The Involvement of the Cas9 Gene in Virulence of Campylobacter jejuni

Abstract

Campylobacter jejuni is considered as the leading cause of gastroenteritis all over the world. This bacterium has the CRISPR-cas9 system, which is used as a gene editing technique in different organisms. However, its role in bacterial virulence has just been discovered; that discovery, however, is just the tip of the iceberg. The purpose of this study is to find out the relationship between cas9 and virulence both phenotypically and genotypically in C. jejuni NCTC11168. Understanding both aspects of this relationship allows for a much deeper understanding of the mechanism of bacterial pathogenesis. The present study determined virulence in wild and mutant strains by observing biofilm formation, motility, adhesion and invasion, intracellular survivability, and cytotoxin production, followed by the transcriptomic analysis of both strains. The comparative gene expression profile of wild and mutant strains was determined on the basis of De-Seq transcriptomic analysis, which showed that the cas9 gene is involved in enhancing virulence. Differential gene expression analysis revealed that multiple pathways were involved in virulence, regulated by the CRISPR-cas9 system. Our findings help in understanding the potential role of cas9 in regulating the other virulence associated genes in C. jejuni NCTC11168. The findings of this study provide critical information about cas9's potential involvement in enhancing the virulence of C. jejuni, which is a major public health threat.

Keywords: Campylobacter jejuni; cas9; differential gene expression; transcriptome analysis; virulence.

Figure 1

(A,B) Standard growth curve of mutant with its parent strain under static and shaking conditions. (A) represents the bacterial growth curve under static conditions, while (B) shows the bacterial growth under shaking conditions. The Y axis shows the growth of C. jejuni NCTC11168, complemented and Δcas9 mutant strains in Log10 (cfu/mL) in static condition. One asterisk (*) represent statistically significant differences at P ≤ 0.05, in comparisons of C. jejuni NCTC11168, complemented and the Δcas9 mutant.

Figure 2

Biofilm formation of C. jejuni NCTC11168 and Δcas9 mutant at different time points. The Y axis is the OD₅₇₀ value of crystal violet in biofilm. The results were obtained from three independent repeats. The asterisk (*) indicates statistical significance at P ≤ 0.05 for the comparison of C. jejuni NCTC11168, complemented, and the Δcas9 mutant.

Figure 3

Adhesion and invasion of C. jejuni NCTC11168 and Δcas9 mutant on macrophage RAW 264.7 cells. The Y axis is the mean of log10 CFU/mL of each strain in the RAW264.7 cell. The results were obtained from three independent repeats. One asterisk (*) and two asterisks (**) represent statistically significant differences at P ≤ 0.05and P ≤ 0.01, respectively, in comparisons of C. jejuni NCTC11168, complemented, and the Δcas9 mutant.

Figure 4

Intracellular survivability of C. jejuni NCTC11168 and Δcas9 mutant in macrophage RAW 264.7 cells. The Y axis shows the percent survival of C. jejuni NCTC11168, complemented and Δcas9 mutant strain in the RAW264.7 cell. The analysis was performed by using the Kaplan-Meier estimate method. The results showed that C. jejuni NCTC11168 and complemented strains had more survivability than Δcas9 mutant strain.

Figure 5

Cytotoxic production of the C. jejuni NCTC11168 and Δcas9 mutant in murine macrophage RAW264.7 cells. The Y axis is the index of cytotoxic production of each strain. The results were obtained from three independent repeats. One asterisk (*) indicates a statistically significant difference at P ≤ 0.05 between C. jejuni NCTC11168, complemented, and the Δcas9 mutant.

Figure 6

Motility of the C. jejuni NCTC 11168 and Δcas9 mutant strains on 0.4% MH agar plates. Motility is positively correlated to the diameter of the growth ring of each strain.

Figure 7

Frozen transmission electron microscopy of the C. jejuni NCTC 11168 and Δcas9 mutant strains. C. jejuni NCTC 11168 (A), Δcas9 mutant (B). The magnification used for TEM images in the capionis is 1μm.

Figure 8

Overview of DEGs and their relationship in Δcas9 mutant strain on the basis of KEGG and STRING protein network analysis. Genes with red arrows indicate down-regulation, while genes with green arrows indicate up-regulation. Black double sided arrows indicate possible gene interaction.