Inhibitory Effect And Mechanism Of Thymol Against Salmonella Type ? Secretion System

Pathogenic Salmonella species arguably are the most important food-borne pathogen in humans and animals caused by food poisoning,they are also important zoonotic pathogens.Salmonella enterica is the leading cause of community-acquired bloodstream infections in many low and middle income countries.These pathogens have a broad range of natural hosts including mammals,reptiles,birds and insects.Salmonella infection often leads to serious animal diseases,causing severe economic losses in the livestock industry.For example,infection by Salmonella is one of the most important infectious disease in the poultry industry.Currently,infections by Salmonella are difficult to control and treatment is often not effective due to the development of resistance to traditional antimicrobial control strategies.Several factors,including the mechanisms of action of traditional antibiotics,their inappropriate use and the lack of antibiotics with novel targets have caused the development of resistance and telorance in an alarming rate.The resistance and telorance development by the pathogens have made the control and treatment of infections caused by Salmonella spp.more and more difficult.Thus,there is an urgent need to develop agents with novel mechnisms of action.Ideally,such agents will not able to or less likely to induce resistance or telorance.Intensive investigation in the past two decades revealed that specialized protein secretion systems are essential pathogenic elements in many important bacterial pathogens.Among these type III secretion systems are among the most widely distributed virulence machineries.It has been well-established that mutations in type III secretion systems(T3SSs)resulted in mutants that are completely non-pathogenic.Many species of Salmonella,one of the most important zoonotic pathogens that cause a wide range of infections in both human and animals contain two distinct type III secretion systems called SPI-1 and SPI-2,responsible for bacterial invasion and intracellular survival and replication,respectively.These two T3 SSs are essential for the virulence of Salmonella species such as Salmonella enterica Typhimurium.Mutants lacking either one of these secretion systems are defective causing diseases.Traditional Chinese medicine has been used successfully for thousands years.The active ingredients in herbs used the treatment are diverse in structure and activity and are widely distributed,thus providing rich resources for the discovery of compounds effective treating infections or at least for lead compounds in new drug discovery.Equally important is that some of these compounds function by targeting bacterial virulence factors or enhancing host immunity but not by bactericidal activity,which make them less likely to induce resistance.To identify natural compounds capable of inhibiting SPI-1-mediated secretion into epithelial cells,we fused the TEM-1-lactamase to the carboxyl end of the well characterized T3SS-1 effector Sip A.Through the reporter system,we found that thymol was able to significantly inhibit the translocation of Sip A.we found that 0.4 m M of thymol did not detectably affect the growth of S.Typhimurium strain SL1344 in bacteriological media.Thymol ability to inhibit T3SS-1,reduced bacterium entry into epithelial cells.Thymol protect mice infection by Salmonella.Consistent with earlier results that thymol did not affect mouse body weight gain,ceca from mice treated with thymol displayed a morphology similar to those from control groups received PBS and those infected with the ?inv A mutant defective in the SPI-1.The ratio between cecum and body weight of these two groups of mice was also similar.In contrast,this ratio was significantly lower in mice infected with wild type S.Typhimurium,which was restored in infected mice treated with thymol.Histological examination after hematoxylin and eosin(H&E)staining showed that ceca from mice infected with wild type bacteria displayed submucosal edema,the loss of goblet cells,damage of the epithelial layer and the infiltration of polymorphonuclear granulocytes(PMN)in the lamina propria;again,these symptoms were alleviated when thymol was given to similarly infected mice.Similarly,thymol treatment significantly reduced the number of bacteria associated with such organs as spleen,liver and cecum,indicating reduced bacterial dissemination in thymol-treated mice.We also measured the status of inflammation in these animals.The amounts of pro-inflammatory cytokines such as IL-1?,IL-6,TNF-? and IFN-?in the ceca were significantly higher in untreated animals infected with wild type bacteria,and thymol treatment significantly reduced the production of these cytokines.Taken together,these results indicate that thymol can provide effective protection against S.Typhimurium infection in mice.Through circular dichroism(CD)spectroscopy and chick chemistry analysis,thymol induces conformational changes in Sip A by directly interacting with two regions of this effector.In reactions containing recombinant Lon and a GST fusion of full-length Sip A purified from cells treated with thymol,The result demonstrate that after undergoing a conformational change,the target proteins of thymol are delivered to the Lon protease for degradation.Generally speaking,we designed a screening strategy to identify compounds from traditional Chinese medicine capable of inhibiting the SPI-1 of Salmonella typhimurium.By using an effector-lactamase fusion reporter,we identified thymol,a monoterpene phenol derivative of cymene,as an effective inhibitor of the T3SS-1 of Salmonella Typhimurium.Our results indicate that thymol destabilizes several virulence factors associated with T3SS-1.Further analyses with Sip A revealed that thymol directly interacts with this protein to induce conformational changes,which make it susceptible to the Lon protease.In an animal infection model thymol effectively protected mice against S.Typhimurium-induced mortality and pathological damages.Thus,thymol represents a class of anti-virulence compounds that function by targeting pathogenic factors for degradation,which can be developed for the control of Salmonella infection.Therefore,this project has both scientific and practical significance.