FUNCTIONAL CHARACTERIZATION OF THE CEST MOTIF (CHAPERONE FOR THE E. COLI SECRETION OF TIR) IN TRYPANOSOMATIDS

CAMILA CENTENO CAMEAN; GABRIEL BRIONES; IGNACIO DURANTE; MARIA DE LOS MILAGROS CAMARA; RAUL TAKADA; CARLOS A BUSCAGLIA

Resistencia

Congreso; XXX Reunion Anual de la Sociedad Argentina de Protozoologia; 2018

Sociedad Argentina de Parasitologia

Using a bioinformatic approach, we identified a small family of proteins restricted to trypanosomatids that present a ´CEST´ domain, as it shows structural homology with CesT proteins (Chaperone for E. coli secretion of Tir). These proteins form a family of chaperones involved in the secretion of virulence factors through the type III secretion system (TIIISS) in enteropathogenic bacteria. Since trypanosomatid CEST motif shown maximal structural homology to protein templates of the Salmonella Typhimurium SicP CesT-type chaperone, which is involved in the TIIISS-mediated secretion of the cytoskeleton-perturbing SptP effector, we selected this organism and this chaperone-effector pair to generate a functional complementation model. We started by characterizing the CEST motifs present in Trypanosoma cruzi TCLP1 (Trypanosomatid CesT-like Protein 1, [1]) and in other 4 molecules from T. cruzi, Trypanosoma brucei, Crithidia fasciculata and Leishmania mexicana. Using biochemical, biological and immuno-cytochemical techniques, we verified that the evaluated CEST motifs were able to revert the phenotypic defects (lack of SptP secretion, impaired mammalian cell infection, etc.) of a S. Typhimurium ΔSicP strain. Interestingly, the most divergent of the evaluated CEST motifs, which belonged to C. fasciculata, was not able to do so. Homology modelling revealed a reasonable superimposition of the buried, hydrophobic ß-sheets and the outer α-helix between the S. Typhimurium SicP chaperone and the CEST motifs from trypanosomatids except for the C. fasciculata CEST motif, which may provide the structural basis for its lack of functional complementation. As to further characterize the C. fasciculata CEST motif, we performed immunoprecipitation and native gel assays upon extracts of transformed bacteria. Analyzing the results we concluded that this motif, although able to dimerize in the cytoplasm of the bacteria (a pre-requisite for CesT chaperone function), it is not able to directly interact with the SptP effector in vivo. Given the socio-economic relevance of the diseases produced by trypanosomatids and the importance of the CesT chaperones in enterobacteria, we hope that the results obtained here may help to understand the basic biology of these parasites and to identify novel drug targets for intervention against these pathogens