Role of heat-stable enterotoxins in the induction of early immune responses in piglets after infection with enterotoxigenic Escherichia coli

Abstract

Enterotoxigenic Escherichia coli (ETEC) strains that produce heat-stable (ST) and/or heat-labile (LT) enterotoxins are cause of post-weaning diarrhea in piglets. However, the relative importance of the different enterotoxins in host immune responses against ETEC infection has been poorly defined. In the present study, several isogenic mutant strains of an O149:F4ac(+), LT(+) STa(+) STb(+) ETEC strain were constructed that lack the expression of LT in combination with one or both types of ST enterotoxins (STa and/or STb). The small intestinal segment perfusion (SISP) technique and microarray analysis were used to study host early immune responses induced by these mutant strains 4 h after infection in comparison to the wild type strain and a PBS control. Simultaneously, net fluid absorption of pig small intestinal mucosa was measured 4 h after infection, allowing us to correlate enterotoxin secretion with gene regulation. Microarray analysis showed on the one hand a non-toxin related general antibacterial response comprising genes such as PAP, MMP1 and IL8. On the other hand, results suggest a dominant role for STb in small intestinal secretion early after post-weaning infection, as well as in the induced innate immune response through differential regulation of immune mediators like interleukin 1 and interleukin 17.

Figure 1. Quantitative detection of STa (A) and LT (B) expression by different isogenic ETEC strains following in vitro culture.

Both toxins were detected by enzyme immunoassays. Mean values ± SD are shown. (A) Samples for STa were tested in triplicate in three independent experiments. (B) LT results are representative for 2 independent experiments. ND  =  below detection limit of 10 ng/ml. WT  =  wild type strain.

Figure 2. Detection of STb expression by different isogenic ETEC strains following in vitro culture.

(A) STb detection with Western blotting. For every GIS26 mutant an equal amount of filtered supernatant was loaded (30 µl), data are representative for 3 independent experiments. (B) In the STb positive strains, STb was quantified by a direct ELISA. Mean values±SD are shown; results are representative for 3 independent experiments. (C) A difference in amount of STb produced between the wild type strain and the GIS26ΔestA mutant was also detected by Western blotting of different dilutions of the supernatant of both strains (20 µl per lane).

Figure 3. Effect of wild type and mutant GIS26 ETEC strains on net fluid absorption (mg/cm²) in 4 h-infected jejunal segments.

Individual data per piglet and the mean from 3 individual experiments are presented.

Figure 4. Effect of STb deletion in GIS26 ETEC strain on net fluid absorption (mg/cm²) in 4 h-infected jejunal segments.

Individual data per piglet and the mean from 5 individual experiments are presented.

Figure 5. Linear regression of qRT-PCR CT ratios versus log2 expression ratios as obtained by microarray analysis for IL1A, IL8, IL17A, PAP, FABP2, TLR4, MMP1, MMP3 and CYP1A1.

The CT values for the genes of interest were normalized for two reference genes RPL4, and GAPDH. The ratios on the x- and y-axis were calculated as the log2 expression value of the experimental sample minus the log2 expression value of the control sample, for qRT-PCR data as well as microarray data. The microarray analysis was performed on pooled samples, and the qRT-PCR analysis on individual samples. The goodness of fit (r²) and P-value are given.