Biological functional identification of holins from Salmonella pullorum phages and their therapeutic efficacy against pullorum disease in chickens

Salmonella pullorum (S.pullorum) is a significant pathogen that seriously endangers the poultry industry. However, its prevention and control mainly rely on antibiotics, which leads to the wide spread of drug-resistant genes and poses a serious public health risk. Phage lytic proteins have received extensive attention in recent years as potential alternatives to antibiotics. In this study, whole genome sequencing and phylogenetic analysis demonstrated that Salmonella Pullorum phage vB_SpuM_23 is most closely related to Salmonella phages SWJM-02 and SalM SPJ41. Two holin proteins (Holin-1 and Holin-P) were identified from the phage genome. Expression in the Escherichia coli system confirmed the broad-spectrum antibacterial activity of Holin-1, Holin-P, and their conjugate holin-p-holin-1, with peak lysis activity observed at pH 6 and 37°C. After expression in Lactococcus lactis (L.lactis) NZ3900, the culture supernatants of the three holins could effectively lyse Salmonella pullorum. In the infection model of chicks, oral administration of recombinant L.lactis containing Holin-P, Holin-1 and Holin-p-holin-1 could significantly reduce the Salmonella load in anal swabs and cecal contents, and significantly inhibit the levels of IL-6 in serum. Particularly importantly, the combined application of Holin-P-Holin-1 demonstrated a synergistic antibacterial effect, which was superior to that of holin alone. The results of this study indicate that phage holin has great potential to be developed into a new type of antibacterial agent in response to bacterial infections.