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A plasmid-based vector system for the cloning and expression of Helicobacter pylori genes encoding outer membrane
W Fischer1, D Schwan, E Gerland
1Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität, München, Germany. schmitt@m3401.mpk.med.uni-muenchen.de
Abstract:
Helicobacter pylori produces a number of proteins associated with the outer membrane, including adhesins and the vacuolating cytotoxin. We observed that the functional expression of such proteins is deleterious to Escherichia coli, the host bacterium used for gene cloning. Therefore, a general method was developed for the functional expression of such genes on a shuttle vector in H. pylori, which has been termed SOMPES (Shuttle vector-based Outer Membrane Protein Expression System). The intact, active gene is reconstituted by recombination in H. pylori from partial gene sequences cloned on an E. coli-H. pylori shuttle vector. This system was established in an H. pylori strain carrying a precise, unmarked chromosomal deletion of the vacA gene, which was constructed by adapting the streptomycin sensitivity system to H. pylori. It is based on the expression of the H. pylori rpsL gene as a counterselectable marker in the genetic background of an rpsL mutant. The utility of this approach is demonstrated by the expression of a recombinant gene encoding vacuolating cytotoxin (vacA) and a recombinant gene encoding an adherence-associated outer membrane protein (alpA) in H. pylori.
Insights
A new Shuttle vector-based Outer Membrane Protein Expression System (SOMPES) enables functional expression of Helicobacter pylori outer membrane proteins in H. pylori. This overcomes challenges with using Escherichia coli for cloning such genes.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Helicobacter pylori outer membrane proteins, like adhesins and vacuolating cytotoxin (VacA), are crucial for its pathogenicity.
- Functional expression of these proteins in Escherichia coli, a common cloning host, is often deleterious, hindering research.
Purpose of the Study:
- To develop a general method for the functional expression of H. pylori outer membrane protein genes directly within H. pylori.
- To establish a shuttle vector-based system for expressing these genes in their native host.
Main Methods:
- Developed the Shuttle vector-based Outer Membrane Protein Expression System (SOMPES) using an E. coli-H. pylori shuttle vector.
- Reconstituted intact, active genes via recombination in H. pylori from partial gene sequences.
- Established a counterselectable marker system in H. pylori using the rpsL gene in an rpsL mutant background.
- Constructed an H. pylori strain with a precise, unmarked chromosomal deletion of the vacA gene.
Main Results:
- Successfully demonstrated the functional expression of a recombinant vacuolating cytotoxin (vacA) gene in H. pylori.
- Successfully demonstrated the functional expression of a recombinant adherence-associated outer membrane protein (alpA) gene in H. pylori.
- Validated the SOMPES system for expressing H. pylori outer membrane proteins in their native host.
Conclusions:
- The SOMPES system provides a robust platform for the functional expression of H. pylori outer membrane proteins.
- This system facilitates the study of H. pylori virulence factors and the development of novel therapeutic strategies.
- Overcomes limitations of traditional cloning methods in E. coli for studying H. pylori outer membrane proteins.