Biological Function And Pathogenic Mechanism Of Single-PilZ Domain C-di-GMP Receptor In Pseudomonas Aeruginosa

The pathogenic bacterium Pseudomonas aeruginosa is notorious for causing acute and chronic infections in humans,animals and plants.The intracellular signaling molecule cyclic di-GMP(c-di-GMP)regulates many processes in bacteria.In recent years,it was reported that c-di-GMP is essential to pathogenesis in Pseudomonas aeruginosa.There are a large number of c-di-GMP receptor proteins were found to be able to regulate pathogenesis,Pil Z domain protein is one of the biggest family of c-di-GMP receptor proteins.However,the understanding of Pil Z proteins in pseudomonas aeruginosa is still very limited,and there is no detailed study about the bacterial pathogenic mechanism regulated by Pil Z proteins.In this study,we choose single-Pil Z domain proteins as the research object,which may help us further reveal the pathogenesis of pseudomonas aeruginosa regulated by the c-di-GMP signaling pathways.We first constructed the four in-frame deletion mutants ?PA0012,?PA2799(hap Z),?PA4324,?PA4608(map Z),and screend their phenotypes.We found that all mutants showed various changed virulence phenotypes compared with the wild type strain PAO1,such as swarming motility,lettuce infection ability,C.elegans killing ability,biofilm formation,et al.And the results show that map Z mutation can affect most of the phenotypes.To further explore the function of Pil Z protein on transcriptional level,we performed RNAseq experiment by using the four single-Pil Z domain mutants.Our RNA-seq data shows that compared with the wildtype strain PAO1,the defection of single-Pil Z protein is likely to cause a large number of genes' differential expression(?PA0012 and ?PA4324),or a small number of genes' differential expression(?map Z),which can affect the cellular physiological process and regulate the pathogenesis of bacteria.We finally decided to do further research of MapZ,which influences the most phenotypes and the least genes.The ability to infect host by P.aeruginosa is dependent on a complex cellular signalling network,which includes a large number of chemosensory signalling pathways that rely on the methyl-accepting chemotaxis proteins(MCPs).We previously found that the second messenger c-di-GMPbinding adaptor MapZ modulates the methylation of an amino acid-detecting MCP by directly interacting with a chemotaxis methyltransferase Che R1.The current study further expands our understanding of the role of MapZ in regulating chemosensory pathways by demonstrating that MapZ suppresses the methylation of multiple MCPs in P.aeruginosa PAO1.The MCPs under the control of MapZ include five MCPs(Aer,Ctp H,Cpt M,Pct A and Pct B)for detecting oxygen/energy,inorganic phosphate,malate and amino acids,and three MCPs(PA1251,PA1608 and PA2867)for detecting unknown chemoattractant or chemorepellent.We used MapZ overexpression strain PAO1/p MapZ and c-di-GMP binding site mutant strain map Z?R13A to performe the chemotaxis and infection assays.Chemotaxis assays showed that overexpression of MapZ hampered the taxis of P.aeruginosa towards chemoattractants and scratch-wounded human cells while the point mutant strain map Z?R13A didn't,so we focus our attention to MapZ overexpression.Mouse infection experiments demonstrated that a dysfunction in MapZ regulation had a profound negative impact on the dissemination of P.aeruginosa and resulted in attenuated bacterial virulence.To further confirm our hypothesis that the phenotypes of PAO1/p MapZ are caused by MapZ overexpression,we first measured the cellular c-di-GMP concentration of MapZ over expression strain and wild type strain by using the HPLC method,the result showed that the cellular c-di-GMP level between the two strains has no significant difference,which indicated that MapZ overexpression influence the pathogenesis of P.aeruginosa doesn't through changing the concentration of intracellular c-di-GMP.And then,we performed RNA-seq experiments,and the data showed that MapZ overexpression didn't affect other genes' expression level.The results imply that by controlling the methylation of various MCPs via the adaptor protein MapZ,c-di-GMP has a profound influence on chemotactic responses and bacterial pathogenesis.Collectively,the experimental work described in this thesis bring us one step closer towards understanding the functional diversity and importance of the single-domain Pil Z proteins in the model organism Pseudomonas aeruginosa,which can provid us a new idea to research the pathogenesis of other pseudomonas.