Escherichia coli expressing EAST1 toxin did not cause an increase of cAMP or cGMP levels in cells, and no diarrhea in 5-day old gnotobiotic pigs

Abstract

Background: Enterotoxigenic Escherichia coli (ETEC) strains are the leading bacterial cause of diarrhea to humans and farm animals. These ETEC strains produce heat-labile toxin (LT) and/or heat-stable toxins that include type I (STa), type II (STb), and enteroaggregative heat-stable toxin 1 (EAST1). LT, STa, and STb (in pigs) are proven the virulence determinants in ETEC diarrhea. However, significance of EAST1 in ETEC-associated diarrheal has not been determined, even though EAST1 is highly prevalent among ETEC strains.

Methodology/principal findings: In this study, we constructed E. coli strains to express EAST1 toxin as the only toxin and studied them in cell lines and five-day old gnotobiotic piglets to determine significance of EAST1 toxin. Data from in vitro studies indicated that EAST1 did not stimulate an increase of intracellular cyclic AMP or GMP levels in T-84 cells or porcine cell line IPEC-J2, nor did it enhance LT or STa toxin of ETEC strains in stimulation of cAMP or cGMP in T-84 cells. In addition, 5-day old gnotobiotic pigs challenged with E. coli strains expressing EAST1 as the only toxin did not developed diarrhea or signs of clinical disease during 72 h post-inoculation.

Conclusion/significance: Results from this study indicated that EAST1 alone is not sufficient to cause diarrhea in five-day old gnotobiotic pigs, and suggest that EAST1 likely is not a virulence determinant in ETEC-associated diarrhea.

Figure 1. EAST1 competitive ELISA to detect EAST1protein expressed from the recombinant strains.

10 ng EAST1-ovalbumin conjugates were coated at each well of a microtiter plate at 37°C overnight. 75 µl supernatants of overnight grown culture, from an equal amount of cells, of each strain, and 75 µl of chicken anti-EAST1 serum (1∶1000; GenWay Biotech, Inc., CA) was mixed and added to each well. HRP-conjugated donkey anti-chicken IgY (1∶2500; GenWay Biotech, Inc., CA) was used as the secondary antibody. OD values were measured at 405 nm after 20 min incubation with peroxidase substrate.

Figure 2. EIA ELISAs to measure stimulation of intracellular cyclic GMP and AMP in T-84 cells.

Supernatants of overnight grown culture (with an equal amount of cells) from each constructed strain were incubated with 1–2×10⁵ T-84 cells. Panel A: Intracellular cGMP levels were measured using an EIA cGMP kit (Assay Designs), with 2 ng purified STa (from Robertson laboratory) was used as a positive control. Panel B: Intracellular cAMP levels were measured using an EIA cAMP kit with 10 ng CT as a positive control.

Figure 3. Ussing chamber assays to detect EAST1 enterotoxic activity.

Panel A: Short-circuit current (I _sc; µA/cm²) changes in monolayers of IPEC-J2 cells after incubation with culture growth of strains 8321, 8322, a negative control 8231, and 4AA medium were measured using a modified Ussing chamber. Panel B: Isc changes in T-84 cells incubated with culture growth of strains 8321, 8322, a negative control 8231, and 4AA medium. Panel C: The p values were calculated by analyzing all 5 data sets, 2 sets of data using IPEC-J2 cells and 3 sets of data using T-84 cells. Forskolin (2 µM) was added to a paired chamber as a positive control.

Figure 4. A microscopic image of piglet ileal section to show colonization of E. coli strain 8321 that expresses EAST1 toxin.

Ileal segments of piglets challenged with strain 8321 were collected at necropsy and fixed at 10% neutral buffered Formalin. Fixed tissues were embedded, sectioned, H&E stained, and examined microscopically using an Olympus IX70 inverted microscope.