Campylobacter jejuni genes Cj1492c and Cj1507c are involved in host cell adhesion and invasion

Abstract

Background: Campylobacter jejuni (C. jejuni) has been assigned as an important food-borne pathogen for human health but many pathogenicity factors of C. jejuni and human host cell responses related to the infection have not yet been adequately clarified. This study aimed to determine further C. jejuni pathogenicity factors and virulence genes based on a random mutagenesis approach. A transposon mutant library of C. jejuni NCTC 11168 was constructed and the ability of individual mutants to adhere to and invade human intestinal epithelial cells was evaluated compared to the wild type. We identified two mutants of C. jejuni possessing altered phenotypes with transposon insertions in the genes Cj1492c and Cj1507c. Cj1492c is annotated as a two-component sensor and Cj1507c is described as a regulatory protein. However, functions of both mutated genes are not clarified so far.

Results: In comparison to the wild type, Cj::1492c and Cj::1507c showed around 70-80% relative motility and Cj::1492c had around 3-times enhanced adhesion and invasion rates whereas Cj::1507c had significantly impaired adhesive and invasive capability. Moreover, Cj::1492c had a longer lag phase and slower growth rate while Cj::1507c showed similar growth compared to the wild type. Between 5 and 24 h post infection, more than 60% of the intracellular wild type C. jejuni were eliminated in HT-29/B6 cells, however, significantly fewer mutants were able to survive intracellularly. Nevertheless, no difference in host cell viability and induction of the pro-inflammatory chemokine IL-8 were determined between both mutants and the wild type.

Conclusion: We conclude that genes regulated by Cj1507c have an impact on efficient adhesion, invasion and intracellular survival of C. jejuni in HT-29/B6 cells. Furthermore, potential signal sensing by Cj1492c seems to lead to limiting attachment and hence internalisation of C. jejuni. However, as the intracellular survival capacities are reduced, we suggest that signal sensing by Cj1492c impacts several processes related to pathogenicity of C. jejuni.

Keywords: Campylobacter jejuni; Host cell response; Mutants; Pathogenicity.

Fig. 1

Genomic locus, transposon insertion sites in C. jejuni NCTC 11168 mutants ::Cj1492c and ::Cj1507c and PCR verification of both mutants and phenotypic analyses of two mutants. a, b Genes are plotted as arrows in order to their genomic locations. Boxed arrows length indicates relative gene sizes. a The location of inserted transposon is indicated in C. jejuni two-component sensor gene (Cj1492c). b The location of the inserted transposon is indicated in C. jejuni regulatory protein gene (Cj1507c). c Agarose gel electrophoresis of genomic DNA of mutants ::Cj1492c, ::Cj1507c and wild type using gene specific primers. With integration of the transposon (1219 base pairs) the DNA fragment sizes of both mutants are around 1400 base pairs in length which are 1.2 kb larger than the target DNA of the wild type (around 200 base pairs). d Growth curve of wild type, ::Cj1492c and ::Cj1507c were assessed by CFU counting. Values are shown as mean value of three independent experiments. Error bars represent SD. Statistical comparison with the wild type: *p ≤ 0.05, unpaired t-test. e Differential swarming ability of mutated strains was normalised with the wild type strain and is presented as percentage of swarming halos of wild type (100%). Columns show median value of five biological replicates and bars indicate maximum and minimum range. *p ≤ 0.05; ***p ≤ 0.001; ****p ≤ 0.0001, unpaired t-test

Fig. 2

Adhesion, invasion and internalisation levels of C. jejuni in HT-29/B6 cells and immunofluorescent detection of intracellular C. jejuni. Adhesion (a) of three tested strains to HT-29/B6 was detected at 1 h p.i.. For determination of invasion (b), the monolayer of HT-29/B6 was incubated with gentamicin for an additional 2 h prior to lysis. Experiments were performed at least three independent times in triplicate. The relative adhesion and invasion index were normalised with wild type and are presented as percentage of the adhesion and invasion index of wild type, respectively. Results show the mean value ± SD. *p ≤ 0.05; ***p ≤ 0.001, unpaired t-test. c HT-29/B6 cells and immunofluorescently labelled wild type, ::Cj1492c and ::Cj1507c (green, indicated by white arrows) and untreated control cells are shown at 5 h p.i. Cell cytoplasm membrane were stained with WGA-CF^®594 (red) and nuclei with DAPI (blue). Scale bars indicates 25 μm. d The internalisation level of C. jejuni by HT-29/B6 was determined as a ratio of particles of invaded C. jejuni and infected cells by means of immunofluorescence microscopy and automatic particle counting using ImageJ. The relative level of internalisation was normalised with wild type and is presented as percentage of the wild type. Fluorescent particles of each strain were quantified in at least six microphotographs selected randomly for each biological replication and results are presented for four individual experiments. Asterisks show statistically significant differences between ::Cj1492c and ::Cj1507c. *p ≤ 0.05; unpaired t-test

Fig. 3

Intracellular survival of C. jejuni, relative viability of HT-29/B6 cells and RT-qPCR analysis of IL-6 and IL-8 mRNA expression in HT-29/B6 cells after C. jejuni infection. a Intracellular survival, presented as CFU recovery rates of intracellular viable cell counts, was determined as a ratio of viable counts between 5 and 24 h p.i. The relative survival rate was normalised with the wild type and is presented as percentage of the wild type. Results represent at least six individual experiments and asterisks indicate statistically significant differences between mutants and the wild type. *p ≤ 0.05; **p ≤ 0.01, unpaired t-test. b Relative viability of HT-29/B6 cells after infection was measured after 5 h and 24 h of infection and calculated as ratio of number of viable cells compared to the total cell numbers and normalised on the viability of uninfected control group. Values are the mean ± SD of three individual experiments. c Fold changes of IL-6 and IL-8 mRNA expression were calculated relatively to non-infected controls and normalized to housekeeper. Experiments were performed in triplicate and the mean ± SD is presented. Only altered mRNA expression level above or below the dotted lines are defined as regulated. Asterisks indicate statistically significant differences between tested strains and negative controls at each time point. *p ≤ 0.05; **p ≤ 0.01; ***p≤ 0.001, unpaired t-test