Immunological pathogenesis of Bovine E. coli infection in a model of C. elegans

Abstract

Background: Cattle industry is critical for China's livestock industry, whereas E. coli infection and relevant diseases could lead huge economic loss. Traditional mammalian models would be costly, time consuming and complicated to study pathological changes of bovine E. coli. There is an urgent need for a simple but efficient animal model to quantitatively evaluate the pathological changes of bovine-derived E. coli in vivo. Caenorhabditis elegans (C. elegans) has a broad host range of diverse E. coli strains with advantages, including a short life cycle, a simple structure, a transparent body which is easily visualized, a well-studied genetic map, an intrinsic immune system which is conservable with more complicated mammalians.

Results: Here, we considered that O126 was the dominant serotype, and a total of 19 virulence factors were identified from 41 common E. coli virulence factors. Different E. coli strains with diverse pathogenicity strengths were tested in C. elegans in E. coli with higher pathogenicity (EC3/10), Nsy-1, Sek-1 and Pmk-1 of the p38 MAPK signaling pathway cascade and the expression of the antimicrobial peptides Abf-3 and Clec-60 were significantly up-regulated comparing with other groups. E. coli with lower pathogenicity (EC5/13) only activated the expression of Nsy-1 and Sek-1 genes in the p38 MAPK signaling pathway, Additionally, both groups of E. coli strains caused significant upregulation of the antimicrobial peptide Spp-1.

Conclusion: Thirteen E. coli strains showed diverse pathogenicity in nematodes and the detection rate of virulence factors did not corresponding to the virulence in nematodes, indicating complex pathogenicity mechanisms. We approved that C. elegans is a fast and convenient detection model for pathogenic bacteria virulence examinations.

Keywords: Bovine; Caenorhabditis elegans (C. elegans); Escherichia coli (E. coli); Innate immunity; Pathogenicity.

Fig. 1

The survival rates of C. elegans on consecutive time-points after a particular E.coli strain infection. Figure legend: L4 stage wild-type N2 C. elegans were used as model animals. Three duplicates (20 nematodes each, total n = 60) were carried out in this study. Testing animals were transferred to fresh plates daily and stimulated with a picking needle for their responses. The number of survival nematodes was recorded. The survival rate of nematodes = number of live worms per day/60 × 100%. In Fig. 1, the dark blue line represents those nematodes fed on E. coli 3. The pink line represents nematodes fed E. coli 10. The purple line represents nematodes feeding on E. coli 5. The light blue line represents nematodes feeding on E. coli 13. The green line represents nematodes feeding on E. coli OP50

Fig. 2

Expression of immune genes in p38MAPK signaling pathway. Figure legend: the graph-a represents tir-1 gene expression changes in nematode after infected with E. coli strains of diverse pathogenicity. The graph-b represents changes of nsy-1 gene expression. The graph-c represents changes of sek-1 gene expression. The graph-d represents changes in pmk-1 gene expression. The graph-e represents changes of skn-1 gene expression. Differences within and between groups were calculated separately and plotting by GraphPad Prism software. ‘ns’: non-significant differences (P ≥ 0.05). *: significant difference (P ≤ 0.05). **: significant difference (P ≤ 0.01). ***: significant difference (P ≤ 0.001). ****: significant difference (P ≤ 0.0001)

Fig. 3

Expression of Dbl-1 of TGF-β signaling pathway. Figure Legend:‘ns’: non-significant differences (P ≥ 0.05). *: significant difference (P ≤ 0.05). **: significant difference (P ≤ 0.01). ***: significant difference (P ≤ 0.001). ****: significant difference (P ≤ 0.0001)

Fig. 4

Expression of immune genes of insulin-like signaling pathway. Figure legend: a Two strains, the E. coli 3 and 10, both highly express Daf-16 gene after infection while this gene expresses relevant low in the other two strains, E. coli 5 and 13. There is no significant variances between E. coli 3 and 10, or between E. coli 5 and 13. But the Daf-16 gene expression was significantly different among the two groups. b The change in Age-1 gene expression is not significant after the infection of E. coli 3 and 10, but are statistical significantly differently either between E. coli 5 and 13, or among E. coli 3 and E. coli 5, 13 and also among E. coli 10 and E. coli 5, 13. It reflecting different pathogenicity among E. coli strains. ‘ns’: non-significant differences (P ≥ 0.05). *: significant difference (P ≤ 0.05). **: significant difference (P ≤ 0.01). ***: significant difference (P ≤ 0.01)

Fig. 5

Expression of antimicrobial peptide genes. Figure legend: a represents the expression change of antimicrobial peptide gene Lys-7 in nematode after infected with E. coli strains of different pathogenicity. b the expression changes of Clec-60 gene after E. coli infection. c the expression changes of Clec-85 infection. d the expression changes of Abf-3 after infection. E the expression changes of Abf-2 gene after E. coli infection in nematodes. F the expression changes of Spp-1 gene after infection. ‘ns’: non-significant differences (P ≥ 0.05). *: significant difference (P ≤ 0.05). **: significant difference (P ≤ 0.01). ***: significant difference (P ≤ 0.001). ****: significant difference (P ≤ 0.0001)