| Listeria monocytogenes(L.monocytogenes)is a Gram-positive,facultative anaerobic bacterium that is widely present in nature,and an important foodborne zoonotic disease bacteria.The bacterium has a strong environmental adaptability.Food contaminated with Listeria monocytogenes may cause diseases such as gastroenteritis,sepsis,meningoencephalitis.The bacteria can even cross the blood-fetal barrier,causing adverse pregnancy outcomes such as abortion,stillbirth,and fetal encephalitis.For the treatment of Listeria monocytogenes infection,the combined therapy of β-lactam antibiotics and aminoglycoside antibiotics is commonly used in clinical practice.Although clinical Listeria monocytogenes still has high sensitivity to both of these two antibiotics,it can still cause a lethality of 20%-30% in the immunocompromised population.So there is still a need to develop other anti-infection methods.The pathogenicity of Listeria monocytogenes depends on secreting a series of virulence factors.Among them,listeriolysin O(listeriolysin O,LLO)is a key virulence factor to assist Listeria monocytogenes to finish the intracellular life cycle.As an intracellular parasitic pathogen,Listeria monocytogenes has a complete cell survival cycle,and LLO is an important participant in the process of bacterial escape from phagocytosis and cell to cell spread.All of the clinically isolated Listeria monocytogenes have hemolytic activity,and the bacteria lost is pathogenicity in cell-level infection tests and mice infection tests after LLO knockout,and is cleared by the host immune system in vivo.It can be considered that the structural integrity and normal function of LLO determine the pathogenicity of Listeria monocytogenes.In this study,a specific small-molecule inhibitor of LLO,cryptotanshinone,which could significantly reduce the hemolytic activity of the LLO protein and bacterial culture supernatant after co-treatment,was screened based on a hemolysis assay.According to the results of molecular simulation docking judgment,it was most likely to bind to the fourth domain of the LLO molecule,thereby inhibiting the process of recognition and binding of the hemolysin protein to the cell membrane.At the same time,it has been proved that cryptotanshinone could significantly reduce the assemble process of LLO from water-soluble monomers into water-insoluble oligomers,thereby inhibiting the pore-forming activity of LLO with an oligomerization assay.Cryptotanshinone also exhibited the pore-forming activity of two other cholesterol-dependent cytolysins(CDCs)family proteins,SLY and PLY,with a similar inhibitory effect.The determination of the minimum inhibitory concentration and growth curves,as well as assays such as real-time PCR and Western Blot,together showed that the effective concentration of cryptotanshinone addition had little to no effect on the bacterial survival and growth,or the hly gene transcription,but significantly inhibited the LLO protein level in both culture supernatant and bacterial pellets.Cryptanthinone addition significantly inhibited LLO induced macrophage damage,significantly restricted the proliferation of Listeria monocytogenes in macrophages,and inhibited macrophage damage caused by Listeria monocytogenes infection.The process of host cells degrading phagocytic bacteria is accompanied by the production of oxidase,causing oxidative stress damage to the host cells.Results proved that cryptotanshinone can significantly promote the nuclear transfer of Nrf2 in J774 macrophages,and promote the expression of a series of antioxidant-related genes regulated by Nrf2 / ARE,mainly like HO-1.Adding Cryptotanshinone into the infection system also significantly activated the Nrf2 signaling pathway.At the same time,the addition of cryptotanshinone inhibited the activation of NLRP3 inflammasomes and the production of infection-induced mature IL-1β release into the co-infectious system.This process may also be associated with the effect of drugs on the formation of ASC dimers.Besides,Listeria monocytogenes infection caused a strong,LLO-dependent activation of the MAPK signaling pathway,while treatment with cryptotanshinone inhibited the phosphorylation of MAPKs and significantly reduced the secretion of two pro-inflammatory factors,TNF-α and IL-6,which performed an anti-inflammatory role.Listeria monocytogenes cause severe infection in pregnant women which will induce miscarriage and fetal infection,which is one of the reasons that seriously harms public health and safety.In this study,we used Listeria monocytogenes to infect the human placental chorionic cell line BeWo cells,which proved that the addition of cryptotanshinone could reverse the decrease of tight proteins,Occludi‘’n and ZO-1,in wild type Listeria monocytogenes infection in cells caused,and inhibited intracellular bacterial multiplication.The ability of cryptotanshinone pretreated bacteria to internalize cells was significantly reduced,and the formation of biofilms was also suppressed.Therapeutic tests on Listeria monocytogenes-infected mice have shown that oral cryptotanshinone administration inhibited the increase of the wet/ dry weight ratio in mice liver tissue caused by infection,relieved the pathological damage of target organs,reduced the liver damage of infected mice and bacterial burden in livers,while significantly inhibiting the infection-induced malondialdehyde(MDA)increased in liver tissue,and improved the survival rate of lethal dose bacterial infected mice,which played a certain therapeutic effect.To sum up,cryptotanshinone was an effective inhibitor of LLO activity.It could inhibit a series of LLO-dependent cell damage and inflammatory reactions during Listeria monocytogenes infection.cryptotanshinone was also an effective antioxidant and played a certain role in relieving anti-oxidative stress in both of the in vitro and in vivo experiments.Besides,with its extensive inhibition of CDCs,it can be speculated that cryptotanshinone may also have a therapeutic effect on other Gram-positive bacterial infections.Therefore,cryptotanshinone can be used and further developed as a potential anti-infective drug or as an antibiotic synergist. |
