A research group led by Prof. GAO Lizeng from the Institute of Biophysics of the Chinese Academy of Sciences (CAS) and the CAS Nano-Engineering Laboratory utilized a newly discovered mechanism, in which ferrous iron and polysulfide coordinate bacterial death with hallmarks similar to ferroptosis, as a promising strategy to combat bacterial infection. The study was published in Nano Today.

Ferroptosis is an iron-dependent oxidation cellular death. It is featured with lipid peroxidation induced by iron or lipid oxidase, together with an exhaustion of glutathione (GSH). It can lead to a contraction of mitochondria and an increase in membrane density. Studies have shown that ferroptosis is closely related to neurological disorders, tumors and heart diseases.

Current studies on ferroptosis have mainly centered on eukaryotic systems such as the cells of animals and plants, which are modulated precisely by cellular pathways. There is no reason to blame bacteria for ferroptosis since such molecular pathways have not been found in bacteria and other eukaryotic systems.

In 2007, Prof. GAO found that iron oxide nanozymes have enzyme-like activity similar to peroxidase, and then applied these nanozymes to remove oral biofilm of bacteria and eliminate intracellular Salmonella. His group found that some nanozymes can produce considerable antibacterial effects without the presence of hydrogen peroxide. In other words, the utility of iron sulfide nanozymes alone can eliminate various bacteria in an efficient manner. Their early investigations showed that iron sulfide nanozymes can produce hydrogen polysulfide which was assumed to be critical for bacteria killing, however, the use of polysulfide alone has a limited effect. 

In this study, apart from expounding the biological mechanism of how an iron-based nano-enzyme material alone eliminates bacteria, the researchers found for the first time that ferroptosis happens to bacteria as well.

Iron sulfide nanozyme materials such as Fe₃S₄ will generate hydrogen sulfide and a large amount of iron and form a solution featuring ferrous iron and polysulfide when they undergo oxygen-sulfide exchange reaction in water. To better monitor the anti-bacteria property of iron sulfide nanozyme materials, their aqueous suspension was utilized to make nano-decoction through the techniques of high-pressure sterilization and centrifugal treatment similar to the procedure of preparing traditional Chinese medicines. Such nano-decoction can kill up to 90 percent of escherichia coli and staphylococcus aureus within five minutes, while it will take antibiotics such as vancomycin about 24 hours to achieve an equivalent therapeutic effect.

Further studies showed that the nano-decoction can destroy the integrity of bacteria, lead to a leakage of their content, trigger their lipid peroxidation, and suppress the activity of complex I and II in the respiratory chain of bacteria. Transcriptome analysis found that multiple metabolic pathways have been restrained and that the oxidation of GSH into oxidized glutathione (GSSG) will exhaust glutathione in the elimination of bacteria.

The researchers concluded that such a death was similar to ferroptosis. Ferroptosis inhibitors such as Ferrostatin-1 were found with the property to suppress bacterial death induced by nano-decoction. Iron chelators can exercise similar effects in the process. Bio-chemical experiment showed that nano-decoction has necessary reaction activity to oxidize GSH into GSSG, with polysulfide playing a major role.

Based on above-mentioned findings, the researchers thus argued that ferrous iron and polysulfide in nano-decoction can coordinate to induce bacterial death with hallmarks similar to ferroptosis. It is noted that such a ferroptosis-like death in bacteria depends on ferrous iron rather than ferric iron. It is only applicable to bacteria, has no effect on fungi, and achieves an optimal effect in aqueous solutions. The anti-bacteria effect has been curtailed because of the existence of chelators and GSH in relevant medium.

Interestingly, the nano-decoction can only suppress staphylococcus aureus and MRSA (methicillin-resistant staphylococcus aureus) in macrophages without triggering cytotoxicity to host cells. Ferrous iron and polysulfide from nano-decoction can restrain bacteria but fail to induce cellular ferroptosis when they enter into macrophages. Therefore, the researchers attributed different results to the fact that the antioxidant system of bacteria is simple and easier to be killed and that eukaryotic system of cells boasts rich antioxidant system and higher GSH and can thus sustain the simulation of nano-decoction.

Animal experiments indicated that intravenous injection of nano-decoction can dramatically reduce staphylococcus aureus in blood and cells and prolong the survival period of septic mice. More importantly, nano-decoction has an equivalent therapeutic effect to vancomycin for infectious pneumonia treatment induced by staphylococcus aureus.

The researchers believed that they had found an antibacterial alternative for anti-infection therapy. In other words, bacteria can be killed in a ferroptosis- like manner. Combining with previous findings related to the mechanism in which iron oxide nano-enzyme induce oxidant death of viral lipids, they stressed that ferroptosis can happen to both bacteria and viruses, though there is no relevant signal pathway modulation mechanism. Nevertheless, such a channel can still be used to develop new anti-bacterial and anti-virus drugs.

The ferroptosis-like death in bacteria is probably shared by various inorganic nano-antibiotic materials and thus can act as a guidance to the development of anti-bacterial materials.