Expression of Helicobacter pylori vacuolating toxin in Escherichia coli.
VacA is a secreted toxin that plays a role in Helicobacter pylori colonization of the stomach and that contributes to the pathogenesis of peptic ulcer disease. Studies of VacA structure and function have been hindered by the lack of an efficient system for expression and genetic manipulation of this toxin. In this study, we developed methodology for expression of a functionally active VacA toxin in Escherichia coli. We then used a high-throughput screen to analyze a library of mutant toxins with pentapeptide insertions and identified six mutants that lacked the capacity to induce vacuolation of HeLa cells. The capacity to analyze VacA in this heterologous-expression system should greatly facilitate efforts to elucidate the structure and function of this toxin.
McClain
Mark S
MS
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
Cover
Timothy L
TL
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D016480:Helicobacter pylori / Q000235:genetics / Q000472:pathogenicity*; D006801:Humans; D015151:Immunoblotting; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D011994:Recombinant Proteins; C074689:VacA protein, Helicobacter pylori