Investigation On The Key Moieties Of Type Four Pilin And Exopolysaccharides To Mediate Their Specific Recognition And Interaction In Myxococcus Xanthus

As a model strain of myxobacteria,Myxococcus xanthus is a bacterium capable of performing sophisticated cellular behaviors,which are throughout all aspects of its life cycle,e.g.,cellular movement,predatory behavior,programmed cell death,fruiting body development and myxospore differentiation.M.xanthus moves over solid surfaces via two motility systems:adventurous motility for individual cell movement,and social(S)motility for coordinated group movement.S motility plays an essential role in the social lifestyle of M.xanthus,therefore,it is of great theoretical and methodological importance to understand the S motility mechanism.S motility in M.xanthus is mechanistically equivalent to the twitching motilitiy in Pseudomonas aeruginosa,which is driven by type IV pili(TFP).In addition,S motility requires another cell surface structure,exopolysaccharies(EPS).As the molecular engines that power S motility,TFP in M.xanthus function by extending the pili at one cell pole,attaching to the EPS on the surface of substratum or another cell and retracting to pull the cell forward in a 'slip-stick' manner.TFP are composed of several thousand subunits of PilA or pilin,and EPS is the binding target for TFP in S-motility.The specific interaction between PilA and EPS is one of the key questions to really understand S motility mechanism.In the current dissertation,we sought to investigate the key moieties of TFP and EPS to mediate their specific recognition and interaction in M.xanthus.In chapter 2,several assays for in vitro analysis of PilA-EPS interaction were developed based on surface plasmon resonance(SPR)method,which had direct coupling,multi-cycle dynamic capture,single cycle dynamics capture and multi-cycle dynamic competition modes for detection.Using the developed assay on direct coupling mode,the interaction between PilA and chitosan that had different molecular weight was detected,which validated our analysis method.The soluble and insoluble EPS were isolated and purified from the cells of M.xanthus DK1622 following different extraction procedures.Both of these EPS samples showed week interaction on the direct coupling detection mode,which might due to the deficiency of capture method.While the direct coupling is widely used in SPR,it still has nonspecific adsorption and inconformity of protein coupling direction,which lead to protein binding sites of being shielded.Therefore,we improved the detection assay to multi-cycle dynamics capture and single cycle dynamics capture modes.As expected,the positive signal of EPS and PilA interaction response was significantly increased.In chapter 3,we investigated the key moieties in EPS responsible for the interaction with PilA.After detecting the interaction between all monosaccharide residues in EPS with PilA protein,the results showed that only amino monosaccharides,especially glucosamine,clearly interact with PilA,neither unmodified monosaccharides nor N-acetyl modified amino monosaccharides bind PilA protein.These observations were further confirmed by subsequent analysis using isothermal titration calorimetry.Moreover,the SPR completion experiments also suggested that glucosamine interacts with PilA protein and repels the EPS molecules from the EPS-PilA complex.In chapter 4,we analyzed the key sites in PilA prote responsible for the recognition of EPS.According to the bioinformatics analysis,22 amino acid residues in PilA are possible to be involved in EPS-recognition.Three amino acid residues(Y69,W146,W181)were mutated to Alanine residue using site-specific mutagenesis,respectively,and the mutant PilA proteins were purified after heterologous expression.In SPR detection,both W146A and W181A mutant proteins showed the similar biding abilities with the wild type PilA,while the Y69A mutant did not interact with glucosamine,which indicated that Tyr69 might be the key site in PilA protein for the interaction with EPS.In the background of M.xanthus DK10410,a strain deleting pilA gene,three mutant full-length pilA genes(Y69,W146,W181)were inserted in the original position of pilA gene on chromosome,respectively.On the surface of solid agar,S motility of three strains was eliminated.Except DK10410::PilAw181A,DK10410::PilAY69A and DK10410::PilAw146A exhibited the diminished movement in methyl cellulose assay.Western blot showed that all of the three strains did not produce surface pili,while DK10410::PilAY69A and DK10410::PilAw146A had whole-cell pilin.We hypothesized that DK10410::PilAw146A produce PilA while fail to assemble the TFP,DK10410::PilAw181A does not produce any pilin protein,whereas DK10410::PilAY69A produce a small amount of PilA without efficient assembling.