Modelos in vitro e in vivo e anticorpos recombinantes para o estudo da toxina termolábil produzida por Escherichia coli enterotoxigênica

Abstract

Enterotoxigenic Escherichia coli (ETEC) is one of the main bacterial pathotypes in- volved in diarrhea, mainly affecting children under 5 years old and travelers to areas where this pathogen is endemic. Heat-labile type I toxin (LT-I), one of the main virulence factors of this pathotype, is associated with diarrhea in humans and is described as a potent mucosal and systemic immune adjuvant. Early diagnosis, therapeutic approaches, and in vitro and in vivo models are the main concerns in diarrhea caused by ETEC. Different in vitro and in vivo models have already been described, however, its use does not always allow screening of biomolecules and more systemic studies with the LT toxin. In addition, the generation of specific and high-affinity recombinant antibodies for diagnosis and therapy are of great importance. Thus, the present work aimed to establish strategies for the generation of recombinant antibodies such as scFv and Fab and evaluate and validate Caco-2 cells and zebrafish as models for studies with LT-I. Caco-2 cells were seeded in the 62nd passage and at different cells density per microplate well. The internalization of LT-I conjugated to FITC was visualized in Caco-2 cells at a density of 3x104 cells per well, both associated with the cell membrane and with the nucleus, thus demonstrating its retrograde transport and validating the use of this model. In a zebrafish, the fish embryo test revealed the sensitivity of embryos to different concentrations of LT-I, as well as evident malformation phenotypes, mainly in the pericardial and yolk region. Systemically, migration of the FITC labeled toxin was observed in the cardiac region, yolk, and intestine, suggesting the observed phenotypes of cardiac edema (100%), absence of swim bladder (100%), yolk edema (80%), in addition to growth delay in larvae, were caused by the toxin. None of these phenotypes were observed in the control group of animals. There was also a decrease in heart rate during the larvae' survival kinetics, showing the cardiotoxic effect of LT-I. In ELISA assays the scFv-LT obtained in the present study was reactive against the purified antigen as well to LT-I producing strains supernatant, but not to LT-I-non-producing strains. For the Fab-LT, a synthetic library was constructed, but no LT-I-reactive clones were generated. Therefore, herein we established in vitro and in vivo models that allowed us to validate and demonstrate unknown characteristics of the LT-I concerning its relationship with the host. Moreover, the generation of scFv-LT will allows us to employ recombinant antibodies for ETEC diagnosis avoiding animals immunization.