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.

Keywords: Campylobacter; Innolysin; antibacterials; endolysin; food safety; prophage binding.

FIGURE 1

Domains of the tail fiber region of CJIE1-like prophages and construction of campycins. (A) Domains of H-fiber C. jejuni prophage CJIE1-like in comparison to prophage Mu and R2-pyocin. The H-fiber of CJIE1-like prophages harbors an N-terminal DUF3751 domain similar to the P. aeruginosa PAO1 R2-pyocin tail fiber encoded by prf15 (Williams et al., 2008). The gene downstream of the H-fiber gene is predicted to encode a DUF4376 domain as found in the U gene that functions as a chaperone for the Mu phage tail fiber (Haggard-Ljungquist et al., 1992). (B) For construction of campycin, the DUF3751 domain encoded by P. aeruginosa PAO1 R2-pyocin was fused with the C-terminus of the CAMSA2147 H-fiber.

FIGURE 2

Antibacterial activity of campycin against Campylobacter CAMSA2147. Campycin was expressed with or without chaperone and mixed with CAMSA2147. TN50 buffer was used instead of campycins as a negative control. Samples were incubated for 3 h in microaerobic conditions at 37°C. Colony forming units per milliliter (cfu/ml) were counted. Experiments were performed in triplicates. Error bars represent the standard deviations of the mean. *Significant reduction at P < 0.05.

FIGURE 3

Construction of Innolysins Cj. Innolysin Cj1 was constructed by fusing the C-terminus of phage T5 endolysin to the tail fiber protein H of Campylobacter CAMSA2147 CJIE1-like prophage. These two domains were fused by using a linker in between composed of 14 amino acids (GAGAGAGAGAGAGA). The same configuration was used for Innolysin Cj2 but instead of the whole H-fiber, the C-terminal part of the H-fiber was used, excluding the N-terminal DUF3751 domain.

FIGURE 4

Muralytic activity of Innolysins Cj. Enzymatic activities of Innolysins Cj1 and Cj2 were tested on outer membrane-permeabilized P. aeruginosa substrate in triplicate. Cleared lysates of cells expressing Innolysins Cj were used for the assay. Cleared lysates of cells expressing either the phage T5 endolysin (T5 Lys) or carrying the empty vector pET-28a (+) were used as positive and negative controls, respectively. Enzymatic activities were depicted as units per milliliter (U/ml) without normalization for product expression yield. Error bars represent the standard deviations of the mean. *Significant muralytic activity at P < 0.05.

FIGURE 5

Antibacterial spectrum of Innolysins Cj against C. jejuni strains. The bacterial log reduction of different C. jejuni strains was determined after application of 1 mg/ml purified Innolysin Cj1 or Innolysin Cj2 and exposed for 45 min at 20°C. Average logarithmic reductions of treated cells were measured compared to the cells treated with 20 mM HEPES-NaOH (pH 7.4) (Innolysins buffer solution). The C. jejuni strains were derived from the Statens Serum Institute (SSI) collection isolated from poultry and pork in Denmark, showing different multilocus sequence typing (MLST) and different MLST clonal complexes (CC) (Table 2). The average logarithmic reduction was calculated based on three independent experiments. Error bars represent the standard deviations of the mean. *Significant reduction at P < 0.05.