Document Detail

Fluorescence resonance energy transfer microscopy of the Helicobacter pylori vacuolating cytotoxin within mammalian cells.
MedLine Citation:
PMID:  12065526     Owner:  NLM     Status:  MEDLINE    
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.
David C Willhite; Dan Ye; Steven R Blanke
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Infection and immunity     Volume:  70     ISSN:  0019-9567     ISO Abbreviation:  Infect. Immun.     Publication Date:  2002 Jul 
Date Detail:
Created Date:  2002-06-14     Completed Date:  2002-07-30     Revised Date:  2013-04-18    
Medline Journal Info:
Nlm Unique ID:  0246127     Medline TA:  Infect Immun     Country:  United States    
Other Details:
Languages:  eng     Pagination:  3824-32     Citation Subset:  IM    
Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204-5001, USA.
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MeSH Terms
Bacterial Proteins / genetics,  metabolism*
Bacterial Toxins / genetics,  metabolism*
Cell Line
Cytotoxins / genetics,  metabolism*
HeLa Cells
Helicobacter pylori / genetics,  metabolism*
Microscopy, Fluorescence
Recombinant Fusion Proteins / genetics,  metabolism
Spectrometry, Fluorescence
Grant Support
Reg. No./Substance:
0/Bacterial Proteins; 0/Bacterial Toxins; 0/Cytotoxins; 0/Recombinant Fusion Proteins; 0/VacA protein, Helicobacter pylori

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