Mycobacterium tuberculosis Eis protein initiates suppression of host immune responses by acetylation of DUSP16/MKP-7 Kyoung Hoon Kima, Doo Ri Anb, Jinsu Songa, Ji Young Yoona, Hyoun Sook Kima, Hye Jin Yoona,HaNaImb, Jieun Kimb, Do Jin Kima, Sang Jae Leea, Ki-Hye Kimc, Hye-Mi Leec, Hie-Joon Kima, Eun-Kyeong Joc, Jae Young Leed, and Se Won Suha,b,1 Departments of aChemistry and bBiophysics and Chemical Biology, College of Natural Sciences, Seoul National University, Seoul 151-742, Korea; cDepartment of Microbiology, Chungnam National University School of Medicine, Daejeon 301-747, Korea; and dDepartment of Life Science, Dongguk University-Seoul, Seoul 100-712, Korea Edited by David Eisenberg, University of California, Los Angeles, CA, and approved April 3, 2012 (received for review December 9, 2011) The intracellular pathogen Mycobacterium tuberculosis (Mtb) components and play an essential role in the regulation of innate causes tuberculosis. Enhanced intracellular survival (Eis) protein, immune signaling during mycobacterial infection (6). Because secreted by Mtb, enhances survival of Mycobacterium smegmatis intracellular survival of Mtb plays a central role in its pathogenesis (Msm) in macrophages. Mtb Eis was shown to suppress host im- (7), it is important to understand the survival strategies of this mune defenses by negatively modulating autophagy, inflamma- bacterium within macrophages. Mtb has evolved a number of tion, and cell death through JNK-dependent inhibition of reactive highly effective survival strategies inside the macrophage (8). The oxygen species (ROS) generation. Mtb Eis was recently demon- best-characterized survival mechanism of Mtb is the inhibition of strated to contribute to drug resistance by acetylating multiple phagosomal maturation and autophagy, between which a func- amines of aminoglycosides. However, the mechanism of enhanced tional overlap was suggested (8–11). Both processes involve sev- intracellular survival by Mtb Eis remains unanswered. Therefore, eral steps, including fusion with lysosomes, and a number of we have characterized both Mtb and Msm Eis proteins biochemi- protein factors, such as Beclin 1 and vacuolar sorting protein 34 BIOCHEMISTRY cally and structurally. We have discovered that Mtb Eis is an effi- ɛ (VPS34), the class III phosphatidylinositol 3-kinase (12). The cient N -acetyltransferase, rapidly acetylating Lys55 of dual- identification and characterization of mycobacterial proteins that specificity protein phosphatase 16 (DUSP16)/mitogen-activated play a role in facilitating intracellular survival remain a priority for protein kinase phosphatase-7 (MKP-7), a JNK-specific phosphatase. α the development of new antituberculosis drugs. In contrast, Msm Eis is more efficient as an N -acetyltransferase. The Rv2416c gene of Mtb H37Rv strain was found to enhance We also show that Msm Eis acetylates aminoglycosides as readily intracellular survival of Mycobacterium smegmatis (Msm) in the as Mtb Eis. Furthermore, Mtb Eis, but not Msm Eis, inhibits LPS- human macrophage-like cell line U-937, and thus it was desig- induced JNK phosphorylation. This functional difference against nated as eis (enhanced intracellular survival) (7). The expression DUSP16/MKP-7 can be understood by comparing the structures of of its protein product directly correlated with the enhanced two Eis proteins. The active site of Mtb Eis with a narrow channel mycobacterial survival in U-937 cells (7). The Mtb Eis protein is fi seems more suitable for sequence-speci c recognition of the pro- produced during human tuberculosis infection and is released tein substrate than the pocket-shaped active site of Msm Eis. We into the culture medium (3). The sigma factor SigA was shown to propose that Mtb Eis initiates the inhibition of JNK-dependent bind to the eis promoter in the W-Beijing strain of Mtb, and the autophagy, phagosome maturation, and ROS generation by acety- activation of the Mtb eis gene correlated with increased SigA lating DUSP16/MKP-7. Our work thus provides insight into the levels and enhanced intracellular survival (13). Treatment of T mechanism of suppressing host immune responses and enhancing cells with Mtb Eis inhibited ERK1/2, JAK pathway, and sub- mycobacterial survival within macrophages by Mtb Eis. sequent production of TNF-α and IL-4 (14). Mtb Eis negatively regulated the secretion of TNF-α and IL-10 by primary human Rv2416c | lysine acetylation | antituberculosis drug monocytes in response to infection with the pathogen (15). Recently Mtb Eis was shown to suppress host innate immune early one-third of the world’s population is infected with defenses by negatively modulating inflammation, autophagy, and NMycobacterium tuberculosis (Mtb). This pathogenic bacte- cell death in a redox-dependent manner (16). The reported data rium causes tuberculosis, which claims the lives of millions of indicate that Mtb Eis plays an essential role in regulating both the people every year (1). Tuberculosis has also become a global early generation of reactive oxygen species (ROS) and in- health issue owing to the increased incidences of multidrug-re- flammatory responses in macrophages (16). It was also found that sistant and extensively drug-resistant strains of Mtb (2). This makes a search for targets of new antituberculosis drugs urgent. Mtb is a highly successful human pathogen, surviving and mul- Author contributions: K.H.K., D.R.A., E.-K.J., J.Y.L., and S.W.S. designed research; K.H.K., tiplying within the human macrophage cells of the infected D.R.A., J.S., J.Y.Y., H.S.K., H.J.Y., H.N.I., J.K., D.J.K., S.J.L., K.-H.K., and H.-M.L. performed people (3). Therefore, treatment of tuberculosis is difficult, re- research; J.S., H.-J.K., E.-K.J., and S.W.S. contributed new reagents/analytic tools; K.H.K., quiring many months of taking a combination of antibiotics. Mtb J.S., J.Y.Y., H.S.K., H.J.Y., H.-J.K., E.-K.J., J.Y.L., and S.W.S. analyzed data; and K.H.K., J.S., E.-K.J., J.Y.L., and S.W.S. wrote the paper. has the ability to persist in the form of a long-term asymptomatic The authors declare no conflict of interest. infection, referred to as latent tuberculosis (4). Latent tubercu- losis becomes activated when the body’s immune system is This article is a PNAS Direct Submission. weakened. As a result, tuberculosis is the major cause of death Data deposition: The crystallography, atomic coordinates, and structure factors reported in this paper have been deposited in the Protein Data Bank, www.pdb.org [PDB ID code among immuno-compromised AIDS patients (5). 3RYO (M. tuberculosis Eis, in complex with acetyl CoA), 3UY5 (M. tuberculosis Eis, apo), In mycobacterial infection, host innate immune responses may and 3SXN (M. smegmatis Eis, in complex with CoA)]. play a crucial role in early protection against Mtb infection, leading 1To whom correspondence should be addressed. E-mail: [email protected]. to establishment of effective adaptive immunity to tuberculosis This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (6). Additionally, MAPK pathways are activated by Mtb or its 1073/pnas.1120251109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1120251109 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 abrogated production of both ROS and proinflammatory cyto- aminoglycosides as quickly as, or more rapidly than, Mtb Eis (Fig. kines by Mtb Eis depends on its N-acetyltransferase domain in S2). Steady-state kinetic parameters, as measured by Km and kcat the N terminus (16). Enhanced macrophage survival by Mtb Eis values (Table S1), indicate that the aminoglycoside acetyltrans- was found to occur through the regulation of ROS signaling, ferase activity of Msm Eis is comparable to or higher than that of which was JNK-dependent but was not p38- or ERK1/2-de- Mtb Eis. This result cannot explain the enhanced intracellular pendent (16). Forced expression of dual-specificity protein survival of mycobacteria by Mtb Eis. phosphatase 16 (DUSP16), also called MAPK phosphatase- To understand the observed catalytic properties, we have de- 7(MKP-7), suppressed activation of MAPKs in COS-7 cells in the termined and compared the crystal structures of both Mtb and order of selectivity, JNK >> p38 > ERK, suggesting that Msm Eis proteins (Table S2 and SI Results and Discussion). The DUSP16/MKP-7 works as a JNK-specific phosphatase (17). crystal structure of selenomethionine-substituted Mtb Eis in the A bioinformatic analysis predicted that the Mtb Eis protein con- acetyl CoA-bound form was determined by de novo phasing using tains a single acetyltransferase domain of the GCN5-related N- the single anomalous diffraction data to 2.80 Å. This model was acetyltransferase (GNAT) superfamily in the amino terminus (15). used to solve the structures of Mtb Eis in the apo form at 2.46 Å The acetyltransferase domain is predicted to cover residues 9−160 of and Msm Eis in the CoA-bound form at 2.03 Å by molecular re- the 408-residue protein and contains a variant of the characteris- placement. The overall monomeric and hexameric structures of tic sequence motif (V/I-x-x-x-x-Q/R-x-x-G-x-G/A) for acetyltrans- Mtb and Msm Eis proteins are similar to each other (Fig. S3). That ferases at positions between 93 and 103 (93VAPTHRRRGLL103) is, each monomer of both Eis proteins comprises three “structural” (Fig. S1) (15, 18). Our sequence numbering of Mtb Eis follows domains, and six subunits are associated to form a hexamer of 32 the current EXPASY UniProtKB/Swiss-Prot database; six residues symmetry. “Structural” domain 1 adopts the GNAT fold, as pre- 1MPQSDS6 at the amino terminus are missing from other databases, dicted. Unexpectedly, “structural” domain 2 is also folded into the owing to a different translation initiation at Val7.