The MgtC virulence factor of Salmonella enterica serovar Typhimurium activates Na(+),K(+)-ATPase

Abstract

The mgtC gene of Salmonella enterica serovar Typhimurium encodes a membrane protein of unknown function that is important for full virulence in the mouse. Since mgtC is part of an operon with mgtB which encodes a Mg(2+)-transporting P-type ATPase, MgtC was hypothesized to function in ion transport, possibly in Mg(2+) transport. Consequently, MgtC was expressed in Xenopus laevis oocytes, and its effect on ion transport was evaluated using ion selective electrodes. Oocytes expressing MgtC did not exhibit altered currents or membrane potentials in response to changes in extracellular H(+), Mg(2+), or Ca(2+), thus ruling out a previously postulated function as a Mg(2+)/H(+) antiporter. However, addition of extracellular K(+) markedly hyperpolarized membrane potential instead of the expected depolarization. Addition of ouabain to block the oocyte Na(+),K(+)-ATPase completely prevented hyperpolarization and restored the normal K(+)-induced depolarization response. These results suggested that the Na(+),K(+)-ATPase was constitutively activated in the presence of MgtC resulting in a membrane potential largely dependent on Na(+),K(+)-ATPase. Consistent with the involvement of Na(+),K(+)-ATPase, oocytes expressing MgtC exhibited an increased rate of (86)Rb(+) uptake and had increased intracellular free [K(+)] and decreased free [Na(+)] and ATP. The free concentrations of Mg(2+) and Ca(2+) and cytosolic pH were unchanged, although the total intracellular Ca(2+) content was slightly elevated. These results suggest that the serovar Typhimurium MgtC protein may be involved in regulating membrane potential but does not directly transport Mg(2+) or another ion.

FIG. 1.

Expression of MgtC and MgtC-EGFP in oocytes. Water or cRNA for MgtC or MgtC-EGFP was injected into oocytes (columns). The expression of the proteins was visualized using a peptide antibody against MgtC or EGFP autofluorescence (rows). The data shown are from oocytes injected 3 to 7 days previously. Similar distributions were obtained at different times after injection (data not shown).

FIG. 2.

Lack of effect of pH or [Mg²⁺] in MgtC-expressing oocytes. (A) Rate of acidification in MgtC-expressing (top two panels) or control (bottom two panels) oocytes. (B) Current voltage relationships in MgtC-expressing (•, ▪) or control (○, □) oocytes were determined at extracellular pHs of 5.5 and 7.5 as described in Materials and Methods. (C) The current voltage relationship in oocytes expressing MgtC was determined at 0 and 1 mM extracellular Mg²⁺ at pH 5.5: without Mg²⁺ (○), with 1 mM Mg²⁺ (•). The data in all three panels are representative of at least six similar experiments.

FIG. 3.

Membrane potential effects in oocytes expressing MgtC or MgtC-EGFP. In most experiments, two oocytes were measured simultaneously within the same chamber to directly compare water-injected control oocytes with MgtC-expressing oocytes. (A) The effect of ouabain and of alterations in extracellular [K⁺] on membrane potential was measured as described in Methods in oocytes injected with water (dashed line) or cRNA for MgtC (solid line). A representative experiment is shown. See text for replication and statistical evaluation. Ouabain is very long acting due to its affinity for Na⁺,K⁺-ATPase; therefore, there is still sufficient ouabain present during the second addition of K⁺ to provide continued inhibition. (B) The effect of alterations in extracellular [K⁺] on membrane potential was measured in oocytes injected with water (dashed line), MgtC-cRNA (dotted line), or MgtC-EGFP cRNA (solid line). The experiment shown is representative of three separate experiments that gave identical results. The average V_m for MgtC-EGFP expressing oocytes did not significantly differ from that for MgtC expressing or water-injected oocytes (data not shown, see Table 2).

FIG. 4.

⁸⁶Rb⁺ uptake in oocyte expressing MgtC. (A) Time course of ⁸⁶Rb⁺ uptake in water-injected (○)and MgtC-injected (•) oocytes. Uptake of 2 μM ⁸⁶Rb⁺ was measured as described in Materials and Methods using two oocytes per point determined in duplicate. (B) Uptake of 35 μM ⁸⁶Rb⁺ was measured after 20 min incubation as described in Materials and Methods with three oocytes per point determined in duplicate. For each panel, the error bars show the range of the duplicate determinations. Each experiment is representative of three similar experiments.