Manganese blocks intracellular trafficking of Shiga toxin and protects against Shiga toxicosis - PubMed (original) (raw)

Manganese blocks intracellular trafficking of Shiga toxin and protects against Shiga toxicosis

Somshuvra Mukhopadhyay et al. Science. 2012.

Abstract

Infections with Shiga toxin (STx)-producing bacteria cause more than a million deaths each year and have no definitive treatment. To exert its cytotoxic effect, STx invades cells through retrograde membrane trafficking, escaping the lysosomal degradative pathway. We found that the widely available metal manganese (Mn(2+)) blocked endosome-to-Golgi trafficking of STx and caused its degradation in lysosomes. Mn(2+) targeted the cycling Golgi protein GPP130, which STx bound in control cells during sorting into Golgi-directed endosomal tubules that bypass lysosomes. In tissue culture cells, treatment with Mn(2+) yielded a protection factor of 3800 against STx-induced cell death. Furthermore, mice injected with nontoxic doses of Mn(2+) were completely resistant to a lethal STx challenge. Thus, Mn(2+) may represent a low-cost therapeutic agent for the treatment of STx infections.

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Figures

Fig. 1

Fig. 1

Manganese specifically blocks STxB trafficking. (A) STxB transport for the indicated times in HeLa cells untreated or pretreated with 500 μM Mn2+ for 4 hours. Scale bar, 10 μm. (B and C) Percentage of cellular STxB or CTxB in the Golgi at the indicated times postinternalization (mean ± SE; 20 cells per point). See fig. S2 for CTxB images. (D) Normalized cell viability by methylthiazolylphenyl-tetrazolium bromide (MTT) assay. Mn2+ was 500 μM for 12 hours (mean ± SE; n = 3 experiments).

Fig. 2

Fig. 2

Manganese alters sorting at endosome, causing STxB degradation. (A) STxB localization compared with Rab5-GFP or Rab7-GFP at the indicated times postinternalization in Mn2+-treated cells. Scale bar, 4 μm. Arrows indicate overlap. (B) Endosomal tubulation of STxB in comparison with EGF at the indicated times postinternalization. See fig. S7A for full images. Scale bar, 1 μm. (C) Percentage of endosomes per cell with STxB tubules at the indicated times (mean ± SE; 10 cells per point; 100 to 150 endosomes per cell). See fig. S7B for EGF tubules. (D) Cellular STxB at the indicated times normalized to the control at 0 hours postinternalization (mean ± SE; 15 cells per point). See fig. S12 for images.

Fig. 3

Fig. 3

GPP130 is the cellular Mn2+ target and directly binds STxB to mediate its endosome-to-Golgi trafficking. (A) STxB localization 30 min postinternalization in Mn-treated cells expressing the indicated constructs. Endogenous GPP130 was detected using an antibody to the acidic domain. Scale bar, 10 μm. (B) Percentage of cellular STxB in Golgi from (A). Data for control and Mn2+ groups without GPP130 transfection are replotted from Fig. 1B (mean ± SE; 15 cells each). (C and D) Quantitation of His-STxB recovery after incubation with the indicated concentrations of either glutathione S-transferase (GST) or GST-GPP13036–247 and the corresponding Coomassie-stained gels. (E and F) Coomassie-stained gels and quantitation of His-STxB recovery after incubation with 5 μM of the indicated GST constructs (mean ± SE; n = 3 experiments).

Fig. 4

Fig. 4

Treatment with Mn2+ protects against STx1-induced death. (A) Cell viability by MTT assay after 24 hours exposure to STx1 at the indicated concentrations. Mn2+ sample was pretreated with 500 μM for 4 hours and then Mn2+ was reduced to 125 μM during STx1 exposure (mean ± SE; n = 3 experiments). (B) LD50 with boxed 95% confidence interval from (A). (C) Fraction of mice surviving injection of Mn2+ at the indicated concentration (n = 4 mice per dose). (D) Body weight change after 96 hours of daily Mn2+ injections at the indicated concentrations (n = 4 mice per dose, except n = 2 for 100 mg/kg). Only animals with depressed body weight exhibited locomotive or any other abnormalities. (E) Body weight change of mice injected with STx1 only (n = 6 mice) or STx1 and Mn2+ at the indicated concentration (n = 6 mice for 50 mg/kg Mn2+ and 4 for other doses). Final weight recorded on day of death or on day 8 for survivors. (F) Survival curves from (E). (G) Histology of kidneys by toluidine blue staining (scale bar, 100 μm; inset, 4×) and electron microscopy (scale bar, 10 μm).

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