Table 1.
Oligonucleotide primers used for sequencing, cloning of the C. jejuni gene cj0268c with and without His-tag, generation of the knockout mutant and screening of genomic DNA samples for the presence of cj0268c.
Figure 1.
Generation of a cj0268c-knockout mutant and a complemented mutant in C. jejuni strain NCTC 11168.
(1) parental strain NCTC 11168, (2) cj0268c knockout mutant (NCTC 11168::cj0268c), (3) complemented knockout mutant (NCTC 11168::cj0268c-comp-cj0268c). (a) Genome arrangements of the bacterial strains under investigation. Primers Cj0268cF and Cj0268cR for the amplification ofcj0268c are indicated by arrowheads. The primer KanF that binds to the 5′-end of the kanamycin resistance cassette is shown by an arrow. (b) Verification of the native gene, the respective mutant and the complemented mutant strain by PCR. (1) PCR analysis with Cj0268cF + Cj0268cR-primers detect the native gene of 1090 bp. (2) aphA-3 insertion in gene cj0268c mediating kanamycin resistance in mutant strain NCTC 11168::cj0268c was verified by the amplification of a 2914 bp PCR- amplicon applying primers Cj0268cF+R. (3) the complemented mutant was verified by PCR analysis with primers Cj0268cF+R to detect the native gene (1090 bp) and with primers KanF + Cj0268cR which amplify a PCR product of 2168 bp.
Figure 2.
1. wild type strain NCTC 11168, 2. mutant strain NCTC 11168::cj0268c, 3. complemented mutant NCTC 11168::cj0268c-comp-cj0268c. Gentamicin protection assays with the strains under investigation using (a) Caco2 cells and (b) PCC-cells. The assays on Caco2 cells with the parental strain, the knockout mutant and the complemented knockout mutant confirmed the infection-deficient phenotype to be due to the functional loss of cj0268c. Four independent experiments have been carried out, respectively. The standard deviations are indicated. (a) Taking the number of C. jejuni wild type strain NCTC 11168 colonies recovered as 100%, the mean value of cj0268c-mutant colonies (NCTC 11168::cj0268c) accounted for 62% (±2.41), whereas the percentage of obtained colonies from the complemented strain (NCTC 11168::cj0268c-comp-cj0268c) was 93% (±2.04). Since the P-value for the mutant was less than 0.001, the reduced invasion capacity was significant. (b) The relevance of cj0268c for invasion is not restricted to human cells, since it could also be shown for the invasion of PCC-cells. Compared to the invasion capacity of parental strain NCTC 11168 (100%), the percentage of colonies obtained from mutant strain NCTC 11168::cj0268c was only 62% (±2.36, P<0.0007). However, complementation of the mutant strain with cj0268c restored the infectivity of NCTC 11168::cj0268c-comp-cj0268c which was similar to that of the parental strain (96%, ±4.96).
Figure 3.
Assays to test for adhesion, autoagglutination and motility of the strains under investigation.
(1) parental strain NCTC 11168, (2) cj0268c knockout mutant (NCTC 11168::cj0268c), (3) complemented knockout mutant (NCTC 11168::cj0268c-comp-cj0268c). (a) Loss of cj0268c reduces the capability of C. jejuni to adhere to Caco2 cells. Defining the number of wild type strain NCTC 11168 colonies obtained performing adhesion assays as 100%, the mean value of colonies from the mutant NCTC 11168::cj0268c was 60.9% (±5.18, P<0.0039) in contrast to NCTC 11168::cj0268c-comp-cj0268c with a recovery rate of 96.4% (±1.77). (b, c) Gene cj0268c does neither impair the property of C. jejuni to autoagglutinate nor the motility of the pathogen. By performing corresponding tests, no differences among the bacterial strains under investigation could be detected. Both, the percentage of autoagglutinated bacteria and the motility zones did not show any significant differences between wild type, mutant and complemented mutant.
Figure 4.
Detection of Cj0268c in E. coli and altered adherence.
1. E. coli DH5α/pRRC, 2. E. coli DH5α/pRRC-cj0268cHis. (a) Immunoblot with a monoclonal antibody against the His-tag detected a protein with a molecular weight of 41 kDa corresponding to Cj0268c. The protein was exclusively found in the lysate of the Cj0268c-expressing E. coli. The respective Coomassie staining served as a loading control. (b) Adherence assays on Caco2 cells with Cj0268c-expressing E. coli and E. coli transformed with plasmid pRRC alone. Taken the number of recovered E. coli colonies from the strain harbouring pRRC without cj0268c as 100%, the mean value of the adherence capacity of E. coli DH5α/pRRC-cj0268cHis was 201.7% (±6.44, P<0.0039).
Figure 5.
Flow cytometric measurements of permeabilized and non-permeabilized E. coli cells expressing Cj0268c (pRRC-cj0268c) or transformed with an empty vector (pRRC).
Bacteria were immunolabelled with a monoclonal anti-pentaHis primary antibody and a R-phycoerythrin-conjugated secondary antibody. E. coli cells of both populations were gated according to the forward scatter and the side scatter (dot plots, left panel). Permeabilized E. coli cells harbouring the empty plasmid pRRC without gene cj0268c (upper panel) possess a considerable lower fluorescence intensity as compared to an E. coli population which express Cj0268c (lower panel). To verify the specificity of binding of anti-pentaHis, a control staining with the secondary antibody only was included (right panel).
Figure 6.
(1) Parental strain NCTC 11168, (2) cj0268c knockout mutant (NCTC 11168::cj0268c), (3) complemented knockout mutant (NCTC 11168::cj0268c-comp-cj0268c). The numbers of colonies of the respective fourth dilution plated onto blood agar are shown. The cj0268c knockout mutant shows a clearly diminished resistance to Triton X-100. When defining the number of wild type colonies as 100%, the mean value of colonies recovered from the corresponding cj0268c-mutant was 21.3% (±9.02, P<0.0039). After complementation of the mutant with an intact copy of cj0268c the percentage of colonies obtained increased to 91.3%.