Requirement for N-ethylmaleimide-sensitive factor activity at different stages of bacterial invasion and phagocytosis.
Bacterial invasion, like the process of phagocytosis, involves extensive and localized protrusion of the host cell plasma membrane. To examine the molecular mechanisms of the membrane remodeling that accompanies bacterial invasion, soluble NSF attachment protein receptor (SNARE)-mediated membrane traffic was studied in cultured cells during infection by Salmonella typhimurium. A green fluorescent protein-tagged chimera of VAMP3, a SNARE characteristic of recycling endosomes, was found to accumulate at sites of Salmonella invasion. To analyze the possible role of SNARE-mediated membrane traffic in bacterial infection, invasion was measured in cells expressing a dominant-negative form of N-ethylmaleimide-sensitive factor (NSF), an essential regulator of membrane fusion. Inhibition of NSF activity did not affect cellular invasion by S. typhimurium nor the associated membrane remodeling. By contrast, Fcgamma receptor-mediated phagocytosis was greatly reduced in the presence of the mutant NSF. Most important, dominant-negative NSF significantly impaired the fusion of Salmonella-containing vacuoles with endomembranes. These observations indicate that the membrane protrusions elicited by Salmonella invasion, unlike those involved in phagocytosis, occur via an NSF-independent mechanism, whereas maturation of Salmonella-containing vacuoles is NSF-dependent.
Coppolino
M G
MG
Cell Biology Programme, Research Institute, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
Kong
C
C
Mohtashami
M
M
Schreiber
A D
AD
Brumell
J H
JH
Finlay
B B
BB
Grinstein
S
S
Trimble
W S
WS
D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000648:ultrastructure; D006224:Cricetinae; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008264:Macrophages / Q000382:microbiology; D008565:Membrane Proteins / Q000378:metabolism; D009154:Mutation; D052066:N-Ethylmaleimide-Sensitive Proteins; D010587:Phagocytosis; D012486:Salmonella typhimurium / Q000472:pathogenicity*; D013744:Tetanus Toxin / Q000494:pharmacology; D014162:Transfection; D014617:Vacuoles / Q000382:microbiology; D051040:Vesicle-Associated Membrane Protein 3; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D008565:Membrane Proteins; D013744:Tetanus Toxin; D051040:Vesicle-Associated Membrane Protein 3; D033921:Vesicular Transport Proteins; D049452:Green Fluorescent Proteins; D052066:N-Ethylmaleimide-Sensitive Proteins