title: Fluorescence resonance energy transfer microscopy of the Helicobacter pylori vacuolating cytotoxin within mammalian cells.
abstract: The Helicobacter pylori vacuolating cytotoxin (VacA) binds and enters mammalian cells to induce cellular vacuolation. To investigate the quaternary structure of VacA within the intracellular environment where toxin cytotoxicity is elaborated, we employed fluorescence resonance energy transfer (FRET) microscopy. HeLa cells coexpressing full-length and truncated forms of VacA fused to cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) were analyzed for FRET to indicate direct associations. These studies revealed that VacA-CFP and VacA-YFP interact within vacuolated cells, supporting the belief that monomer associations at an intracellular site are important for the toxin's vacuolating activity. In addition, the two fragments of proteolytically nicked VacA, p37 and p58, interact when coexpressed within mammalian cells. Because p37 and p58 function in trans when expressed separately within mammalian cells, these data suggest that the mechanism by which these two fragments induce vacuolation requires direct association. FRET microscopy also demonstrated interactions between mutant forms of VacA, as well as wild-type VacA with mutant forms of the toxin within vacuolated cells. Finally, a dominant-negative form of the toxin directly associates with wild-type VacA in cells where vacuolation was not detectable, suggesting that the formation of complexes comprising wild-type and dominant-negative forms of toxin acts to block intracellular toxin function.
authors.affiliation: Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204-5001, USA.
authors.forename: David C
authors.initials: DC
authors.lastname: Willhite
authors.forename: Dan
authors.initials: D
authors.lastname: Ye
authors.forename: Steven R
authors.initials: SR
authors.lastname: Blanke
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003603:Cytotoxins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D016480:Helicobacter pylori / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008322:Mammals; D008856:Microscopy, Fluorescence; D016296:Mutagenesis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014617:Vacuoles
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003603:Cytotoxins; D011993:Recombinant Fusion Proteins; C074689:VacA protein, Helicobacter pylori