In silico study, cloning and functional analysis of CjS8 protein from Campylobacter jejuni

Abstract

In Gram-negative bacteria, protein secretion plays an important part in pathogenesis. Secretory proteins perform a variety of important role for bacterial survival in the environment and its involvement to cause disease to human. We have identified specific surface protein of Campylobacter jejuni. Genomic and protein analysis using bioinformatics tools on that protein reveals the presence of signal peptide at N-terminal of its peptide sequence, thus signifies this protein is highly potential acted as secreted protein. The size of the protein was calculated as 24.1 kDa. The availability of complete genome sequences of C. jejuni has allowed this study to make predictions about the composition of bacterial secreted protein that has good similarity in their sequence homology. The prediction of others C. jejuni secreted proteins were performed based on 24.1 kDa and several enterobacteriaceae pathogenic bacteria proteins sequences using a set of internet-based programs, including BLAST, ORF Finder and SignalP v 5. In silico analysis in this study identified the secreted protein of S8 family serine peptidase in C. jejuni genome and designated as CjS8. To investigate the involvement of CjS8 in the pathogenesis of C. jejuni infection, a C-terminal fragment of CjS8 was successful amplified, cloned and expressed using a TOPO expression vector. Rabbit polyclonal serum was raised against the purified recombinant CjS8 protein. The CjS8 null-mutant was constructed in C. jejuni by natural transformation and allelic exchange. PCR analysis and immunoblot of whole cell lysates with Ab_CjS8 (polyclonal antibody against CjS8) showed that CjS8 is naturally expressed in C. jejuni but not in the null mutant. In a strain survey on clinical isolates of C. jejuni, using the PCR and immunoblot analysis. Data showed that the CjS8 was presence in twenty out of twenty-three clinical isolates. Importantly, this study revealed the functional analysis results, that showed the CjS8 mutant was shown to affect the C. jejuni ability to adhere to the host cells (Caco-2 cells). Invasion was also affected by CjS8 mutant strain as well as the biofilm formation and motility. Thus, as a conclusion the CjS8 was the one, among a few secreted proteins described in C. jejuni and may represent a novel virulence factor. These results will be important in furthering our understanding of Campylobacter biology and pathogenesis.