| Summary: | 碩士 === 國立成功大學 === 生物化學研究所 === 96 === Streptococcus pyogenes, also known as group A streptococci (GAS), is a member of Gram positive bacteria and a common human pathogen in the environment. GAS causes a wide variety of diseases, including pharyngitis, impetigo, scarlet fever, cellulitis, toxic shock syndrome, necrotizing fasciitis, rheumatic fever, and acute glomerulonephritis. In this study I determined 3D structures of two virulence factors from GAS, including streptopain (SPE B) and SPy0985, by NMR spectroscopy. SPE B is a cysteine protease and can digest many kinds of substrates such as host proteins, proteoglycans, to help GAS to release surface virulent proteins like C5a peptidase and M protein, and then cause severe injury in the infected host. It is known that phage infection can enhance bacterial virulence, such as bacterial adhesion, colonization, invasion, resistance to immune defenses, exotoxin production, sensitivity to antibiotics, and transmissibility among humans. SPy0985 is a phage-associated hypothetical protein and contains 88 amino acid residues with a superantigen signature sequence at the positions of 49-75. In contrast to X-ray structure of 40-kDa zymogen SPE B (zSPE B), NMR structure of 28-kDa mature SPE B (mSPE B) found the interactions between the residues in the catalytic loop (V189 and A196) and the residues in the C-terminal loop (A231 and A238), which is unobserved in X-ray structure, and H195, the catalytic residue, is located at catalytically competent position. The structural differences are due to the insertion of N89p from the prodomain of zSPE B X-ray structure displacing H195 from the catalytically competent position. This was observed from NMR spectra that H�� of H195 interacted with the HN of G213, H�� of W214, and H�� of F197. The resulting 3D structures of mSPE B were deposited to Protein Data Bank (PDB) with the accession code 2JTC. I also have successfully expressed SPy0985 in E. coli and purified to homogeneity with a yield of 30 mg/L. The experimental molecular weight of recombinant Spy0985 was 9782.3 Da with a deviation of <1 compared with the calculated value. Molecular weight determination by gel filtration chromatography showed that Spy0985 is a tetramer. The results of peripheral blood mononuclear cell proliferation assay suggested that Spy0985 may act as a superantigen. Structural and functional analysis also showed that Spy0985 required calcium ion for its stability and activity. The 1H, 15N, and 13C resonance assignments of Spy0985 were obtained by analyzing triple resonance spectra. NMR analysis showed that Spy0985 consisted of two ��-helices at C terminal region, and a hydrophobic core packed by Y17, Y23 and Y36 at N terminal region. These studies will extend our understanding of the molecular basis of SPE B and SPy0985 in GAS infection.
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