Deciphering Toxin-Antitoxin System Regulated Bacterial Stress Response And Persister Cell Formation Via Single-cell Analysis

Bacterial toxin-antitoxin(TA)are commonly existed systems in environmental microorganisms and pathogens.A typical TA system consists of two adjacent genes that encode a labile antitoxin(protein or RNA)and a stable toxin(protein).The antitoxin and toxin work synergistically to regulate the cell processes such as DNA replication,translation,and cell wall synthesis,which farther influence the development of environmental adaptation,and persistent bacteria.It is indicated that the expression of a TA system is highly heterogeneous among individual cells,and a specific subpopulation could be responsible for the formation of multidrug persistent bacteria(typically 10-4 to 10"6 of the bacterial population).Therefore,the investigations concerning the mechanism and expression of the TA systems are desired to find new drug targets,reduce or eliminate the persistent bacteria,alleviate the bacterial resistance,and thus reduce the clinical recurrent infections and chronic diseases.Most studies of TA systems have been conducted at the gene expression and transcriptional levels,such as quantitative real-time reverse-transcription PCR(qRT-PCR),Northern analysis,and so on.However,it has been reported that the TA systems are mainly regulated at the protein level.Therefore,focusing only on transcript analysis might lead researchers to overlook some key factors that can cause persistence formation.Western blot(WB)analysis is the most utilized method for protein expression analysis.However,the bulk method will mask the heterogeneous expression of TA system among bacterial individuals,which will miss the abnormal expression of TA system in a minority of persistent bacteria,and can not revel the accurate mechanism of TA system.Current approach to confirm and quantify TA expression in individual bacteria is fluorescence microscopy.However,routine application of microscopy to the investigation of TA system is hampered due to the tedious procedures and limited statistical power.Therefore,it is necessary to develop a sensitive,rapid,and high-throughput method to measure the abundance of TA proteins at the single-cell level.Using a laboratory-built nano-flow cytometer(nFCM)for multiparameter analysis and abiarsenical-tetracysteine(FlAsH-TC)system for live-cell labeling,we developed a sensitive method that enables the detection of toxin or/and antitoxin expression at the single bacteria level.Based on the detection method we deciphered the antitoxin-regulated bacterial stress response,and explored the relationship between the ratio of antitoxin to toxin and persister cells.This dissertation consists of the following sections.The first chapter systematically introduced the TA systems and the roles they play in stress response and persister cell formation.The research methods and progress for the TA systems are reviewed.Finally,the ideas and research contents of-this thesis was introducedIn chapter two,we deciphered the antitoxin-regulated bacterial stress response via single-cell analysis.Using MqsR/MqsA as model TA system;we developed an nFCM-TC-FlAsH based method for rapid and simultaneous determination of bacterial growth rate and MqsA expression level.We revealed for the first time that under native promoter and in the absence of environmental stress,there exist two populations of bacteria with high or low-level of antitoxin MqsA expression.Under environmental stresses such as bile acid stress,heat shock,and amino acid starvation,the two populations of bacteria respond differently in the regulation of MqsA degradation or production.Meanwhile,the rebound of MqsA production upon amino acid stress was observed.By coupling the bacterial growth and MqsA expression,we found that,under environmental stress,the response of bacterial growth was consistent with MqsA production but with an approximate 60-min lag.Overall,the results of the present study indicate that stochastic elevation of MqsA level facilitates bacterial survival,and the two populations with distinct phenotypes empower bacteria to deal with the fluctuating environments.The as-developed method will provides an advanced platform for the heterogeneity analysis of TA systems.In the third chapter,an nFCM-TA-double fluorescence method was developed for simultaneous detection of toxin and antitoxin at the single-cell level,which enable us to obtain T/A ratio and its relationship with the formation of persister cell.Based on the nFCM-TC-FlAsH system,we detected the trace expression of toxin MqsR under native promoter at the single-cell level.Employing biarsenical dye and immunofluorescence to specifically label MqsR-TC and MqsA-His respectively,the toxin and antitoxin expressions at low levels were detected simultaneously at the single-cell level.In order to reveal the relationship between the ratio of T/A and the formation of persister cells in the presence of rifampicin,we analyzed the toxins and antitoxin levels at the single-cell level vis nFCM-TA-double fluorescence method.We found that there were two populations before rifampicin treatment:one population with low levels of MqsA and MqsR,and the other population with a high level of MqsA and a low level of MqsR.The first population disappeared after rifampicin treatment.Remarkably,a third population with a high level of MqsR(25 times higher than the low expression subgroup of MqsR)and a low level of MqsA was emerged at 12 h during rifampicin treatment.This proportion increased with the extending of the action time of rifampicin,and reached to 24.7%at 24 hour.Combining nFCM-TC-FlAsH strategy with membrane-impermeant nucleic acid dye propidium iodide(PI),the MqsR and viability of single bacteria were analyzed simultaneously.It is interesting that bacterial subpopulation with a high toxin MqsR level showed PI fluorescence intensity between that of live bacteria and dead bacteria,and looks more like live bacteria by confocal microscopy imaging,indicating that the population with a high level of MqsR and a low level of MqsA was still biologically active.In the presence of rifampicin,the antitoxin MqsA decreased followed by the increase of the toxin MqsR,both of which contributed to the increase of T/A.Combined with persister frequency analysis,we found that the increased T/A enable the bacteria to withstand high concentrations of antibiotics.Chapter four are summary and prospects.The present investigations of the TA systems in regulating stress response and persister cell formation arc summarized.The further researches on the mechanism of TA systems in the bacterial persistence,and the interaction of TA systems were put forward.