Bacteriophage-encoded endolysins degrading the bacterial peptidoglycan are promising antibacterials for combating antibiotic-resistant bacteria. However, endolysins have limited use against ...Gram-negative bacteria, since the outer membrane prevents access to the peptidoglycan. Here, we present Innolysins, an innovative concept for engineering endolysins to exert antibacterial activity against Gram-negative bacteria. Innolysins combine the enzymatic activity of endolysins with the binding capacity of phage receptor binding proteins (RBPs). As proof-of-concept, we constructed 12 Innolysins by fusing phage T5 endolysin and RBP Pb5 in different configurations. One of these, Innolysin Ec6 displayed antibacterial activity against Escherichia coli only in the presence of Pb5 receptor FhuA, leading to 1.22 ± 0.12 log reduction in cell counts. Accordingly, other bacterial species carrying FhuA homologs such as Shigella sonnei and Pseudomonas aeruginosa were sensitive to Innolysin Ec6. To enhance the antibacterial activity, we further constructed 228 novel Innolysins by fusing 23 endolysins with Pb5. High-throughput screening allowed to select Innolysin Ec21 as the best antibacterial candidate, leading to 2.20 ± 0.09 log reduction in E. coli counts. Interestingly, Innolysin Ec21 also displayed bactericidal activity against E. coli resistant to third-generation cephalosporins, reaching a 3.31 ± 0.53 log reduction in cell counts. Overall, the Innolysin approach expands previous endolysin-engineering strategies, allowing customization of endolysins by exploiting phage RBPs to specifically target Gram-negative bacteria.
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
. Here, we have expanded the Innolysin concept to target
. As no
phage RBP had been identified so far, we first showed that the H-fiber originating from a CJIE1-like prophage of
CAMSA2147 functions as a novel RBP. By fusing this H-fiber to phage T5 endolysin, we constructed Innolysins targeting
(Innolysins Cj). Innolysin Cj1 exerts antibacterial activity against diverse
strains after
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
CAMSA2147 led to 1.63 ± 0.46 and 1.18 ± 0.10 log reduction of cells, respectively, confirming that Innolysins Cj can kill
. 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
phage RBPs may further increase the repertoire of new Innolysins Cj targeting distinct receptors and working as antibacterials against
phages are divided into two genera;
and
, showing only limited intergenus homology. Here, we aim to identify the lytic genes of both genera using two representative phages (F352 and F379) from our ...collection. We performed a detailed in silico analysis searching for conserved protein domains and found that the predicted lytic genes are not organized into lysis cassettes but are conserved within each genus. To verify the function of selected lytic genes, the proteins were expressed in
, followed by lytic assays. Our results show that
phages encode a typical signal peptide (SP) endolysin dependent on the Sec-pathway for translocation and a holin for activation. In contrast,
phages encode a novel endolysin that does not belong to currently described endolysin groups. This endolysin also uses the Sec-pathway for translocation but induces lysis of
after overexpression. Interestingly, co-expression of this endolysin with an overlapping gene delayed and limited cell lysis, suggesting that this gene functions as a lysis inhibitor. These results indicate that
phages regulate lysis timing by a yet undescribed mechanism. In conclusion, we found that the two
phage genera control lysis by two distinct mechanisms.
Poultry meat is the main source of Campylobacter jejuni foodborne disease. Currently, no effective control measures prevent C. jejuni from contaminating poultry meat. However, post-harvest phage ...treatment is a promising biocontrol strategy that has not yet been explored. Here we identified phages capable of reducing C. jejuni at chilled temperature by a systematic screening of unique phages of our collection consisting of flagellotropic phages and phages dependent on capsular polysaccharides (CPSs) for infection. Interestingly, CPS phages showed varied killing efficiencies at 5 °C in vitro, ranging from insignificant reduction to 0.55 log reduction. In contrast, none of the flagellotropic phages significantly reduced C. jejuni counts at low temperature. Phage adsorption at 5 °C showed that flagellotropic phages bind reversibly and less efficiently to C. jejuni than CPS phages, which may explain why flagellotropic phages did not reduce C. jejuni. The CPS phages tested showed similar binding capacities. Thus, the varying effectiveness of CPS phages to reduce C. jejuni counts may be attributed to other stages of the phage life cycle than phage binding. Finally, a phage cocktail of the two most effective phages (F356 showing 0.49 and F357 showing 0.55 log reductions, respectively) led to a 0.73 log reduction of C. jejuni on artificially contaminated chicken skin. Our study shows that poly-phage treatment at 5 °C can be more effective against C. jejuni compared to single phage application. A thorough understanding of phage-host interactions is prerequisite to further advance phage application as a post-harvest biocontrol strategy against C. jejuni.
•Lytic phages showed different efficiency in reducing C. jejuni at low temperature.•Flagellotropic phages may be less suitable for reducing C. jejuni in food settings.•Capsular phages were most efficient and a phage cocktail improved this efficiency.•A cocktail of two capsular phages reduced C. jejuni by 0.73 log on chicken skin.•Elucidation of phage-host dynamics is necessary for optimization of phage treatment.
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