Membrane potential changes after infection of monocytes by Toxoplasma gondii.
Membrane potential changes in host cell plasma membrane were analyzed and the parasitophorous vacuole membrane (PVM) potential was characterized after infection by Toxoplasma gondii. Human monocytes infested by T. gondii were stained with two membrane potential sensitive dyes, DiOC(6)(3) carbocyanine and DiSBAC(2)(3) bis-oxonol, before fluorescence emission analysis by confocal laser scanning microscopy. After 24 and 48 h of infection, 34 and 39%, respectively, of monocytes showed several parasites (from two to six) per cell. At these infection times, significant decreases in cytoplasmic emissions were observed for both DiOC(6)(3) and DiSBAC(2)(3). Thus, hyperpolarisation of the host plasma membrane would occur consecutively to infection. Inside the parasitophorous vacuole, the fluorescence intensity of DiOC(6)(3) and DiSBAC(2)(3) increased significantly from 6 to 24 h after infection and the PVM became less polarised. Involvement of different ATPases in the membrane potential of infected monocytes was evaluated with ouabain, DCCD, omeprazole and sodium orthovanadate, ATPase inhibitors. All inhibitors induced a depolarisation of the plasma membrane. In the parasitophorous vacuole compartment, DCCD, omeprazole and sodium orthovanadate but not ouabain caused a significant depolarisation of the PVM, suggesting that H(+), H(+)/K(+) and P-type ATPases were at the origin of the PVM potential. This is the first report showing the presence of ion transporters in the T. gondii PVM and the existence of at least two members of the P-type family of ion pumps: an electrogenic H(+)ATPase and an electroneutral H(+)/K(+) ATPase.
Bouchot
A
A
Laboratoire de Parasitologie-mycologie-Faculté de Médecine-UPRES EA 2070-IFR53, Reims, France.
Millot
J M
JM
Charpentier
S
S
Bonhomme
A
A
Villena
I
I
Aubert
D
D
Pinon
J M
JM
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000502:physiology; D000818:Animals; D002232:Carbocyanines / Q000494:pharmacology; D002462:Cell Membrane / Q000502:physiology; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006096:Gramicidin / Q000494:pharmacology; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009000:Monocytes / Q000276:immunology / Q000469:parasitology* / Q000502:physiology*; D009853:Omeprazole / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D014122:Toxoplasma / Q000502:physiology*; D014638:Vanadates / Q000494:pharmacology
D002232:Carbocyanines; D004791:Enzyme Inhibitors; D006096:Gramicidin; D014638:Vanadates; D004024:Dicyclohexylcarbodiimide; C007392:3,3'-dihexyl-2,2'-oxacarbocyanine; D010042:Ouabain; D000251:Adenosine Triphosphatases; D009853:Omeprazole