Purification and characterization of oligopeptide permease A in Streptococcus pyogenes
| Autor: | Chao-Jie Yang, 楊朝傑 |
|---|---|
| Rok vydání: | 2006 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 94 Streptococcus pyogenes (group A Streptococcus, GAS) is known to be the causation of diseases in broad range symptoms that include streptococcal toxic shock syndrome, sepsis, and necrotising fasciitis. Oligopeptide permease (OPP) is a membrane-associated complex of five proteins (OppABCDF) belonging to the ABC-transporter family. OppA is an outer membrane-associated lipoprotein. OppB and OppC are transmembrane proteins. OppD and OppF are predicted as inner membrane-associated ATPases. The oligopeptide transport system has been described that involved in nutrient acquisition, sensing of extracellular signaling molecules, influence of adherence, and ecto-ATPase of OppA. However, the biological functions of OPP in S. pyogenes are unclear. The aim of this study was to demonstrate the functions of OppA in S. pyogenes. The opp genes were analyzed by PCR and presented in every strain so far we tested it (including 19 emm types of 41 strains). The full length of recombinant OppA (r-OppA) protein incubated with peptides mixture purified from TSBY broth can cause mobility shift on the native cationic gel. The high concentration of 4 mM KKKKK peptide and 2.5 mM VYIHPE peptide could cause the band of r-OppA disappeared on the native cationic gel. The FITC-labeled peptides of KKKKK and VYIHPE incubated with A20 (wild type) and SW569 (oppA mutant). The fluorescence intensity could be decreased in SW569. These results suggested that OppA could bind to oligopeptide. In order to find the peptide-binding domain of OppA, the six different truncated r-OppA proteins were constructed and purified. We found that the truncated r-OppA proteins could not bind to oligopeptide. This result suggested that the intact structure of OppA might be required for the peptide binding ability. In addition, we found that the r-OppA of S. pyogenes had no ATPase activity. The adhesion ability of OppA was determined by the five independent assays including Western Blot, ligand overlay assay, CELISA, scanning electron microscopy and competitive adhesion assay. None of assay could demonstrate that r-OppA can bind to host cells. However, OppA could enhance the invasion activity of S. pyogenes in the invasion assay and antibody inhibited invasion assay. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
| Externí odkaz: |
|