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. One is putative HP0339 gene of H. pylori strain ATCC 26695
Data bank searches
Similarity search genomic databases for phage T4 lysozyme
2 phage lysozymes
2 hypothetical proteins
4 homologues
One is putative HP0339 gene 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 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.

11. Randomly amplified polymorphobic DNA (RAPD)

12. Clinical H. pylori strains from 38 patients
RAPD & PCR Results
Clinical H. pylori strains from 38 patients
30 RAPD types
21 HP0339 positive strains
9 HP0339 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, a HP0339 gene carrying 24 extra nucleotides
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
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. M. lysodeikticus cells (G+)
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
lys
HP0339
control
HP0339
H. pylori murein sacculi
M. 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 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. Thank You For your Attention！
~~ THE END ~~
Thank You For your Attention！

32. lys
T4 lys