The anti-adhesive and anti-invasive effects of recombinant azurin on the interaction between enteric pathogens (invasive/non-invasive) and Caco-2 cells.

Anti-adhesion therapy and anti-adhesin immunity are meant to diminish the interaction between pathogens and host tissues, either by prevention or by exclusion of bacterial adhesion and entrance to cells. Azurin is a scaffold protein possessing antiviral, antiparasitic, and anticancer activities. The purpose of the present study was to determine the effect of recombinant Azurin (rAzurin) on the adhesion and invasion capacity of invasive (Shigella sonnei, Shigella flexneri, Campylobacter jejuni) and non-invasive (Vibrio cholerae) enteric bacteria to cells. The non-toxic dose of rAzurin and the best MOI (Multiplicity of Infection) of bacterial species was assessed by MTT assay. Bacterial species were used at MOIs of 20:1 and Azurin was applied at the concentrations of 5 and 25 µg/mL and added to Caco-2 cells in competition and replacement assay to assess the anti-adhesion and anti-invasion properties of rAzurin. The protein caused significant decrease in the adhesion rate of S. sonnei, S. flexneri, C. jejuni, and V. cholerae strains to Caco-2 cells by 43, 39, 72, and 38% in competition and 45, 46, 75, and 48% in replacement assays, respectively. Also, S. sonnei, S. flexneri, and C. jejuni strains invasion rate was reduced to 50, 50, and 70% in anti-invasion assay, respectively. The inhibitory effect of Azurin against C. jejuni and V. cholerae strains adhesion was more significant (p < .001) compared to Shigella spp. (p < .05) which may be due to smaller size of the former bacteria. On the contrary, in invasion assay, rAzurin showed a greater inhibitory effect against Shigella spp. (p < .001) compared to C. jejuni (p < .05), which may probably be due to the interaction of rAzurin with several effectors or ligands, involved in Shigella invasion and internalization. The findings of the present study opens new insights of rAzurin as a new and potent candidate for reducing or probably preventing enteric bacterial attachment, invasion, and pathogenesis.

Influence of Heterologously Expressed azurin from Pseudomonas aeruginosa on the Adhesion and Invasion of Pathogenic Bacteria to the Caco-2 Cell Line.

This study proposed to investigate the effect of azurin on the major stages of pathogenesis (adhesion and invasion) of intestinal bacterial pathogens (Salmonella spp. and Escherichia coli) and epithelial pathogens (Staphylococcus aureus and Pseudomonas aeruginosa) on the human colorectal adenocarcinoma (Caco-2) cell line. Azurin protein was produced by cloning the azurin gene into pET21a and heterologous expression in E. coli BL21. The protein was purified using affinity chromatography and confirmed by Western blotting. The purified protein was evaluated by three experiments of adhesion and invasion assays, including exclusion, competition, and replacement. Azurin was observed to significantly inhibit the attachment and invasion of S. aureus, Salmonella spp., and E. coli, while no such inhibitory effects were observed on P. aeruginosa. In fact, the protein increased the adhesion of P. aeruginosa to the cell. In conclusion, our study proposes that azurin is a potential prophylactic or preventive helper candidate to inhibit the attachment and invasion of pathogenic bacteria to host cells and reduce the progression of the infection process. Our study also reveals the involvement of azurin in bacteria-host cell interactions, providing novel and important insights toward the elucidation of its biological function in this field. Thus, this study provides new opportunities to use azurin as an adjunct therapy against critical stages of infection by a wide range of pathogenic bacteria.