Study On The Mechanism Of Toxin-antitoxin System Mediated Formation Of Persister

Persister is a phenotypic variant of bacteria that survive lethal doses of antibiotics and can continue to reproduce when normal culture conditions are restored.In recent years,more and more researchers attribute the problem of incurable chronic infections to the presence of persister.Current studies on the formation mechanism of persistent bacteria have addressed many important physiological processes and related genes,but due to the complexity of persistent bacteria,we have not been able to fully clarify the formation mechanism,and further studies are still needed.The toxin-antitoxin(TA)system is one of the most important parts of the persister formation mechanism.The TA system usually consists of a stable toxin protein and an unstable antitoxin,which can be divided into six different types according to the nature of the antitoxin,among which the type II TA system is most closely related to the formation of persister.In the type II TA system,the antitoxin is an unstable protein;when in a normal growth environment,the toxin is neutralized by the antitoxin;and when the bacteria are under stressful environments such as antibiotics,the unstable antitoxin will be degraded by proteases,resulting in overexpression of toxins,which will affect physiological processes such as DNA replication and protein synthesis,thereby inhibiting the growth of bacteria.Since hip A was the first TA system gene found to be associated with the formation of persister bacteria,hip BA is currently the most widely studied.In this paper,we first constructed the toxin protein Hip A overexpression plasmid using molecular cloning and introduced different inducible promoters for it,at the same time fused the hip A gene with the red fluorescent protein m Cherry,and the target sequences were verified by DNA sequencing.Then the toxicity and fluorescence signals were examined by inducing toxin protein overexpression,and it was demonstrated that overexpression of toxin protein could reduce the cell growth rate in our system,and there was a gradient effect of gradual decrease of cell growth rate with the gradual increase of toxin protein in the system with Plac-lac UV5 as promoter.In order to make the system with reduced basal expression of toxin proteins,we changed the start codon of lac I and the promoter of lac I to optimize this system by increasing the expression of the blocking protein Lac I.We then investigated the mechanism of toxin protein-mediated persister formation by adjusting the expression of toxin protein and inducing Hip A overexpression in different initial states,we found that toxin protein-mediated persister formation was dependent on the initial state of the cells.For cells in stable initial state,overexpression of toxin protein could easily affect the growth rate of the cells and the proportion of persister formation;however,the expression level of toxin protein was not linearly correlated with the formation of persister bacteria,and when the expression level of toxin protein exceeded a certain threshold,the excess toxin would lead to cell death,thus reducing the persistent rate.In contrast,for the more active cells in their initial state,even if the same amount of toxin protein expression was induced,it would only have a small effect on the growth rate of cells and not on the formation of persister.For both MG1655 and DH5α strains,the same induction conditions also produced different results.In order to further investigate the formation of persister bacteria under this condition,we analyzed the Hip A overexpression plasmids induced by different promoters under the logarithmic phase.And found that in the system containing the p BAD promoter,due to the high induction efficiency of the promoter,the growth of cells soon came to a stagnant state after the addition of the inducer.While this phenomenon could not be seen in the Plac system,which required a longer time and a higher concentration of the inducer to be induced.In addition,the induction of toxin protein overexpression in the Plac system was carried out under different culture conditions,also at plateau stage,and it was found that in the more nutrient-rich EZRich medium it was difficult to induce the production of persister cells due to its excessive cell growth rate and inability to accumulate toxin protein.This result suggests that the toxin protein-mediated formation of persistent bacteria is not only related to the initial state of the cells,but also to the rate of toxin protein accumulation.In summary,this paper takes the toxin protein Hip A as the entry point to study the effect of its overexpression on the formation of persister bacteria,revealing a new strategy for the formation of persister,and provides ideas for further research on the formation mechanism of persistent bacteria and the design of antibacterial drugs.