia in the FLY model employed. In conclusion, the results suggest that FLY facilitates in vivo infection by T. cruzi and concurs with other factors to improve parasite survival to such an extent that might influence the progression of pathology in Chagas' disease.