Conclusion
These data suggest that IONzymes can increase ROS levels to promote the antibacterial effects of acid autophagic vacuoles, and thus suppress the establishment and survival of invading intracellular S. Enteritidis. As a result, IONzymes may be a novel alternative to current antibiotics for the control of intractable S. Enteritidis infections.
Methods
The antibacterial activities of IONzymes against planktonic S. Enteritidis, intracellular S. Enteritidis in Leghorn Male Hepatoma-derived cells (LMH), and liver from specific pathogen free (SPF) chicks were investigated by spread-plate colony count method and cell viability assay. Changes in levels of microtubule-associated protein light chain 3 (LC3), a widely used marker for autophagosomes, were analyzed by immunoblotting, immunofluorescence, and electron microscopy. Reactive oxygen species (ROS) production was also assessed in vitro. High-throughput RNA sequencing was used to investigate the effects of IONzymes on liver transcriptome of S. Enteritidis-infected chicks.
Results
We demonstrated that IONzymes had high biocompatibility with cultured LMH cells and chickens, which significantly inhibited intracellular S. Enteritidis survival in vitro and in vivo. In addition, co-localization of IONzymes with S. Enteritidis were observed in autophagic vacuoles of LMH cells and liver of chickens infected by S. Enteritidis, indicating that IONzymes mediated antibacterial reaction of S. Enteritidis with autophagic pathway. We found ROS level was significantly increased in infected LMH cells treated with IONzymes, which might enhance the autophagic elimination of intracellular S. Enteritidis. Moreover, orally administered IONzymes decreased S. Enteritidis organ invasion of the liver and prevented pathological lesions in a chicken-infection model. Non-target transcriptomic profiling also discovered IONzymes could change hepatic oxidation-reduction and autophagy related gene expressions in the S. Enteritidis infected chickens.