Metabolic Mechanism And Toxic Effects Of Glycerol-3-phosphate In Pseudomonas Aeruginosa PAO1

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen,which can cause a variety of life-threatening acute or chronic infections.It is the main cause of further deterioration in patients with cystic fibrosis.The extensive repertoire of virulence factors and the intrinsic resistance to antibiotics of P.aeruginosa present a huge challenge in treating infection induced by the strain.P.aeruginosa can use glycerol produced by the degradation of phosphatidylcholine(PC)as the energy source for growth.Glycerol metabolism may play an important role in the adaptation of P.aeruginosa to the pulmonary environment of CF patients.Glycerol-3-phosphate(G3P),the product of glycerol phosphorylation,can be dehydrogenated to produce dihydroxyacetone phosphate(DHAP)to enter the central metabolic pathway.It is also the key precursor for cell membrane and storage lipids synthesis.However,the excess phosphorylated metabolites are usually toxic and it has been reported that the excessive accumulation of G3P has negative effects on the growth and physiological phenotypes of both prokaryotes and eukaryotes.In recent years,more and more studies have shown that physiological metabolism is also closely related to the sensitivity of bacteria to antibiotics and the production of virulence factors.Finding drug targets in bacterial metabolic pathways to develop narrow spectrum antibiotics has become a new hotspot in the field of prevention and therapy of drug-resistant bacteria.In this thesis,the metabolic mechanism and toxic effects of G3P in P.aeruginosa were studied to provide theoretical basis and data support in the development of narrow spectrum antibiotics targeting G3P metabolism for against P.aeruginosa infections.In chapter 2,different mutants of P.aeruginosa type strain PAO1 as a model,with deletions of genes associated with glycerol metabolism were constructed and the anabolism,catabolism,and transport mechanisms of G3P in P.aeruginosa PAO1 were revealed.G3P can be derived from the phosphorylation of glycerol and the reduction of DHAP,which are catalyzed by glycerol kinase GlpK and NAD(P)+-dependent G3P dehydrogenase GpsA,respectively.G3P catabolism is catalyzed by FAD-dependent G3P dehydrogenase GlpD.G3P can be imported into cells via the G3P transporter GlpT.In addition,GlpR,a G3P operon repressor,uses G3P instead of glycerol as the effector.GlpR represses the expression of glycerol and G3P metabolism-related genes in P.aeruginosa PAO1 by binding to glpFK and glpD promoter regions.In chapter 3,the physiological phenotypes of the glycerol metabolic genes knockout strains of P.aeruginosa PAO1 were analyzed.It was found that the phenotype of glpD mutant strain changed significantly in rich medium.The respiratory tract of CF patients is a glycerol-rich environment.Further research found that P.aeruginosa PAO1 could co-utilize glycerol in the presence of the favourable carbon source,succinic acid or glucose,and the glpD knockout strain had intracellular and extracellular accumulation of G3P during growth in medium containing succinic acid/glucose and glycerol,and its growth was severely inhibited.The accumulation of G3P led to the decrease of pyocyanin and twitching and swimming motility,as well as the reduction of the resistance to kanamycin and oxidative stress of the glpD mutant strain.GlpD has the potential to be a therapeutic target against P.aeruginosa infection.In chapter 4,three putative G3P phosphatases in P.aeruginosa PAO1 including PA0562,PA3172 and PA2067 were identified by sequence alignment.Subsequent research found that overexpression of PA0562 and PA3172 contributed to alleviate the G3P stress,while their deletion strengthened the G3P stress.PA0562 and PA3172 were expressed,purified,and characterized.The apparent Km values of PA0562 and PA3172 were 7.114±1.305 mM and 5.634 ± 0.208 mM,while the corresponding Vmax were estimated to be 47.410 ± 4.102 U·mg-1 and 18.060 ± 0.305 U·mg-1,respectively.Analysis of the substrate spectra of PA0562 and PA3172 revealed that they both had relaxed specificity to G3P and also had catalytic activity for other phosphorylated metabolites such as glucose-6-phosphate,ribose-5-phosphate and fructose-6-phosphate.In summary,this study revealed the metabolism and regulatory mechanism of G3P in P.aeruginosa PAO1 and found that G3P accumulation caused inhibition of growth,decreases of various virulence factors,and reduction of the tolerance to oxidative or antibiotic stress.Two G3P phosphatases with G3P as the substrate were identified and proved to play a certain role in alleviating the pressure of G3P.This study has certain guiding significance for revealing the G3P metabolic mechanism in microorganisms and for the prevention and treatment of infection caused by P.aeruginosa.