The Function And Mechanism Of Mt-PemIK In Toxin-antitoxin System Of Mycobacterium Tuberculosis

Objectives Through bioinformatics,in vitro,and in vivo experiments,to identify a pair of new TA systems,construct an H37Rv△Pem K knockout strain,and observe the effect of mt-Pem IK on the growth of Mycobacterium smegmatis and Mycobacterium tuberculosis(Mtb).Based on this work,we aim to reveal the mt-Pem IK function and mechanism in Mtb,complementing the function of Mtb toxin-antitoxin system,and further explaining the mechanism of Mtb persister and pathogenicity.Methods First,a pair of new TA systems with featured structures supposed to be of traditional TA systems were screened in the Mycobacterium tuberculosis genome by the bioinformatics method.The function and interaction mechanism of the new pair were verified by Mycobacterium smegmatis as a model organism,and the accordance with the traditional TA system was analyzed.Second,the H37Rv△Pem K knockout strain was constructed by phage mediated homologous recombination,which was then verified by PCR,real-time fluorescence quantitative PCR,and sequencing.The growth curves of wild strain and knockout strain were drawn and analyzed in liquid and solid culture under normal and different stress conditions.Third,the models of RAW264.7 cells infected by the wild strain and knockout strain were constructed,respectively.Differences in CFU count,cytokine level,and apoptosis level were compared.At last,animal models infected by wild strain and knockout strain were constructed,respectively.The differences of clinical manifestations,pathological morphology of lung and spleen,CFU count,and cytokine level were compared.Results 1.A pair of new TA systems,called mt-Pem IK,which includes mt-Pem K and mt-Pem I,has been found in the Mtb genome.When mt-Pem K was introduced into Mycobacterium smegmatis and induced to express,Pem K protein had the toxic effect of inhibiting the growth of Mycobacterium smegmatis.The protein Pem I,an antitoxin protein,could antagonize the toxicity of Pem K.It was proved that mt-Pem IK was atypical type II TA system.2.DNA and RNA of the knockout strain,H37Rv△Pem K,were extracted.The knockout strain was successfully confirmed by PCR,real-time fluorescence quantitative PCR,and sequencing.3.Under normal culture conditions,there was no significant difference between the growth of wild and knockout strain.However,under various stress conditions,the growth of wild and knockout strain was significantly inhibited.Under the condition of acid PH,high osmotic pressure,and anaerobic,the growth of knockout strain was slower than that of wild strain.4.The vitro cell experiment showed that the CFU count in the wild strain groups was higher than that of knockout strain groups at 0h,24 h,and 48 h after infection.At 24 h after infection,the IL-1α and IL-10 levels in the wild strain groups were the highest,which was statistically significant compared with that of the knockout strain groups,but the peak of secretion was delayed in the knockout strain groups.The highest IL-6 level was found in both groups at 4h after infection,and the difference was statistically significant at 24 h after infection.The highest IL-12 level was found in the wild strain groups at 48 h after infection,while that of the knockout strain groups at 72 h after infection.The highest TNF-α level was found in the wild and knockout groups at 0h after infection.After 72 h of infection,IFN-γ level in the wild strain groups increased significantly,which was significantly higher than that of knockout strain ones.As the extension of infection time,the apoptosis rate in two groups increased gradually.At24 h after infection,the level of apoptosis induced by knockout strain was higher,the difference between the two groups was statistically significant.5.The animal experiment showed that in the first two months of infection,the growth rate of body weight,degree of tissue inflammation reaction and CFU colony count in the knockout strain groups were all aggravated compared with those in the wild strain groups.In the first one and two months of infection,the plasma IL-6 and IL-10 levels in the knockout strain groups were mainly increased,while in the third month of infection,the plasma IFN-γ level was mainly increased in the wild strain groups.Conclusions In this study,a new TA system named mt-Pem IK was identified,H37Rv△Pem K was successfully constructed by high titer phage mediatedrecombinant gene engineering technology.Under the stress condition,TA system may be involved in the growth regulation of Mtb.Moreover,the conditions of mt-Pem IK regulation were related to acid p H,osmotic pressure and anaerobic stress conditions.The vitro cell experiment showed that the survival ability and virulence of wild strain were stronger than that of knockout strain.The animal experiment showed that mtPem K may regulate the growth of Mtb by affecting its virulence.