Bacterial persisters are small non-genetic phenotypic variants of the bacterial population(typically 10-4 to 10-6)that can survive exposure to lethal doses of antibiotics.Persister is increasingly recognized as another major cause of antibiotic treatment failure and relapsing infections.Toxin-antitoxin system(TA system)plays an important role in the formation of multi-drug tolerant persisters.Deciphering the key role of TA systems in persister formation would be helpful for the development of new strategies to eliminate persisters and control the persister formation.TA system is expected to become a new target of the antibacterial drug to alleviate chronic infectious diseases and bacterial resistance caused by bacterial persistence.The TA system directly regulates the formation of bacterial persisters at the protein level.It is crucial to explore the relationship between the expression of TA systems and persisters formation at the protein level.TA system contains two homologous molecules:toxin and antitoxin.The expression of toxin and antitoxin protein is low and highly diverse in individual bacteria,and the abundance of persisters is extremely low.Therefore,revealing the relationship between the heterogeneous expression of the TA system and the formation of low abundance persisters at the single bacterial level is the problematic point at present.In our laboratory,we had built a nano-flow cytometer(nFCM)in 2009 for singlemolecule fluorescence detection.nFCM is a powerful technique for the rapid,quantitative,sensitive,and multiparameter analysis of single cells:In this dissertation,we labeled the toxin and antitoxin with the tetracysteine-biarsenical TC-FlAsH system and immunofluorescence technology,respectively,and we employed the nFCM to detect the expression of toxin and antitoxin.Based on the dual fluorescence method,we explored the critical biological mechanisms of the TA system that produce bacterial persistence.This dissertation consists of the following sections.In chapter one,the role of the TA system in bacterial persistence and the current research status are introduced,and the importance of single-cell analysis is emphasized.In the end,the research plan and main contents of this dissertation are put forward.Chapter two describes the relationship between toxin protein level and persister formation at the single-cell level.We selected MqsR/MqsA TA system as the research object,and we developed an nFCM-TC-FlAsH method for detection of toxin MqsR expression under native promoter at the single-bacteria level.On this basis,we analyzed the expression of toxin MqsR during the formation of persister induced by rifampicin(RIF).We found two populations after RIF treatment,one population with low expression of MqsR,and the other population with high expression of MqsR.To further determine the relationship between the high level of MqsR and persister,we isolated persister cells and found that the expression of MqsR in persister was higher than the negative control,which proved that the population with high expressed of MqsR was persister.Moreover,we found for the first time that the highly expressed toxin MqsR was heterogeneous in persister cells.There existed two populations with a low or high level of MqsR in persister cells,and the proportion of the two populations was 39.7%and 60.3%,respectively.The abuse of antibiotics makes the bacteria in the body or environment accumulate resistance factors,which promotes the evolution of bacterial resistance.To explore the role of the TA system in this process,we prolonged the RIF treatment time to 0.5,3,12,and 24 h,and analyzed the expression of MqsR.We found that there was a population with a high expression of MqsR at 0.5 h and 24 h.We didn’t find the same result at 3 h and 12 h,but the expression of MqsR was higher than 0 h.The high expression of MqsR may be related to bacterial persistence,so we tested the persister frequency at a different time of RIF treatment.We found the persister frequency increased with the prolonging of rifampicin exposure time,which was not associated with the high expression of MqsR.Furthermore,we induced the resistance evolution of bacteria after RIF treatment through daily ampicillin antibiotic treatments.We found that the bacteria after 0.5 h and 24 h RIF treatment developed resistance fastly,because that bacteria contained a population with high expression of MqsR at 0.5 h and 24 h.We found that bacteria after 12 h RIF treatment also developed resistance because the overall expression level of MqsR in bacteria is high at 12 h.The above research results indicate that there is a certain relationship between the high expression of toxin protein MqsR and the evolution of bacterial resistance.In summary,through the singlecell detection of the toxin MqsR,we revealed that the bacteria promote persister formation by regulating the heterogeneous expression of the toxin protein,and the heterogeneous expression of toxin promotes the evolution of bacterial resistance.In chapter three,we simultaneously detected the expression of toxin MqsR and antitoxin MqsA,and explored the relationship between the toxin/antitoxin ratio and persister formation.Combining with the tetracysteine-biarsenical TC-FlAsH system,immunofluorescence staining,and nFCM,we developed a nFCM-TA-double fluorescence method for the detection of toxin MqsR and antitoxin MqsA at the singlecell level.Using RIF to induce persister formation,we detected the expression of MqsR and MqsA.The expression of MqsR increased and the expression of MqsA decreased after RIF treatment,whereas the persister frequency increased.These results suggested that bacteria increased the toxin/antitoxin ratio to promote persister formation.Under tetracycline or CCCP treatment,bacteria also regulated the formation of persister by increasing the ratio of MqsR/MqsA,but the increase of persister frequency is small because of the small change of toxin/antitoxin ratio.Moreover,under long-term RIF treatment,the antitoxin MqsA production decreased continuously,while the toxin MqsR production continued to increase.Based on the above results,we deciphered the molecular mechanism of the TA system mediating persister formation:bacteria regulate the expression of toxins and antitoxins simultaneously,and a fraction of bacteria highly express toxin and degrade antitoxin,increasing toxin/antitoxin ratio,so that toxin predominates and promotes persister formation.In chapter four,we summarized the research results,and put forward the prospects. |