Toxoplasma gondii macrophage migration inhibitory factor shows anti- Mycobacterium tuberculosis potential via AZIN1/STAT1 interaction

Abstract

Mycobacterium tuberculosis (MTB) is a pathogenic bacterium, belonging to the family Mycobacteriaceae, that causes tuberculosis (TB). Toxoplasma gondii macrophage migration inhibitory factor (TgMIF), a protein homolog of macrophage migration inhibitory factor, has been explored for its potential to modulate immune responses during MTB infections. We observed that TgMIF that interacts with CD74, antizyme inhibitor 1 (AZIN1), and signal transducer and activator of transcription 1 (STAT1) modulates endocytosis, restoration of mitochondrial function, and macrophage polarization, respectively. These interactions promote therapeutic efficacy in mice infected with MTB, thereby presenting a potential route to host-directed therapy development. Furthermore, TgMIF, in combination with first-line TB drugs, significantly inhibited drug-resistant MTB strains, including multidrug-resistant TB. These results demonstrate that TgMIF is potentially a multifaceted therapeutic agent against TB, acting through immune modulation, enhancement of mitochondrial function, and dependent on STAT1 and AZIN1 pathways.

Fig. 1.. TgMIF interacts with CD74, STAT1, and AZIN1.

(A) Bacterially purified 6xHis-TgMIF validation via Coomassie blue (CB) staining (left) or Western blotting with αHis (right). IB, immunoblotting. (B) Expression of pro-inflammatory cytokines (TNF-α, IL-6, and IL-12p40) and anti-inflammatory cytokines (IL-10, TGF-β1, and IL-4) in BMDMs treated with rTgMIF (5 μg/ml). The data shown are the means ± SD of five experiments. (C) Identification of STAT1, AZIN1, and CD74 using MS analysis in THP-1 cell lysates expressed with TgMIF or vector. (D) Binding partner identification using protein microarrays against rTgMIF. A, affinity; S, specificity. (E) Interaction with CD74, STAT1, and AZIN1 in BMDMs or THP-1 stimulated with rTgMIF (5 μg/ml) for the indicated times. WCL, whole cell lysate. (F) Western blot analysis of STAT1 and AZIN1 and their interactions in the nuclear and cytoplasmic fractions of Raw264.7 cells. (G) Schematic diagrams of the interaction of TgMIF and CD74, STAT1, and AZIN1. The data are representative of four independent experiments with similar results [(E) and (F)]. Full-length images of the blots presented in fig. S8.

Fig. 2.. Mapping TgMIF binding sites.

(A) Exploration of the CD74 binding site of TgMIF using GST-vector or GST-TgMIF and its truncated constructs. (B) Identification of the TgMIF binding site of CD74 using GST-vector or GST-CD74 and its truncated constructs. (C) Exploration of TgMIF binding sites on STAT1 or STAT1 on TgMIF using GST-vector, GST-TgMIF, or GST-STAT1 and its truncated constructs. (D) Identification of TgMIF binding sites on AZIN1 or AZIN1 on TgMIF using GST-vector, GST-TgMIF, or GST-AZIN1 and its truncated constructs. The data are representative of four independent experiments with similar results [(A) to (D)]. Full-length images of the blots presented in fig. S8.

Fig. 3.. Endocytosis pathway of TgMIF and its interaction with CD74.

(A) Effect of endocytosis inhibitors (10 mM methyl-β-cyclodextrin, cholesterol chelator; 1 μM amiloride, inhibits Na+/H+ exchange; 10 μM cytochalasin D, inhibitor of actin polymerization) on TgMIF expression in BMDMs. MFI, mean fluorescence intensity; DMSO, dimethyl sulfoxide. (B) Inhibition of TgMIF expression by shCD74 treatment in BMDMs. (C) Binding specificity of the TgMIF peptide (1 μM) with CD74. (D) Inflammatory responses of TgMIF peptide (1 μM) in BMDMs. The data are representative of four independent experiments with similar results [(B) and (C)]. Statistical significance was determined by the Student’s t test with Bonferroni adjustment (*P < 0.05; ***P < 0.001). Full-length images of the blots presented in fig. S8.

Fig. 4.. TgMIF modulates mitochondrial dynamics and metabolism through AZIN1.

(A to C) AZIN1-dependent regulation of mitochondrial fission and fusion gene expression by TgMIF (1, 5, and 10 μM). (D and E) AZIN1-dependent modulation of ATP synthase–related gene expression by TgMIF. (F) Cellular ATP production in MTB-infected BMDMs is dependent on TgMIF and AZIN1. (G) Restoration of mitochondrial respiration and ECARs by MTB infection with TgMIF and AZIN1. Oligo, oligomycin; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; 2-DG, 2-deoxyglucose. (H) Measurement of mtDNA content and mitochondria complex V activity in MTB-infected macrophages. (I) Number of CFU of MTB. Statistical significance was determined by the Student’s t test with Bonferroni adjustment (*P < 0.05; **P < 0.01; ***P < 0.001). ns, not significant. Full-length images of the blots presented in fig. S8.

Fig. 5.. TgMIF-induced STAT1-dependent macrophage polarization enhances host defense.

(A) Differential expression of macrophage polarization markers in TgMIF-treated BMDMs. (B) Expression of phosphorylation of STAT1, STAT3, and STAT6 in BMDMs treated with rTgMIF (5 μg/mL) for the indicated times. (C) Expression of M1 macrophage polarization markers and phosphorylation of STAT1 by amino acid variants of the TgMIF peptide. (D) The expression of macrophage polarization markers changes depending on various concentrations of the TgMIF (1, 5, and 10 μM) or 9R-TgMIF K76D (10 μM). (E) STAT1-dependent M1 and M2 macrophage polarization regulation by TgMIF. (F) Inflammatory cytokine production in MTB-infected BMDMs is dependent on TgMIF and STAT1. (G) Number of CFU of MTB. Statistical significance was determined by the Student’s t test with Bonferroni adjustment (*P < 0.05; ***P < 0.001). Full-length images of the blots presented in fig. S8.

Fig. 6.. Therapeutic effects of TgMIF on lung injury and immune response in MTB-induced mice.

(A and B) Therapeutic effects of TgMIF peptide and its mutants, TgMIF MT1 (CD74-binding mutant) and TgMIF MT2 (STAT1-binding mutant), against lung injury in MTB-induced mice (n = 25). i.n., intranasally. UN, untreated. (C) Effect of TgMIF peptide on the restoration of mitochondrial function in MTB-infected mouse lung lysates. (D) Modulation of mitochondrial function-alterations in mitochondrial respiration and ECARs by TgMIF treatment. (E) Alteration in macrophage polarization marker expression in MTB-infected mice treated with TgMIF peptide. (F) Inflammatory cytokine production in the lungs of MTB-infected mice following TgMIF treatment. Statistical significance was determined by the Student’s t test with Bonferroni adjustment (*P < 0.05; **P < 0.01; ***P < 0.001).

Fig. 7.. TgMIF treatment affects the TB survival rate after MTB infection.

The efficacy of TgMIF on MTB-induced mortality is dependent on STAT1 and AZIN1 (n = 35). Significant differences in comparison relative to the control mice are indicated (log-rank test).

Fig. 8.. Therapeutic synergy of TgMIF with TB drugs against drug-resistant TB strains.

(A) Effect of the combination of TgMIF with INH (0.05, 0.1, and 0.5 μg/ml), PZA (5, 10, and 20 μg/ml), and RFP (0.025, 0.1, and 0.5 μg/ml) on MTB growth inhibition in human MDMs. Low (red circle), medium (green circle), and high (yellow circle) concentration of drugs. MOI, multiplicity of infection. (B) Inhibition of MTB growth in the lungs of MTB-infected mice by the combination of INH (15 mg/kg) and TgMIF (10 μg) (n = 35). p.o., per os. (C and D) Reduction in bacterial colony counts by TgMIF in drug-resistant TB strains. (E and F) Restoration of mitochondrial function in lungs infected with MDR-TB following TgMIF treatment. (G) Regulation of macrophage polarization markers by TgMIF in MDR-infected lungs. (H) Induction of inflammatory cytokine production by TgMIF in MTB-infected macrophages. Statistical significance was determined by the Student’s t test with Bonferroni adjustment (**P < 0.01; ***P < 0.001).