Proteína ErpY-like de Leptospira spp.: da ciência básica à aplicação no diagnóstico

Abstract

Leptospirosis is a zoonosis caused by pathogenic bacteria from the genus Leptospira. This disease can affect several animals, being considered a public health problem and responsible for losses in livestock. The "gold standard" for leptospirosis diagnosis is microscopic agglutination test (MAT). However, it has limitations, such as a high number of cross-reactions, it is laborious and time-consuming. For this reason, the search for alternatives to leptospirosis control becomes necessary. The ErpY-like protein was reported as a potential virulence factor of Leptospira interrogans, and thus possibly involved in the infection process and important in the development of new diagnostic tests and as a vaccine antigen. In this way, the objective of this work was the ErpY-like protein production in its recombinant form, its in silico characterization and application in diagnosis. For this, the protein was evaluated through bioinformatics tools and the presence of the erpY-like gene in different serovars of Leptospira spp. investigated by PCR. ErpY-like protein was produced in recombinant (rErp-like) form using a prokaryotic heterologous system (Escherichia coli) and its antigenicity assessed in an ELISA (Enzyme-Linked Immunosorbent Assay) indirect assay with serum from infected animals. Mice were immunized with rErpY-like protein to produce polyclonal antibodies (pAbs), which were titrated in indirect ELISA and tested for reactivity with the recombinant and native L. interrogans forms in indirect ELISA and indirect immunofluorescence. Finally, the rErpY-like protein was tested in the development of an indirect ELISA for the diagnosis of swine leptospirosis. In PCR, the erpY-like gene was amplified in all tested serovars. ErpY-like protein was successfully expressed using heterologous system, expected size of 13 kDa and yield of 25 mg.L-1. Its antigenicity was confirmed in an indirect ELISA assay, where the recombinant protein reacted with naturally infected human, swine and canine sera. The produced pAbs bound to native ErpY-like protein, both in whole cells and lysates of leptospires. In indirect immunofluorescence, the pAbs reaction was also observed, fluorescently labeling the native ErpY-like protein in Leptospira. In addition, the use of rErpY-like protein in the indirect ELISA was sensitive and specific for the diagnosis of swine leptospirosis, with sensitivity of 96.8% and specificity of 100%. These results demonstrated that ErpY-like protein is a promising target in the development of trials for the control of leptospirosis.