PEPTIDE FOR TREATING SEPSIS DERIVED FROM Rv2626c PROTEIN OF MYCOBACTERIUM TUBERCULOSIS

Abstract

The present invention relates to a use of a peptide containing an amino acid sequence represented by SEQ ID NO: 1 derived from the Rv2626c protein of Mycobacterium tuberculosis for treating sepsis. The peptide is excellent in inhibiting the inflammatory response induced by macrophages and is excellent in inhibiting the increase in the number of bacterial colonies, and thus can be usefully used for the treatment of sepsis.

Claims

1. A peptide for treating sepsis, wherein the peptide comprises an amino acid sequence represented by SEQ ID NO: 1.

2. The peptide of claim 1, wherein the peptide for treating sepsis comprises an amino acid sequence in which the amino acid sequence represented by SEQ ID NO: 1 is repeated 2 to 15 times.

3. The peptide of claim 1, wherein the peptide for treating sepsis comprises a cell penetrating peptide linked to an N-terminus of the amino acid sequence represented by SEQ ID NO: 1.

4. The peptide of claim 3, wherein the cell penetrating peptide is one selected from a group consisting of HIV-TAT (SEQ ID NO: 3), TAT (SEQ ID NO: 5), dNP2 (SEQ ID NO: 6), VP22 (SEQ ID NO: 7), MPG (SEQ ID NO: 8), PEP-1 (SEQ ID NO: 9), EB1 (SEQ ID NO: 10), transportan (SEQ ID NO: 11), p-Antp (SEQ ID NO: 12), hCT (18-32) (SEQ ID NO: 13), KLA (SEQ ID NO: 14) and oligoarginine (SEQ ID NO: 15).

5. The peptide of claim 1, wherein the peptide for treating sepsis includes a tuftsin peptide (SEQ ID NO: 2) linked to one end of the amino acid sequence represented by SEQ ID NO: 1.

6. The peptide of claim 5, wherein the tuftsin peptide is included repeatedly from 1 to 20 times.

7. The peptide of claim 1, wherein the peptide for treating sepsis is a fusion peptide in which a cell penetrating peptide, an amino acid sequence represented by SEQ ID NO: 1 and a tuftsin peptide are sequentially linked, or a fusion peptide in which the cell penetrating peptide, the tuftsin peptide and the amino acid sequence represented by SEQ ID NO: 1 are sequentially linked.

8. The peptide of claim 7, wherein each of the cell penetrating peptide, the amino acid sequence represented by SEQ ID NO: 1, and the tuftsin peptide are linked to one another with a peptide linker.

9. The peptide of claim 8, wherein the peptide linker is one selected from a group consisting of GG (SEQ ID NO: 16), (GGGGS)n (n=1-5) (SEQ ID NO: 17) and (EAAAK)n (n=1-5) (SEQ ID NO: 18).

10. A pharmaceutical composition for treating sepsis comprising the peptide for treating sepsis of claim 1 as an active ingredient.

11. A food composition for preventing or alleviating sepsis comprising the peptide for treating sepsis of claim 1 as an active ingredient.

12. A method for treating sepsis, comprising a step of administering the peptide for treating sepsis of claim 1 to a subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] These and/or other aspects, features, and advantages of the present invention will become apparent and more readily appreciated from the following description of example embodiments, taken in conjunction with the accompanying drawings of which:

[0040] FIG. 1A is a schematic diagram of Rv2626c cloned from the pET-23b (−) vector, FIG. 1B is a result of identifying the rRv2626c peptide (16 and 36 kDa) derived from the purified Mycobacterium tuberculosis by SDS-PAGE and immunoblotting, and FIG. 1C is a result of measuring the level of cytotoxicity after treatment of macrophages with the rRv2626c peptide;

[0041] FIG. 2A is a result of identifying the intracellular distribution location of rRv2626c by confocal microscopy after treatment of macrophages with the rRv2626c peptide, and FIG. 2B is a result of identifying the intracellular fraction analysis after treatment of macrophages with the rRv2626c peptide;

[0042] FIG. 3A is a result of identifying the cytokine production level after treatment of macrophages with the rRv2626c peptide, FIG. 3B is a result of identifying the cytokine production level after treatment of TLR2.sup.−/−, TLR4.sup.−/−, MyD88.sup.−/−, IRAK1.sup.−/−, TRAF6.sup.−/−, or TBK1.sup.−/− macrophages with the rRv2626c peptide, and FIG. 3C is a result of identifying endocytosis after treatment of TLR2.sup.−/− or TLR4.sup.−/− macrophages with the rRv2626c peptide;

[0043] FIG. 4A is a result of identifying the production level of inflammatory cytokines by treating macrophages with lipopolysaccharide (LPS) to induce an inflammatory response and then treating the macrophages with the rRv2626c peptide, and FIG. 4B is a result of identifying the production level of inflammatory cytokines by treating macrophages with Pam.sub.2CSK.sub.4 to induce an inflammatory response and then treating the macrophages with the rRv2626c peptide;

[0044] FIG. 5 is a result of identifying the activation of signal molecules involved in the inflammatory response of macrophages by treating LPS-treated macrophages with the rRv2626c peptide;

[0045] FIG. 6 is a result of identifying the intracellular binding partner of the rRv2626c peptide;

[0046] FIG. 7A is a result of identifying the binding of the rRv2626c peptide to TRAF6 by immunoprecipitation in HEK 293T cells, and FIG. 7B is a result of identifying the binding of the rRv2626c peptide to TRAF6 by immunoprecipitation in THP-1 cells;

[0047] FIG. 8 is a result of identifying the intracellular localization of the rRv2626c peptide and TRAF6 in macrophages;

[0048] FIG. 9A is a result of identifying the domain of the rRv2626c peptide interacting with TRAF6, and FIG. 9B is a result of identifying the amino acid sequence essential for interaction with TRAF6 in the CBS2 domain of the rRv2626c peptide;

[0049] FIG. 10A is a result of identifying the domain of TRAF6 involved in the interaction with rRv2626c, and FIG. 10B schematically shows the interaction between rRv2626c and TRAF6;

[0050] FIG. 11A is a result of identifying the change in the ubiquitination state of TRAF6 by rRv2626c with HA-ubiquitin (HA-Ub), FIG. 11B is a result of identifying the change in the ubiquitination state of TRAF6 by rRv2626c with lysine(K)48-linked ubiquitin (K48-Ub), and FIG. 11C is a result of identifying the change in the ubiquitination state of TRAF6 by rRv2626c with K63-linked ubiquitin (K63-Ub);

[0051] FIG. 12A is a result of identifying the change in the ubiquitination state of TRAF6 by rRv2626c with K48-Ub and K63-Ub, and FIG. 12B is a result of identifying the change in the ubiquitination state of TRAF6 by Tat-CBS2 (123-131) with K48-Ub and K63-Ub;

[0052] FIG. 13A shows the structure of the cell penetrating peptide-tuftsin-rRv2626c conjugate designed in one embodiment of the present invention, FIG. 13B is a result of purifying Rv2626c-CA (active conformation) and Rv2626c-DN (TRAF6 binding capacity loss form) peptides, and FIG. 13C is a result of identifying the cytotoxicity of the Rv2626c-CA and Rv2626c-DN peptides;

[0053] FIG. 14 is a result of identifying the production level of inflammatory cytokines by treating macrophages inducing inflammatory responses by treatment with LPS with the Rv2626c-CA or Rv2626c-DN peptide;

[0054] FIG. 15 is a result of identifying the binding to TRAF6 and the ubiquitination status of TRAF6 after treating LPS-treated macrophages with the Rv2626c-CA or Rv2626c-DN peptide;

[0055] FIGS. 16A, 16B, and 16C are results of identifying the change in mortality after administration of different doses of rRv2626c-CA or Rv2626c-DN peptides to mice having induced sepsis;

[0056] FIG. 17 is a result of identifying the level of cytokines in the serum after administration of different doses of rRv2626c-CA or Rv2626c-DN peptides to mice having induced sepsis;

[0057] FIG. 18 is a result of performing biopsies after administration of different doses of rRv2626c-CA or Rv2626c-DN peptides to mice having induced sepsis;

[0058] FIG. 19 is a result of identifying the binding to TRAF6 and the ubiquitination status of TRAF6 in splenocytes after administration of different doses of rRv2626c-CA or Rv2626c-DN peptides to mice having induced sepsis;

[0059] FIG. 20 is a result of identifying the in vivo distribution and residence time after administration of the fluorescent rRv2626c-CA peptide to mice having induced sepsis;

[0060] FIG. 21 is a result of identifying the cell types into which the rRv2626c-CA peptide is absorbed in the lungs, liver and spleen after administration of the rRv2626c-CA peptide to mice having induced sepsis;

[0061] FIG. 22 is a result of identifying the cell types into which the rRv2626c-CA peptide is absorbed by immunoprecipitation in the lungs, liver and spleen after administration of the rRv2626c-CA peptide to mice having induced sepsis;

[0062] FIG. 23A is a result of identifying the change in the number of bacterial colonies in to blood and peritoneal fluid after administration of the rRv2626c-CA peptide to mice induced to sepsis, and FIG. 23B is a result of identifying the degree of bacterial death in blood and peritoneal fluid after administration of the rRv2626c-CA peptide to mice induced to sepsis;

[0063] FIGS. 24A and 24B are results of identifying the number of immune cells recruited in the spleen and lung after administration of the rRv2626c-CA peptide to mice induced to sepsis;

[0064] FIG. 25 is a result of identifying the types of macrophages recruited in the spleen and lungs after administration of the rRv2626c-CA peptide to mice induced to sepsis;

[0065] FIG. 26 is a result of identifying the expression levels of scavenger receptor-A (SR-A), Fc receptor (FcR), innate immune receptors (TLR2 and TLR4), and neutrophil receptors (Neuropilin-1 (NRP1) and CXCR2) after treatment of TRAF6 expressing macrophages with rRv2626c-CA; and

[0066] FIG. 27 schematically shows the regulatory mechanism of the TLR signaling pathway mediated by rRv2626c and the sepsis treatment mechanism therethrough.

DETAILED DESCRIPTION

[0067] Hereinafter, preferred examples of the present invention are presented to aid in the understanding of the present invention. However, the following examples are provided only to facilitate a better understanding of the present invention and the scope of the present invention is not limited to the following examples.

[0068] Experimental Methods

[0069] 1. Mice and Cell Culture

[0070] Wild-type C57BL/6 mice were purchased from Samtaco Bio Korea (Gyeonggi-do, Korea). Primary bone marrow-derived macrophages (BMDMs) were isolated from C57BL/6 mice and cultured in DMEM supplemented with M-CSF (R&D Systems, 416-ML) for 3 to 5 days. BMDMs (TLR2.sup.−/−, TLR4.sup.−/−, MyD88.sup.−/−, IRAK1.sup.−/−, TRAF6.sup.−/−, and TBK1.sup.−/−) of C57BL/6 mice were provided by Dr. Cheol-Ho Lee (Laboratory Animal Center, Korea Research Institute of Bioscience and Biotechnology; Daejeon, Korea) and the experiment was conducted. HEK293T (ATCC-11268; American Type Culture Collection), RAW264.7 (ATCC TIB-71) and THP-1 (ATCC-TIB-202) cells were cultured in DMEM supplemented with 10% FBS (Gibco, USA), sodium pyruvate, nonessential amino acids, penicillin G (100 IU/ml), and streptomycin (100 μg/ml) or in RPMI1640 (Gibco). Transfection was performed transiently using calcium phosphate (Clonetech, USA) in 293T according to manufacturer's instructions. Stable cell lines of RAW264.7 and THP-1 were generated by transfection using standard selection methods with Lipofectamine 3000 (Invitrogen, USA) and G418 (400-800 μg/ml).

[0071] 2. Recombinant Protein Construction

[0072] In order to obtain Rv2626c (GenBank accession no. NP 217142.1) and CPP-Tuftsin-Rv2626c CBS2 (aa 123-131) protein of recombinant MTB H37Rv strain, Rv2626c amino acids (1-143) and CPP sequence (GRKKRRQRRRPQ), macrophage target sequence (TKPR), and CBS2 (LPEHAIVQF), each of the above sequences was cloned into the pRSFDuet-1 vector (Novagen) using the N-terminal 6×His tag according to the company's recommended protocol. Then, the vector was introduced into Escherichia coli BL21 (DE3) pLysS, and expression was induced to collect and purify the recombinant protein. Purified rRv2626c was dialyzed using a permeable cellulose membrane, and lipopolysaccharide (LPS) contamination was tested by Limulus amebocyte lysate assay (BioWhittaker). The rGRA7 protein experimented in the present invention contains less than 20 pg/ml of LPS.

[0073] 3. Reagents and Antibodies

[0074] LPS (Escherichia coli O111:B4) and BLP (Escherichia coli Braun Lipoprotein) (Pam2CSK4) were purchased from Invivogen. Phospho-(Ser473)-AKT, phospho-(Thr202/Tyr204)-p42/44, phospho-(Thr180/Tyr182)-p38, phospho-(Thr183/Tyr185)-SAPK/JNK, phospho-(Ser32/36)-IκB-α, K63-Poly-Ub (D7A11), and K48-Poly-Ub (D9D5) antibodies were purchased from Cell Signaling Technology (Danvers, Mass., USA). IκB-α (C-21), TRAF6 (H-274), Lamin B1 (B-10), Tubulin (5F131), CD68 (KP1), F4/80 (BM8), CD3 (PC3/188A), CD19 (5J25-C1), Ub (P4D1), His (His17), HA (12CA5), Flag (D-8), GST (B-14), Myc (9E10), and Actin (I-19) antibodies were purchased from Santa Cruz Biotechnology.

[0075] 4. Plasmid Design

[0076] HA-tagged ubiquitin (Ub), K48-related (linkage) specific ubiquitin (K48-Ub), and K63-related specific ubiquitin (K63-Ub) plasmids were purchased from Addgene. Plasmids containing all of TRAF6 and modified plasmids have been described in the related art (Infect Immun 84: 339-50). Plasmids encoding different regions of Rv2626c (1-143, 8-65, and 73-131, see Table 1) were amplified from the full-length Rv2626c cDNA by PCR amplification, and were generated by sub-cloning into a pEBG derivative encoding an N-terminal GST epitope tag between BamHI and NotI. All plasmids were derived from the pEBG-GST mammalian fusion vector and the pEF-IRES-Puro expression vector for excessive and stable expression in mammalian cells. All plasmid sequences were analyzed with the ABI PRISM 377 automatic DNA sequencer to identify whether they were 100% identical to the original sequences. FIG. 1A shows a schematic diagram of Rv2626c cloned from the pET-23b (−) vector.

[0077] 5. Peptides

[0078] Tat-linked Rv2626c peptides were synthesized by Peptron (Korea) and purified in the form of an acetate salt to avoid abnormal reactions in cells. The amino acid sequence of each peptide is shown in Table 1. The LPS concentration measured by Limulus amebocyte lysate assay (BioWhittaker) is contained in the peptide used in the experiment as less than 3-5 pg/ml.

TABLE-US-00001 TABLE 1 Names Sequences (From N to C) Tat RRRQRRKKRGY Tat-Rv2626c-(73-131) RRRQRRKKRGY-G-LARDSIYYVDA NASIQEMLNVMEEHQVRRVPVISEH RLVGIVTEADIARHLPEHAIVQF Tat-Rv2626c-(73-82) RRRQRRKKRGY-G-LARDSIYYVD Tat-Rv2626c-(83-92) RRRQRRKKRGY-G-ANASIQEMLN Tat-Rv2626c-(93-102) RRRQRRKKRGY-G-VMEEHQVRRV Tat-Rv2626c-(103-112) RRRQRRKKRGY-G-PVISEHRLVG Tat-Rv2626c-(113-122) RRRQRRKKRGY-G-IVTEADIARH Tat-Rv2626c-(123-131) RRRQRRKKRGY-G-LPEHAIVQF Tat-Rv2626c-(E125K) RRRQRRKKRGY-G-LPKHAIVQF Tat-Rv2626c-(E125Q) RRRQRRKKRGY-G-LPQHAIVQF Tat-Rv2626c-(H126D) RRRQRRKKRGY-G-LPEDAIVQF Tat-Rv2626c-(H126Q) RRRQRRKKRGY-G-LPEQAIVQF

[0079] 6. GST pulldown, Immunoblot and Immunoprecipitation Experiments

[0080] Cells were collected, and cells were lysed with NP-40 buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA) supplemented with a protease inhibitor (Roche) at a concentration of 1% (v/v). After centrifugation, the supernatant was reacted with protein A/G beads at 4° C. for 2 hours. The pretreated lysate was mixed with a 50% suspension of glutathione-binding sepharose beads (Amersham Biosciences) and reacted at 4° C. for 4 hours. After the reaction was completed, the precipitate was washed extensively with a lysis buffer. Proteins bound to glutathione beads were boiled for 5 minutes and eluted with SDS loading buffer.

[0081] For immunoprecipitation, cells were collected and lysed with NP-40 buffer to which a protease inhibitor was added. Cell lysate and protein A/G agarose beads were reacted at 4° C. for 1 hour to pre-clear the unnecessary reaction. Thereafter, 1-4 μg of antibody was added to 1 ml of the cell lysate, reacted at 4° C. for 8 to 12 hours, and protein A/G agarose beads were treated for 6 hours. The immunoprecipitates were then washed with lysis buffer, boiled for 5 minutes, and eluted with SDS loading buffer.

[0082] For immunoblotting, the polypeptide was denatured by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a PVDF membrane (Bio-Rad). After the PVDF membrane was reacted with a specific antibody, antibody binding was visualized by chemiluminescence (ECL; Millipore), and the signal was detected with a Vilber chemiluminescence analyzer (Fusion SL 3; Vilber Lourmat).

[0083] 7. Protein Purification and Mass Spectrometry

[0084] In order to identify the protein binding to rRv2626c, THP-1 cells were lysed with NP-40 buffer to which a protease inhibitor was added after treatment with or without rRv2626c for 30 minutes. After centrifugation, the supernatant was reacted with protein A/G beads at 40° C. for 2 hours to perform pre-clearing. The pre-cleared cell lysate was mixed with α-His antibody conjugated with agarose beads at 4° C. for 4 hours. Thereafter, the precipitates were washed with a lysis buffer, and the protein bound to the beads was eluted and then separated from a Nupage 4-12% Bis-Tris gradient gel (Invitrogen). After silver staining (Invitrogen), specific protein bands were extracted by ion-trap mass spectrometry, and amino acid sequences were determined by tandem mass spectrometry and database search.

[0085] 8. Quantitative Real-Time Polymerase Chain Reaction (RT-PCR) Total RNA was extracted from cells with RNeasy RNA extraction mini-kit (Qiagen). cDNA was synthesized with the Enzynomix kit (Enzynomix), and quantitative PCR was performed with a gene-specific primer set (Bioneer) and SYBR green PCR master mix (Roche). Real-time PCR was performed using QuantStudio™ 3 (ABI) according to manufacturer's instructions. Data were normalized to expression of (3-actin. The relative expression level was calculated using the delta-delta Ct method. The sequences of the primers are shown in Table 2.

TABLE-US-00002 TABLE 2 Genes Sequences mCD86 Forward gcacgtctaagcaaggtcac Reverse catatgccacacaccatccg miNOS Forward ccccgctactactccatcag Reverse ccactgacacttcgcacaaa mCD163 Forward tgtgaccatgctgaggatgt Reverse ctcgaccaatggcactgatg mArg1 Forward ctgagctttgatgtcgacgg Reverse tcctctgctgtcttcccaag mβ-Actin Forward aagtgtgacgttgacatc Reverse gatccacatctgctggaagg

[0086] 9. Confocal Fluorescence Microscopy

[0087] Immunofluorescence analysis was performed by confocal fluorescence microscopy. Cells were fixed on coverslips with 4% (w/v) paraformaldehyde dissolved in PBS, and cells were permeabilized with PBS containing 0.25% (v/v) Triton X-100 at 25° C. for 10 minutes. TRAF6 or His was detected by reacting with the primary antibody diluted in 1/100 at 25° C. for 1 hour. After washing, TRAF6 or His was reacted with a fluorescently labeled secondary antibody at 25° C. for 1 hour. Slides were checked with a laser scanning confocal microscope (model LSM 800; Zeiss).

[0088] 10. Cell Sorting

[0089] The cytosol and mitochondria were separated using the mitochondria fractionation kit (Active Motif, 40015). The intracellular fractionated protein was dissolved in a buffer containing 2% SDS, boiled with 2x reducing sample buffer, and then used for SDS-PAGE.

[0090] 11. MTT Assay

[0091] After culturing the cells of each experimental group for a specified time, 5 mg/ml of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution was added to the medium and cultured for an additional 4 hours. After removing all the media, the same volume of DMSO (dimethyl sulfoxide) solution was added to dissolve formazan for 15 minutes. Cell viability was identified by measuring the absorbance at 540 nm with a UV/VIS spectrophotometer.

[0092] 12. Flow Cytometry

[0093] Flow cytometry data were collected at FACSCanto (BD Biosciences, San Diego, Calif.) and analyzed with FlowJo software (Tree Star, Ashland, Oreg.). In order to identify the expression of cell surface proteins, cells and mAb were reacted at 4° C. for 20 to 30 minutes, and the cells were fixed with Cytofix/Cytoperm solution (BD Biosciences). The mAb clones used were as follows: NK1.1 (PK136, eBioscience), LY6G (1A8-Ly6g, eBioscience), SR-A (PSL204, eBioscience), FcR (MAR-1, eBioscience), TLR2 (6C2, eBioscience), TLR4 (HTA125, eBioscience), NRP1 (3DS304M, eBioscience) and CXCR2 (eBio5E8-C7-F10, 5E8-C7-F10, eBioscience).

[0094] 13. ELISA (Enzyme-Linked Immunosorbent Assay)

[0095] The levels of TNF-α, IL-6, IL-1(3, IL-12p40 and IL-10 were identified in cell culture supernatants and mouse serum with the BD OptEIA ELISA set (BD Pharmingen) according to the manufacturer's recommended procedure.

[0096] 14. Sepsis Induction and Checking Number of Bacteria

[0097] Cecal ligation and puncture (CLP) was performed on 6-week-old C57BL/6 female mice (Samtaco Bio). Specifically, mice were anesthetized with pentothal sodium (50 mg/kg, i.p.) and a small abdominal midline incision was made to expose the cecum. The cecum was then connected under the ileocecal valve, the surface was punctured twice with a 22-gauge needle, and the abdomen was sutured. The survival rate of mice was checked every day for 7 days, and all animals were bred in a pathogen-free environment.

[0098] The number of bacteria was checked as follows. Blood and peritoneal lavage fluid were collected from mice through cardiac foramen at defined times after CLP, and blood was serially diluted. Then, 5 μl of each diluted solution was plated into a blood agar plate, and the bacteria were cultured at 37° C. for 24 hours. The number of bacteria was calculated by counting colony forming units per total peritoneal lavage fluid or blood.

[0099] 15. Tissue Analysis

[0100] For tissue analysis, the spleen, liver and lungs of mice were fixed in 10% formalin and placed in paraffin. Paraffin sections (4 μm) were cut and stained with hematoxylin and eosin (H&E). Histopathological scores (0 to 4) were scored independently for each organ section by a pathologist based on the severity of inflammation and the number and distribution of inflammatory cells in the tissue without prior knowledge of the treatment group.

[0101] 16. In Vivo Imaging

[0102] rRv2626c-CA/Cy5.5 was prepared by adding streptavidin-binding Cy5.5 dye to rRv2626c-CA, and rRv2626c-CA/Cy5.5 was administered to CLP mice by intraperitoneal injection (i.p). In order to study tissue biodistribution, mice were sacrificed at different time zones after administration, and major organs were imaged with an IVIS spectrum-CT in vivo imaging system (PerkinElmer, Inc.).

[0103] 17. Statistical Analysis

[0104] All data were analyzed using Student's t-test with Bonferroni adjustment or ANOVA for multiple comparisons and described as mean±SD. Statistical analysis was performed with the SPSS (version 12.0) statistical software program (SPSS, Chicago, Ill., USA). Differences were considered significant at p<0.05. For survival, data were graphed and analyzed by the product restriction method of Kaplan and Meier using the log-rank (Mantele-Cox) test for comparison using GraphPad prism (version 5.0, La Jolla, Calif., USA).

[0105] Experiment Results

[0106] 1. In Macrophages, rRv2626c Regulates the Inflammatory Response Through the TLR Signaling Pathway.

[0107] In order to test the role of Rv2626c in the early immune response of macrophages, His-tagged rRv2626c was constructed. Purified rRv2626c (16, 36 kDa) was identified by SDS-PAGE and immunoblotting (FIG. 1B). The cytotoxicity induced by rRv2626c in macrophages did not show a significant difference (FIG. 1C). In addition, it was identified that rRv2626c was distributed in the cytoplasm of macrophages through the results of confocal microscopic analysis and cell fraction analysis (FIGS. 2A and 2B).

[0108] In a previous study, the present inventors identified that rRv2626c was attached to the cell surface and located in macrophages to regulate NF-κB and promote cytokine production. Accordingly, in order to examine the functional effect of rRv2626c in macrophages, the production amount of cytokine induced by rRv2626c was identified by ELISA.

[0109] As a result of identification, rRv2626c decreased the production of pro-inflammatory cytokines (TNF-α, IL-6 and IL-12p40) and increased the production of anti-inflammatory cytokines (IL-10) (FIGS. 3A and 3B). Those results were dependent on the TLR2-MyD88-TRAF6-IRAK1 pathway, not the TRIF-dependent (TBK1) pathway. In addition, rRv2626c was internalized into macrophages, and His-tagged rRv2626c was not detected only in cell lysates of rRv2626c-treated TLR2.sup.−/− BMDMs (FIG. 3C). These results suggest that rRv2626c is internalized into macrophages via TLR2, and that a TLR2-MyD88-dependent pathway is important in the inflammatory response mediated by rRv2626c.

[0110] 2. rRv2626c Inhibits TLR-Mediated Inflammatory Response in Macrophages.

[0111] In order to identify the functional role of Rv2626c in the TLR-mediated inflammatory signaling pathway, it was investigated whether Rv2626c inhibited the production of inflammatory cytokines induced by TLR4 (LPS) or TLR2/6 (Pam2CSK4). As a result of identification, rRv2626c attenuated the production of TLR-mediated pro-inflammatory cytokines (TNF-α, IL-6 and IL-12p40) and, conversely, promoted the production of anti-inflammatory cytokines (IL-10) (FIG. 4). Based on the above results, the present inventors hypothesized that rRv2626c would enter macrophages via TLR2 and that intracellular rRv2626c would perform any function to regulate signaling pathways induced by TLR ligands.

[0112] Since the PI3K, MAPK, and NF-κB signaling pathways are presently known to be involved in inflammatory signals of LPS-induced macrophages, it was investigated whether the activation of signaling proteins in these pathways is affected by rRv2626c. As a result, it was identified that LPS-induced phosphorylation of AKT, MAPK and IκBα and degradation of IκBα were inhibited by pretreatment with rRv2626c (FIG. 5). Taken together, these results suggest that rRv2626c acts as a modulator to inhibit TLR ligand-induced activation in PI3K, MAPK, and NF-κB signaling pathways, thereby inhibiting pro-inflammatory cytokine production and promoting anti-inflammatory cytokine production in macrophages.

[0113] 3. Rv2626c Interacts with TRAF6.

[0114] The present inventors hypothesized that rRv2626c would modulate the LPS-induced TLR signaling pathway through binding to innate immune signaling molecules. In order to identify the binding partner of rRv2626c, THP-1 cells were treated with His-tagged rVector or rRv2626c, cultured, and co-immunoprecipitated with His-agarose beads.

[0115] As a result of mass spectrometry, several endogenous proteins predicted to bind rRv2626c, including TLR2 (100 kDa), IRAK1 (80 kDa), RIP1 (70 kDa), TRAF6 (60 kDa), MAPK11 (41 kDa) and TOLLIP (32 kDa) were identified (FIG. 6). Immunoprecipitation results in 293T cells (ectopic expression of TRAF6) and macrophages showed that rRv2626c interacted strongly with TRAF6, although transiently (30 minutes to 60 minutes) (FIG. 7). In addition, according to confocal microscopy images of rRv2626c and TRAF6, rRv2626c was mainly co-located with endogenous TRAF6 in the cytoplasm of macrophages (FIG. 8). These results suggest that rRv2626c directly interacts with TRAF6, a signaling molecule of the TLR signaling pathway, at both endogenous and ectopic levels.

[0116] 4. The CBS2 Domain of Rv2626c Interacts with the N-Terminal Region of TRAF6 and Attenuates Poly-Ubiquitination of TRAF6.

[0117] As a result of identifying which domain of Rv2626c interacts with TRAF6 in HEK 293T cells, it was identified that the CBS2 domain of Rv2626c and TRAF6 bound (FIG. 9A).

[0118] In addition, the 9 to 10 amino acids sequences unit of the CBS2 domain was designed to find the detailed region of Rv2626c that mediates interaction with TRAF6, and in order to prevent proteolysis, a plurality of Tat-CBS2 peptides was prepared by adding the retro-inverso peptide HIV-1 Tat sequence to the 9 to 10 amino acids sequences of Rv2626c (Table 1). With this peptide, the minimum residue of Rv2626c that interacts with TRAF6 in 293T cells was identified.

[0119] As a result of the experiment, it was found that the Tat-CBS2 (123LPEHAIVQF131) peptide effectively blocked the TRAF6 and Rv2626c interaction, whereas the Tat peptide itself could not be blocked (FIG. 9B). In particular, 125EH126 amino acids with electrically charged side chains at 9 amino acids (123LPEHAIVQF131) of CBS2 were essential for interaction with TRAF6 and were important for minimal interaction. In addition, it was identified that the RING domain of TRAF6 is required for interaction with Rv2626c through detailed mapping of various truncation mutations of Rv2626c with TRAF6 (FIG. 10A). Taken together, these results suggest that Rv2626c is directly associated with the TRAF6 N-terminal RING domain via the CBS2 domain (aa 123-131), particularly a subregion of the CBS2 domain (FIG. 10B).

[0120] Since poly-ubiquitination of TRAF6 is important for activation of TRAF6 and downstream signaling pathways, it was investigated whether the interaction between Rv2626c and TRAF6 regulates the ubiquitination status of TRAF6.

[0121] As a result of western blotting, it was identified that expression of HA-ubiquitin (HA-Ub) caused poly-ubiquitination of Flag-TRAF6, and that ubiquitination of TRAF6 was attenuated by Rv2626c expression in a treatment concentration-dependent manner (FIG. 11A).

[0122] It is known that K48-linked ubiquitination mediates proteasome degradation of ubiquitin substrates, whereas K63-linked ubiquitination is essential for activation of NF-κβ and downstream signaling pathways. Immunoprecipitation was performed using HA-tagged lysine48-linked ubiquitin (K48-Ub) and K63-linked ubiquitin (K63-Ub) plasmids to determine the type of ubiquitin chain regulated by Rv2626c.

[0123] As a result, it was identified that overexpression of K48-Ub and K63-Ub induced ubiquitination in TRAF6 through ubiquitin binding, respectively (FIGS. 11B and 11C). In addition, although the treatment concentration of Rv2626c increased, the K48-Ub chain had no significant effect, but the K63-Ub chain was strongly inhibited in a manner dependent on the treatment concentration of Rv2626c (FIG. 12). These results suggest that Rv2626c and TRAF6 bind, and Rv2626c inhibits poly-ubiquitination of K63-Ub of TRAF6, which regulates activation of TRAF6 and downstream signaling pathways.

[0124] 5. The Tuftsin-Linked Rv2626c Peptide was Designed to Target Macrophages.

[0125] Tuftsin, four natural immunomodulatory peptides (Thr-Lys-Pro-Arg) derived from amino acid residues 289-292, a proteolytic product of IgG in the spleen, is known as phagocytosis-stimulating factors. Accordingly, the present inventors designed a cell penetrating peptide/tuftsin-conjugated Rv2626c peptide containing amino acids 123-131 of Rv2626c for macrophage targeting and immune response regulation (FIG. 13A).

[0126] In order to evaluate the function of peptides Rv2626c-CA (active conformation) and Rv2626c-DN (TRAF6 binding capacity loss form, including E125Q and H126Q mutations) peptides in macrophages, His-tagged rRv2626c-CA and Rv2626c-DN were produced in bacteria and purified by affinity chromatography. Purified rRv2626c-CA and Rv2626c-DN were approximately 10 kDa in size and were identified by SDS-PAGE and immunoblotting (FIG. 13B). As a result of MTT analysis, no significant difference in cytotoxicity was observed between rRv2626c-CA and Rv2626c-DN in BMDM as compared to the control group (rVehicle) (FIG. 13C).

[0127] In addition, it was identified whether rRv2626c-CA could mimic the pharmacological and biological profile of rRv2626c. As a result of identification, consistent with the activity of rRv2626c alone, rRv2626c-CA also modulated the inflammatory response in macrophages in a treatment concentration-dependent manner. Surprisingly, rRv2626c-CA exhibited an IC.sub.50 value of 10 ng/ml, a 250-fold increase compared to the IC50 of rRv2626c-WT of 2.5 μg/ml (FIG. 14). In addition, treatment with rRv2626c-CA dramatically inhibited the LPS-induced interaction between endogenous TRAF6 and Rv2626c and polyubiquitination of TRAF6 in Rv2626c-expressing cells (FIG. 15). In particular, no significant difference was observed in the inflammatory response, TRAF6 binding and polyubiquitination in the experimental group treated with rRv2626c-DN (TRAF6 binding loss) to macrophages or control group (rVehicle; tuftsin treatment) (FIGS. 14 and 15). Accordingly, rRv2626c-CA serves as a selective and potent inflammatory modulator for macrophage-targeted immune responses.

[0128] 6. rRv2626c-CA Protects Mice from Systemic Sepsis.

[0129] The present inventors investigated whether rRv2626c-CA protected mice from sepsis induced by several microorganisms.

[0130] First, the efficacy of rRv2626c-CA on CLP-induced mortality in mice was tested. When CLP mice were treated with rRv2626c-CA, 4 times, through intraperitoneal injection after 0, 6, 12 and 18 hours, the mortality was reduced in a dose-dependent manner. In particular, when rRv2626c-CA was administered to mice at a dose of 100 μg/kg, 90% of mice did not die. Surprisingly, rRv2626c-CA had an IC50 value of 20 μg/kg, which is 1000 times higher than the IC50 value of rRv2626c-WT (CPP+rRv2626c full length) (FIGS. 16A and 16B). No significant difference was observed in the survival graph in the rRv2626c-DN (TRAF6 binding loss) administration group or the control group (rVehicle; tuftsin treatment) (FIG. 16C).

[0131] Consistent with mortality data values, serum concentrations of pro-inflammatory cytokines such as TNF-α, IL-6, IL-1β, and IL-12p40 were significantly lowered in mice administered with rRv2626-CA. However, the concentration of IL-10 was significantly increased in mice administered with rRv2626c-CA (FIG. 17). This tendency was also similarly observed in H&E staining, and thus it was identified that in mice administered with rRv2626c-CA, the infiltration of immune cells was small and damage to the lungs, liver, and spleen was reduced (FIG. 18).

[0132] Next, it was identified whether rRv2626c-CA had in vivo pharmacological activity. In vivo detection of TRAF6 binding and its ubiquitination activity may be important for the evaluation of rRv2626c-CA in the discovery of candidate drugs for treating fatal inflammatory diseases.

[0133] Consistent with in vitro data (FIG. 12), rRv2626c-CA interacted with TRAF6 and decreased the level of ubiquitination of TRAF6 in splenocytes (FIG. 19A). After administration of fluorescent rRv2626c-CA (rRv2626c-CA/Cy5.5), as a result of identifying the biodistribution and pharmacokinetics in the body of mice, rRv2626c-CA/Cy5.5 remained in the body for 72 hours, and the highest concentration was observed in the liver 18 and 24 hours after administration (FIG. 20). In addition, the tissue distribution of therapeutic rRv2626c-CA was in macrophages, not T cells and B cells of the lung, liver, and spleen (FIGS. 21 and 22). Taken together, these results suggest that rRv2626c-CA is not rapidly degraded in the body and circulates for a considerable period of time, and has potential as a therapeutic agent to ameliorate CLP-induced sepsis in macrophages.

[0134] 7. rRv2626c-CA Enhances Bacterial Clearance.

[0135] CLP-induced mortality is correlated with the number and amount of bacterial colonies in peripheral blood and peritoneal fluid. For identification, as a result of intraperitoneal injection of rRv2626c-CA to CLP mice, the number of bacterial colonies in both peritoneal fluid and blood was significantly reduced (FIG. 23A). However, a direct killing effect of rRv2626c-CA on bacteria was not observed (FIG. 23B).

[0136] Since it has been reported that the bactericidal effect in the sepsis model is mainly mediated by the recruitment of neutrophils, it was investigated whether rRv2626c-CA increased the recruitment of immune cells in the sepsis model induced by CLP. In the rRv2626c-CA administration group, the number of macrophages recruited to the spleen and lungs increased significantly, but in the control group (rVehicle) and the rRv2626c-DN administration group, the number of macrophages did not increase (FIG. 24). In particular, macrophage recruitment by rRv2626c-CA increased type 2 macrophages and decreased type 1 macrophages (FIG. 25).

[0137] In order to investigate how rRv2626c-CA contributes to macrophage recruitment, the expressions of phagocytic receptor-A (scavenger receptor-A, SR-A), Fc receptor, innate immune receptor (TLR2, TLR4), and neutrophil receptor (Neuropilin-1 and CXCR2) were identified in macrophages under the presence of TRAF6. As a result of identification, rRv2626c-CA increased the expressions of phagocytic receptor-A and Fc receptor (FIG. 26). These results strongly suggest the differentiation and recruitment of type 2 macrophages induced by rRv2626c-CA, phagocytosis, and antibacterial effect by enhanced phagocytosis.

[0138] Through the experimental results so far, the present inventors have identified the following facts: (1) Rv2626c C-terminal amino acid 123-131 sequences including two electrically charged residues were required for binding of Rv2626c and TRAF6. (2) Rv2626c inhibited TLR4 inflammatory signaling in macrophages by interacting with the RING domain of TRAF6 and inhibiting K63-linked polyubiquitination of TRAF6. (3) A tuftsin-binding Rv2626c peptide (123LPEHAIVQF131) targeting macrophages in vitro and in vivo was designed and expressed, and thus (4) newly developed rRv2626c-CA has significantly improved efficacy and selectivity compared to rRv2626c-WT. (5) rRv2626c-CA effectively eliminated sepsis-inducing bacteria by differentiation and recruitment of type 2 macrophages and enhanced phagocytosis. (6) rRv2626c-CA protected mice from infection by various microorganisms, which means that rRv2626c-CA of the present invention may be used as a therapeutic agent suitable for treating sepsis and other microorganism-mediated diseases.

[0139] FIG. 27 shows the regulatory mechanism of the TLR signaling pathway mediated by rRv2626c and the sepsis treatment mechanism therethrough.

[0140] Hereinbefore, the description of the present invention is for illustrative purposes only. It will be understood by those skilled in the technical field to which the present invention pertains that the present invention may be embodied in various other specific forms without departing from the technical ideas or essential characteristics of the present invention. Therefore, the disclosed examples should be considered in an illustrative rather than a restrictive perspective in all aspects.