Identification And Neutralization Mechanism Of The Novel Toxin-antitoxin System HipBST From Legionella Pneumophila

Toxin-Antitoxin system（TA）is widely identified in bacteria,archaea and other prokaryotes,and plays an important role in resisting phage infection and responding to environmental stress.The TA system usually consists of a toxin that inhibits bacterial growth and an antitoxin that counteracts the toxicity of the toxin.In pathogenic bacteria,the TA system often mediates the formation of persistent bacteria that render many conventional antibiotic treatments ineffective.（1）We found that Lpg2370 which is an effector protein of Legionella pneumophila shares 20%homology with the C-terminus of Hip A.In particular,the P-loop that responsible for binding ATP is highly conserved.（2）Lpg2370 was found to be autophosphorylated in autophosphorylation experiment,and it was found to be autophosphorylated at the S54 site by mass spectrometry.These results indicate that Lpg2370 is a serine/threonine protein kinase rather than an E3 ligase.Subsequently,we analyzed the structure of Lpg2370,which also confirmed that Lpg2370 has a typical kinase structure similar to Hip A＿C.Considering that the sequence and structure of Lpg2370 are similar to Hip A＿C and hip A form a Type II TA system with its upstream hip B.We unexpectedly found genes lpg2369 and lpg2368 in the same operon as lpg2370 by analyzing the position of lpg2370.Lpg2369 and Lpg2368 have higher sequence homology with Hip A＿N and HipB,respectively.These results suggest that the functions of these three genes may be related to the TA system.Subsequently,we found that overexpression of Lpg2370 in Legionella can significantly inhibit the growth of Legionella,and co-expression of Lpg2369 and Lpg2368 can relieve the growth inhibition of Lpg2370 on Legionella.In addition,In addition,we found that Lpg2370 and Lpg2369 can form a complex,and can further form a ternary complex with Lpg2368 through size exclusion chromatography experiments and ITC experiments.These results demonstrate that Lpg2370-Lpg2369-Lpg2368 constitute a three-component TA system.（3）Interestingly,a similar three-component TA system has just recently been identified in pathogenic E.coli O127.Moreover,it was found that three-component HipBST TA system widely exists in a variety of pathogenic bacteria including pneumococcus through evolutionary analysis.Both toxins and antitoxins in HipBST TA are derived from the C-terminus and N-terminus of the toxin Hip A in HipBA but the neutralization mechanism of the novel HipBST TA system remains unclear.To further elucidate the neutralization mechanism of this three-component HipBST TA system,we also solved the structures of the p Hip T_Lp-Amp-Pnp complex and the Hip T_Lp-Hip S_Lp complex.By comparing multiple structures of HipBST,we found that the P-loop in Hip T_Lp became anα-helix after Hip S_Lp combined with Hip T_Lp.And by comparing with the structure of p Hip T_Lp-AMP-PNP complex,we found that the changed conformation of P-loop leads to conflict with the binding position of ATP.Through ITC and Thermal shift experiments,it was further confirmed that Hip T_LPcan bind to ATP,but when it forms a Hip T_Lp-Hip S_Lp complex,it completely loses the ability to bind ATP.So far we have elucidated the molecular mechanism of toxin neutralization in the HipBST TA system from Legionella pneumophila.In conclusion,we identified the effector protein Lpg2370 in Legionella pneumophila as a Hip A-like protein kinase and together with its upstream Lpg2368,Lpg2369 constitute a three-component type II TA system HipBST,and elucidated the molecular mechanism of neutralization of this system.