Iron oxide nanozyme suppresses intracellular <i>Salmonella</i> Enteritidis growth and alleviates infection <i>in vivo</i>

Abstract

<b>Rational:</b><i>Salmonella</i> Enteritidis (<i>S.</i> Enteritidis) is a globally significant zoonotic foodborne pathogen which has led to large numbers of deaths in humans and caused economic losses in animal husbandry. <i>S.</i> Enteritidis invades host cells and survives within the cells, causing resistance to antibiotic treatment. Effective methods of elimination and eradication of intracellular <i>S.</i> Enteritidis are still very limited. Here we evaluated whether a new intracellular antibacterial strategy using iron oxide nanozymes (IONzymes) exerted highly antibacterial efficacy via its intrinsic peroxidase-like activity <i>in vitro</i> and <i>in vivo.</i><b>Methods:</b> The antibacterial activities of IONzymes against planktonic <i>S.</i> Enteritidis, intracellular <i>S.</i> 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 <i>in vitro</i>. High-throughput RNA sequencing was used to investigate the effects of IONzymes on liver transcriptome of <i>S</i>. Enteritidis-infected chicks. <b>Results:</b> We demonstrated that IONzymes had high biocompatibility with cultured LMH cells and chickens, which significantly inhibited intracellular <i>S.</i> Enteritidis survival <i>in vitro</i> and <i>in vivo</i>. In addition, co-localization of IONzymes with <i>S.</i> Enteritidis were observed in autophagic vacuoles of LMH cells and liver of chickens infected by <i>S.</i> Enteritidis, indicating that IONzymes mediated antibacterial reaction of <i>S.</i> 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 <i>S.</i> Enteritidis. Moreover, orally administered IONzymes decreased <i>S.</i> 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 <i>S.</i> Enteritidis infected chickens. <b>Conclusion:</b> 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 <i>S.</i> Enteritidis. As a result, IONzymes may be a novel alternative to current antibiotics for the control of intractable <i>S.</i> Enteritidis infections.