Chicken extracellular fatty acid-binding protein specifically binds to enterobactin to inhibit the growth of Campylobacter

Abstract:

[Background] Campylobacter is a genus of major foodborne pathogens that colonize the chicken intestine at high levels. The chicken innate immune response to Campylobacter colonization remains to be fully elucidated. [Objective] To investigate the activity of extracellular fatty acid binding protein (Ex-FABP) related to chicken innate immunity in inhibiting the growth of Campylobacter by specifically binding to enterobactin, providing a theoretical basis for revealing the function of the host innate immunity against Campylobacter infection. [Methods] We knocked out the entB gene from Escherichia coli BL21(DE3) to construct a strain that did not secrete enterobactin. Chicken Ex-FABP and human lipocalin-2 (Lcn2) were expressed in the engineered strain and purified. Western blotting was employed to examine the binding of the expressed proteins to enterobactin. The inhibitory activities of Ex-FABP and Lcn2 against E. coli, Salmonella, and their mutant strains with entB knockout were verified under iron-rich and iron-limited culture conditions, respectively. Then, the inhibitory activities of Ex-FABP and Lcn2 against Campylobacter derived from different hosts were examined by addition of enterobactin. [Results] Ex-FABP and Lcn2 were successfully expressed in the constructed BL21(DE3) mutant with entB knockout. The in vitro binding assays confirmed that the expressed proteins could specifically bind to enterobactin. Under the iron-limited culture condition, Ex-FABP and Lcn2 significantly inhibited the growth of E. coli AN102 capable of secreting enterobactin but had no inhibitory effects on E. coli ATCC25922, Salmonella CVCC1806/CVCC1800, and corresponding mutant strains with entB knockout. Finally, it was confirmed that Ex-FABP and Lcn2 inhibited the growth of five Campylobacter strains derived from different host species by specifically binding to enterobactin. [Conclusion] Chicken Ex-FABP has a similar function to Lcn2. Under iron-limited conditions, it can specifically bind to enterobactin and inhibit bacterial growth by blocking the enterobactin-mediated iron uptake, thus triggering the host innate immune function. The findings provide a theoretical basis for developing new approaches to control Campylobacter.

Key words:extracellular fatty acid binding protein (Ex-FABP);lipocalin-2 (Lcn2);enterobactin;entB gene;Campylobacter