Abstract
Mycobacterium tuberculosis PtpA, a secreted tyrosine phosphatase essential for tuberculosis pathogenicity, could be an ideal target for a drug against tuberculosis, but its active-site inhibitors lack selectivity over human phosphatases. Here we found that PtpA suppressed innate immunity dependent on pathways of the kinases Jnk and p38 and the transcription factor NF-κB by exploiting host ubiquitin. Binding of PtpA to ubiquitin via a region with no homology to human proteins activated it to dephosphorylate phosphorylated Jnk and p38, leading to suppression of innate immunity. Furthermore, the host adaptor TAB3 mediated NF-κB signaling by sensing ubiquitin chains, and PtpA blocked this process by competitively binding the ubiquitin-interacting domain of TAB3. Our findings reveal how pathogens subvert innate immunity by coopting host ubiquitin and suggest a potential tuberculosis treatment via targeting of ubiquitin-PtpA interfaces.
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Acknowledgements
We thank J. Han (Xiamen University), W.R. Jacobs (Albert Einstein College of Medicine) and F. Shao (National Institute of Biological Sciences, Beijing, China) for plasmids. Supported by the National Basic Research Programs of China (2012CB518700, 2014CB744400 and 2012CB910300), National Natural Science Foundation of China (81371769 and 91319303), the Ministry of Health and the Ministry of Science and Technology of China (2013ZX10003006) and the Chinese Academy of Sciences (KJZD-EW-L02).
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J.W., B.-X.L. and P.-P.G. performed the majority of the experiments with assistance of J.L. and Q.W.; C.H.L. designed the study; C.H.L., J.W., X.-B.Q. and G.F.G. collected and analyzed the data; C.H.L. and X.-B.Q. wrote the manuscript. All authors discussed the results and commented on the manuscript.
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Supplementary Figure 1 Mtb PtpA suppresses the NF-κB-activation and Jnk and p38 pathways but not the Erk pathway.
(a) Activation of Erk pathway is not inhibited by Mtb PtpA. HEK293T cells were transfected with wild-type (WT) or mutant (D126A) Mtb PtpA. Erk pathway was stimulated by co-expression of constitutively active RasV12 in HEK293T cells. The luciferase reporter assay for Elk reporter was performed, and the related proteins were analyzed by immunoblot analysis. (b) Overexpression of PtpA in M. smeg matis reduces phosphorylation of IκBα, Jnk and p38, but not of Erk, in U937 cells infected for 0–48 h with indicated M. smeg matis strains as analyzed by immunoblot analysis, and quantified by densitometry. The relative levels of phosphorylated IκBα, Jnk and p38 were obtained by normalizing to the loading control (β-actin). (c) PtpA is mainly secreted into the cytosol of the infected U937 cells. U937 cells were infected with BCG at a multiplicity of infection (MOI) of 10:1. At 4 h post-infection, cells were collected for fractionation to obtain the cytosolic fraction, the pellet containing the U937 nuclei and BCG, and the bacteria (after excluding macrophage nuclei and cell debris from pellet) for immunoblot analysis of PtpA expression. *P < 0.05 and **P < 0.01 (two-tailed unpaired t-test). Data are representative of one experiment with at least three independent biological replicates (a,b; mean and s.e.m., n = 3).
Supplementary Figure 2 Deletion of PtpA in BCG increases IL-12p40 production in infected mice, reduces acid-fast bacilli accumulation in lungs, and mitigates liver pathology of the infected mice.
(a) Deletion of PtpA in BCG increases the protein levels of IL-12p40 in mice infected intratracheally with 2.0 × 106 CFU of wild-type BCG, BCG△PtpA, or BCG (△PtpA + PtpA) for 0–20 days. IL-12p40 protein levels in serum from the infected mice were analyzed by ELISA assay. (b) Lung sections of mice infected with BCG△PtpA had a marked reduction of acid-fast bacilli at day 12 post-infection. Ziehl-Neelsen staining for acid-fast bacilli (arrows) was performed for lung sections of the mice infected as in (a). Scale bars, 5 μm. (c, d) Deletion of PtpA in BCG reduces total cellular and neutrophilic infiltration in the livers of the mice infected as in (a). Enlarged views of the boxed regions in (c) are shown in (d). Foci of cellular infiltration and neutrophils (with a lobulated nucleus) are indicated by arrows in (c) and (d), respectively. Liver sections from BCG-infected mice were stained with hematoxylin and eosin. Scale bars, 200 μm (c) or 20 μm (d). (e) The △PtpA mutant strain cultivated in 7H9 media under laboratory conditions exhibits no growth defect as compared to the wild-type BCG strain. A total of 1 × 106 CFU of mycobacterial strains were inoculated into Middlebrook 7H9 broth supplemented with 10% oleic acid-albumin-dexrose-catalase (OADC) and 0.05% Tween-80 and cultivated at 37 °C, and then three sets of serial 10-fold dilutions of the culture from each time point were plated on 7H10 agar for bacterial colony forming unit (CFU) counting after 3–4 weeks of incubation at 37 °C. *P < 0.05 and **P < 0.01 (two-tailed unpaired t-test). Data are representative of one experiment with two independent biological replicates (a – d; mean and s.e.m., n = 6) or are representative of one experiment with at least three independent biological replicates (e; mean and s.e.m., n = 3).
Supplementary Figure 3 Ubiquitin binding stimulates the phosphatase activity of Mtb PtpA toward its substrates p-Jnk and p-p38.
(a, b) Co-immunoprecipitation of Mtb PtpA with Jnk (a) and p38 (b) from the lysates of HEK293T cells tranfected with Flag-PtpA or its mutant (D126A). (c,d) Mtb PtpA by itself possesses little phosphatase activity towards purified p-Jnk (c) or p-p38 (d) in vitro. p-Jnk or p-p38 was treated with purified Mtb PtpA, followed by phosphospecific immunoblot assays of p-Jnk or p-p38. (e) Mtb PtpA interacts with ubiquitin (Ub) in the yeast two-hybrid assay. Yeast strains were transformed with indicated plasmids in which TAK1-TAB2 interaction serves as a positive control. Left, high-stringency. Right, low-stringency. (f,g) Ubiquitin stimulates the phosphatase activity of Mtb PtpA, but not PtpA (D126A), towards p-Jnk (f) and p-p38 (g) as shown in immunoblot analysis. p-Jnk (f) and p-p38 (g) were titrated with purified wild-type Mtb PtpA or PtpA D126A mutant proteins in the presence of 1.5 μM ubiquitin. (h) Ubiquitin stimulates the phosphatase activity of Mtb PtpA as analyzed by para-nitrophenyl phosphate (pNPP) phosphatase assay. The specific activity for Mtb PtpA + Ub was 2.96 ± 0.4 U/g (mean ± s.e.m.). (i,j) Mutation at A140E abolishes the phosphatase activity of Mtb PtpA towards p-Jnk (i) and p-p38 (j) in the presence of ubquitin as shown in immunoblot analysis. p-Jnk (i) and p-p38 (j) were titrated with purified wild-type Mtb PtpA or PtpA A140E mutant proteins in the presence of 1.5 μM ubiquitin. *P < 0.05 and **P < 0.01 (two-tailed unpaired t-test). Data are representative of one experiment with at least three independent biological replicates (h; mean and s.e.m., n = 3).
Supplementary Figure 4 Binding to ubiquitin is required, but not sufficient, for stimulation of PtpA phosphatase activity.
(a) The full-length M.smegmatis (M. smeg) PtpA T140A mutant, but not its wild-type form, could precipitate ubiquitin. GST-tagged proteins (9 μg) were used to precipitate 3 μg of free ubiquitin (His6-Ub) in the in vitro precipitation (Ppt) assay. (b) The putative UIM-like (UIML) region of the M.smegmatis PtpA T140A mutant, but not its wild-type form, could precipitate ubiquitin. GST-tagged proteins (6 μg) were used to precipitate 3 μg of free ubiquitin (His6-Ub). (c) The phosphatase activity of M.smegmatis PtpA T140A mutant, but not its wild-type form, could be stimulated by ubiquitin. The specific activity for M. smegmatis PtpA (T140A) + Ub was 15.31 ± 0.62 U/g (mean ± s.e.m.). (d) Both full-length Mtb PtpA L146A mutant and its UIML region could precipitate ubiquitin. GST-tagged full-length Mtb PtpA L146A mutant protein (9 μg) or its UIML region (6 μg) were used to precipitate 3 μg of free ubiquitin. (e) Mutation at L146A abolishes the phosphatase activity of Mtb PtpA stimulated by ubiquitin as shown in pNPP phosphatase assay. The specific activity for Mtb PtpA + Ub was 3.42 ± 0.15 U/g. *P < 0.05 and **P < 0.01 (two-tailed unpaired t-test). Data are representative of one experiment with at least three independent biological replicates (c,e; mean and s.e.m., n = 3).
Supplementary Figure 5 Mtb PtpA binds to Ile44 in ubiquitin for stimulation of its phosphatase activity toward p-Jnk and p-p38.
(a) GST-fused UIML region of Mtb PtpA could precipitate wild-type ubiquitin, but not its I44A mutant. (b) Mutation at I44A abolishes the ubiquitin-stimulated phosphatase activity of Mtb PtpA as shown in the pNPP phosphatase assay. The specific activity for Mtb PtpA + Ub was 4.30 ± 0.10 U/g. (c,d) Mutation at I44A abolishes the ubiquitin-stimulated phosphatase activity of Mtb PtpA against p-Jnk (c) and p-p38 (d) as shown in immunoblot analysis. (e,f) Ubiquitin activates the Mtb PtpA-mediated dephosphorylation of p-Jnk (e) or p-p38 (f) as analyzed by immunoblot analysis. *P< 0.05 and **P< 0.01 (two-tailed unpaired t-test). Data are representative of one experiment with at least three independent biological replicates (b; mean and s.e.m., n = 3).
Supplementary Figure 6 Leu146 is critical to the activation of Mtb PtpA by ubiquitin.
(a) Mutation at A140E abolishes the ubiquitin-stimulated phosphatase activity of Mtb PtpA towards p-VPS33B as shown in immunoblot analysis. p-VPS33B was titrated with purified wild-type Mtb PtpA or PtpA A140E mutant protein in the presence of 1.5 μM ubiquitin. (b) Mutation at I44A abolishes the ubiquitin-stimulated phosphatase activity of Mtb PtpA against p-VPS33B as shown in immunoblot analysis. (c) Mutation at A140E abolishes PtpA-mediated inhibition of phagosome acidification. The indicated mycobacterial strains were labeled with pH-sensitive fluorescent dye (pHrodo) and used to infect U937 cells. Phagosomal pH of the infected macrophages was measured with FACS. Wild-type BCG and the complemented strain △PtpA + PtpA maintained a phagosomal pH of 6.2 ~ 6.7, whereas phagosomes of △PtpA, △PtpA + D126A and △PtpA + A140E strains were acidified to pH 4.5 ~ 5.5. *P < 0.05 and **P < 0.01 (significant difference compared with wild-type BCG by two-tailed unpaired t-test). (d) A model for activation of the catalytic activity of Mtb PtpA by ubiquitin. The active site of Mtb PtpA (red and orange) is located between the α helixes 1 and 2, both of which are closely adjacent to α helix 5. Binding of ubiquitin to the UIML region on α helix 5 may thus change the structure of the active site of PtpA, leading to activation of its phosphatase activity. Hydrophobic Leu146, which is sandwiched by two other hydrophobic residues (Phe25 on α helix 1 and Phe113 next to α helix 4), is critical to the activation, probably by bridging α helix 5 with α helix 1 through a hydrophobic interaction. (e) Kinetic analysis of phosphatase activity of Mtb PtpA and its mutants. The small molecule substrate pNPP and the protein substrates p-Jnk, p-p38 and p-VPS33B were used as the substrates, and the released inorganic phosphate was measured by the nonradioactive molybdate dye-based phosphatase assay. The Lineweaver-Burk plots were used to determine the Michaelis-Menten kinetic parameters (kcat and Km). Data are representative of one experiment with at least three independent biological replicates (c; mean and s.e.m., n = 3).
Supplementary Figure 7 Mtb PtpA binds TAB3 and blocks it from binding K63-linked ubiquitin chains.
(a) Mtb PtpA interacts with TAB3 in the yeast two-hybrid assay. Yeast strains were transformed with indicated plasmid combinations in which TAK1-TAB2 interaction serves as a positive control. Left, high-stringency. Right, low-stringency. (b) Mutation of Mtb PtpA at D126A does not interfere with the binding between PtpA and TAB3-NZF. GST-tagged proteins (10 μg) were used to precipitate 10 μg of purified PtpA (D126A) protein. Precipitation of the PtpA mutant by GST-TAB3-NZF was eventually analyzed by immunoblot analysis. (c) The NZF domain of TAB3, but not its D690T and S691A mutant form, could precipitate ubiquitin. On the contrary, the T671D and A672S mutant form of the NZF domain of TAB2, but not its wild-type form, could precipitate ubiquitin. GST-tagged proteins (10 μg) were used to precipitate 10 μg purified PtpA (D126A) protein. (d) Mtb PtpA precipitates free ubiquitin (His6-Ub), but not Lys 63-linked ubiquitin (K63-Ub) chains, as analyzed by in vitro precipitation assay. (e) Mtb PtpA blocks TAB3-NZF from binding K63-Ub chains as shown in the in vitro precipitation assay. GST-tagged proteins (10 μg) were used to precipitate 2 μg of K63-Ub chains. Data are representative of one experiment with at least three independent biological replicates.
Supplementary Figure 8 TAB3 is critical to the PtpA-mediated suppression of cytokine production.
(a) TAB3 siRNA-2 could efficiently reduce the expression of TAB3 in both HEK293T and U937 cells as shown by immunoblot analysis. (b) Quantitative PCR analysis showed that TAB3 knockdown abolishes the PtpA-mediated down-regulation of IL1B mRNA expression in U937 cells infected with the indicated mycobacterial strains for 0–48 h. (c,d) ELISA showed that TAB3 knockdown abolishes the PtpA-mediated reduction in TNF protein (c) and IL-1β (d) released by U937 cells infected as in (b). (e) Model mechanisms by which Mtb PtpA modulates innate immune signaling. Quantitative PCR results are presented relative to expression of the control gene Gapdh (in b). *P < 0.05 and **P < 0.01 (two-tailed unpaired t-test). Data are representative of one experiment with at least three independent biological replicates (b–d; mean and s.e.m., n = 3).
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Wang, J., Li, BX., Ge, PP. et al. Mycobacterium tuberculosis suppresses innate immunity by coopting the host ubiquitin system. Nat Immunol 16, 237–245 (2015). https://doi.org/10.1038/ni.3096
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DOI: https://doi.org/10.1038/ni.3096
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