Heat-Labile Toxin from Enterotoxigenic Escherichia coli Causes Systemic Impairment in Zebrafish Model

Abstract

Heat-labile toxin I (LT-I), produced by strains of enterotoxigenic Escherichia coli (ETEC), causes profuse watery diarrhea in humans. Different in vitro and in vivo models have already elucidated the mechanism of action of this toxin; however, their use does not always allow for more specific studies on how the LT-I toxin acts in systemic tracts and intestinal cell lines. In the present work, zebrafish (Danio rerio) and human intestinal cells (Caco-2) were used as models to study the toxin LT-I. Caco-2 cells were used, in the 62nd passage, at different cell concentrations. LT-I was conjugated to FITC to visualize its transport in cells, as well as microinjected into the caudal vein of zebrafish larvae, in order to investigate its effects on survival, systemic traffic, and morphological formation. The internalization of LT-I was visualized in 3 × 10⁴ Caco-2 cells, being associated with the cell membrane and nucleus. The systemic traffic of LT-I in zebrafish larvae showed its presence in the cardiac cavity, yolk, and regions of the intestine, as demonstrated by cardiac edema (100%), the absence of a swimming bladder (100%), and yolk edema (80%), in addition to growth limitation in the larvae, compared to the control group. There was a reduction in heart rate during the assessment of larval survival kinetics, demonstrating the cardiotoxic effect of LT-I. Thus, in this study, we provide essential new depictions of the features of LT-I.

Keywords: Caco-2 cells; cardiotoxic effect; heat-labile toxin; systemic effects; zebrafish.

Figure 1

Fluorescence microscopy at the concentration of 3 × 10⁴ Caco-2 cells per well: (a) Caco-2 cells, phase contrast; (b) Cell nucleus stained with DAPI, in blue; (c) Actin of the cell wall stained with rhodamine-phalloidin, in red; (d) Overlay of all fluorescence images.

Figure 2

Fluorescence microscopy of LT-I-/FITC interaction with Caco-2 cells for 7 h, visualized with magnification of 63×: (a) Caco-2 cells, phase contrast; (b) Cell nucleus stained with DAPI, in blue; (c) Actin of the cell wall stained with rhodamine phalloidin, in red; (d) LT-I/FITC toxin; (e) Overlay of all fluorescence images; (f) Orthogonal analysis of LT-I/FITC toxin in Caco-2 cells, visualizing the toxin marked with FITC (→) associated with both the cell membrane and the nucleus; (g) 3D analysis of LT-I/FITC toxin in Caco-2 cells, evidencing the presence of toxin marked with FITC (→) in the cell membrane and nucleus.

Figure 3

Fluorescence microscopy of systemic traffic of LT-I toxin in zebrafish larvae up to 96 hpi, visualized at 40× magnification: (a–d) FITC (control) by fluorescence microscopy—(a) 24 hpi; (b) 48 hpi; (c) 72 hpi; (d) 96 hpi; (e–h) LT-I/FITC by fluorescence microscopy—(e) 24 hpi; (f) 48 hpi; (g) 72 hpi; and (h) 96 hpi. Visualized using a Lumar V12 stereomicroscope with Axiocam MRC REV 3. Fluorescence deconvolved using the AxioVision^® software (Carl Zeiss, Germany), using the following calibration parameters: 0.8× lenses, 52× magnification, −0.10 brightness, 5.78 contrast, and gamma 2.20.

Figure 4

Diagram of zebrafish intestine, showing intestinal bulb, mid intestine, and posterior intestine.

Figure 5

Survival kinetics and malformation events of zebrafish larvae microinjected with the toxin LT-I (n = 31) and FITC (n = 12) (control): (a) Larvae survival kinetics; and (b) Malformation events observed at 96 hpi in zebrafish larvae with LT-I toxin.

Figure 6

Malformation events observed in larvae injected with the LT-I/FITC toxin and FITC (as control) at different times. Visualized using a Leica M205C LASV 4.11 with software magnification of 28× and 80×: (a–d) Larvae microinjected with FITC at (a) 24 hpi; (b) 48 hpi; (c) 72 hpi; and (d) 96 hpi; and (e–h) larvae microinjected with LT-I/FITC at (e) 24 hpi; (f) 48 hpi; (g) 72 hpi; and (h) 96 hpi. Yellow dotted: heart; light blue dotted: yolk; purple dotted: larva length; and dark blue dotted: intestine.

Figure 7

Measurement of heart circumference, yolk, and length of zebrafish larvae injected with toxin LT-I (n = 31) and FITC (n = 12) (control) at different times of analysis: (a) yolk circumference, (b) larval length, and (c) heart circumference. All values express the mean ± SEM. The differences were considered statistically significant when * p < 0.01, as determined using the GraphPad Prism software (GraphPad Software, v6.02, 2013, La Jolla, CA, USA).

Figure 8

Heart rate at different times of microinjection of zebrafish larvae with toxin LT-I/FITC (n = 31) and FITC (control-n = 12). (a) 24 hpi; (b) 48 hpi; (c) 72 hpi; (d) 96 hpi. All values express the mean ± SEM. The differences were considered statistically significant when p < 0.01 as calculated using the GraphPad Prism (Graph Pad Software, v6.02, 2013, La Jolla, CA, USA).