This study indicates that preservation of the CRISPR array in C. jejuni is strongly associated with the risk of developing GBS. In addition, the probability of developing GBS following C. jejuni infection increases significantly with the CRISPR array length. Moreover, we found the Campylobacter bacteriophage DA10 is targeted by the CRISPR-Cas system of the majority of C. jejuni strains with a preserved CRISPR array. Moreover, this study identified 38 novel CTs and 20 novel spacer sequences in the CRISPR array, and demonstrates that GBS-related C. jejuni strains contain unique CT patterns compared to enteritis- and healthy control-related strains.
The type II CRISPR-Cas system has previously been reported to be involved in endogenous gene expression and gene regulation, respond to envelope stress and regulate other physiological processes in bacteria 25,35. The ability of the Cas9 endonuclease to regulate the virulence genes of bacteria containing the type II CRISPR-Cas system were described in experimental studies of C. jejuni 15,36, Francisella novicida 37 and Neisseria meningitides 38. C. jejuni strains harbouring the Cas9 gene were found to be more virulent than strains that do not harbour Cas9 or contain a degenerated Cas9 gene 15. Recently, Dugar et al. reported that C. jejuni Cas9 can regulate gene expression by targeting endogenous mRNA 39. However, the relationship between CRISPR array preservation and bacterial virulence or pathogenesis had not previously been assessed. This study provides the first evidence that preservation of the CRISPR array in C. jejuni is associated with the development of GBS, which suggests a preserved CRISPR array may contribute to virulence or immunogenicity by controlling the expression of bacterial genes that increase the risk of developing GBS.
The CRISPR array length depends on the number of spacers acquired by the bacteria (Fig. 4). In this study, we observed longer C. jejuni CRISPR arrays were associated with a higher probability of developing GBS. This observation can potentially be explained by the findings of Martynov et al., who reported that bacteria maintain the optimum numbers of spacers to gain an evolutionary benefit 40. Additionally, Levin’s mathematical model predicted that a preserved CRISPR array provides a competitive advantage to bacteria by facilitating long-term co-evolutionary arms races between phage and bacteria 41. This study supports the mathematical prediction that bacteria prefer to maintain an optimum CRISPR array length.
We also confirmed that CRISPR array-based typing had more discriminatory power and sensitivity than PCR-based subtyping due to the highly polymorphic CRISPR arrays of enteritis- and healthy control-related C. jejuni strains; thus, CRISPR array-based typing may represent a superior genotyping method 42. CRISPR-based genotyping of C. jejuni 28,42−44 and other bacteria were previously reported 45–47. Cardenas et al. and Yeh et al. found CRISPR typing of C. jejuni had sufficient discriminatory power (SID) to be considered a useful genotyping method 28,44. Our study provides additional evidence to suggest that CRISPR typing could represent a useful molecular detection tool for epidemiological source identification during outbreaks of GBS or C. jejuni-induced enteritis.
A major part of the genome of the recently discovered Campylobacter phage DA10 was found to be attacked by the C. jejuni CRISPR array 48,49. Similarly, most of our CRISPR array-containing C. jejuni strains acquired spacers from the Campylobacter phage DA10.
This study has several limitations. Even though we used PCR-based CRISPR typing, the discriminatory power of CRISPR typing was not compared with traditional genotyping or serotyping methods such as pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) or flaA serotyping. Additionally, the sample size was relatively small and the significance of the association between C. jejuni CRISPR array preservation and the development of GBS needs to be verified by an experimental study.
Overall, this study reveals that preservation of the CRISPR array in C. jejuni may provide an additional evolutionary benefit during the immunopathogenesis of GBS, which establishes a link between CRISPR array preservation and the risk of disease development. Further studies of larger, multicentric cohorts and genomic analysis of the entire C. jejuni CRISPR-Cas system using an experimental study design are required to confirm the role of the C. jejuni CRISPR array in bacterial virulence and the pathogenesis of GBS.