Visualization of vacuolar acidification-induced transcription of genes of pathogens inside macrophages.
The objective of these studies was to analyze the role of the ionic environment of phagosomal vacuoles in the control of pathogens by macrophages. Digital imaging and flow cytometry were used to follow the induction of the phoP promoter of Salmonella enterica Typhimurium within live macrophages. Manipulating the Mg2+ concentration within the Salmonella-containing vacuole (SCV) was without effect on the early induction of PhoPQ. Moreover, direct measurement of [Mg2+] within the SCV using nanosensor particles showed that, during this initial period of phoP activation, the concentration of the divalent cation is rapidly regulated and stabilizes around 1 mm. Extrusion of other divalent cations via the Nramp1 efflux pump was similarly ruled out as an important contributor to the activation of the regulon. By contrast, induction of PhoP was greatly attenuated when the pH gradient across the SCV membrane was dissipated. A second, more modest pH-independent component of PhoP induction was unmasked by inhibition of the vacuolar proton pump. This second component was eliminated by pretreatment of cells with IFNgamma, even though the cytokine augmented the overall PhoP response. These findings demonstrate the existence of at least three separate activators of phoP transcription: resting and IFNgamma-stimulated pH-sensitive components, plus a pH-independent component.
Martin-Orozco
Natalia
N
Cell Biology Program, Hospital for Sick Children and Department of Biochemistry, University of Toronto, Toronto, Ontario M5G 1X8, Canada.
Touret
Nicolas
N
Zaharik
Michael L
ML
Park
Edwin
E
Kopelman
Raoul
R
Miller
Samuel
S
Finlay
B Brett
BB
Gros
Philippe
P
Grinstein
Sergio
S
D000143:Acids / Q000494:pharmacology; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D027682:Cation Transport Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D002413:Cations, Divalent / Q000737:chemistry; D002460:Cell Line; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007371:Interferon-gamma / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology* / Q000187:drug effects / Q000382:microbiology*; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D018345:Mice, Knockout; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012480:Salmonella Infections / Q000382:microbiology; D012486:Salmonella typhimurium / Q000187:drug effects / Q000235:genetics*; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects; D014617:Vacuoles / Q000737:chemistry* / Q000382:microbiology*; D000099173:Natural Resistance-associated Macrophage Protein 1
D000143:Acids; D001426:Bacterial Proteins; D027682:Cation Transport Proteins; D002413:Cations, Divalent; D007371:Interferon-gamma; D008274:Magnesium; D011994:Recombinant Proteins; D000099173:Natural Resistance-associated Macrophage Protein 1; C061064:PhoP protein, Bacteria