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Characterization of Helicobacter pylori dapE and construction of a conditionally lethal dapE mutant.

ABSTRACT: Helicobacter pylori colonizes the human gastric mucosa and causes gastritis, ulceration, or gastric cancer. A previously uncharacterized region of the H. pylori genome was identified and sequenced. This region includes a putative operon containing three open reading frames termed gidA (1,866 bp), dapE (1,167 bp), and orf2 (753 bp); the gidA and dapE products are highly homologous to other bacterial proteins. In E. coli, dapE encodes N-succinyl-L-diaminopimelic acid desuccinylase, which catalyzes the hydrolysis of N-succinyl-L-diaminopimelic acid to L-diaminopimelic acid (L-DAP) and succinate. When wild-type H. pylori strains were transformed to select for dapE mutagenesis, mutants were present when plates were supplemented with DAP but not with lysine; orf2 mutants were selected without DAP supplementation. Consistent with the finding that GidA is essential in Escherichia coli, we were unable to obtain a gidA mutant in H. pylori despite evidence that insertional mutagenesis had occurred. The positions of gidA, dapE, and orf2 suggest that they form an operon, which was supported by slot blot RNA hybridization and reverse transcriptase PCR studies. The data imply that the H. pylori dapE mutant may be useful as a conditionally lethal vaccine.

SUBMITTER: Karita M

PROVIDER: S-EPMC175598 | biostudies-other | 1997 Oct

REPOSITORIES: biostudies-other

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Characterization of Helicobacter pylori dapE and construction of a conditionally lethal dapE mutant.

Karita M M Etterbeek M L ML Forsyth M H MH Tummuru M K MK Blaser M J MJ

Infection and immunity 19971001 10

Helicobacter pylori colonizes the human gastric mucosa and causes gastritis, ulceration, or gastric cancer. A previously uncharacterized region of the H. pylori genome was identified and sequenced. This region includes a putative operon containing three open reading frames termed gidA (1,866 bp), dapE (1,167 bp), and orf2 (753 bp); the gidA and dapE products are highly homologous to other bacterial proteins. In E. coli, dapE encodes N-succinyl-L-diaminopimelic acid desuccinylase, which catalyzes ...[more]

PMID: 9317022

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Project description:The N-terminal sequence of a protein, originally described as an adhesin of Helicobacter pylori, was used in an oligonucleotide-based screening procedure of an H. pylori plasmid library in Escherichia coli. Five independent plasmid clones were isolated, all mapping to the same chromosomal region and encoding the H. pylori catalase. The gene, designated katA, comprises 1,518 nucleotides and encodes a putative protein of 505 amino acids with a predicted Mr of 58,599. A second open reading frame, orf2, encoding a putative 32,715-Da protein of unknown function, follows katA. The transcriptional start site of katA mRNA was determined, but no typical consensus promoter sequence was present. A potential binding site for the Fur protein is located upstream of katA. When introduced into the catalase-deficient E. coli double-mutant UM255, the cloned gene readily complemented E. coli for catalase activity. H. pylori KatA is highly homologous to catalases in both prokaryotes and eukaryotes, with the highest homology being shown to Bordetella pertussis (64.9%), Bacteroides fragilis (59.8%), and Haemophilus influenzae (57.9%) catalases. Transposon insertion mutants were generated in three independent H. pylori strains by TnMax5-mediated transposon shuttle mutagenesis. In contrast to the wild-type strains, no significant catalase-specific enzymatic activity could be detected in the mutant strains, consistent with the fact that no additional katA-homologous gene copies were found in the H. pylori chromosome. No significant difference between wild-type and mutant strains for binding to epithelial cells was apparent, suggesting that KatA is not involved in H. pylori adhesion. The cloning and genetic characterization of katA are essential steps for further investigation of the role of catalase in the defense of H. pylori against oxygen-dependent killing mechanisms by polymorphonuclear granulocytes, a process not well understood for this chronically persisting pathogen.

Project description:AIM:To construct a recombinant vector which can express outer membrane protein (OMP) with M(r)18,000 and heat shock protein A (HspA) from Helicobacter pylori (H. pylori) in E. coli BL21, and to exploit the possibility for obtaining the vaccine conferring protection from H. pylori infection. METHODS:The target gene of HspA was amplified from H. pylori chromosome by PCR, and then inserted into the prokaryotic expression vector pET32a (+) by restrictive endonuclease enzyme kpn I, BamH I simultaneously. The recombinant vector was used to sequence, and then together with pET32a (+)/Omp(18), digested by restrictive endonuclease enzyme Hind III and BamH I simultaneously. pET32a(+)/HspA and Omp(18) were recovered from 1 % agarose gel by gel kit, and ligated with T(4) ligase by BamH I digested viscidity end. The recombinant plasmid of pET32a(+)/HspA/Omp(18) was transformed and expressed in E. coli BL21 (DE3) under induction of IPTG. After purification, its antigenicity of the fusion protein was detected by Western blot. RESULTS:Enzyme digestion analysis and sequencing showed that the target genes were inserted into the recombinant vector, composed of 891 base pairs, encoded objective polypeptides of 297 amino acid residues. Compared with GenBank reported by Tomb et al, there were 1.3 % and 1.4 % differences in obtained H. pylori nucleotide sequence and amino acid residues, respectively. SDS-PAGE analysis showed that relative molecule mass (M(r)) of the expressed product was M(r) 51,000, M(r) of protein expressed by pET32a (+) was about M(r) 20,000, and soluble expression product accounted for 18.96 % of total bacterial protein. After purification with Ni(+2)-NTA agarose resins, the purification of recombinant fusion protein was about 95 %. Western blot showed that recombinant fusion protein could be recognized by the patients' serum infected with H. pylori and anti-Omp(18) monoclone, suggesting that this protein had good antigenicity. CONCLUSION:The gene coding for H. pylori M(r)18,000 OMP and HspA was cloned and expressed successfully. The results obtained lay the foundation for development of H. pylori protein vaccine and a quick diagnostic kit.

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Characterization of Helicobacter pylori dapE and construction of a conditionally lethal dapE mutant.

Publications

Characterization of Helicobacter pylori dapE and construction of a conditionally lethal dapE mutant.

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