| In the late1920s, the discovery of penicillin opened up a new era of antibiotictreatment and many infectious diseases have been effectively controlled from then on.However, with the misuse and abuse of antibiotics, bacterial resistance is more andmore serious and a large number of drug-resistant bacteria, multi-resistant bacteriaand even super-resistant strains emergence. These problems have restricted thesurvival and development of human. Our country is one of the most seriousantibiotics-abuse countries where the problem of bacterial resistance is more severecompared to other countries. Each year about80,000people died due to the adversereaction of antibiotic abuse, and the number is increasing progressively every year.Therefore, how to solve the problem of bacterial resistance and find novel drug targetsis imminent. It has been proposed that the biological effect caused by antibiotics atsubinhibitory concentrations (less than therapeutic dose) is an important factor inclinical bacterial resistance. The study of antibiotic-effect at subinhibitoryconcentrations will not only play an important role in the analysis of bacterialresistance mechanism, but also have great significance for guiding clinical dosage andadministration time.In this study, we found a clear enhancement of violacein production at the edge ofthe kanamycin inhibition zone when strain CV12472was grown on plates. Our datashowed that this enhancement was caused by kanamycin at subinhibitoryconcentrations and violacein production was promoted when the concentration ofantibiotics was1/4,1/6and1/8of MICs. A peak induction occurred in the presence ofkanamycin at1/6MIC. The amount of violacein was increased by25%at thisconcentration. Similar results were observed in1/4MIC of amikacin,1/2MIC ofgentamycin,1/16MIC of tetracycline, and1/8MIC of erythromycin, but the effect is not as obvious as1/6MIC of kanamycin. In Chromobacterium violaceum, thesynthesis of violacein is under the control of QS. Thus, we analyzed the violaceinsignal molecule synthase gene and signal molecule production with quantitativereal-time PCR and signal molecule report strain, respectively. The results showed thatkanamycin at1/6MIC enhanced the transcription of signal molecule synthetase geneand the production of QS signaling molecules N-acyl-L-homoserine lactones. At thesame time, antibiotics promoted the QS controlled virulence, including chitinaseproduction and biofilm formation. Besides, a positive flagellar activity and increasedbacterial clustering ability were found to be related to the antibiotic-induced biofilmformation. These results indicated that antibiotics at subinhibitory concentrationsimproved the QS behavior of C. violaceum.Cyclic di-guanosine monophosphate (c-di-GMP) is the second messenger inbacteria and triggers wide-ranging physiological changes interdisciplinary with QS,such as cell movement and biofilm formation, which suggests that c-di-GMP signalsystem and QS signal system are not separated. In this thesis, we transferred thec-di-GMP synthetase gene adrA of E. coli into C. violaceum successfully withconjugal transfer and the QS behavior of it was inhibited obviously when overexpressed adrA. Quantitative real-time PCR analysis showed that adrA decreased thegene transcription level of the violacein signal molecule synthase. Signal moleculequantitative analysis was consistent with these results. At the same time, violaceinsynthesis and chitinase production were both inhibited. These results indicated thatadrA regulate QS behavior of C. violaceum by increasing the c-di-GMP synthesis.The traditional cell c-di-GMP monitoring method is using radioisotopes ofc-di-GMP synthesis substrate GTP tag, and then detecting the intracellular amount ofisotope with thin layer chromatography and autoradiography. However, this methodrequests high experimental condition, operation is complicated and it is difficult toreflect intracellular c-di-GMP content in real-time. To solve this problem, we built thec-di-GMP riboswitch monitoring model according the principle of c-di-GMP acting asriboswitch ligand molecular regulates gene expression. Then, we used this model tomonitor the intracellular level of c-di-GMP changes and determine whether antibiotics regulate bacterial QS system through c-di-GMP. Here we cloned six c-di-GMPriboswitch genes from the genomes of Vibrio cholera VC16961, Aeromonashydrophila and Bacillus cereus, and constructed six c-di-GMP content-changedetection models in vivo using sacB and lacZ biological reporter gene successfully.Through function verification and structure optimization, we got a strong specificityand high sensitivity c-di-GMP riboswitch biological detection model Ahy-1. Usingthis model, we found that antibiotics at subinhibitory concentrations decreased thelevel of c-di-GMP and antibiotics regulated QS behavior through c-di-GMP signalpathway in C. violaceum.In summary, we proved that antibiotics at subinhibitory concentrations improvedthe QS behavior of C. violaceum, demonstrated c-di-GMP regulation of QS behavior,constructed a strong specificity and high sensitivity c-di-GMP riboswitch biologicaldetection model and found that antibiotics at subinhibitory concentrations regulatedQS behavior through c-di-GMP signal pathway in C. violaceum. Our findings in thisthesis provided a new theoretical basis to solve clinical bacterial resistance anddiscover new drug targets for developing new anti-infective drugs. Meanwhile, itprovided valuable reference for antibacterial drugs in clinical use. |
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