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

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.

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.

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.

T4 Lysozyme PDB ID：2LZM 164 aa catalytic triad

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

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)

putative HP0339 gene of H. pylori strain ATCC 26695

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

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.

Randomly amplified polymorphobic DNA (RAPD)

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

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.

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

Lysozyme-like gene expression in vivo (RT-PCR) HPTS142, HPTS65 and HPTS36 are lysozyme-positive strains.

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.

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

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

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

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

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

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

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

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

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

Phage T4 lysozyme HP0339 product lys product 8 insertion：LKENHRAL

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.

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.

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.

Future Work Further functional characterization, such as in vivo localization and crystallographic structure analyses, will be performed.

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lys T4 lys