Table 1.
Primer sequences used for the deletion of enterotoxin genes in ETEC strain GIS26.
Table 2.
Primer sequences used for qRT-PCR.
Table 3.
Enterotoxin genotype and phenotype of GIS26 mutants used in this study.
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.
Table 4.
Summary of differentially expressed probe sets of all mutant strains and control versus wild type ETEC strain.
Table 5.
Transcripts down-regulated by ETEC. Microarray data expressed as a log2 fold-change of PBS versus ETEC-infected small intestinal segments at 4 h (for full list see Table S1).
Table 6.
Transcripts up-regulated by ETEC. Microarray data expressed as a log2 fold-change of ETEC-infected versus PBS treated small intestinal segments at 4 h (for full list see Table S1).
Table 7.
Microarray data expressed as a log2 ratio of PBS and mutant ETEC-infected versus wild type ETEC-infected (WT) small intestinal segments at 4 h (Group I, transcripts in common for the three comparisons where differential regulation was found).
Table 8.
Microarray data expressed as a log2 fold-change of PBS and mutant ETEC-infected versus wild type ETEC-infected (WT) small intestinal segments (Group II, transcripts differentially regulated by both mutant ETEC strains).
Table 9.
Microarray data expressed as a log2 fold-change of PBS and mutant ETEC-infected versus wild type ETEC-infected (WT) small intestinal segments at 4 h (Group III, transcripts differentially regulated in the PBS/WT comparison and in one of both mutant strains).
Table 10.
Microarray data expressed as a log2 fold-change of PBS and mutant ETEC-infected versus wild type ETEC-infected (WT) small intestinal segments at 4 h (Group IV, differentially regulated transcripts exclusively found in one of the mutant strain comparisons).
Table 11.
Microarray data expressed as a log2 fold-change of PBS and mutant ETEC-infected versus wild type ETEC-infected (WT) small intestinal segments (Group V, differentially regulated transcripts only found in the comparison WT/PBS).
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.