Study On The Mechanisms Of Nucleoside Diphosphate Kinase In Modulating Pseudomonas Aeruginosa Virulence And Pathogenicity

Pseudomonas aeruginosa is a leading opportunistic pathogen that causes nosocomial infections,such as bacteremia,urethritis and pneumonia,in individuals suffering from immune deficiency,severe burns and cystic fibrosis.P.aeruginosa contains a large bacterial genome(6.3 million base pairs,6.3 Mbp),in which more than 5,570 ORFs(Open Reading Frames)have been predicated.At present,the known functional ORFs(nearly 54.2%)are involved in regulating protein secretion,two-component regulatory system,outer membrane formation,molecule metabolism/transportation,antibiotic resistance and bacterial surface molecule generation.The huge and complex genome composition provide s the crucial molecular basis for the regulation of P.aeruginosa virulence and adaptation.Among the diverse ORFs in P.aeruginosa,the protein secretion-associated ORFs are critical in manipulating bacterial virulence and host pathogenicity.P.aeruginosa has five types of protein secretion systems,termed types I-Ⅲ and types V-VI secretion systems.The type I secretion system(T1SS)and type II secretion system(T2SS)secrete bacterial proteins directly into the external environment.The alkaline protease secreted by T1 SS,and elastase,phospholipase C and alkaline phosphatase secreted by T2 SS,are re sponsible for aggravating tissue injury,while the lipase secreted by T2 SS is participated in promoting lipid hydrolysis.The type Ⅲ secretion system(T3SS)participates in regulating cellular events,such as cell apoptosis,proliferation,phagocytosis and inflammation,by injecting T3 SS effector proteins Exo S,Exo T(both possess ADP-ribosyl transferase and Rho GTPase-activating protein activities),Exo Y(adenylate cyclase)or/and Exo U(phospholipase)directly into the cytoplasm of eukaryotic cells.The t ype V secretion system(T5SS)and type VI secretion system(T6SS)promote tissue damage and hemagglutination by the synthesis of esterase,hemagglutinin(T5SS)and phospholipase D(T6SS).In addition,P.aeruginosa aggravates host pathogenicity through the production of exoproduts including pyocyanin,pyoverdin,alginate and the regulation of motilities(e.g.swarming)and biofilm formation.Even though the mechanisms underlying the virulence and pathogenicity of P.aeruginosa have been reported extensively,there still remains a great challenge to do the in-depth investigations owing to the fact that the virulence regulatory mechanisms of P.aeruginosa are sophisticated and diverse.Nucleoside diphosphate kinase(Ndk)is a crucial housekeeping enzyme in reg ulating nucleotide metabolism in both prokaryotes and eukaryotes.Even though its roles in promoting alginate production in P.aeruginosa and regulating host phagocytosis and inflammatory response in certain bacteria have been reported,the global effects and mechanisms of Ndk in tuning virulence and host pathogenicity remain elusive.In the previous investigation,we found that the expression level of ndk gene is significantly inhibited,while those levels of genes encoding T3 SS are upregulated in the mous e models of acute lung infection,suggesting that Ndk is a host-responsive regulator that might manipulate P.aeruginosa virulence and pathogenicity during infection.Therefore,to investigate the roles and underlying mechanisms of Ndk during acute lung infection caused by P.aeruginosa,the corresponding mutant strains have been utilized to treat the BABL/c mice intranasally and the adenocarcinomic human alveolar basal epithelial A549 cells.In addition,to observe the global effects of Ndk on gene expression,especially the virulence-associated genes,we did a transcriptomic analysis using the PAO1 and PAO1 ndk gene null-mutant strain(△ ndk).Then,the methods including RT-q PCR,Western blot,and phenotype analyses,have been utilized to confirm the results obtained from transcriptome sequencing.To further investigate the possible roles and mechanisms of Ndk in modulating quorum sensing(QS)system,exoproducts generation and T3 SS activation,the corresponding gene mutant and complementary strains have been constructed,and their related phenotypes have been detected.The research schemes and major results obtained in this study have been outlined as follows:The roles of Ndk in regulating host pathogenicity of P.aeruginosa.The mice were intranasally infected with the P.aeruginosa mutant strains constructed by the Red homologous recombination method,and their mortality,lung tissue edema,injury and inflammation were observed.In addition,the A549 cells were infected with the mutant strains,and cell viability,apoptosis,membrane permeability and cellular translocation of T3 SS effector proteins were analyzed by the methods,including CCK8,flow cytometry,immunofluorescence staining,and Western blot.Results showed that deprivation of ndk gene aggravates T3SS-mediated mouse mortality,lung injury and inflammation,and enhances cytotoxicity,cell apoptosis or cell membrane permeability by increasing T3 SS protein expression or T3 SS effector protein cellular translocation.The global effects of Ndk on gene expression of P.aeruginosa.The global gene expression levels were analyzed by transcriptome sequencing using PAO1 and △ ndk strains.The gene expression differences were evaluated by the bioinformatic analyses,including gene ontology(GO)annotation and kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment.The results showed that null-mutation of P.aeruginosa ndk gene plays extensively roles in regulating the expression of genes related to bacterial substance metabolism,molecule transportation and the syntheses of virulence determinants including exoproducts and T3 SS.The effects of Ndk on regulating virulence-associated phenotypes of P.aeruginosa.The virulence-associated determinants of PAO1,△ ndk,and △ ndk+(ndk gene complementary strain)strains,including toxic factors and proteases,T3 SS,motilities and biofilm formation,were analyzed by RT-q PCR,Western blot and phenotype analyses.Results showed that the null-mutation of ndk gene inhibits the expression of genes encoding elastase,alkaline phosphatase and phenazine,suppresses the production of exoproducts including elastase,pyocyanin and pyoverdine,and impairs the swarming motility and biofilm formation,whereas the deprivation of ndk gene increases the levels of genes and proteins of T3 SS.The role of Ndk in regulating P.aeruginosa QS system.The las,rhl and pqs QS system-related expression of gene encoding synthetases/regulators and productions of signal molecules of the PAO1,△ ndk,and △ ndk+ strains were analyzed by RT-q PCR and signal molecules reporter strain method or high performance liquid chromatography(HPLC).Results demonstrated that ndk gene deprivation suppressed the expression levels of genes encoding the las,rhl and pqs synthetases(e.g.las I,rhl I and pqs A genes),regulators(e.g.las R,rhl R and pqs R genes),and the productions of 3-oxo-C12-HSL,C4-HSL,and PQS signal molecules.Ndk modulates virulence-associated phenotypes of P.aeruginosa through the regulation of QS system.To investigate the role of QS system in Ndk-mediated regulation of P.aeruginosa virulence,the exogenous 3-oxo-C12-HSL,C4-HSL or PQS signal molecules was added to the culture supernatant of the △ ndk strain,and the exoproducts and T3 SS levels in the △ ndk strain were analyzed.Results revealed that the supplement of C4-HSL enhances the production of elastase and pyocyanin in the △ ndk strain,whereas the addition of C4-HSL or PQS decreases the gene and protein levels of T3 SS in the △ ndk strain.