Lawsonia intracellularis exploits β-catenin/Wnt and Notch signalling pathways during infection of intestinal crypt to alter cell homeostasis and promote cell proliferation

Lawsonia intracellularis is an obligate intracellular bacterial pathogen that causes proliferative enteropathy (PE) in pigs. L. intracellularis infection causes extensive intestinal crypt cell proliferation and inhibits secretory and absorptive cell differentiation. However, the affected host upstream cellular pathways leading to PE are still unknown. β-catenin/Wnt signalling is essential in maintaining intestinal stem cell (ISC) proliferation and self-renewal capacity, while Notch signalling governs differentiation of secretory and absorptive lineage specification. Therefore, in this report we used immunofluorescence (IF) and quantitative reverse transcriptase PCR (RTqPCR) to examine β-catenin/Wnt and Notch-1 signalling levels in uninfected and L. intracellularis infected pig ileums at 3, 7, 14, 21 and 28 days post challenge (dpc). We found that while the significant increase in Ki67+ nuclei in crypts at the peak of L. intracellularis infection suggested enhanced cell proliferation, the expression of c-MYC and ASCL2, promoters of cell growth and ISC proliferation respectively, was down-regulated. Peak infection also coincided with enhanced cytosolic and membrane-associated β-catenin staining and induction of AXIN2 and SOX9 transcripts, both encoding negative regulators of β-catenin/Wnt signalling and suggesting a potential alteration to β-catenin/Wnt signalling levels, with differential regulation of the expression of its target genes. We found that induction of HES1 and OLFM4 and the down-regulation of ATOH1 transcript levels was consistent with the increased Notch-1 signalling in crypts at the peak of infection. Interestingly, the significant down-regulation of ATOH1 transcript levels coincided with the depletion of MUC2 expression at 14 dpc, consistent with the role of ATOH1 in promoting goblet cell maturation. The lack of significant change to LGR5 transcript levels at the peak of infection suggested that the crypt hyperplasia was not due to the expansion of ISC population. Overall, simultaneous induction of Notch-1 signalling and the attenuation of β-catenin/Wnt pathway appear to be associated with the inhibition of goblet cell maturation and enhanced crypt cell proliferation at the peak of L. intracellularis infection. Moreover, the apparent differential regulation of apoptosis between crypt and lumen cells together with the strong induction of Notch-1 signalling and the enhanced SOX9 expression along crypts 14 dpc suggest an expansion of actively dividing transit amplifying and/or absorptive progenitor cells and provide a potential basis for understanding the development and maintenance of PE

Characterization and Differential Gene Expression between Two Phenotypic Phase Variants in <em>Salmonella enterica</em> Serovar Typhimurium

<div><p><em>Salmonella enterica</em> serovar Typhimurium strain 798 has previously been shown to undergo phenotypic phase variation. One of the phenotypes expresses virulence traits such as adhesion, while the other phenotype does not. Phenotypic phase variation appears to correlate with the ability of this strain to cause persistent, asymptomatic infections of swine. A new method to detect cells in either phenotypic phase was developed using Evans Blue-Uranine agar plates. Using this new assay, rates of phenotypic phase variation were obtained. The rate of phase variation from non-adhesive to adhesive phenotype was approximately 10⁻⁴ per cell per generation while phase variation from the adhesive to the non-adhesive phenotype was approximately 10⁻⁶ per cell per generation. Two highly virulent <em>S.</em> Typhimurium strains, SL1344 and ATCC 14028, were also shown to undergo phase variation. However, while the rate from adhesive to non-adhesive phenotype was approximately the same as for strain 798, the non-adhesive to adhesive phenotype shift was 37-fold higher. Differential gene expression was measured using RNA-Seq. Eighty-three genes were more highly expressed by 798 cells in the adhesive phenotype compared to the non-adhesive cells. Most of the up-regulated genes were in virulence genes and in particular all genes in the <em>Salmonella</em> pathogenicity island 1 were up-regulated. When compared to the virulent strain SL1344, expression of the virulence genes was approximately equal to those up-regulated in the adhesive phenotype of strain 798. A comparison of invasive ability demonstrated that strain SL1344 was the most invasive followed by the adhesive phenotype of strain 798, then the non-adhesive phenotype of strain 798. The least invasive strain was ATCC 14028. The genome of strain 798 was sequenced and compared to SL1344. Both strains had very similar genome sequences and gene deletions could not readily explain differences in the rates of phase variation from non-adhesive to the adhesive phenotype.</p> </div

Alignment and comparison of the genomes of <i>S.</i> Typhimurium strains 798 and SL1344.

<p>The results are visualized using a dot plot prepared using BLAST.</p

Comparison of expression of the SPI1 genes.

<p>RNA-Seq data representing results from <i>S. enterica</i> i518, 1519, SL1344, and LT2 are graphically compared and aligned with the known SPI1 genes.</p

Growth of i518 and i519 in LB With 1% Salt and LB Without Added Salt.

<p>Growth curves of i518 and i519 were repeated on three separate occasions. Spectrophotometric readings were taken every thirty minutes. i518 grown in LB-0: ⧫ i518 grown in LB-H: ▴, i519 grown in LB-0: ▪, i519 grown in LB-H: • Shown is a single representative graph.</p

Results of invasion assays using Henle 407 cells.

<p>Invasion assays were performed using the strains indicated. All final results represent the mean of 3 replicates. Error bars are +/− one standard deviation. The data is expressed as the percent of bacterial cells that invaded the Henle 407 cells. The <i>hilA</i> mutants were each used as negative controls as HilA is required for expression of genes involved in invasion.</p

Rate of phenotypic phase variation between different strains of <i>S.</i> Typhimurium.

*<p>Significant at ≤0.03 compared to strain 798.</p

Image of i518 and i519 colonies on an Evans Blue Uranine agar plate.

<p>Colonies of i518 are dark and colonies of i519 are light.</p