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. 2022 Jun 13;23(12):6589.
doi: 10.3390/ijms23126589.

Intestinal Epithelial Cells Modulate the Production of Enterotoxins by Porcine Enterotoxigenic E. coli Strains

Haixiu Wang  1 , Eric Cox  1 , Bert Devriendt  1
Affiliations

Intestinal Epithelial Cells Modulate the Production of Enterotoxins by Porcine Enterotoxigenic E. coli Strains

Haixiu Wang et al. Int J Mol Sci. .

Abstract

Enterotoxigenic Escherichia coli (ETEC) strains are one of the most common etiological agents of diarrhea in both human and farm animals. In addition to encoding toxins that cause diarrhea, ETEC have evolved numerous strategies to interfere with host defenses. These strategies most likely depend on the sensing of host factors, such as molecules secreted by gut epithelial cells. The present study tested whether the exposure of ETEC to factors secreted by polarized IPEC-J2 cells resulted in transcriptional changes of ETEC-derived virulence factors. Following the addition of host-derived epithelial factors, genes encoding enterotoxins, secretion-system-associated proteins, and the key regulatory molecule cyclic AMP (cAMP) receptor protein (CRP) were substantially modulated, suggesting that ETEC recognize and respond to factors produced by gut epithelial cells. To determine whether these factors were heat sensitive, the IEC-conditioned medium was incubated at 56 °C for 30 min. In most ETEC strains, heat treatment of the IEC-conditioned medium resulted in a loss of transcriptional modulation. Taken together, these data suggest that secreted epithelial factors play a role in bacterial pathogenesis by modulating the transcription of genes encoding key ETEC virulence factors. Further research is warranted to identify these secreted epithelial factors and how ETEC sense these molecules to gain a competitive advantage in the early engagement of the gut epithelium.

Keywords: enterotoxigenic Escherichia coli; enterotoxins; gut epithelium.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Figure 1
Figure 1
Factors secreted by gut epithelial cells significantly altered transcript levels of enterotoxins in ETEC strains. The mRNA expression levels of enterotoxins were assessed by qPCR in ETEC strains. These strains were cultured in CAYE medium, IPEC-J2 differentiation medium (Diff), or apical medium obtained from intestinal epithelial monolayers (IEC). The mRNA expression level of the targeted genes was normalized to three reference genes and presented as the fold change to CAYE medium. Data are presented as the mean ± SD. ** p < 0.01, *** p < 0.001.
Figure 2
Figure 2
Porcine strains differ in their ability to secrete enterotoxins in response to secreted epithelial factors. ETEC strains were grown in CAYE medium, IPEC-J2 differentiation medium (Diff), and IEC-conditioned medium. Periplasmic LT production (A) and secretion levels (B) were quantified by GM1-ELISA. (C) The pSTa secretion levels were quantified by an in-house competitive ELISA. (D) The secreted STb levels expressed by ETEC strains were evaluated by Western blotting and quantified by ImageJ. Data are presented as the mean ± SD. *** p < 0.001.
Figure 3
Figure 3
The mRNA expression levels of components of T2SS and T1SS and the key transcriptional regulator CRP. gspD and yghG genes were selected as key components of the T2SS system, while tolC and dsbA genes were selected as key components of the T1SS system. Transcript levels were assessed by qPCR in the ETEC strains. The mRNA expression level was normalized to three reference genes. Data are presented as the mean ± SD. ** p < 0.01, *** p < 0.001.
Figure 4
Figure 4
The mRNA expression levels of enterotoxin genes, crucial components of T1SS, T2SS, and the key transcriptional regulator CRP, were assessed by qPCR in ETEC strains grown in IEC- and heat-treated IEC-conditioned medium. The mRNA expression level was normalized to three reference genes. Data are presented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 4
Figure 4
The mRNA expression levels of enterotoxin genes, crucial components of T1SS, T2SS, and the key transcriptional regulator CRP, were assessed by qPCR in ETEC strains grown in IEC- and heat-treated IEC-conditioned medium. The mRNA expression level was normalized to three reference genes. Data are presented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 5
Figure 5
Heat treatment of media conditioned by epithelial cells (IEC)-affected secretion of enterotoxins. ETEC strains were grown in IEC-conditioned medium (IEC) or heat-treated IEC-conditioned medium (IEC-heat). Periplasmic LT production (A) and secretion levels (B) were quantified by GM1-ELISA. (C) The pSTa secretion levels were quantified by an in-house competitive ELISA. (D) The secreted STb levels expressed by ETEC strains were quantified by ImageJ. Data are presented as the mean ± SD. *** p < 0.001.
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