鸡细胞外脂肪酸结合蛋白特异性结合肠杆菌素抑制弯曲菌生长

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

摘要:

【背景】弯曲菌(Campylobacter)是一种重要的食源性病原细菌，在鸡肠道内高水平定殖，鸡天然免疫在该菌定殖中的作用机制尚未解析。【目的】探究鸡天然免疫相关的细胞外脂肪酸结合蛋白(extracellular fatty acid-binding protein, Ex-FABP)通过特异性结合肠杆菌素抑制弯曲菌生长的活性，为宿主发挥天然免疫功能提供理论基础。【方法】通过敲除大肠杆菌(Escherichia coli) BL21(DE3)的entB基因构建不分泌肠杆菌素的表达宿主菌，原核表达纯化鸡Ex-FABP蛋白和人源脂钙蛋白2 (lipocalin-2, Lcn2)，Western blotting鉴定表达蛋白与肠杆菌素特异性结合，分别在富铁和限铁培养条件下，验证Ex-FABP和Lcn2对大肠杆菌、沙门氏菌(Salmonella)及entB基因突变株的抑菌活性，继而通过外源添加肠杆菌素鉴定Ex-FABP和Lcn2对不同宿主源弯曲菌的抑菌活性。【结果】利用构建的宿主菌BL21(DE3)的entB基因突变株，成功表达和纯化Ex-FABP和Lcn2蛋白，体外结合试验证实表达蛋白可与肠杆菌素特异性结合。在限铁培养条件下，Ex-FABP和Lcn2对仅分泌肠杆菌素的大肠杆菌AN102具有显著抑制生长的作用，而对大肠杆菌ATCC 25922、沙门氏菌CVCC 1806、CVCC 1800及对应的entB缺失株均无抑制作用。最后证实Ex-FABP和Lcn2通过特异性结合肠杆菌素对5株不同宿主来源的弯曲菌均有显著抑制生长的作用。【结论】鸡Ex-FABP蛋白具有与Lcn2类似的功能，即在限铁环境下可特异性结合肠杆菌素，通过阻断细菌利用肠杆菌素摄取铁的途径来抑制其生长，发挥宿主天然免疫功能，为开发新型弯曲菌防控策略提供了理论依据。

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.