Campycins are novel broad spectrum antibacterials killing Campylobacter jejuni by targeting the essential major outer membrane protein (MOMP)

Pyocins are high molecular weight bacteriocins produced by Pseudomonas aeruginosa that can be retargeted to new bacterial species by exchanging the pyocin tail fibers with bacteriophage receptor binding proteins (RBPs). Here, we develop retargeted pyocins called campycins as new antibacterials to specifically and effectively kill the major foodborne pathogen Campylobacter jejuni. We used two diverse RBPs (H-fibers) encoded by CJIE1 prophages found in the genomes of C. jejuni strains CAMSA2147 and RM1221 to construct Campycin 1 and Campycin 2, respectively. Together Campycin 1 and 2 could target all C. jejuni strains tested due to complementary antibacterial spectrums. In addition, both campycins led to more than 3 log reductions in C. jejuni counts under microaerobic conditions at 42{degrees}C, whereas the killing efficiency was less efficient under anaerobic conditions at 5{degrees}C. We furthermore discovered that both H-fibers used to construct the campycins bind to the essential major outer membrane protein (MOMP) present in all C. jejuni, in a strain specific manner. Protein sequence alignment and structural modelling suggest that the highly variable extracellular loops of MOMP form the binding sites of the diverse H-fibers. Further in silico analyses of 5000 MOMP sequences suggest that the protein fall into three major clades predicted to be targeted by either Campycin 1 or Campycin 2. Thus, campycins are promising antibacterials against C. jejuni expected to broadly target numerous strains of this human pathogen found in nature and agriculture. IMPORTANCECampylobacter jejuni is the leading cause of bacterial foodborne gastroenteritis and responsible for more than 800 million cases globally each year posing a continuous risk to human health and a huge economic and societal burden. Here, we developed re-targeted R2 pyocins (campycins) as novel antibacterials against C. jejuni by using the receptor binding proteins of CJIE1 prophages observed in many C. jejuni genomes. Notably, campycins broadly target the highly variable yet essential major outer membrane protein (MOMP), and result in more than 3-log reductions in C. jejuni counts under conditions promoting bacterial growth. We therefore propose that campycins have the potential to lower C. jejuni colonization levels in the chicken gut, the main reservoir and cause of human disease, representing a novel efficient antibacterial solution specifically developed to target this widespread foodborne pathogen.