Role of 3-phosphoinositides in the maturation of Salmonella-containing vacuoles within host cells.
Salmonella typhimurium invades mammalian cells and replicates within a vacuole that protects it from the host's microbicidal weapons. The Salmonella-containing vacuole (SCV) undergoes a remodelling akin to that of the host cell's endocytic pathway, but SCV progression is arrested prior to fusion with lysosomes. We studied the role of phosphatidylinositol 3-kinase (PI3-K) in SCV maturation within HeLa cells. Phosphatidylinositol 3-phosphate (PI3P), monitored in situ using fluorescent conjugates of FYVE or PX domains, was found to accumulate transiently on the SCV. Wortmannin prevented PI3P accumulation and the recruitment of EEA1 but did not affect the association of Rab5 with the SCV. Importantly, inhibition of PI3-K also impaired fusion of the SCV with vesicles containing LAMP-1. Rab7, which is thought to be required for association of LAMP-1 with the SCV, still associated with SCV in wortmannin-treated cells. We have therefore concluded that a 3-phosphoinositide-dependent step exists following recruitment of Rab7 to the SCV. The data also imply that 3-phosphoinositide-dependent effectors of Rab5 are not an absolute requirement for recruitment of Rab7. Despite failure to acquire LAMP-1, the SCV persists and allows effective replication of Salmonella within wortmannin-treated host cells. These findings imply that PI3-K is involved in the development of the SCV but is not essential for intracellular survival and proliferation of Salmonella.
Scott
Cameron C
CC
Division of Cell Biology, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada.
Cuellar-Mata
Patricia
P
Matsuo
Tsuyoshi
T
Davidson
Howard W
HW
Grinstein
Sergio
S
D000730:Androstadienes / Q000494:pharmacology; D015703:Antigens, CD / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins / Q000378:metabolism; D015335:Molecular Probes; D019255:NADPH Oxidases / Q000378:metabolism; D010716:Phosphatidylinositols / Q000502:physiology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012486:Salmonella typhimurium / Q000302:isolation & purification*; D014617:Vacuoles / Q000201:enzymology / Q000382:microbiology*; D000077191:Wortmannin; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins
D000730:Androstadienes; D015703:Antigens, CD; D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D015335:Molecular Probes; D010716:Phosphatidylinositols; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011993:Recombinant Fusion Proteins; D000090802:rab7 GTP-Binding Proteins; C000716463:rab7 GTP-binding proteins, human; D049452:Green Fluorescent Proteins; D019255:NADPH Oxidases; D020691:rab GTP-Binding Proteins; D000077191:Wortmannin