Data_Sheet_2_Developing Innolysins Against Campylobacter jejuni Using a Novel Prophage Receptor-Binding Protein.PDF

Campylobacter contaminated poultry remains the major cause of foodborne gastroenteritis worldwide, calling for novel antibacterials. We previously developed the concept of Innolysin composed of an endolysin fused to a phage receptor binding protein (RBP) and provided the proof-of-concept that Innolysins exert bactericidal activity against Escherichia coli. Here, we have expanded the Innolysin concept to target Campylobacter jejuni. As no C. jejuni phage RBP had been identified so far, we first showed that the H-fiber originating from a CJIE1-like prophage of C. jejuni CAMSA2147 functions as a novel RBP. By fusing this H-fiber to phage T5 endolysin, we constructed Innolysins targeting C. jejuni (Innolysins Cj). Innolysin Cj1 exerts antibacterial activity against diverse C. jejuni strains after in vitro exposure for 45 min at 20°C, reaching up to 1.30 ± 0.21 log reduction in CAMSA2147 cell counts. Screening of a library of Innolysins Cj composed of distinct endolysins for growth inhibition, allowed us to select Innolysin Cj5 as an additional promising antibacterial candidate. Application of either Innolysin Cj1 or Innolysin Cj5 on chicken skin refrigerated to 5°C and contaminated with C. jejuni CAMSA2147 led to 1.63 ± 0.46 and 1.18 ± 0.10 log reduction of cells, respectively, confirming that Innolysins Cj can kill C. jejuni in situ. The receptor of Innolysins Cj remains to be identified, however, the RBP component (H-fiber) recognizes a novel receptor compared to lytic phages binding to capsular polysaccharide or flagella. Identification of other unexplored Campylobacter phage RBPs may further increase the repertoire of new Innolysins Cj targeting distinct receptors and working as antibacterials against Campylobacter.

受弯曲杆菌（Campylobacter）污染的家禽仍是全球食源性胃肠炎的主要致病源，亟需开发新型抗菌制剂。我们此前提出了由内溶素（endolysin）与噬菌体受体结合蛋白（RBP）融合构成的Innolysin概念，并完成了Innolysin对大肠杆菌（Escherichia coli）具有杀菌活性的概念验证。本研究将Innolysin概念拓展至靶向空肠弯曲杆菌（Campylobacter jejuni）。由于目前尚未有针对空肠弯曲杆菌的噬菌体RBP被鉴定，我们首先证实：源自空肠弯曲杆菌CAMSA2147的CJIE1样前噬菌体（prophage）的H纤维可作为新型RBP。通过将该H纤维与T5内溶素（T5 endolysin）融合，我们构建了靶向空肠弯曲杆菌的Innolysins Cj。在20℃条件下体外孵育45分钟后，Innolysin Cj1对多种空肠弯曲杆菌菌株均表现出抗菌活性，对CAMSA2147的菌落数可实现1.30 ± 0.21 log的对数减少量。通过筛选由不同内溶素构成的Innolysins Cj文库以考察其生长抑制活性，我们筛选得到另一极具潜力的抗菌候选物Innolysin Cj5。将Innolysin Cj1或Innolysin Cj5应用于被空肠弯曲杆菌CAMSA2147污染、冷藏于5℃的鸡肉表皮，可分别使菌落数减少1.63 ± 0.46 log和1.18 ± 0.10 log，证实了Innolysins Cj可在原位杀灭空肠弯曲杆菌。尽管Innolysins Cj的受体仍有待鉴定，但其RBP组分（H纤维）所识别的受体，与结合荚膜多糖（capsular polysaccharide）或鞭毛（flagella）的裂解性噬菌体所识别的受体均存在差异。进一步发掘未被研究的弯曲杆菌噬菌体RBP，或可扩充靶向不同受体、可作为抗弯曲杆菌抗菌制剂的新型Innolysins Cj的储备库。