Developing Innolysins Against Campylobacter jejuni Using a Novel Prophage Receptor-Binding Protein

Abstract

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.

Application of Bacteriophages to Limit Campylobacter in Poultry Production

“…For example, phage endolysins have a broader host specificity than lytic phages ( Siringan et al, 2011 ). Along these lines, Zampara et al (2021) suggested the application of phage-derived enzymes instead of lytic phages to overcome some of the problems inherent with intact phages. Endolysins are phage-programmed enzymes that destroy the peptidoglycan layer when externally added leading to osmotic imbalance and cell death ( Zampara et al, 2021 ).…”

“…Along these lines, Zampara et al (2021) suggested the application of phage-derived enzymes instead of lytic phages to overcome some of the problems inherent with intact phages. Endolysins are phage-programmed enzymes that destroy the peptidoglycan layer when externally added leading to osmotic imbalance and cell death ( Zampara et al, 2021 ). Although endolysins have successfully been used in an antibacterial application and can exhibit low development of resistance, their application is currently more suitable toward Gram-positive bacteria, as Gram-negative bacteria contain an outer membrane that inhibits entry of endolysins to the peptidoglycan layer ( Gutiérrez and Briers, 2021 ).…”

“…Based on Zampara et al (2020) reporting of a successful bactericidal effect of Innolysins against E. coli , they ( Zampara et al, 2021 ) applied the same concept against C. jejuni . Zampara et al (2021) demonstrated that the H-fiber derived from a C. jejuni integrated elements (CJIEs)-1 like prophage CAMSA2147 functions as a unique RBP. Zampara et al (2021) designed Innolysins that target C. jejuni by fusing the H-fiber and T5 endolysin.…”

“… Zampara et al (2021) demonstrated that the H-fiber derived from a C. jejuni integrated elements (CJIEs)-1 like prophage CAMSA2147 functions as a unique RBP. Zampara et al (2021) designed Innolysins that target C. jejuni by fusing the H-fiber and T5 endolysin. Zampara et al (2021) noted that the application of Innolysins on chicken skin at 5°C contaminated with C. jejuni CAMSA2147 led to an average of 1.4 log 10 reduction of bacteria, indicating that Innolysins can eradicate C. jejuni in situ .…”

Genomic characterization of four novel bacteriophages infecting the clinical pathogen Klebsiella pneumoniae

“…12–14 The study of phages and their genomes is therefore inherently valuable to advance our understanding in a diversity of fields including molecular biology, ecology, evolution, bacterial pathogenesis, biotechnology, and health. Understanding phage genomes will certainly create opportunities to translate novel phage proteins and phages themselves into potent biotechnological 15 and medical tools. 16 Here we isolated and sequenced the genomes of four novel phages infecting Klebsiella pneumoniae, an increasingly relevant pathogen identified by the World Health Organization as priority for the development of new antibiotics.…”