MtsABC is important for manganese and iron transport, oxidative stress resistance, and virulence of Streptococcus pyogenes

Abstract

MtsABC is a Streptococcus pyogenes ABC transporter which was previously shown to be involved in iron and zinc accumulation. In this study, we showed that an mtsABC mutant has impaired growth, particularly in a metal-depleted medium and an aerobic environment. In metal-depleted medium, growth was restored by the addition of 10 microM MnCl(2), whereas other metals had modest or no effect. A characterization of metal radioisotope accumulation showed that manganese competes with iron accumulation in a dose-dependent manner. Conversely, iron competes with manganese accumulation but to a lesser extent. The mutant showed a pronounced reduction (>90%) of (54)Mn accumulation, showing that MtsABC is also involved in Mn transport. Using paraquat and hydrogen peroxide to induce oxidative stress, we show that the mutant has an increased susceptibility to reactive oxygen species. Moreover, activity of the manganese-cofactored superoxide dismutase in the mutant is reduced, probably as a consequence of reduced intracellular availability of manganese. The enzyme functionality was restored by manganese supplementation during growth. The mutant was also attenuated in virulence, as shown in animal experiments. These results emphasize the role of MtsABC and trace metals, especially manganese, for S. pyogenes growth, susceptibility to oxidative stress, and virulence.

FIG. 1.

Growth of WT (AP1) and mutant (RJ1) mtsABC strains under different environmental conditions. Experiments were performed at least three times with duplicate samples, and representative growth curves are shown. (A) Growth in THY under aerobic conditions. (B) Comparison of WT and mutant growth in THY or metal-depleted THY (Cx-THY) in a 5% CO₂ atmosphere. (C) Growth in a 5% CO₂ atmosphere in Cx-THY with 50 μM supplements of the following metal salts: ferric citrate, FeSO₄, MnCl₂, CuCl₂, ZnCl₂. (D) Growth in a 5% CO₂ atmosphere in Cx-THY supplemented with increasing concentrations (0 to 50 μM) of MnCl₂.

FIG. 2.

Bacterial incorporation of radioactive metal ion isotopes during the exponential phase of growth in Cx-THY. (A) Accumulation of ⁵⁵Fe in the presence or absence of 10 μM concentrations of competing metal salts. AP1 accumulation (a) was competed with ferric citrate (c), FeCl₃ (d), FeSO₄ (e), Na citrate (f), MnCl₂ (g), CuCl₂ (h), and ZnCl₂ (i). Results for the mtsABC mutant RJ1 are seen in bar b. In this context, accumulation is defined as the radioactivity found in the bacterial pellet as a fraction of the total radioactivity added. The bars represent the means of three experiments performed with duplicate samples. Standard deviations are represented by the error bars. (B) Dose-dependent competition of ⁵⁵Fe accumulation by MnCl₂ and ferric citrate. Data points are means of two experiments performed with triplicate samples. Standard deviations are represented by the error bars. (C) Dose-dependent competition of ⁵⁴Mn accumulation by MnCl₂ and ferric citrate. Data points are means of two experiments performed with triplicate samples. Standard deviations are represented by the error bars.

FIG. 3.

Comparison of WT and mutant sensitivity to oxidative stress conditions. (A) WT (AP1) and mutant (RJ1) bacteria were grown in a 5% CO₂ atmosphere until the A₆₂₀ was 0.5, and 5 mM hydrogen peroxide was added. Samples were collected at various time points, and viable counts were performed. Results are presented as the percentages of viable bacteria at a certain time point compared with the number of bacteria prior to challenge (time zero). Data points represent means of five experiments, and error bars indicate the standard deviations. (B) Growth of WT (AP1) and mutant (RJ1) in the presence or absence of paraquat. ON cultures were reinoculated in fresh THY containing 0, 2, or 10 mM paraquat. MnCl₂ or ferric citrate was added to some samples at a concentration of 30 μM. The A₆₂₀ was recorded after 15 h of incubation. Results represent the means of four experiments performed with duplicate samples. Standard deviations are indicated by the error bars.

FIG. 4.

Analysis of SOD activity and sodA transcription. (A) Cell extracts were prepared from WT (AP1) and mutant (RJ1) bacteria grown on THY plates in ambient air, with or without 50 μM MnCl₂ supplementation. Twenty micrograms of protein per sample was separated by native PAGE. Gels were then subjected to an assay where SOD activity is visualized as clear zones. The experiment was performed three times, and the image shown represents the highest observed activity in RJ1 (without manganese supplementation). (B) Northern blot analysis of total RNA from WT and mutant strains. A ³²P-labeled 620-bp probe corresponding to sodA was hybridized with the membrane. The experiment was performed twice with identical results.

FIG. 5.

Homology modeling of MtsA. Overall fold of MtsA seen from a view orthogonal to the pseudosymmetry axis, with the N-terminal (membrane associated) region facing down. The β strands are shown in green, and the α helices are shown in red. The arrow indicates the approximate location of the putative metal-binding pocket.