| In this century,bacterial drug resistance is one of the most important menaces for human health.With the increasing spread of antibiotic-resistant bacteria,searching for new drug targets has become the focus of researchers.The design of antibiotics against specific molecular targets of pathogens might prevent the spread of bacterial drug resistance.Previous studies had focused on the role of single regulator in controlling expression of virulence genes,and generally paid attention to the development of drug targets by using virulence factors of pathogens as narrow-spectrum targets.Recent researches have gradually proposed a new view that bacterial metabolism plays a key role in pathogens virulence,and metabolism is closely related to the sensitivity of bacteria to antibiotics and the production of virulence factors.Finding drug targets in bacterial metabolic pathways has become a new hotspot in the field of prevention and therapy of drug-resistant bacteria.Pseudomonas aeruginosa is a widely distributed conditional pathogen that can cause a variety of acute or chronic infections which may be potentially life-threatening,especially in the patients with compromised immune defenses.It is one of the main pathogens of nosocomial infections,and has caught more attention in clinical medical treatment.P.aeruginosa can be found in the lungs of nearly all adult cystic fibrosis(CF)patients and has a high rate of multidrug-resistant strains due to chronic exposure to antibiotics.Because of the irrational use of antibiotics,multidrug-resistant P.aeruginosa has been increasing in recent years which has brought enormous challenges to the prevention and treatment of clinically relevant infections.It has been reported that the metabolism of P.aeruginosa has important functions on its virulence and pathogenicity.Therefore,the study of specific metabolic pathways or key metabolic functional enzymes of P.aeruginosa may provide new insights and theoretical basis in the development of new drug targets for against P.aeruginosa infections.In this research,we investigated the 2,3-BD degradation pathway on the one hand to study effects of the special mechanism of uptake and decomposition of exogenous substances by P.aeruginosa on its virulence and pathogenicity,and the specific enzyme malate:quinone oxidoreductase MqoB in the central metabolic pathway on the other hand to study the effects of the central metabolic pathway in P.aeruginosa on its virulence and pathogenicity and the its feasibility to be as a drug target.2,3-BD is a primary microbial metabolite with three stereoisomeric forms:(2R,3R)-2,3-BD,meso-2,3-BD,and(2S,3S)-2,3-BD.Microbial 2,3-BD metabolism can influence the physiological metabolism of various organisms in symbiotic habitat.It has been reported that 2,3-BD can increase the virulence of P.aeruginosa and influence the lung microbiome.Compared to the biosysthesis of 2,3-BD,there are still several key steps in the catabolic mechanism of 2,3-BD unclarified which restricted the development of studies about the physiological process related to 2,3-BD catabolism.In the second chapter of this thesis,on the basis of that P.aeruginosa PAO1 can utilize all the three stereoisomeric forms of 2,3-BD,we analyzed the role of two 2,3-butanediol dehydrogenases(2,3-BDH)in dehydrogenation of three stereoisomers of 2,3-BD to form acetoin(AC),revealed the mechanism of oxidative cleavage of AC to form acetyl-CoA and acetaldehyde by acetoin dehydrogenase enzyme system(AoDH ES),and parsed the downstream metabolic pathway of the cleaved product of AC to expounded 2,3-BD catabolism pathway in P.aeruginosa integratedly.Concretely,three 2,3-BD stereoisomers were first dehydrogenized to form two AC stereoisomers,(3S)-AC and(3R)-AC.In detail,(2R,3R)-2,3-BD was converted to(3R)-AC,while meso-2,3-BD was converted to(3S)-AC by(2R,3R)-2,3-BDH.(2S,3S)-2,3-BD was converted to(3S)-AC by(2S,3S)-2,3-BDH and quinoprotein ethanol dehydrogenase ExaA.(3S)-AC and(3R)-AC were then converted to acetyl-CoA and acetaldehyde by AoDH ES.Acetaldehyde was finally converted to acetyl-CoA by acetaldehyde dehydrogenase and acetyl-CoA synthetase and subsequently introduced into the glyoxylate cycle for further metabolism.Moreover,encoding genes of(2R,3R)-2,3-BDH,(2S,3S)-2,3-BDH,El and E2 components of AoDH ES belong to a newly identified 2,3-butanediol utilization(bdu)operon.AcoR,located adjacent to the bdu operon,plays a critical role in activating the operon using acetaldehyde,one of the cleavage products of acetoin,as its direct effector.We finally identified that 2,3-BD or AC is not a direct signal molecule enhancing P.aeruginosa virulence.Otherwise 2,3-BD is utilized as a carbon source through the integrated 2,3-BD catabolism pathway,accompanied with higher biomass and quorum-sensing signal molecule production and finally enhances virulence of P.aeruginosa.The stability and balance of the central metabolic pathway has global regulatory effects on the microbial survival and physiological phenotype.L-malic acid is a key intermediate metabolite of several central metabolic pathways such as the TCA cycle and the glyoxylate cycle.In Pseudomonas syringae and Plasmodium,the key enzyme of L-malic acid oxidation,malate:quinone oxidoreductase(MQO),has been shown to play an important role in virulence of pathogens and can serve as a potential drug target.In the third chapter of this thesis,we studied the function of malate:quinone oxidoreductase MqoB in metabolism and virulence of P.aeruginosa PAO1.In P.aeruginosa PAO1,MqoB participates in oxidation of L-malic acid and plays an important role in carbon metabolism through glyoxylate cycle.In addition,mutation of MqoB led to decreased pyocyanin synthesis and reduced infectivity of P.aeruginosa.According to transcriptomics and metabolomics analysis,it was found that loss of MqoB function led to alterations of metabolic state of P.aeruginosa.The expression level of lots of metabolic related genes had changed.According to the analysis of the expression of key quorum sensing proteins,it was found that loss of MqoB function led to the hierarchy quorum sensing network change in P.aeruginosa.The expression of pqs quorum sensing system was significantly inhibited,and the synthesis of its signal molecule PQS was obviously decreased.We speculated that as a key enzyme in the central metabolic pathway,mutation of MqoB led to the metabolic imbalance of P.aeruginosa,which affected the supply of precursor for synthesis of autoinducers of quorum sensing system,leading to abnormal expression of quorum sensing system especially the synthesis of key signal molecule PQS,and finally influenced the virulence and pathogenicity of P.aeruginosa.Meanwhile,ferulenol,the inhibitor of MQO,could effectively inhibit the pyocyanin production of P.aeruginosa,which proved that MqoB has the potential to be as a drug target. |
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