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Retargeted Pyocins: A novel tool for combating major food borne pathogens and exploiting phage-host interactions

Periodic Reporting for period 1 - PYANO (Retargeted Pyocins: A novel tool for combating major food borne pathogens and exploitingphage-host interactions)

Reporting period: 2016-06-01 to 2018-05-31

Campylobacter jejuni and Salmonella are the top-ranking foodborne pathogens of Europe. The economic burden resulting from these bacterial infections is substantial and has been estimated to exceed EUR 5.4 billion each year, due to loss in productivity, hospitalization, medicinal costs and management of associated chronic diseases. The problem is further accentuated by the increase in resistance to antibiotics commonly used in treatment of human C. jejuni and Salmonella infections. To tackle antimicrobial resistance development, the European Commission has emphasized an urgent need for developing new effective antimicrobials or alternatives for treatment.

Natural predators of bacteria, bacteriophages, as well as phage-based therapeutics are promising alternatives to combat pathogenic bacteria. Being obligate parasites of the bacteria, phages bind to, infect and kill specific bacterial species, subspecies, serovars or even particular strains during their propagation. Their ability to recognize a bacterium is dependent on Receptor Binding Proteins (RBPs) found in the distal tip of phage tail structure. The RBP binds to a specific receptor on the bacterial surface thereby ensuring precision in host recognition. This astonishing specificity is a result of evolution and natural selection over millions of years which secures binding of the phage to a potential host. Due to the immense diversity of phages, RBPs present a rich source of biotechnological tools for specific targeting of any bacterial species with outstanding precision.

PYANO aimed at discovering novel RBPs and to develop alternative phage-based therapeutics to control C. jejuni and Salmonella. PYANO combined these objectives by exploring R-type pyocins (hereafter referred as pyocins) as a novel, advanced and versatile approach. Pyocins are highly-ordered bacteriocins of pseudomonads that resembles a headless phage with a contractile tail. Like phages, these “nanoscale-injectors” bind to a bacterial receptor via tail fibers that determine the host specificity. Once specifically bound, the tail contracts and pyocins exert their highly potent bactericidal activity by punching a hole on the bacterial envelope that dissipates the membrane potential and kills the bacterial cell. Importantly, by fusing novel RBPs from different phages to the fibers of pyocins, the bactericidal activity of the chimeric pyocin can be directed towards bacteria that are recognized by the fused RBP. Therefore, successful binding of the novel RBP can be confirmed by clearance of the bacterial cells, which also allows high throughput screening of chimeric pyocins on an array of relevant bacterial strains. Moreover, chimeric pyocins are advantageous with regards to the phages as they circumvent the necessity of genome replication, and the immediate bactericidal activity from puncturing of the cell envelope prohibits development of bacterial resistance. These features consolidate chimeric pyocins as a powerful, cost-effective and phage-derived, green and biodegradable alternative to conventional antimicrobials.
While approaching to the post-antibiotic era due to overwhelming global antibiotic resistance crisis, phages and phage-based pathogen control is progressively being recognized as a promising alternative or complementation to the current therapeutic options. Since there is an enormous diversity of phages, it is hard to predict their bacterial host, thus forming a major bottleneck in their applicability. PYANO documented the host range of more than 100 different phages using in total 150 C. jejuni and Salmonella serovars that were originating from poultry and pork meat. This has generated an RBP-receptor recognition dataset that is invaluable for future development of phage-based alternative antimicrobials. Moreover, complementing the phage host range with rigorous comparative bioinformatics allowed selection of the best candidate RBPs to construct chimeric pyocins with a broad bacterial killing activity. Overall, PYANO created two chimeric pyocins that killed 98% of the tested C. jejuni strains, and one other chimeric pyocin with bactericidal activity towards 92% of Derby, 75% of Typhimurium, 69% monophasic variant, 100% of Dublin, 100% of Enteritidis and 32% of various other tested Salmonella serovars.
Collectively, PYANO supplied a proof-of-concept and three novel antimicrobials for broad, yet specific biocontrol of top-ranking bacterial food borne pathogens of Europe. Owing to the superb bactericidal activity of pyocins combined with the specificity and documentation of RBPs discovered, PYANO opens up new avenues for applicability of chimeric pyocins in settings that encompasses different levels of farm-to-fork cascade, thus improving public health. It is expected that the created chimeric pyocins will be of extraordinary value for producers of foods of animal origin.