Host mechanisms involved in cattle Escherichia coli O157 shedding: a fundamental understanding for reducing foodborne pathogen in food animal production

Abstract

The host mechanisms involved in Escherichia coli O157 super-shedding in cattle is largely unknown. In this study, the comparison of transcriptomes of intestinal tissues between super-shedders (SS) and cattle negative for E. coli O157 (NS) was performed, aiming to identify genes that are potentially associated with super-shedding. In total, 16,846 ± 639 (cecum) to 18,137 ± 696 (distal jejunum) were expressed throughout the intestine, with the expressed genes associated with immune functions more pronounced in the small intestine. In total, 351 differentially expressed (DE) genes were identified throughout the intestine between SS and NS, with 101 being up-regulated and 250 down-regulated in SS. Functional analysis revealed DE genes were involved in increased T-cell responses and cholesterol absorption in the distal jejunum and descending colon, and decreased B-cell maturation in the distal jejunum of SS. RNA-Seq based SNP discovery revealed that the mutations in seven DE genes involved in leukocyte activation and cholesterol transportation were associated with E. coli O157 shedding. Our findings suggest that T-cell responses and cholesterol metabolism in the intestinal tract may be associated with super-shedding phenomenon, and the SNPs in the DE genes are possibly associated with the observed gene expression difference between SS and NS.

Figure 1

Heatmaps for core transcriptome of all tissues. The genes in core transcriptomes were first aggregated into 100 clusters using k-means clustering. DU, duodenum; PJ, proximal jejunum; DJ, distal jejunum; CE, cecum; SC, spiral colon; DC, descending colon.

Figure 2

Principle component analysis for core transcriptome of all tissues. The red circles indicate non-shedders that are clustered together based on their gene expression profiles. The expression values of each gene were scaled by subtracting mean expression value then dividing by standard deviation. The cluster analysis was performed using k-mean algorithm. DU, duodenum; PJ, proximal jejunum; DJ, distal jejunum; CE, cecum; SC, spiral colon; DC, descending colon.

Figure 3

Function analysis for core transcriptome of cattle intestinal tissues. (A) IPA (Ingenuity Pathway Analysis) functional analysis of the core transcriptome of intestinal tissues. The heatmap shows scaled values of −log₁₀(p-value) indicated with blue colours: the darker the blue, the more likelihood that a function is associated with the core transcriptome of a tissue. (B) Functional analysis for differentially expressed (DE) genes. Results obtained from IPA downstream analysis for DE genes of each tissue. Positive z-scores were indicated with blue bars and negative z-scores with brown bars. The absolute values of z-scores were plotted, and those with a threshold greater than 2 hr, considered to indicate a difference in functionality between super-shedders and non-shedders. A positive z-score indicates enhancement, while a negative z-score indicates a reduction in function. DJ, distal jejunum; CE, cecum; SC, spiral colon; DC, descending colon; for differentially expressed genes identified in the other tissues, no IPA functional terms were enriched.

Figure 4

Differentially expressed genes between super-shedders and non-shedders. (A) Number of differentially expressed (DE) genes identified in intestinal regions. DE genes: FDR < 0.05, log2(fold change) <−1 or >1, cpm ≥1 in at least 50% of SS and NS. (B) and (C) are Heatmaps for log₂(counts per million) of differentially expressed genes in all tissues: (B) up-regulated differentially expressed genes in super-shedders; (C) down-regulated differentially expressed genes in super-shedders. Scaled log2(counts per million) was indicated by red and blue colors, and red indicates higher expression level, while blue colour indicates lower expression level. The column labels indicate super-shedders and non-shedders: NS, non-shedders; SS, super-shedders. The row labels indicate the tissue where differentially expressed genes were identified. DU, duodenum; PJ, proximal jejunum; DJ, distal jejunum; CE, cecum; SC, spiral colon; DC, descending colon.

Figure 5

(A) SNPs detected from RNA-Seq data. The x-axis shows the location of a SNP on the genes, and y-axis shows the −log2(p-values). The SNPs that shows association with super-shedding phenomena are presented above the red line (p-value < 0.05). (B) Cholesterol quantification results.

Figure 6

Possible host and E. coli O157 interactions in super-shedder cattle. (A) Changes in functions of different intestinal regions of super-shedders. (B) Proposed potential host mechanism involved in lipid metabolism, host immune responses that could be associated with E. coli O157 colonisation in the gut of cattle.