Anthrax lethal toxin inhibits translation of hypoxia-inducible factor 1α and causes decreased tolerance to hypoxic stress

Abstract

Hypoxia is considered to be a contributor to the pathology associated with administration of anthrax lethal toxin (LT). However, we report here that serum lactate levels in LT-treated mice are reduced, a finding inconsistent with the anaerobic metabolism expected to occur during hypoxia. Reduced lactate levels are also observed in the culture supernatants of LT-treated cells. LT inhibits the accumulation of hypoxia-inducible factor (HIF)-1α, a subunit of HIF-1, the master regulator directing cellular responses to hypoxia. The toxin has no effect on the transcription or protein turnover of HIF-1α, but instead it acts to inhibit HIF-1α translation. LT treatment diminishes phosphorylation of eIF4B, eIF4E, and rpS6, critical components of the intracellular machinery required for HIF-1α translation. Moreover, blockade of MKK1/2-ERK1/2, but not p38 or JNK signaling, lowers HIF-1α protein levels in both normoxic and hypoxic conditions, consistent with a role for MKK1 and MKK2 as the major targets of LT responsible for the inhibition of HIF-1α translation. The physiological importance of the LT-induced translation blockade is demonstrated by the finding that LT treatment decreases the survival of hepatocyte cell lines grown in hypoxic conditions, an effect that is overcome by preinduction of HIF-1α. Taken together, these data support a role for LT in dysregulating HIF-1α and thereby disrupting homeostatic responses to hypoxia, an environmental characteristic of certain tissues at baseline and/or during disseminated infection with Bacillus anthracis.

Keywords: Bacillus; Bacterial Pathogenesis; Hypoxia; Hypoxia-inducible Factor; Infectious Diseases; MAP kinases (MAPKs); Protein Stability; Toxins; Translation; mTOR.

FIGURE 1.

Anthrax LT treatment causes a reduction in lactate accumulation. A, levels of serum lactate in untreated mice (control, n = 13) or mice treated with LT for 24 h (n = 9) and 48 h (n = 7). B, levels of lactate in the supernatants collected from HepG2 cells (5 × 10³) cultured under normoxic or hypoxic conditions for 24 h in the presence (LT) or absence (M) of LT. C, uptake of glucose by HepG2 cells (5 × 10³) pretreated with (LT) or without (M) LT for 3 h, and then subsequently cultured in normoxic or hypoxic conditions for 24 h. Error bars shown in A–C denote standard deviation generated from three independent experiments. *, statistical analysis was performed using the two-tailed Student's t test.

FIGURE 2.

Anthrax LT treatment blocks the induction of HIF-1α and its target genes. A, differential expression of HIF-1α target genes in HepG2 cells pretreated for 3 h with (LT) or without (M) LT, and then cultured in normoxic or hypoxic conditions for 20 h. Gene expression is indicated by a color code that ranges from light green (4 or more fold lower than reference levels in normoxic cells cultured in medium alone) to bright red (4 or more fold greater than the reference levels). B, Western blotting analysis of HIF-1α expression in various cell lines treated with (LT) or without (M) LT in normoxic or hypoxic conditions as indicated. Shown is one representative of three independent experiments showing the same trend. C, HepG2 cells were cultured for 3 h in medium alone (M) or with medium containing a combination of wild-type PA and mutated lethal factor lacking protein cleavage activity (LTm), and they were then cultured in normoxic or hypoxic conditions for an additional 4 h. Shown are Western blotting results from one of two independent experiments exhibiting the same trend. The intensity of protein bands in B and C was quantified using Fluorchem Q software; relative amounts are shown below each lane.

FIGURE 3.

Anthrax LT treatment does not affect HIF-1α gene transcription and protein stability. A, quantitative PCR analysis of HIF-1α mRNA expression in HepG2 cells cultured in medium alone (M) or supplemented with LT for 1–3 h in normoxic conditions (three left columns) or pretreated with LT or medium alone (M) for 3 h, and then cultured in hypoxic conditions for an additional 4 h (two right columns). B and C, Western blotting analysis of HIF-1α protein levels in HepG2 cells pretreated in normoxic conditions with DMSO, MG132, ALLN, and/or lactacystin (Lact) for 30 min and then incubated with or without LT for 7 h as indicated. D, turnover of HIF-1α protein in HepG2 and Hepa1c1c7 cells cultured in the presence or absence of LT in hypoxic conditions. Cells were cultured in hypoxic conditions for 4 h with or without LT during the last 2 h. Cells were subsequently treated with CHX for 0, 0.5, and 1–4 h as shown. HIF-1α protein levels were determined by Western blotting. E, quantification of HIF-1α protein levels. β-Actin was used as a loading control. The intensity of protein bands in B and C was quantified using Fluorchem Q software; relative amounts are shown below each lane. Data shown are representative of three (A–C) or two (D and E) independent experiments.

FIGURE 4.

Anthrax LT treatment inhibits HIF-1α protein synthesis. A, Western blotting analysis of HIF-1α protein levels in HepG2 cells cultured in the presence or absence of LT and/or MG132 for the indicated periods of time. B, Western blotting analysis of HIF-1α protein levels in HepG2 cells, which were treated with CHX for 4 h in the presence or absence of wild-type LT or a protease-deficient LT mutant (LTm) for the last 2 h. Cells were subsequently washed twice with PBS and then incubated with MG132 for 0–3 h in the absence of LT or LT mutant as indicated. The intensity of protein bands in A and B was quantified using Fluorchem Q software; relative amounts are shown below each lane. C, polysomal distribution of HIF-1α (left panel) and β-actin (right panel) mRNA in HepG2 cells cultured in the presence or absence of LT for 3 h and then cultured in hypoxic conditions for 4 h. The treated cells were extracted with polysome extraction buffer, and the supernatants were fractioned through a 10–50% sucrose gradient. HIF-1α and β-actin mRNA levels in each gradient fraction were determined by quantitative PCR and plotted as a percentage of the total HIF-1α and β-actin mRNA levels in each sample. The peak in fraction 4 corresponds to monosomes, whereas the peak in fractions 8–10 corresponds to polysomes with active translation.

FIGURE 5.

Anthrax LT treatment disrupts signal pathways critical for protein synthesis. HepG2 cells were pretreated with (LT) or without (M) LT for 3 h and then cultured under normoxic or hypoxic conditions for an additional 4 h. Cell lysates were subjected to Western blotting analysis for assessing protein levels of various MKKs (A), total and phosphorylated ERK1/2 and p38 (B), total and phosphorylated MNK and RSK (C), total and phosphorylated eIF4E and eIF4B (D), total and phosphorylated p70^S6K and rpS6 (E), and total and phosphorylated eIF2A (F). The intensity of protein bands in C–F was quantified using Fluorchem Q software; relative amounts are shown below each lane. Data shown are representative of three independent experiments.

FIGURE 6.

Blockade of MKK1/2-ERK1/2, mTOR, MNK, and/or RSK signaling pathways have differential effects on HIF-1α protein levels in normoxic versus hypoxic conditions. HepG2 cells were pretreated with the vehicle control (DMSO) or the MAPK inhibitors U0126, SB203580, and SP600125 individually (A); with U0126 (U) plus SB203580 (SB) or the mTOR inhibitors PP242 and rapamycin (Rapa) (B); or with various combinations of an MNK inhibitor (Mi), RSK inhibitor (Ri), and/or PP242 (C) for 3 h. Following pretreatment in these conditions, cells were cultured in normoxic or hypoxic conditions for an additional 4 h. HIF-1α protein levels in treated cells were determined by Western blotting. β-Actin was used as a loading control. The intensity of protein bands in A–C was quantified using Fluorchem Q software; relative amounts are shown below each lane. Data shown are representative of three independent experiments.

FIGURE 7.

Preinduction of HIF-1α correlates with protection from LT-dependent cytotoxicity during hypoxic stress. A, Western blot assay of HIF-1α protein in HepG2 cells that were treated with LT either 3 h prior to (LT-3) or at the initiation (LT) of the incubation under normoxic or hypoxic conditions for 12 h. Cells grown in medium alone (M) are shown as a control. The intensity of protein bands in A was quantified using Fluorchem Q software; relative amounts are shown below each lane. B, cytotoxicity was assessed in HepG2 (left panel) or Hepa1c1c7 hepatocytes (right panel) treated with LT either 3 h prior to (LT-3) or at the initiation (LT) of the incubation under hypoxia for 24 h. Cells grown in medium alone (M) are shown as a control. Cytotoxicity was determined by the detection of the release of intracellular lactate dehydrogenase into the culture supernatants. Data shown are representative of three independent experiments.