Construction Of Staphylococcus Aureus Transposon Library And Studies Of Mutants With Altered Persistence

Persisters/persister cells are a subpopulation typically different from the normalbacterial population, which are able to survive from treatment of lethal concentrationantimicrobials. Because of the existence of persisters, two-phase of the antimicrobialskilling curves are usually observed, with a rapid initial decrease in CFU followed by aslower decline. Since persisters’ difference from genetic resistance with a altered minimuminhibitory concentration, when the surviving persisters are reinoculated in fresh mediumand cultured, theres is still only a portion of subpopulation being persisters. Persisters areconsidered to be closely associated with biofilm, as well as a culprit of refractory chronicinfectious diseases. So the great importance of persisters in clinical anti-infection treatmenthas been realized, and it is suggested that persistence is an even more serious problem thanresistance in clinic settings.S. aureus is one of the important nosocodial infection and community acquiredinfection pathogens, which can induce mild pyogenic infections in skin, mucosa and so on.More serious, it can also results in fatal diseases such as bacteremia, toxic shock syndrome,etc. Recently, as the development of antimicrobial resistance, S. aureus infections tend topresent higher morbidity, stronger resistance, more difficulties on therapy, and highermortality. In consideration of the importance of S. aureus and persisters in clinic notedabove, investigation of formation mechanisms on S. aureus persisters appears to be greatsignificance to control S. aureus infections. Although early had been discovered inStaphylococcus sterilization experiments in1944by Bigger, E. coli, P. aeruginosa, M.tuberculosis were usually chosen as the study subjects for a long period.To investigate the formation mechanisms of persisters, one of the most importantworks is to identify persisters-related genes. In previous studies, researchers had identifiedmany genes associated with bacterial persistence through transposon library, single-geneknockout mutants library and expression library of E. coli or P. aeruginosa. Here, weplanned to construct a S. aureus transposon library for screening of genes related to S. aureus persistence. Meanwhile, the S. aureus transposon library can be used in otherscreening experiments as well.Many of the genes involved in the metabolism of bacteria are known to be related tobacterial persistence, which suggested that bacterial metabolism activities play a vital rolein bacterial persistence, including energy production, amino acids metabolism, G3Pmetabolism, stringent responses, and so on. The glpD gene, encodes a glyceral dehyde3-phosphate dehydrogenase, had been proved to be associated with E. coli persistersregulation, however, whether it has a positive or negative role in the process is controversial.By now, no report has been presented in researching the role of glpD on S. aureus persistersregulation. Therefore, we focused on the relationship between glpD and S. aureus persisters,and tried to reveal the mechanism of S. aureus persisters formation.In the section one of this thesis, we took advantage of Tn917to construct a transposonlibrary of S. aureus Newman, which was then used to screen strains with altered persistence.Meanwhile, a DAP-resistance strain was also screened from the library. In the section two,we constructed a glpD deletion mutant, and tested its persistence compared with the wildtype. The results, to our knowledge, led us to firstly interpret the role of glpD in S. aureuspersisters. The main works and results in this thesis are concluded as follow：1. Construction and application of S. aureus transposon library(1) Construction of Tn917S. aureus transposon libraryThe temperature sensitive shuttle plasmid pID408with Tn917was electrotransformedinto S. aureus RN4220in which the plasmid was modified and then electrotransformed intoS. aureus Newman. Transposition in S. aureus Newman was induced by high temperatureand erythromycin, as a result, the transposon library was got. Then the valuation of librarywas made, the number of strains in this library was far more enough for regular screeningexperiments, and the insertion randomness was also verified to be good, so the applicationvalue of the S. aureus transposon library was fully proved.(2) Screening for strains with altered persistence, and the finding of persistence-associated gene pyrDT18strain is a mutant with Tn917insertion in pyrD which is involved in bacterialrespiration and pyrimidine synthesis. Because of its defection in pyrimidine synthesis, T18may show SCVs (small clone variations) in specific conditions and changed sensitivity to some antimicrobials. T18s (T18small) is a derived strain of T18acquired with CIPtreatment. After cultured for24h or36h, the persistences of wild type, T18and T18s strainswere compared. The results showed that:①In the timepoint of24h, CFU of T18was alittle less than wild type and T18s, however, after8h treatment of daptomycin (DAP), thenumber of CFU in T18was the most, which indicated higher persistence of T18s.②Comparing the persistence of36h and24h cultures of these three strains, respectively.Persistence of T18decreased significantly, while persistence of T18s did not showsignificant change. Notably, persistence of wild type increased by about1000times.③T18s showed a prolonged lag phase timescale when compared with wild type and T18, thischaracteristic was considered to be associated with increased persistence. However, T18showed decreased persistence. The results①and②indicated that there are persistencedifferences among wild type, T18and T18s, and pyrD was associated with S. aureuspersistence. Some gene change(s) in T18s compensated the altered persistence in T18s tosome extent. Meanwhile, the different persistence changes in24h and36h cultures of wildtype, T18and T18s provided us good materials for the following persistence studies. Theresult③suggested that the relationship between the timescale of lag phase and thepersistence needed further investigation.(3) A DAP-resistance strain was screened from library, and the resistancemechanism was related to acuAC transcription unit.In this study, a insertion of Tn917in the-35region of the acuAC transcription unitpromoter was found in DAP-resistance strain. The RT-PCR results also showed that theexpression levels of AcuA and AcuC in DAP-resistance strain are obviously lower thanthose of the wild type. These results indicated that the destruction of acuAC transcriptionmay associated with the resistance of DAP, which has not been reported. The further studieson the mechanism of how the disorder of acuAC transcription unit resulted in DAPresistance would probably expand our understanding of DAP resistance and open up a newdirection in DAP-resistance studies.2. Relationship between glpD and bacterial persistence(1) Research statusglpD is closely related to bacterial metabolism for its coded product glyceral dehyde3-phosphate dehydrogenase. In the past researches in E. coli, there was a controversial conclusion on the question that whether glpD deletion would increase or decrease bacterialpersistence. We hope to answer this question in advantage of S. aureus.(2) Construction of glpD knockout strain in S. aureus NewmanThe glpD knockout plasmid was constructed with plasmid pYT3. The cloningfragment including chloramphenicol-resistance gene (cat) and the left and right arms ofglpD on its two ends respectively was cloned into pYT3. The glpD of knockout mutant wasreplaced by cat by the way of homologous recombination, which was verified bymulti-primer PCR.(3) The influences of glpD deletion to S. aureus persistenceFor the great differences between exponential and stationary phase bacterial persisters,the persistence tests were done during both phases in this part. To choose the appropriateantimicrobials for the experiments, various kinds of antimicrobials were used in thepreliminary experiments. Finally, CIP and vancomycin (VAN) were selected for theexponential phase persistence tests because of their stabilities and significant differences inwild type and△glpD persisters formation. For the stationary phase persistence tests, onlyDAP showed an obvious bactericidal effect on S. aureus in stationary phase. In theexponential phase persistence tests, It was found that when the cultures were treated with20MIC CIP for8h, the persistence of△glpD strain increased by approximately10timescompared to that of the wild type. When20MIC VAN was used for8h, the persistence of△glpD strain increased by about20-40times, while when the concentration of VAN wasraised to100MIC, the persistence of△glpD strain increased by about40-100times. In thestationary phase persistence tests, the persistence of△glpD strain decreased byapproximately1000times when the cultures were treated with100MIC DAP. So ourresults showed that, in S. aureus, glpD deletion increased bacterial persistence inexponential phase but substantially decreased bacterial persistence in stationary phase.In conclusion, we constructed a Tn917transposon library with verified applicationvalue. Utilizing this library, we acquired strains with altered persistence and found thatpyrD was associated with S. aureus persisters formation. At the same time, aDAP-resistance strain was screened out from this library, and this mutant was believed tobe defective in acuAC transcription unit. This screening experiment also verified theapplication value of the trasposon library. On the other hand, we detected the relationship of S. aureus persistence and glpD through constructing a glpD knockout mutant. The resultsshowed that glpD deletion increased persistence in exponential phase and decreasedpersistence in stationary phase on the contrary. To our knowledge, this is the first reportindicating that a gene can played opposite roles on bacterial persisters formation inexponential and stationary phase, respectively. These results led us to a betterunderstanding of S. aureus persisters.