Research On Molecular Mechanism Of Specific Interactions Between Type Ivpili And Exopolysaccharides In Myxococcus Xanthus

Myxococcus xanthus is a Gram-negative soil bacterium capable of sophisticated social behaviors, i.e. fruiting body formation and specific predative behavior. A crucialfeature of these behavior is the ability to move in the directionof cell’s long axis on solid surfaces by a flagella-independentmechanism called gliding.M xanthus gliding motility isregulated by the A (adventurous) and the S (social) motility systems, which appear to operate independentlywhile in acoordinated fashion. Unlike the A motility system that allows movement of isolated cellsand does not require cell-cell contact, the S motility system istypically employed for coordinated group movement of cells in M. xanthus, which is mechanistically equivalent to the twitching motility in Pseudomonas aeruginosa and Neisseria gonorrhoeae.In M. xanthus, mutations that abolish S motility normally affect type IV pili(TFP) biogenesis, the exopolysaccharide (EPS) component ofthe extracellular matrix (ECM) or the lipopolysaccharideO-antigen. Further functional studies have shownthat S motility is powered by cycles of TFP extension andretraction and depend on the interaction of TFP with EPS.TFP, composed of thousands of subunits ofprotein called type IV pilin (or PilA), are the molecularengines which enable S motility.They function by extending the pili at one of the cellpoles, which attach to the surfaces of the substratum oranother cell and then retract to pull the cell forward. As the binding targetfor TFP in S-motility,EPS also forms thescaffold of M. xanthus biofilms and fruiting bodies, and mediates cell-cell cohesion and cell-substratum adhesion. Although specific interactions between TFP and EPS havebeen proposed, and some direct observations of the interactionsunder native conditions have been made, there have as yet been no quantitativeanalysis of these processes and the key moieties, both in TFP and EPS, involved in the interaction remain elusive.In this study, we explored the specific interaction between TFP and EPS in M. xanthus using some real-time and label-free detection techiques, and sought to understand the molecular mechanism of how PilA protein recognize and interact with specific sacchrides, e.g. EPS.In Chapter2, we describe the preparation of M. xanthus PilA protein and EPS for the further analysis. As fulllength PilA was extremely difficult to overexpressreproducibly in vitro due to its poor solubility, based on previous studies and our bioinformatics analysis, we constructed an overexpression plasmidcarrying a truncated form of M. xanthus PilA, which contains only the C-terminal domain(amino acids32-208of the mature pilin). In consistent with previous observations, the truncated PilAprotein is sufficient for EPS binding in vitro. Meanwhile, the EPS was isolated from M. xanthus cells and purified to remove any nuleic acids contaiminations. Glycosyl composition analysis of isolated EPS was performed by combined gas chromatography/mass spectrometry (GC/MS) of the per-O-trimethylsilyl (TMS) derivatives of the monosaccharide methyl glycosides produced from the sample by acidic methanolysis. The results revealed the presence of heterogeneous materials containing arabinose, rhamnose, fucose, xylose, mannose, galactose, glucose, N-acetyl galactosamine, N-acetyl glucosamine and N-acetyl mannosamine in different ratios.In Chapter3, the label-free biophysical techiques, i.e. surface plasmon resonance (SPR) and isothermal titration calorimetry(ITC), were employed to investigate the kinetic and thermodynamic characterizes of interaction between the PilA protein and some sacchrides that were revealed the existence in M. xanthus EPS by GC/MS analysis.The experiment results consistently showed that among all the glycosyl residues in EPS, glucosamine exhibited specific interaction with PilA protein, which suggested that TFP might recognize glucosamine as the binding site. Moreover,chitosan, a soluble polymer formed by N-acetyl-gulcosamine and glucosamine, also showed clear interaction with PilA, and the binding ability was decreased according to the increase ofchitosan’s molecular weight. In a competition binding assay, glucosamine could inhibit the interaction between PilA protein andchitosan, which indicated that both glucosamine andchitosan interact with the same site on PilA.After established the potential moieties in EPS responsible for interaction with PilA protein, in Chapter4, we tried to nail down the key sites in PilA dominating the interaction. The M. xanthus PilA protein was analyzed through several different bioinformatical approaches, and22residues were predicted as the candidates for interacting sites with sachcrides. Among them, tryptophan residues at position146and181were most promising, which were revealed according to active sites on the known chitosam degradting enzymes that should share some similarities with PilA considering the glusosamine was also shown as the recognition residue in EPS by PilA. The site-directed mutagenesis was performed to obtain the mutant PilA proteins carring analanine to tryptophan substitution at position146and181, respectively. However, both mutant PilA protiens resembled titration behavior as that of wild-type PilA in ITC experiment, which suggest that tryptophan residues at position146and181might not be crucial in PilA-EPS interaction. We are now in the process to test the other20predicted residues.