Study On The Structure And Function Of A New Type Of Toxin-antitoxin System In Mycobacterium Tuberculosis That Is Detoxified By Phosphorylation

Tuberculosis(TB)is one of the most global health threats and becomes one of the top 10 causes of death.According to the world health organization(WHO)global TB report 2018,the best estimate is that 10.10 million people developed TB disease and TB caused an estimated 1.57 million deaths in 2017.The challenges of TB prevention and control mainly include the following two aspects:on the one hand,the situation of drug resistance of TB is increasingly.According to WHO latest data,there were about 460,000 new cases of multidrug－resistant tuberculosis(MDR-TB)in 2017.MDR-TB is difficult to treat and need long treatment.Hence,there is an urgent need for developing new anti-tubercuiosis drug.Futhermore,the treatment success remains low.On the other hand,latent tuberculosis infection(LTBI)situation is not optimistic.According to WHO latest data,a quarter of the world’s population belongs to the LTBI population,and there are nearly 360 million LTBI people,which are at increased risk for developing active TB.Therefore,strategies for preventing LTBI to develop active TB are urgently needed.Toxin-antitoxin(TA)system is widely identified in the chromosomes or plamids of many bacterial and archeal.These genetic modules typically contain a two-gene operon as bicistronic,which respectively codes for toxins that exert toxic effects and antitoxins that have detoxification functions.More and more evidences show that TA system is involved in bacterial genome stability,biofilm formation,phage resistance,persister formation and virulence.The exploitation of TA systems as an antibacterial strategy via artificial activation of the toxin has been proposed and has considerable potential in multiple pathogenic bacteria including Mycobacterium tuberculosis,which has become a new strategy for developing of anti-tuberculosis drugs.There are at least 79 TA systems in M.tuberculosis,and 11 of TA systems have not been characterized for their biological functions.Three of the 11 TAs,i.e.Rv0836c-Rv0837c,Rv1045-Rv1044,and Rv2827c-Rv2826c were predicted to be type IV TA.In this study,Rv1045-Rv1044 TA locus was characterized structurally and functionally.The genetic organization of the Rv1045-Rvl044 in the H37Rv genome was characterized by an arrangement resembling the bicistronic operon.Then,Rv1045-Rv1044 was identified as a typical TA system by the toxicity and antitoxicity assay,i.e.toxin Rvl045 exerts toxic effects and antitoxin Rv1044 that have detoxification functions.The Co-immunoprecipitation experiment demonstrated that Rv1045 and Rvl044 interact directly with each other.This behavior is atypical for type Ⅳ TA family members.Crystal structure demonstrated that Rv1045 encodes nucleotidyltransferase-like proteins,which encompasses an N-terminal domain(NTD)and a C-terminal domain(CTD).The NTD(1-181aa)exhibits an α/β fold that commonly found in nucleotidyltransferase一like proteins.The long α2 helix is surrounded by seven β-sheet(βl-β7)and five α-helices(α1-α5).We found a large central positively charged cavity formed between NTD and CTD,suggesting the cavity functioned as an NTP binding pocket and a NTase active site.NTP binding assays demonstrate that toxin TglT preferentially binds GTP.All data suggest that Rv1045 functions as a GTP-specific NTase that transfers this nucleotide to a target,resulting in growth inhibition with an unknown mechanism.Interestingly,Rvl045 co-expressed with the antitoxin Rvl044 showed phosphorylation located at S78,compared with the Rvl045 D82A expressed alone.In order to confirm that Rvl045 phosphorylation was modified by antitoxin Rv1044,we have separately analyzed the crystal structure of Rv1045 S78A,Rv1045D82A and Rv1045E146Q co-expressed with/without antitoxin Rv1044.Comparison of 7 crystal structures,we identified that Rv1045 phosphorylation at S78 was related to Rv1044 expression.Subsequently,antitoxin Rvl044 was demonstrated to be involved in the phosphorylation of Rv1045 at S78,while the Rvl045 and mutations co-expressed with/without Rv1044 was identified by LC-MS/MS.At last,Rv1044 was directly involved in the phosphorylation of Rv1045 S78 through in vitro kinase assay,and the phosphorylation of Rv1045 S78 by antitoxin Rvl044 made Rv1045 lost the toxicity.In conclusion,we characterized the putative M.tuberculosis Rv1045-Rv1044 TA locus structurally and functionally,demonstrating that it constitutes a bona fide TA system.While Rv1045 encodes a guanylyltransferase TglT functioning as the toxin.Rvl044 is an atypical serine protein kinase,which adopts a previously unobserved antitoxicity mechanism involving post-translational modification.Rvl044 that specifically phosphorylates the cognate toxin at residue S78,thereby neutralizing its toxicity.In contrary to the previous predictions,we found that Rvl045-Rvl044 do not belong to type Ⅳ TA family because Rvl044 and Rvl045 interact with each other as substrate and kinase.Therefore,we classify them to a novel family type Ⅶ.We found that while Rv 1045 encodes atype Ⅶ guanylyltransferase-like toxin(TglT),which arrests bacteria growth,Rv 1044 encodes a type Ⅶ atypical kinase antitoxin(TakA),which neutralizes the activity of TglT via phosphorylation.Our results enrich the content of TA system and are expected to provide informative data for developing anti-tuberculosis drug targeted Rv1045-Rv1044.