Utilization of alkaline phosphatase fusions to identify secreted proteins, including potential efflux proteins and virulence factors from Helicobacter pylori.
The targeted genomic strategy of random fusions to a partial gene encoding a signal sequence-deficient fragment of bacterial alkaline phosphatase was utilized to screen for secreted proteins in Helicobacter pylori. The rationale for targeting extracytoplasmic proteins was based on the hypothesis that most virulence factors and vaccine candidates are secreted or exported proteins. In addition, extracytosolic proteins represent good potential targets for drug intervention since they are in general more accessible to drugs than are cytoplasmically localized proteins. The application of this strategy to H. pylori allowed the identification of putative virulence factors and novel targets for drug intervention including four putative antibiotic efflux genes. The strategy used here is rapid and technically simple, relatively inexpensive, adaptable to a wide variety of microbes and genetic systems, and selects for expressed and accessible proteins.
Bina
J E
JE
Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada.
Nano
F
F
Hancock
R E
RE
D000469:Alkaline Phosphatase / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D015723:Gene Library; D005798:Genes, Bacterial / Q000235:genetics; D016480:Helicobacter pylori / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014774:Virulence
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase