Speaker: Hsin-Yi Chen 2002/12/10

1. Helicobacter pylori Expresses an Autolytic Enzyme: Gene Identification,Cloning, and Theoretical Protein Structure Eleonora Marsich,1 Pierfrancesco Zuccato,1 Sonia Rizzi,1 Amedeo Vetere,1*Enrico Tonin,2 and Sergio Paoletti1 Journal of Bacteriology, Nov.2002,vol 184 : 6270~6279 Speaker: Hsin-Yi Chen 2002/12/10

2. Introduction • In 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. Autolysins • Many bacteria contain one or more cell wall lytic enzymes. These enzymes are called autolysins. • Diverse：N-acetylglucosaminidases N-acetylmuramidases transglycosylases etc. • Conjectural functions： involved in cell wall turnover; cell separation, competence for genetic transformation, formation of flagella, and sporulation.

4. T4 Lysozyme • An 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.

5. T4 Lysozyme PDB ID：2LZM 164 aa catalytic triad

6. Purpose • To investigate a gene whose predicted product exhibits a high level of homology with T4 lysozyme. • To characterize the gene been found.

7. Data bank searches • Similarity search genomic databases for phage T4 lysozyme 2phage lysozymes 2hypothetical proteins 4 homologues One is putative HP0339gene of H. pylori strain ATCC 26695 (highest homology)

8. putative HP0339 gene of H. pylori strain ATCC 26695

9. CLUSTALW alignment Asterisks -- Matching residues Colons -- Conserved Dots -- semiconserved Highlighted --catalytic triad 27% identical; 52% similarity

10. H. pylori strain screening • H. pylori is characterizedby 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.

11. Randomly amplified polymorphobic DNA (RAPD)

12. RAPD & PCR Results Clinical H. pylori strains from 38 patients 30 RAPD types 21HP0339 positive strains 9HP0339 negative trains

13. DNA sequence comparison • Comparison clinically isolated strains and HP0339 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 and HP0339 T4 lysozyme gene sequence Database searches HP0339 PCR cloning of HP0339 from clinical strains Lys 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. pylori protein extracts ( Zymogram gel analysis ) Protein extracts electrophoresis in SDS-PAGE gels containing M. lysodeikticus cells as a substrate soaked 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 strains HPTS12 and HPTS90 Lanes 3, 4, 5, and 6--crude protein extracts from lysozyme-positive strains HPTS142, HPTS65,HPTS61,and HPTS36 Lane 7--bovine serum albumin and ovalbumin

18. Cloning, over-expression, and purification of the Lys protein HTPS142 thrombin cleavage site EcoRI XhoI glutathioneS- transferase pGEX4T-1 E. coli BL21

19. SDS-PAGE analysis of GST-Lys protein expression Lane 1-- Marker Lane 2--crude extract from nontransformed cells Lane 3--crude extract cells containing nonrecombinant pGEX Lane 4--crude extract from cells containing the pGEX-lys Lane 5--solubilized pellet from nontransformed cells Lane 6--solubilized pellet from cells transformed with the nonrecombinant pGEX Lane 7--solubilized inclusion bodies from cells containing the pGEX-lys

20. GST-Lys protein purified by affinity chromatography Lane 1--markers Lane 2--total proteins before affinity purification Lane 3--total proteins after loading on affinity resin Lane 4--eluate from glutathione- Sepharose 4B resin Lane 5--flowthrough following thrombin digestion of GST- Lys fusion protein bound to glutathione-Sepharose 4B Lane 6--thrombin digest of eluate from glutathione-Sepharose 4B resin

21. Zymogram analysis of hydrolytic activity of lys gene against gram-positive and gram-negative bacteria Cell extract Lys Lys Lys control M. lysodeikticus cells (G+) E. coli murein sacculi (G-) H. pylori sacculi

22. Hydrolytic activity of purified recombinant HP0339 protein HP0339 lys HP0339 control H. pylori murein sacculi M. lysodeikticus cells

23. Molecular modeling HP0339 and lys products • 3D-pssm server： Classified as a member of the phage T4 lysozyme family (Proteins family d119l) • Expectation value of the match is >95%. • Two domains ：one α, one β domain • Model ：Swiss Model, Ramachandran plot check • Refine ：loop library of Swiss-Pdb Viewer

24. Tertiary structures of HP0339 one α domain：residues 38 to 111 one β domain：residues 3 to 27

25. Tertiary structures of lys one α domain：residues 56 to 119 one β domain：residues 3 to 27 insert：residues 46 to 54

26. Phage T4 lysozyme HP0339 product lys product 8 insertion：LKENHRAL

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. Discussion • The 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. Discussion • Structural 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 Work • Further functional characterization, such as in vivo localization and crystallographic structure analyses, will be performed.

31. ~~ THE END ~~ Thank You For your Attention！

32. lys T4 lys