Presentation on theme: "Helicobacter pylori Expresses an Autolytic Enzyme: Gene Identification, Cloning, and Theoretical Protein Structure Eleonora Marsich,1 Pierfrancesco Zuccato,1."— Presentation transcript:
1 Helicobacter pylori Expresses an Autolytic Enzyme: Gene Identification, Cloning, and Theoretical Protein StructureEleonora Marsich,1 Pierfrancesco Zuccato,1 Sonia Rizzi,1 Amedeo Vetere,1*Enrico Tonin,2 and Sergio Paoletti1Journal of Bacteriology, Nov.2002,vol 184 : 6270~6279Speaker: Hsin-Yi Chen2002/12/10
2 IntroductionIn the past few years, we have successfully complete numerous genome sequencing projects.Combining database searches with molecular biology techniques, it is possible to provide a complete description of the role of a gene.
3 AutolysinsMany bacteria contain one or more cell wall lytic enzymes. These enzymes are called autolysins.Diverse：N-acetylglucosaminidasesN-acetylmuramidases transglycosylases etc.Conjectural functions：involved in cell wall turnover; cell separation, competence for genetic transformation, formation of flagella, and sporulation.
4 T4 LysozymeAn enzyme found in T4 phage, with splits the glycosidic bond between certain residues in mucopeptides of bacterial cell walls, resulting in bacteriolysis.A commonβ(1-4)-N-acetylmuramidase.
6 PurposeTo investigate a gene whose predicted product exhibits a high level of homology with T4 lysozyme.To characterize the gene been found.
7 One is putative HP0339 gene of H. pylori strain ATCC 26695 Data bank searchesSimilarity search genomic databases for phage T4 lysozyme2 phage lysozymes2 hypothetical proteins4 homologuesOne is putative HP0339 gene of H. pylori strain ATCC 26695(highest homology)
8 putative HP0339 gene of H. pylori strain ATCC 26695
10 H. pylori strain screening H. pylori is characterized by the high level of genetic variation.Examined bacteria by endoscopy from 38 patients with H. pylori infections and use RAPD analysis for genetic typing of the clinical isolates.
12 Clinical H. pylori strains from 38 patients RAPD & PCR ResultsClinical H. pylori strains from 38 patients30 RAPD types21 HP0339 positive strains9 HP0339 negative trains
13 DNA sequence comparison Comparison clinically isolated strains andHP0339 gene showed 96% identity in the complete sequence, except clinical strains contained an insertion of 24 consecutive nucleotides.We designated this variant form of the HP0339 gene lys.
14 Lys gene, a HP0339 gene carrying 24 extra nucleotides Lys gene and HP0339T4 lysozyme gene sequenceDatabase searchesHP0339PCR cloning of HP0339 from clinical strainsLys gene, a HP0339 gene carrying 24 extra nucleotides
15 Lysozyme-like gene expression in vivo (RT-PCR) HPTS142, HPTS65 and HPTS36 are lysozyme-positive strains.
16 Cell wall lytic activity in H Cell wall lytic activity in H. pylori protein extracts ( Zymogram gel analysis )Protein extracts electrophoresis in SDS-PAGE gels containing M. lysodeikticus cells as a substratesoaked in Milli-Q water then incubated in renaturation buffer with gentle agitation.Staining with 0.1% methylene blue in 0.01% KOH.
17 Zymogram gel analysis 13.5 kDa Lanes 1 and 2--crude protein extracts from lysozyme-negative strainsHPTS12 and HPTS90Lanes 3, 4, 5, and 6--crude protein extracts from lysozyme-positive strains HPTS142, HPTS65,HPTS61,and HPTS36Lane 7--bovine serum albumin and ovalbumin
18 Cloning, over-expression, and purification of the Lys protein HTPS142thrombin cleavage siteEcoRIXhoIglutathioneS-transferasepGEX4T-1E. coli BL21
19 SDS-PAGE analysis of GST-Lys protein expression Lane 1-- MarkerLane 2--crude extract from nontransformed cellsLane 3--crude extract cells containing nonrecombinant pGEXLane 4--crude extract from cells containing the pGEX-lysLane 5--solubilized pellet from nontransformed cellsLane 6--solubilized pellet from cells transformed with the nonrecombinant pGEXLane 7--solubilized inclusion bodies from cells containing the pGEX-lys
20 GST-Lys protein purified by affinity chromatography Lane 1--markersLane 2--total proteins before affinity purificationLane 3--total proteins after loading on affinity resinLane 4--eluate from glutathione-Sepharose 4B resinLane 5--flowthrough following thrombin digestion of GST- Lys fusion protein bound to glutathione-Sepharose 4BLane 6--thrombin digest of eluate from glutathione-Sepharose 4B resin
21 M. lysodeikticus cells (G+) Zymogram analysis of hydrolytic activity of lys gene against gram-positive and gram-negative bacteriaCell extractLysLysLyscontrolM. lysodeikticus cells (G+)E. coli murein sacculi (G-)H. pylori sacculi
22 Hydrolytic activity of purified recombinant HP0339 protein lysHP0339controlHP0339H. pylori murein sacculiM. lysodeikticus cells
23 Molecular modeling HP0339 and lys products 3D-pssm server：Classified as a member of the phage T lysozyme family (Proteins family d119l)Expectation value of the match is >95%.Two domains ：one α, one β domainModel ：Swiss Model, Ramachandran plot checkRefine ：loop library of Swiss-Pdb Viewer
24 Tertiary structures of HP0339 one α domain：residues 38 to 111one β domain：residues 3 to 27
25 Tertiary structures of lys one α domain：residues 56 to 119one β domain：residues 3 to 27insert：residues 46 to 54
27 Conclusion HP0339 gene product is an enzyme with lysozyme activity. Lys gene is 96% identical to HP0339, except the lys gene contained an insertion of 24 extra nucleotides.There’s no apparent activity difference between lys product and HP0339 product.
28 DiscussionThe presence of an autolytic lysozyme in H. pylori is consistent with the hypothesis concerning altruistic behavior.The activity of the Lys lysozyme could be modulated by interactions with a larger complex of other enzymes.
29 DiscussionStructural modifications of the cell wall should play a role in H. pylori pathogenesis if cell wall fragments are released.One possible effect due to the insertion could be modulation of the domain motions and, of the shape of the active cleft.
30 Future WorkFurther functional characterization, such as in vivo localization and crystallographic structure analyses, will be performed.
31 Thank You For your Attention！ ~~ THE END ~~Thank You For your Attention！