Therapeutic Effect And Mechanism Of Amentoflavone Against Necrotic Enteritis And Gas Gangrene Caused By Clostridium Perfringens

Clostridium perfringens(C.perfringens)is a Gram-positive anaerobic bacterium that can cause a variety of diseases in humans and agriculturally important animals.In humans,C.perfringens causes gas gangrene and food poisoning,and ranked as one of the most common bacterial causes of food-borne illness by the Center for Disease Control and Prevention in the United States.In poultry,C.perfringens is identified as the key pathogen causing necrotic enteritis,a disease of economic importance to the poultry industry around the world.A virulence factor possibly involved in bacterial cell adhesion and colonization is the presence of Type IV pili(TFP),surface fibers composed of polymerized proteins(major pilins)and smaller numbers of other proteins(minor pilins).TFP mediates a variety of functions in bacteria,such as gliding motility,biofilm formation,adherence to host cells and DNA uptake.Type IV pilus-dependent adhesion to cells is the first step to the bacterial colonization.Evidence shows that the host cell adherence of the mutant that lacked functional TFP adherence is lower than that of wild-type C.perfringens cells.Other studies indicated that in the presence of pili,C.perfringens can form biofilm on host-cell surfaces and increase the toxin production.Perfringolysin O(PFO)is a cholesterol-dependent cytolysin that produced by nearly all C.perfringens strains.In previous studies,PFO plays synergistically with C.perfringens alpha toxin in the development of necrohemorrhagic enteritis and PFO-mediated cytotoxicity may be important factors in the ability of C.perfringens to survive in host tissues.Therefore,TFP and PFO may be potential targets for developing therapeutic agents in C.perfringens-associated diseases.In this study,firstly,TFP-dependent gliding motility assays and PFO-mediated hemolytic activity assays were employed as screening phenotypes.And,we found that amentoflavone could inhibit both TFP-mediated gliding motility and PFO-mediated hemolytic activity.Then,we investigated the influence of amentoflavone on TFP-dependent biological activity and adhesion by gliding motility,biofilm formation,minimum inhibitory concentration,growth curve and adherence assays.By electron microscope observation,we observed the morphology of pilus on the surface of the tested strains after treatment with amentoflavone.Subsequently,we examined the inhibition effect of amentoflavone to the PFO-mediated hemolytic activity and cytotoxicity.Molecular modelling,mutational assays and oligomerization test were used to clarify the molecular mechanism of action of amentoflavone with PFO.In in vivo assays,we established a mouse gas gangrene model and examined the effects of amentoflavone on the mortality,in vivo survival of C.perfringens and tissue damage.Furthermore,we used an in vivo model of necrotic enteritis induced by challenge with C.perfringens,and flatulence,hemorrhage and feces conditions of broilers were evaluated to further examine the therapeutic effects of amentoflavone on necrotic enteritis in chickens.The effect of amentoflavone on the relative abundance of Clostridiales in the intestine of broilers were also tested.Firstly,we found that amentoflavone had significant inhibitory effects on TFP-mediated gliding motility and PFO-mediated hemolytic activity of C.perfringens.Secondly,our data showed that amentoflavone,which had little antibacterial activity against C.perfringens significantly inhibited the TFP-dependent gliding motility,biofilm formation and adherence to the Caco-2 cell line at 4 or 16 ?g/mL.By electron microscope observation,we found that amentoflavone treated C.perfringens did not express pili on the cell surface,indicating that amentoflavone is a potential TFP inhibitor.Thirdly,amentoflavone could significantly inhibit the hemolytic activity mediated by PFO towards rabbit red blood cells and PFO-dependent cytotoxicity towards Caco-2 cells at 2 or 4 ?g/mL.Through computational biology methods,we found that S190 A,S193A,N199,V375 and N377 are key residues for amentoflavone binding.The inhibitory effect of mutants of the key residues were significantly reduced in hemolytic assays.In PFO oligomerization assays,amentoflavone weakened PFO oligomerization.Furthermore,in a mouse gas gangrene model,the survival time of infected mice was extended,the bacterial survival rate in infected mouse tissues was reduced,and the pathological damage was relieved.Moreover,in chicken in vivo assays,amentoflavone significantly alleviated gut flatulence,hemorrhage and decreased the relative abundance of Firmicutes(based on the phylum level),Clostridia(based on the class level)and Clostridiales(based on the order level).In conclusion,our results clearly demonstrate that amentoflavone is a potential inhibitor of TFP in C.perfringens.Amentoflavone treatment led to the lack of pili on the cell surface and significantly reduced the adherence to host cells.Additionally,amentoflavone is also a PFO inhibitor,which effectively weakened PFO activity by targeting PFO oligomerization.As TFP/PFO-dependent virulence is crucial for the pathogenicity of C.perfringens,amentoflavone may act as a promising leading compound for the treatment of C.perfringens infection,especially for necrotic enteritis and gas gangrene.