WO2010077005A2 - Mycobacteria-derived polypeptide and polynucleotide, and method of screening anti-mycobacterial agent - Google Patents

Abstract

A mycobacteria-derived polypeptide, a mycobacteria-derived polynucleotide, and a method of screening an anti-mycobacterial agent.

Description

MYCOBACTERIA-DERIVED POLYPEPTIDE AND POLYNUCLEOTIDE, AND METHOD OF SCREENING ANTI-MYCOBACTERIAL AGENT

The present invention relates to mycobacteria-derived polypeptide and polynucleotide, and a method of screening an anti-mycobacterial agent.

Tuberculosis (TB) is one of the most common infectious diseases in the world and continues to be a major health problem due to very low protective efficiency of the BCG vaccine, increase in opportunistic infections of AIDS patients, and increase in multi-drug-resistant tuberculosis (MDR-TB) infection that cannot be treated using conventional drugs. In order to overcome such problems, research into developing tuberculosis vaccines and tuberculosis-treating agents has been conducted. However, since the tuberculosis-treating agents developed so far are targeted to well known biosynthesis of mycolic acid, RNA transcription, FASI inhibition, arabino galactan synthesis, or protein synthesis of the mycobacterium tuberculosis, tuberculosis is not efficiently treated. Carbon monoxide dehydrogenase (CO-DH) is a novel target to treat tuberculosis which has been identified by the present inventors. It is expected that proteins related to the expression and activity of CO-DH may be novel targets to treat tuberculosis.

More than 150 types of mycobacteria have been known ()sofar. Among them, genome projects of 17 types of mycobacteria have been completed. Among the 17 types of mycobacteria, 14 types of mycobacteria have CO-DH, indicating that a lot of mycobacteria have CO-DH. In addition, based on a specific NO detoxification by CO-DH of mycobacteria, it was identified that mycobacteria have more CO-DH than other bacteria. In this regard, 7 genes that are well preserved are disposed in the vicinity of CO-DH of mycobacterium tuberculosis and influence the expression and activity of CO-DH. In order to search for targets to treat tuberculosis other than tuberculosis-treating agents or candidate tuberculosis-treating agents that are currently used, gene products influencing the expression and activity of CO-DH that commonly exists in mycobacterium tuberculosis, detoxifies NO, and have NO-DH activity have been targeted analyzed.

The present invention provides mycobacteria-derived polypeptide and polynucleotide.

The present invention provides a primer or probe specifically hybridized to a mycobacteria-derived nucleotide molecule.

The present invention provides an antibody specifically binding to a mycobacteria-derived polypeptide.

The present invention provides a recombinant vector including a mycobacteria-derived nucleotide sequence and promoter and a cell transformed thereby.

The present invention provides a method of screening an anti-mycobacterial agent.

According to an aspect of the present invention, there is provided mycobacteria-derived polypeptide and polynucleotide.

According to an aspect of the present invention, there is provided a primer or probe specifically hybridized to a mycobacteria-derived nucleotide molecule.

According to an aspect of the present invention, there is provided an antibody specifically binding to a mycobacteria-derived polypeptide.

According to an aspect of the present invention, there is provided a recombinant vector including a mycobacteria-derived nucleotide sequence and promoter and a cell transformed thereby.

According to an aspect of the present invention, there is provided a recombinant vector including a method of screening an anti-mycobacterial agent.

The mycobacteria-derived polypeptide and polynucleotide and the method of screening an anti-mycobacterial agent according to the present invention may be applied to develop an anti-mycobacterial agent that does not have side effects in the human body.

The above and other features and advantages of the present invention will become more apparent by describing in detail exemplary embodiments thereof with reference to the attached drawings in which:

FIG. 1 shows a method of obtaining Mycobacterium sp. strain JC1 cutI ^- mutant. Mycobacterium sp. strain JC1 cutI ^- mutant was selected using pSW60 having a mutated cutI gene having a middle portion deleted and a sacB gene and hygR gene for selecting a mutant.

FIG. 2 shows PCR results to identify Mycobacterium sp. strain JC1 cutI ^- mutant. Primers for analysis were cutI-F(5'-CCGAGACCATCGACTGGGTG-3') and cutI-R(5-GCGCATTACCGTGCGACGTG-3'). A PCR product having the length of 1,772-bp was obtained from Mycobacterium sp. strain JC1 wild type, and a PCR product having the length of 1,183-bp was obtained from Mycobacterium sp. strain JC1 cutI ^- mutant. Lane 1 is a size marker to identify the size of the PCR products, Lanes 2 to 7 show the PCR results of Mycobacterium sp. strain JC1 cutI ^- mutant candidates. As a result of PCR, candidates of lanes 2, 4, and 6 were identified as Mycobacterium sp. strain JC1 cutI ^- mutants.

FIG. 3 is a graph showing the growth of Mycobacterium sp. strain JC1 cutI ^- mutant using glucose. In order to identify phenotyes of Mycobacterium sp. strain JC1 cutI ^- mutant, Mycobacterium sp. strain JC1 wild type and Mycobacterium sp. strain JC1 cutI ^- mutant were cultured in a SMB-glucose medium to obtain growth curves. There was no difference between Mycobacterium sp. strain JC1 wild type and Mycobacterium sp. strain JC1 cutI ^- mutant with glucose.

FIG. 4 is a graph showing the growth of Mycobacterium sp. strain JC1 cutI ^- mutant using CO. In order to identify phenotyes of Mycobacterium sp. strain JC1 cutI ^- mutant, Mycobacterium sp. strain JC1 wild type and Mycobacterium sp. strain JC1 cutI ^- mutant were cultured in a SMB-CO medium to obtain growth curves. Mycobacterium sp. strain JC1 cutI ^- mutant could not grow with CO.

FIG. 5 is a graph showing a complementation test of Mycobacterium sp. strain JC1 cutI ^- mutant. As a result of the complementation test of Mycobacterium sp. strain JC1 cutI ^- mutant, it was identified that Mycobacterium sp. strain JC1 cutI ^- mutant could not grow with CO because of cutI mutation.

FIG. 6 shows that CO-DH protein is not expressed in Mycobacterium sp. strain JC1 cutI ^- mutant. Lane 1 and 2 show CBB staining results. It was identified that other proteins were similarly expressed in Mycobacterium sp. strain JC1 cutI ^- mutant ( Lane 2, 4 and 6) but CO-DH was not expressed in Mycobacterium sp. strain JC1 cutI ^- mutant when compared with Mycobacterium sp. strain JC1 wild type( Lane 1, 3 and 5). Lane 3 and 4 show CO-DH active staining results. It was identified that Mycobacterium sp. strain JC1 cutI ^- mutant did not have CO-DH activity since CO-DH was not expressed in Mycobacterium sp. strain JC1 cutI ^- mutant according to the western blot results using Mycobacterium sp. strain JC1 CO-DH antibody(Lane 5 and 6).

FIG. 7 shows the transcription of cutBCA in Mycobacterium sp. strain JC1 cutI ^- mutant. It was identified that transcription of cutBCA did not occur in Mycobacterium sp. strain JC1 cutI ^- mutant according to northern blot using cutB as a probe. Lane 1 and 2 show the total RNA extracted. Lane 3 and 4 show the northern blot results using cutB as a probe. Lane 1 shows the total RNA extracted from Mycobacterium sp. strain JC1. Lane 2 shows the total RNA extracted from Mycobacterium sp. strain JC1 cutI ^- mutant. The RNA was extracted after Mycobacterium sp. strain JC1 and Mycobacterium sp. strain JC1 cutI ^- mutant were cultured to an exponential growth phase in a SMB-glucose medium.

FIGS. 8 to 11 are graphs showing activity of cutBCA promoter in Mycobacterium sp. strain JC1 cutI ^- mutant. As a result of identifying the activity of cutBCA promoter in Mycobacterium sp. strain JC1 cutI ^- mutant during the growth phases, copyI cutBCA and copyII cutBCA did not have activity. Since it is known that the expression of CO-DH increases in a stationary phase in Mycobacterium sp. strain JC1,the activity of cutBCA promoter in Mycobacterium sp. strain JC1 cutI ^- mutant was identified in growth phases(#1, #2 and #3 of FIG. 8),mid-exponential growth phase (FIG. 9),early-stationary phase(FIG. 10),and mid-stationary phase(FIG. 11).

FIG. 12 is a graph showing inhibition of growth of Mycobacterium sp. strain JC1 cutI ^- mutant by NO. The survival rate of Mycobacterium sp. strain JC1 cutI ^- mutant(-○-)was reduced by about 80% compared to that of Mycobacterium sp. strain JC1 wild type(-●-)as a result of measuring resistance of Mycobacterium sp. strain JC1 and Mycobacterium sp. strain JC1 cutI ^- mutant against NO using SNP generating NO.

Hereinafter, the present invention will now be described more fully with reference to the accompanying drawings, in which exemplary embodiments of the invention are shown

According to an embodiment of the present invention, there is provided a mycobacteria-derived polypeptide having an amino acid sequence of SEQ ID NO: 2.

A macrophage is an immune cell eliminating pathogens invading a human body. The macrophage swallows the pathogens and produces various compounds, e.g., nitric oxide (NO), to digest and break down the pathogens. However, some pathogenic bacteria may live and proliferate in the macrophage by inhibiting the functions of NO or avoiding the influence of NO using a natural NO inhibitor produced by the macrophage or by self-producing an enzyme detoxifying NO. It is known that pathogenic mycobacteria contain an enzyme such as NO dioxygenase or peroxynitritase which oxidize NO in the macrophage. It is also known that carbon monoxide dehydrogenase according to an embodiment of the present invention oxides NO.

The “mycobacteria” used herein are classified as aerobic acid fast gram-positive bacteria with a thick cell wall that is waxy, hydrophobic, and rich in mycolic acid. Mycobacteria may be classified into non-pathogenic mycobacteria that grow fast and pathogenic mycobacteria that grow slow. The non-pathogenic mycobacteria may be Mycobacterium fortuitum, Mycobacterium parafortuitum, Mycobacterium vaccae, Mycobacterium flavescens, Mycobacterium phlei, Mycobacterium cuneatum, Mycobaterium gastri, Mycobacterium ID-Y, Mycobacterium flavescens, Mycobacterium neoaurum, Mycobacterium peregrinum, Mycobacterium diernhoferi, Mycobacterium smegmatis, Mycobacterium wolinsky, Mycobacterium sp. strain JC1, or the like. The pathogenic mycobacteria may be Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium avium, Mycobacterium ulcerans, Mycobacterium abscessus, Mycobacterium chelonae, Mycobacterium asiaticum, Mycobacterium porcinum, or the like. It has been reported that quite a large number of these mycobacteria use carbon monoxide as an only carbon source and energy source. Researchers have found features of mycobacteria based on the non-pathogenic mycobacteria having genetic features similar to those of the pathogenic mycobacteria for convenience of cultivation and safety.

The “carboxydobacteria” used herein indicates bacteria that grow using carbon monoxide as a sole carbon source and energy source.

The carbon monoxide dehydrogenase (CO-DH) is a key enzyme that oxidizes carbon monoxide into carbon dioxide in carboxydobacteria according to the following reaction: CO + H₂O→CO₂+2H₊+2e^-. In addition, the carbon monoxide dehydrogenase oxidizes toxic nitrogen monoxide into non-toxic nitrogen dioxide in mycobacteria. According to this mechanism, pathogenic mycobacteria may be alive in a macrophage. Thus, the carbon monoxide dehydrogenase may be used together with nitric oxide dehydrogenase (NO-DH). In addition, a structural gene of the carbon monoxide dehydrogenase of Mycobacterium sp. strain JC1 may be represented by “cutBCA”. The “Mycobacterium sp. strain JC1”that will be used in the following examples belongs to mycobacteria and also belongs to carboxydobacteria. 16S rRNA gene of Mycobacterium sp. strain JC1 shares at least 96% similarity with 16S rRNA gene of the other mycobacteria and up to 99% similarity with 16S rRNA of some mycobacteria, e.g., M. peregrinum, M. wolinsky, etc. Mycobacterium sp. strain JC1 also contains 10-methyl C_18:0 that is also referred to as tuberculostearic acid and has fatty acid distribution that is similar to that of the other mycobacteria.

The mycobacteria-derived polypeptide according to the present embodiment has an amino acid sequence SEQ ID NO: 2. The polypeptide having an amino acid sequence of SEQ ID NO: 2 is a protein activating the transcription of carbon monoxide dehydrogenase gene or the expression of the carbon monoxide dehydrogenase.

The mycobacteria-derived polypeptide according to the present embodiment may have an amino acid sequence representing substantial identity to the amino acid sequence. The substantial identity indicates at least 60% of homology, preferably 80% of homology, and more preferably 90% of homology, when the amino acid sequence and another sequence are aligned so as to maximally correspond to each other and the aligned sequences are analyzed using an algorithm that is commonly used in the art. Aligning methods to compare sequences are well known in the art. Various methods and algorithms for alignment are disclosed by Smith and Waterman, Adv. Appl. Math. 2:482(1981); Needleman and Wunsch, J. Mol. Bio. 48:443(1970); Higgins and Sharp, Gene 73:237-44(1988);Higgins and Sharp, CABIOS 5:151-3(1989);Corpet et al.,Nuc. Acids Res. 16:10881-90(1988);Huang et al.,Comp. Appl. Bio Sci. 8:155-65(1992) and Pearson et al.,Meth. Mol. Biol. 24:307-31(1994). NCBI Basic Local Alignment Search Tool (BLAST) may be accessed at ,the webpage of the National Center for Biological Information(NCBI) and may be used with a sequence analysis program such as blastp, blasm, blastx, tblastn, and tblastx. A method of comparing sequence homology using this program may be found at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

The mycobacteria-derived polypeptide may also include amino acid sequence variants in addition to those having natural amino acid sequence. The mycobacteria-derived polypeptide variants refer to proteins having a different sequence since by deletion, insertion, non-conservative substitution, or conservative substitution of at least one amino acid residue of the natural amino acid sequence of the mycobacteria-derived polypeptide or a combination thereof. Amino acid substitutions in proteins and peptides which do not change activities of molecules are known in the art. The common amino acid substitutions are found between Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly. The amino acid may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, and farnesylation.

The mycobacteria-derived polypeptide or their variants may be extracted from natural resources, synthesized, or prepared using a recombination process based on DNA sequences.

According to an embodiment of the present invention, there is provided a mycobacteria-derived polynucleotide having (a) a nucleotide sequence coding the amino acid having SEQ ID NO: 2; a nucleotide sequence complementary to the nucleotide sequence of (a); or a nucleotide sequence obtained by hybridizing the nucleotides of (a) and (b) under stringent conditions.

The “nucleotide” used herein is a double-stranded or single-stranded deoxyribonucleotide or ribonucleotide. The nucleotide may include a RNA genome sequence, a cDNA, a RNA sequences transcribed thereby, and analogs of natural nucleotides unless specifically stated otherwise.

The nucleotide according to the present embodiment includes not only a nucleotide coding the amino acid having SEQ ID NO: 2 but also its complementary sequence. The complementary sequence may include a perfectly complementary sequence and a substantially complementary sequence. The “substantially complementary sequence” used herein is a sequence that may be hybridized to the nucleotide of SEQ ID NO: 1 under stringent conditions that are known in the art. The “stringent conditions” used herein are disclosed in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001) and Haymes, B. D.,et al.,Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington,D.C.(1985), and may be determined by controlling temperature, ionic strength(concentration of a buffer solution), the existence of a compound such as an organic solvent, and hybridized sequence. For example, the stringent conditions may be obtained by a) washing with 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50℃, or b) hybridizing in a hybridization buffer solution (50% formamide, 2 x SSC and 10% dextran sulfate) at 55℃ and washing with EDTA-containing 0.1 x SSC at 55℃.

The nucleotide sequence may include a nucleotide sequence coding the amino acid having SEQ ID NO: 2 or a nucleotide sequence hybridized with a nucleotide sequence complementary to the nucleotide sequence coding the amino acid having SEQ ID NO: 2 under stringent conditions. The stringent conditions may be obtained by hybridizing in a hybridization buffer solution (50% formamide, 2 x SSC and 10% dextran sulfate) at 55℃ and washing with EDTA-containing 0.1 x SSC at 55℃.

Meanwhile, the nucleotide sequence may include a part, i.e., a fragment, of the nucleotide sequence coding the amino acid having SEQ ID NO: 2. For example, the mycobacteria-derived nucleotide may include a nucleotide having SEQ ID NO: 1.

According to an embodiment of the present invention, there is provided a primer or probe specifically hybridized to the mycobacteria-derived nucleotide.

The “primer” used herein is a single-stranded oligonucleotide functioning as an origin of polymerization of template DNA under an appropriate conditions (i.e., 4 types of different nucleoside triphosphates and polymerases) at a suitable temperature and in a suitable buffer solution. The length of the primer may vary according to various factors, for example, temperature and the use of the primer, but the primer may have 15 to 30 nucleotides. Generally, a short primer molecule may form sufficiently stable hybrid complexes with a template at a low temperature.

The sequence of the primer is not required to be perfectly complementary to a part of the sequence of the template. The primer may have sufficient complementarity to be hybridized with the template and perform intrinsic functions of the primer. Thus, a primer set is not required to be perfectly complementary to the sequence of the nucleotide that is a template. The primer set may have sufficient complementarity to be hybridized with the sequence and perform intrinsic functions of the primer. The primer may be designed based on cDNA sequence of the nucleotide, for example, using a program for designing primers (PRIMER 3 program).

Meanwhile, the primer according to the present embodiment may be hybridized or annealed to a part of the template to form a double-strand structure. Conditions for hybridizing nucleotides suitable for forming the double-stranded structure are disclosed by Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001) and Haymes, B. D.,et al.,Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington,D.C.(1985).

The “probe” used herein is a deoxyribonucleotide or ribonucleotide which may be hybridized to a specific nucleotide sequence. Preferably, the probe may be a single strand for increasing efficiency of hybridization. The probe may be a deoxyribonucleotide.

The probe according to the present embodiment may include a perfectly complementary sequence to the nucleotide or a substantially complementary sequence that does not inhibit specific hybridization. Conditions suitable for the hybridization are described above.

According to an embodiment of the present invention, there is provided an antibody specifically binding to a mycobacteria-derived polypeptide.

The antibody specifically binding to a mycobacteria-derived polypeptide according to the present embodiment may be a polyclonal or monoclonal antibody, and preferably a monoclonal antibody. The antibody specifically binding to a mycobacteria-derived polypeptide may be prepared using a method that is commonly used in the art, for example, fusion, recombination of DNA, or phage antibody library. Methods of preparing antibody are disclosed by Harlow, E. and Lane, D., Using Antibodies:A Laboratory Manual, Cold Spring Harbor Press, New York, (1999); and Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY(1991), which are incorporated herein by reference. For example, hybridoma cells producing a monoclonal antibody may be prepared by fusing an immortal cell line with an antibody-producing lymphocyte, which is well known in the art. The polyclonal antibody may be prepared by administering the mycobacteria-derived polypeptide antigen to an animal by injection, collecting an anti-serum from the animal, and isolating an antibody from the anti-serum using affinity chromatography.

According to an embodiment of the present invention, there is provided a recombinant vector having a nucleotide sequence coding the mycobacteria-derived polypeptide and a promoter operatively linked to the mycobacteria-derived polynucleotide.

In the recombinant vector, the mycobacteria-derived nucleotide is operatively linked to the promoter. The “operatively linked” used herein indicates a functional binding between a nucleotide expression controlling sequence (e.g., promoter sequence) and other nucleotide sequence, and the nucleotide expression controlling sequence may control transcription and/or translation of the other nucleotide sequence.

The vector may be a vector for cloning or expression. In addition, a prokaryotic cell or a eukaryotic cell may be used as a host cell for the vector. Since the polynucleotide is derived from bacteria, a prokaryotic cell may be used as a host cell for the convenience of cultivation.

For example, if the vector is an expression vector and a prokaryotic cell is used as a host cell, the vector may include a promoter capable of initiating transcription, such as tac promoter, lac promoter, lacUV5 promoter, lpp promoter, p_L ^λ promoter, p_R ^λ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, a ribosome-binding site to initiate translation, and a transcription/translation termination sequence. If E. coli is used as a host cell, a promoter and operator sites of biosynthetic pathway of E. coli tryptophan and left-side promoter of phage λ(e.g.,p_L ^λ promoter) may be used as a controlling site.

Meanwhile, the recombinant vector according to the present embodiment may be prepared using plasmid, such as pSC101, ColE1, pBR322, pUC8/9, pHC79, pUC19, and pET, phage, such as λgt4λB, λ-Charon, λΔz1 and M13, or virus, such as SV40, which are widely used in the art.

The recombinant vector according to the present embodiment may be fused to another sequence in order to facilitate purification of mycobacteria-derived protein expressed from the recombinant vector. For example, the fused sequence may be glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA) and hexahistidine (6x His, Qiagen, USA), and preferably 6x His. Due to the additional sequence for the purification, protein expressed in the host cell may be quickly and efficiently purified through affinity chramoatography.

Meanwhile, the expression vector is a selectable marker that may include an antibiotic-resistant gene commonly used in the art. For example, the expression vector may include genes resistant to ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, hygromycin and tetracycline.

According to an embodiment of the present invention, there is provided a cell transformed by the recombinant vector.

In order to prepare the transformed cell according to the present embodiment, a genome DNA and its transcripts may be used. A method of preparing transcripts is known in the art. In order to prepare transcripts using the recombinant vector, the vector is required to be linearized.

The host cell for transcription may be any host cell that is known in the art. For example, E. coli JM109, E. coli BL21(DE3), E. coli RR1, E. coli LE392, E. coli B, E. coli X1776, E. coli W3110, Bacillus sp. strains such as Bacillus subtilis and Bacillus thuringiensis, intestinal flora such as Salmonella tryphimurium, Sarratia marcescens and various pseudomonas, and bacteria.

If the host cell is a prokaryotic cell, the vector may be transported to the host cell during the transformation using a CaCl₂ method, a Hanahan method, and electroporation.

According to an embodiment of the present invention, there is provided a method of screening an anti-mycobacterial agent, the method including: (a) contacting a sample to be assayed to a cell including the mycobacteria-derived polypeptide or the mycobacteria-derived polynucleotide; and (b) measuring the activity of the polypeptide, the amount of transcribed or expressed polynucleotide, wherein if the activity of the polypeptide, or the amount of transcribed or expressed polynucleotide is down-regulated, the sample is determined to be an anti-mycobacterial agent.

According to the method, a substance to be screen is contacted to a cell containing the mycobacteria-derived polypeptide or the mycobacteria-derived polynucleotide.

The “sample” used herein is an unknown substance to be screened in order to detect whether the sample influences the activity of the polypeptide and the amount of transcribed or expressed polynucleotide. The sample may include chemicals, nucleotides, antisense-RNA, small interference RNA (siRNA), and natural extract, but is not limited thereto.

The cell may include any cell containing the mycobacteria-derived polypeptide or the mycobacteria-derived polynucleotide, for example, transformed E. coli or wide type mycobacteria. Examples of the transformed microorganism or wild type mycobacteria used for the screening are as follows.

The mycobacteria used in the present embodiment may include Mycobacterium fortuitum, Mycobacterium parafortuitum, Mycobacterium vaccae, Mycobacterium flavescens, Mycobacterium phlei, Mycobacterium cuneatum, Mycobaterium gastri, Mycobacterium ID-Y, Mycobacterium flavescens, Mycobacterium neoaurum, Mycobacterium peregrinum, Mycobacterium diernhoferi, Mycobacterium smegmatis, Mycobacterium wolinsky, Mycobacterium sp. strain JC1, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium avium, Mycobacterium ulcerans, Mycobacterium abscessus, Mycobacterium chelonae, Mycobacterium asiaticum or Mycobacterium porcinum, preferably, Mycobacterium vaccae, Mycobacterium flavescens, Mycobacterium phlei, Mycobaterium gastri, Mycobacterium flavescens, Mycobacterium neoaurum, Mycobacterium smegmatis, Mycobacterium sp. strain JC1, Mycobacterium tuberculosis, Mycobacterium bovis, or Mycobacterium avium, and more preferably, Mycobacterium smegmatis, Mycobacterium sp. strain JC1 or Mycobacterium tuberculosis.

The sample that is assayed by the screening may be a single compound or a mixture of compounds, for example, natural extract or cell or tissue culture. The sample may be obtained from a library of synthesized or natural compounds. A method of obtaining the library is well known in the art. The library of the synthesized compounds may be purchased from Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA) and Sigma-Aldrich (USA), and the library of the natural compounds may be purchased from Pan Laboratories (USA) and MycoSearch (USA).

Then, the activity of the polypeptide and the amount of transcribed or expressed polynucleotide in the cell are measured. If the activity of the polypeptide or the amount of transcribed or expressed polynucleotide is down-regulated, the substance may be determined as an anti-mycobacterial agent.

The amount of polypeptide may be measured using various analytical methods. For example, the amount of polypeptide may be measured by various immunoanalytical formats using antibodies specifically binding to the polypeptide. The immunoanalysis may be conducted by various quantitative immunoanalytical protocols. The immunoanalytical format may include a radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, sandwich assay, immunofluorescence and immunoaffinity purification, but is not limited thereto.

For example, if the screening method is conducted for a radioimmunoassay, an antibody marked with a radioisotope such as C¹⁴,I¹²⁵,P³²and S³⁵ may be used to detect the polypeptide.

If the screening method is conducted for an ELISA, the method may include: coating degradation products of an unknown cell on the surface of a solid substrate; reacting a polypeptide-specific antibody, as a primary antibody, with the degradation products of the cell; reacting the resultants with a secondary antibody binding to an enzyme; and measuring the activity of the enzyme.

The solid substrate may be a hydrocarbon polymer (for example, polystyrene and polypropylene), glass, metal or gel, and preferably microtiter plate.

The enzyme bound to the secondary antibody may include an enzyme catalyzing color development, fluorescence, luminescence, or infrared light interactance, but is not limited thereto. For example, alkaline phosphatase, β-galactosidase, horseradish peroxidase, luciferase, and cytochrome P₄₅₀ may be used as the enzyme. If the enzyme binding to the secondary antibody is alkaline phosphatase, the substrate may be a color developing substrate such as bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate, and enhanced chemifluorescence (ECF). If the enzyme is horseradish peroxidase, the substrate may be chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, bis-N-methylacryidinium nitrate (lucigenin), resorufin benzyl ether, luminol, 10-acetyl-3,7-dihydroxyphenoxazine (amflex red), p-phenylenediamine-HCl and pyrocatechol (HYR), tetramethylbenzidine (TMB), 2,2‘-Azine-di[3-ethylbenzthiazoline sulfonate] (ABTS), o-phenylenediamine(OPD) and naphthol/pyronine, glucose oxidase, nitroblue tetrazolium(t-NBT) and phenzaine methosulfate(m-PMS).

If the screening method is conducted for a capture-ELISA, the method may include: coating an antibody of the polypeptide, as a capturing antibody, on the surface of a solid substrate; reacting the capturing antibody with a cell sample; reacting the resultants with a detecting antibody binding to a label generating a signal and specifically reacting with the polypeptide; and measuring the signal generated by the label.

The detecting antibody includes a label generating a detectable signal. The label may include chemicals (e.g., biotin), enzymes (alkaline phosphatase, β-galactosidase, horseradish peroxidase, luciferase, and cytochrome P₄₅₀),radio active materials(C¹⁴,I¹²⁵,P³² and S³⁵), fluorescent materials(fluorescein), luminescent materials, chemiluminescent materials and fluorescence resonance energy transfer(FRET) material,but is not limited thereto.

In the ELISA and capture-ELISA, the activity of the enzyme or the signal may be measured using a method that is well known in the art. By detecting the signal, the polypeptide may be quantitatively analyzed. If biotin is used as the label, the signal may be efficiently detected using streptavidin. If luciferase is used, the signal may be efficiently detected using luciferin.

If the signal from the polypeptide in the sample is weaker than that of non-treated control, the sample is determined as an anti-mycobacterial agent.

The amount of transcribed or expressed polynucleotide may be measured using methods that are well known in the art. For example, the amount of transcribed or expressed polynucleotide may be measured by hybridization using RT-PCR, northern blotting, cDNA microarray or insituhybridization.

By the RT-PCR protocol, the total RNA is isolated from the cell, and a single-stranded cDNA using an oligo dT primer (e.g., prokaryotic cell) or a primer specific to a gene to be identified (e.g., eukaryotic cell), and a reverse transcriptase. Then, a PCR is conducted using the single-stranded cDNA as a template and a polynucleotide-specific primer set. Then, PCR products are subjected to electrophoresis, and bands obtained thereby are analyzed to measure the change of the amount of expressed cutIgene.

If the activity of the polypeptide or the amount of transcribed or expressed polynucleotide is down-regulated, the sample may be determined to be an anti-mycobacterial agent.

According to the present embodiment, the anti-mycobacterial agent may be a substance for preventing or treating Buruli ulcer, lymphadenitis, or tuberculosis.

A variety of pathogenic mycobacteria including M. tuberculosis H37Rv that is a mycobacterium tuberculosis have CO-DH(e.g.,M. tuberculosis C, M. tuberculosis CDC1551, M. tuberculosis F11, M. bovis, M. tuberculosis sp. Haarlem, M. marinum, M. ulcerans, or the like). Among them, some bacteria, for example, M. marinum, M. ulcerans, etc., may induce another disease such as Buruli ulcer and lymphadenitis as well as tuberculosis. Whether they are alive or not is determined by eliminating toxicity of NO in microphage so as to induce diseases. The anti-mycobacterial agent may be used to prevent or treat Buruli ulcer and tuberculosis, and preferably to prevent or treat tuberculosis. The tuberculosis screened by the method according to the present embodiment may include ocular tuberculosis, cutaneous tuberculosis, adrenal tuberculosis, renal tuberculosis, tuberculosis of epididymis, tuberculosis of lymph nodes, laryngeal tuberculosis, tuberculosis of middle ear, intestinal tuberculosis and pulmonary tuberculosis, but is not limited thereto.

The anti-mycobacterial agent developed according to the screening method may be efficiently used to treat diseases related to mycobacteria with conventional anti-mycobacterial agents.

The present invention will be described in further detail with reference to the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the invention.

Examples

1. Experimental Method

Strains and cultivation of bacteria

Bacterium used herein was Mycobacterium sp. strain JC1. Bacteria were cultured in a standard mineral base (SMB) medium (Kim et al., J Bacteriol 148,904-911(1981)) used to culture carboxydobacteria while supplying carbon monoxide(30%, v/v) or glucose(0.2%, w/v) at 37℃. E. coli was cultured in a Luria-Bertani(LB) medium for gene manipulation.

Isolation of chromosomal DNA

Chromosomal DNA was isolated using modified Murray and Thompson’s methods. Mycobacterium sp. strain JC1 was shaking-cultured to an exponential growth phase in a SMB medium containing glucose at 37℃ at 200 rpm, and 5 ㎖ of the culture solution was centrifuged (18,000 × g, for 15 minutes, at 4℃) to obtain a precipitate. 567 ㎕ of a TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA [pH 8.0]) was added to the precipitate to prepare a suspension. 10 ㎕ of lysozyme dissolved in a TE buffer to a concentration of 20 ㎎/㎖ and 30 ㎕ of 10% (w/v) SDS solution were added thereto, and the mixture was maintained at 37℃ for 1 hour. 100 ㎕ of 5 M sodium chloride was added to the mixture, and then 80 ㎕ of cetyltrimethyl ammonium bromide (CTAB)/sodium chloride solution, prepared by dissolving 4.1 g of sodium chloride in 80 ㎖ of distilled water, slowly adding 10 g of CTAB thereto while heating, and adding distilled water such that the total volume reached 100 ㎖, was added thereto at 65℃, and the resultant was placed for 10 minutes. A phenol-chloroform-isoamyl alcohol (25:24:1, v/v/v) solution was added thereto, and the resultant was centrifuged (18,000 × g, for 15 minutes, at 4℃) to obtain a supernatant. 3 M sodium acetate (pH 5.2) was added to the supernatant such that the concentration of sodium acetate was set to 0.3 M, and ethanol having twice amount of the supernatant was added thereto. The resultant was maintained at -20℃ for 30 minutes and centrifuged (18,000 × g, for 15 minutes, at 4℃) to obtain a precipitate. The precipitate was washed with 70% (v/v) ethanol, dried, and dissolved in 20 ㎕ of RNase (10 ㎍/㎖)-TE buffer.

Isolation of plasmid DNA

Manuals of DNA-spin^TM plasmid DNA purification kit(Intron) were referred. In order to extract plasmid from E. coli, E. coli was cultured in a LB medium overnight, and 3 ㎖ of the culture solution was added to a test tube and centrifuged (18,000 × g, for 1 minute, at 25℃), and the centrifuged E. coli was suspended in 250 ㎕ of buffer 1 (Intron). 250 ㎕ of buffer 2 (Intron) was added thereto and slowly mixed, and the resultant was maintained at room temperature for 2 minutes. Then, 350 ㎕ of buffer 3 (Intron) was added thereto and slowly mixed, and the resultant was maintained in ice for 2 minutes. The resultant was centrifuged (18,000 × g, for 10 minutes, at 4℃), and a supernatant was carefully collected and passed through a column filled with a silica bead membrane (Intron) while centrifuging (18,000 × g, for 1 minute, at 25℃). The supernatant passed through the column was removed and 700 ㎕ of a washing buffer was passed through the column while centrifuging (18,000 × g, for 1 minute, at 25℃). The washing buffer was completely removed, and the column was dried at room temperature for 2 minutes. 50 ㎕ of distilled water was added thereto and maintained at room temperature for 1 minute. The resultant was centrifuged (18,000 × g, for 1 minute, at 25℃) to obtain a solution containing plasmid.

Isolation of DNA fragment from agarose gel

A mega-spin kit (Intron) was used to isolate DNA fragments from agarose gel. A DNA sample processed with a restriction enzyme was subjected to electrophoresis in agarose gel to identify the site of a desired DNA fragment using UV transilluminator. A desired portion of the agarose gel was cut and placed in a test tube, and 500 ㎕ of a gel extraction buffer (Intron) was added thereto and placed in an oven for 5 minutes. When the agarose gel is dissolved in the gel extraction buffer, they were mixed at room temperature, and then the mixture was passed through an elution column while centrifuging (18,000 × g, for 1 minute, at 25℃). The mixture passed through the elution column was removed and 700 ㎕ of a washing buffer was passed through the elution column while centrifuging (18,000 × g, for 1 minute, at 25℃). The washing buffer passed through the elution column was removed and the elution column was dried at room temperature for 1 minute. After the elution column was completely dried, 30 ㎕ of distilled water was dropped at the center of the elution column and dried at room temperature for 2 minutes. The collecting tube was replaced with a new collecting tube and centrifuged (18,000 × g, for 1 minute, at 25℃) to obtain isolated DNA fragments.

Transformation

E. coli was transformed using a CaCl₂ method, and Mycobacterium sp. strain JC1 was transformed using electroporation, methods which were modified from Parish and Stocker methods. For the electroporation, Gene Pulser^TM and Pulse Controller of Bio-Rad Laboratories, Inc. were used. E. coli competent cells were prepared by inoculating E. coli into a LB liquid culture medium and cultured at 37℃ overnight, inoculating the cultured E. coli into 100 ㎖ of a fresh LB liquid culture medium, shaking-culturing the E. coli for 3 hours, transferring the E. coli to a sterilized test tube for centrifuging when an OD₆₀₀ value was about 0.7, and placing the test tube in ice for 30 minutes. The competent cell was centrifuged (4,000 × g, for 10 minutes, at 4℃), and E. coli collected and re-suspended with 10 ㎖ of a cold 0.1 M CaCl₂ solution and placed in ice for 30 minutes. The suspension was centrifuged (4,000 × g, for 10 minutes, at 4℃) to collect E. coli, a supernatant was removed. 2 ㎖ of cold 0.1 M CaCl₂ solution containing 15%(v/v) glycerol was added to the remaining solution, and 50 ㎕ of each E. coli was divided into sterilized test tubes and stored at -70℃ before use. DNA was mixed with the competent cell and the mixture was placed in ice for 20 minutes, heat-treated at 42℃ for 90 seconds, and cooled in ice for 1 minute. Then, 200 ㎕ of a LB liquid culture medium was added thereto, and the DNA and the competent cell were cultured at 37℃ for 1 hour. The culture solution was spread onto a LB solid culture medium containing antibiotics and cultured at 37℃. 40 ㎕ of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal, 20 ㎎/㎖) and 4 ㎕ of isopropylthio-β-D-galactoside (IPTG, 1 M) were added to a plate for color selection.

To obtain a competent cell for Mycobacterium sp. strain JC1 for electroporation, Mycobacterium sp. strain JC1 was shaking-cultured in a SMB medium containing glucose to an exponential growth phase (OD₆₀₀:0.5), the culture solution was placed in ice for 2 hours, and the resultant was centrifuged(4,000×g, for 10 minutes, at 4℃). The resultant was washed with the same amount of sterilized cold 10% (v/v) glycerol solution and washed twice by reducing the volume by 1/3. The resultant was added to a 10% (v/v) glycerol solution and stored at -70℃ to be used as the competent cell. 100 ㎕ of the competent cell and 300 ng of DNA were added to a 0.2 cm cuvette (Bio-Rad) for transformation, and electroporation was performed at 2.5 ㎸/㎝, 25 ㎌ and 600 Ω for 12 msec. Before transformation, they were placed in ice for 10 minutes. After the transformation, they were placed in ice for 10 minutes. Then, 0.5 ㎖ of a 0.2% (w/v) SMB medium containing glucose was added to a unit cuvette, and they were cultured at 37℃ for 4 hours. Then, transformed Mycobacterium sp. strain JC1 was spread onto a solid SMB medium including hygromycin(75 ㎍/㎖) and 0.2% (w/v) glucose.

Enzyme and reagent

A restriction enzyme was purchased from KOSCO (Seoul, Korea), and T4 DNA ligase was purchased from Invitrogen (Carlsbad, California). The use of the restriction enzyme, the DNA ligation, and DNA recombination are performed based on manufacturer’s instructions. Ampicillin was purchased from Gemini Bio-Product Co. (Calabasas, CA), and hygromycin was purchased from Roche (Mannheim, Germany). The other reagents were purchased from Sigma (St. Louis, MO).

Preparation ofMycobacterium sp. strain JC1 cutI ^- mutant

pJK53 including cutI gene was cleaved by KpnI to obtain fragments having the lengths of 4,020-bp, 2,739-bp, and 589-bp. Among them, fragments having the lengths of 4,020-bp and 2,739-bp were ligated to prepare pSW58 vector including cutI-deleted gene. In order to insert the cutI-deleted gene of pSW58 into pKO(Sherman, D. R. M. Voskuil, D. Schnappinger,R. Liao, M. I. Harrell, and G. K. Schoolnik., Proc. Natl. Acad. Sci. USA 98:7534-7539(2001)),a DNA fragment having the length of 2,167-bp, among DNA fragment obtained by cleaving pSW58 by PstI, was inserted into pKO cleaved using PstI, and the resultant was named pSW60(Table 1 and FIG.1). The prepared pSW60 was introduced into Mycobacterium sp. strain JC1 using electroporation, and a single crossover mutant was obtained using SMB-glucose solid medium containing hygromycin(75 ㎍/㎖). The single crossover mutant was cultured in an SMB-glucose medium not having hygromycin for one week to induce a second homologous recombination. 30 ㎕ of the culture was spread onto a SMB solid medium including 10% (w/v) sucrose to search a double crossover mutant that may grow in a condition including sucrose. Chromosomal DNA was isolated from the obtained double crossover mutant and PCR was conducted. Then, base sequence of amplified products was analyzed to identify that the mutant was cutI ^- mutant.

Polymerization chain reaction (PCR)

PCR was performed using a Master gradient (Eppendorf, Hamburg, Germany). 50 ㎕ of PCR solution includes 2.5 mM MgCl₂, dNTP, a 10x buffer, 20 pmol of primers, an Ex Taq DNA polymerase(Takara, Shiga, Japan) and 100 ng of a DNA template. PCR conditions are as follows: pre-denaturation at 94℃ for 10 minutes, denaturation at 94℃ for 1 minute, annealing at 65℃ for 1 minute, elongation 30 times at 72℃ for 2 minutes, and post-elongation at 72℃ for 10 minutes.

Growth curve

In order to identify a phenotype of Mycobacterium sp. strain JC1 cutI ^- mutant, growth curves of Mycobacterium sp. strain JC1 cutI ^- mutant cultured in a SMB-glucose medium or a SMB-CO medium were obtained using a spectrophotometer(U-2000, Hitachi).

Complementation test

For a complementation test of Mycobacterium sp. strain JC1 cutI ^- mutant, a PCR product having the length of 1,539 bp and including cutI of Mycobacterium sp. strain JC1 was amplified from chromosomal DNA using cutI-CT-F(5'-AAGCTTAGTCCAGTCCGAACCCGAAC-3';underline, HindⅢ recognition site) and cutI-CT-R(5'-GGATCCCGAATAGGAAGCCAGCTTTC-3';underline, BamHI recognition site) primers and ligated to pNBVI cleaved using HindⅢ and BamHI to prepare pSW98 vector(Table 1). The prepared pSW98 was subjected to electroporation for the complementation test of Mycobacterium sp. strain JC1 cutI ^- mutant.

Extraction of protein

In order to obtain an enzyme extract used for CO-DH active staining and western blot, Mycobacterium sp. strain JC1 cultured in a SMB-glucose medium was centrifuged(18,000×g, for 10 minutes, at 4℃) (Eppendorf centrifuge-5403, Hamburg, Germany) and collected, washed twice with a 50 mM Tris-HCl (pH 7.5) buffer solution, and re-suspended with 3 ㎖ of the 50 mM Tris-HCl (pH 7.5) buffer solution. The suspended Mycobacterium sp. strain JC1 was sonicated at 0℃ using a sonicator (Sonics & Materials Inc., Danbury, CT) at 20% amplitude for 3 seconds and paused for 10 seconds. This process was repeated 20 times to homogenize Mycobacterium sp. strain JC1. The homogenized culture solution was centrifuged (18,000 × g, for 30 minutes, at 4℃)(Eppendorf centrifuge-5403) and collected, and a supernatant was used as an enzyme extract.

Quantification of protein

Proteins were quantified using bovine serum albumin (BSA) as a standard protein according to Bradford protein assay.

Electrophoresis

Electrophoresis was performed using a Mighty Small SE245 vertical slab gel device (Amersham Pharmacia Biotech, Arlington Heights, IL) using a modified Laemmli method. In order to analyze a native enzyme, electrophoresis was performed without using SDS (Sigma, St. Louis, MO). A 7.5% (w/v) of acrylamide gel was prepared using a stock solution including 30% (w/v) acrylamide (Sigma) and 0.8% (w/v) N,N'-bis-methylene acrylamide(Sigma)7.5%(w/v). Protein mixed with a buffer was loaded, and the electrophoresis was performed while cooling using an ice water circulation device. Voltages during the electrophoresis were constantly maintained using a power supply apparatus (EPS-310, Amersham Pharmacia Biotech.). 80 V was applied to a stacking gel, and 120 V was applied to a separate gel when the protein was applied to the separate gel.

CBB staining

Protein was stained using a solution including 45% (v/v) methanol, 10% (v/v) acetic acid, and 0.25% (w/v) CBB R-250 for 30 minutes. The stained gel was washed several times with a destaining solution (30% [v/v] methanol and 10% [v/v] acetic acid).

CO-DH active staining

The enzyme extract was subjected to electrophoresis in acrylamide gel not having SDS, and the acrylamide gel was added to a glass tube including a 50 mM Tris-HCl (pH 7.5) buffer solution and maintained therein for 5 minutes to saturate with CO. 1 ㎖ of a solution including 0.05% (w/v) phenazine methosulfate (PMS, Sigma) and 0.25% (w/v) nitroblue tetrazolium (NBT, Sigma) was added thereto, and the resultant was maintained until a clear active staining band was observed. This process was performed while light was blocked.

Western blot

The enzyme extract was subjected to electrophoresis in acrylamide gel not containing SDS, and protein in the acrylamide gel was transferred to a membrane using a Western blotter (Amersham Pharmacia Biotech.) by electricity. The membrane was dipped in 1× PBS (13.7 mM NaCl, 0.27 mM KCl, 0.43 mM Na₂HPO₄·7H₂O and 0.14 mM KH₂PO₄) for 1 minute and shaken using a 10% blocking solution prepared by dissolving 10%[w/v] skim milk in 1×PBS for 1 hour. CO-DH antibody of Mycobacterium sp. strain JC1 obtained from a rabbit was added to the above solution in a ratio of 1:5,000, and the mixture was slowly shaken for 1 hour. The mixture was washed twice with 1× PBS for 15 minutes and slowly shaken using a 10% blocking solution including Zymax^TMGoatanti-Rabbit IgG(H+L)(Zymed, S. Sanfrancisco, California) in a ratio of 1:5,000 for 1 hour. The resultant was washed three times with PBS for 15 minutes, and a mixture of solution A and solution B of WEST-ZOL^TM(Intron, Sungnam, Korea)in a ratio of 1:1 was sprayed to the membrane. Then, the membrane was photosensitized with an X-ray film (Fuji Photo Film Co., Ltd., Tokyo, Japan).

Activity of β-galactosidase

In order to identify the influence of cutI gene on CO-DH promoter activity, Mycobacterium sp. strain JC1 was transformed using pCS6 including a promoter of a putative copyI CO-DH gene having the length of 720-bp that is from 12bp site from 'A' of start codon(ATG) of cutB in the opposite direction of transcription of cutB to 411bp site from 'G' of start codon(GTG) of cutR gene in the transcription direction of cutR, pCS7 including a promoter of a putative copy II CO-DH gene having the length of 760-bp that is from 12bp site from 'A' of start codon(ATG) of cutB in the opposite direction of transcription of cutB to 496bp site from 'A' of start codon(ATG) of cutD gene in the transcription direction of cutD, and pCS8 as a control without having a promoter using electroporation(Lee,Cho-soon, Master’s Thesis, Department of Biology, Yonsei University(2004)). pNBV1 that is used to prepare pCS6, pCS7 and pCS8 was disclosed by Howard et al, Gene 166:181-182(1995). Mycobacterium sp. strain JC1 transformed by pCS6, pCS7 and pCS8 was shaking-cultured in 5 ㎖ of a SMB medium including 10.2% (w/v) glucose and hygromycin (75 ㎍/㎖) at 37℃ at 200 rpm overnight, and 5 ㎖ of the culture solution was inoculated into 500 ㎖ of a SMB including 0.2% (w/v) glucose and hygromycin (75 ㎍/㎖). The inoculated culture solution was cultured at 37℃ at 200 rpm, and re-suspended in 3 ㎖ of 50 mM Tris-HCl (pH 7.5). The suspended Mycobacterium sp. strain JC1 was sonicated using a sonicator(Sonic & Materials, VCX600, Danbury, CT) at 20% amplitude for 3.0 seconds and paused for 9.9 seconds. This process was repeated for 5 minutes to homogenize Mycobacterium sp. strain JC1. Then, the homogenized Mycobacterium sp. strain JC1 was centrifuged (18,000 x g, for 30 minutes, at 4℃) to obtain a supernatant. The supernatant was used as an enzyme extract to measure the activity of β-galactosidase according to a Miller method. 500 ㎕ of a Z buffer (0.1 M sodium phosphate [pH 7.0], 10 mM KCl, 1 mM MgSO₄, 50 mM β-mercaptoethanol) was added to 200 ㎕ of the enzyme extract, and 200 ㎕ of o-nitrophenyl-β-D-galactoside(ONPG[4 ㎎/㎖]) was added thereto as a substrate. Color change of the mixture was measured using absorbance at 420 nm. 1 unit of β-galactosidase was defined as an amount required to produce 1 nmole o-nitrophenol from ONPG per minute,and specific activity of enzyme was represented by β-galactosidase unit per mg of protein per minute.

Survival rate

A survival rate of Mycobacterium sp. strain JC1 and cutI ^- mutants against SNP was measured using a modified Hernandez-Urzua method. 100 ㎕ of each of Mycobacterium sp. strain JC1 and cutI ^- mutants cultured to the mid-exponential growth phase in a SMB-glucose medium was added to a test tube. The survival rate thereof was measured after 0, 30, 60, and 90 minutes, three experimental groups to which 5 mM SNP was added and three control groups were prepared. The experimental groups and control groups were cultured, diluted, and smeared onto a SMB-glucose solid medium. Colony forming units (CFUs) were calculated based on the number of colony to obtain a survival curve. The Mycobacterium sp. strain JC1 was cultured under sterile conditions with light since SNP is degraded by light to generate NO.

Table 1

Plasmid	Genotype or Description	Reference or Source
pBluescriptⅡ SK(+)	2,961-bp plasmid derived from pUC19, Ampr	Stratagene
pNBV1	5.8-kb plasmid derived from p16R1, Hygr	Howard et al.(1995)
pKO	8,366-bp plasmid used as a suicide vector in Mycobacterium sp. strain JC1, Kmr, Hygr, sacB.	Sherman et al.(2001)
pJK53	pBluescriptⅡ SK(+) containing 4.4-kb SacI fragment including cutI gene	The present invention
pSW58	pJK53 self-ligated with 4,020-bp and 2,739-bp KpnI fragments	The present invention
pSW60	pKO containing 2,167-bp PstI fragment from pSW58	The present invention
pSW98	pNBVI containing 1,539-bp a PCR product including cutI gene	The present invention
pCS6	pNBV1 having the length of 9,341 bp and containing a putative copy I CO-DH promoter-lacZ fusion	Lee, Cho-soon (2004)
pCS7	pNBV1 having the length of 9,381 bp and containing a putative copy II CO-DH promoter-lacZ fusion	Lee, Cho-soon (2004)
pCS8	pNBV1 having the length of 8,621 bp and containing promoterless lacZ	Lee, Cho-soon (2004)

2. Results

Comparison of identity between cutBCA and cutI of Mycobacterium sp. strain JC1 and cutBCA and cutI homolog gene of pathogenic mycobacteria

Identity between cutBCA that is a structural gene of carbon monoxide dehydrogenase of Mycobacterium sp. strain JC1 and cutBCA homolog gene of pathogenic mycobacteria was analyzed using a ClustalW program. Referring to Table 2 below, cutBCA of Mycobacterium sp. strain JC1 had very high identity of nucleotide sequence to cutBCA homolog gene of pathogenic mycobacteria such as M. tuberculosis. In addition, identity of cutI according to the present invention to homolog gene of various pathogenic mycobacteria was analyzed in the same manner as described above (Table 3). As a result, it was identified that genes expressing carbon monoxide dehydrogenase of Mycobacterium sp. strain JC1 used in this example and genes related to the expression of carbon monoxide dehydrogenase do not have a big difference from those of pathogenic mycobacteria.

Table 2

Bacteria	Gene	Identity to cutB,cutC and cutA of Mycobacterium sp. strain JC1(%)a
		Identity to cutB	Identity to cutC	Identity to cutA
M. tuberculosis	rv0375c	71	-	-
	rv0374c	-	73	-
	rv0373c	-	-	81
M. bovis	mb0382c	71	-	-
	mb0381c	-	73	-
	mb0380c	-	-	81
M. marinum	mmar_0655	72	-	-
	mmar_0656	-	71	-
	mmar_0657	-	-	82
M. ulcerans	mul_0117	71	-	-
	mul_0116	-	71	-
	mul_0115	-	-	82

^a analysis results obtained using ClustalW program.

Table 3

Bacteria	Gene	Identity to cutI of Mycobacterium sp. strain JC1(%)a
M. tuberculosis	rv0368c	67
M. bovis	mb0375c	67
M. marinum	mmar_0661	70
M. ulcerans	mul_0110	70

^a analysis results obtained using ClustalW program.

Mycobacterium sp. strain JC1 cutI ^- mutants

Mycobacterium sp. strain JC1 cutI ^- mutant was induced by homologous recombination and screened using levan sucrase that is a sacB gene product which is widely used to prepare mutants of mycobacteria. It was identified that Mycobacterium sp. strain JC1 cutI ^-mutant grew well with glucose(FIG. 3) but could not grow with CO(FIG. 4).

Complementation test of Mycobacterium sp. strain JC1 cutI ^- mutant

Complementation test of Mycobacterium sp. strain JC1 cutI ^- mutant was performed according to the method described above in order to identify whether Mycobacterium sp. strain JC1 cutI ^- mutant cannot grow with CO because of cutI mutation or because of other factors. As a result of the complementation test of Mycobacterium sp. strain JC1 cutI ^- mutant, it was identified that Mycobacterium sp. strain JC1 cutI ^- mutant could not grow with CO because of cutI mutation(FIG. 5).

Identification of expression of CO-DH in Mycobacterium sp. strain JC1 cutI ^- mutant

CO-DH enzyme active staining and western blot were performed in order to identify whether Mycobacterium sp. strain JC1 cutI ^- mutant could not grow with CO since CO-DH protein was not expressed or since a cofactor was not properly acting even though the CO-DH protein was expressed. As a result of the CO-DH enzyme active staining and western blot, it was identified that Mycobacterium sp. strain JC1 cutI ^- mutant could not grow with CO since CO-DH protein was not expressed(FIG. 6).

Northern blot and promoter assay were performed in order to identify transcription of CO-DH in Mycobacterium sp. strain JC1 cutI ^- mutant. As a result of the northern blot, CO-DH was not transcribed in Mycobacterium sp. strain JC1 cutI ^- mutant(FIG. 7), and this was verified by the promoter assay(FIGS. 8 to 11). CO-DH of Mycobacterium sp. strain JC1 was expressed while growing with glucose and the expression thereof increased in the late-exponential growth phase. According to the result of the promoter assay, it was identified that the activity of the copy II cutBCA promoter of Mycobacterium sp. strain JC1 wild type increased in the late-exponential growth phase, but the activity of the promoter of Mycobacterium sp. strain JC1 cutI ^- mutant did not increase. Thus, it was identified that CutI is an important factor influencing the transcription of CO-DH.

Survival rate against NO

Resistance of Mycobacterium sp. strain JC1 cutI ^- mutant against NO was identified using SNP generating NO in order to identify whether the Mycobacterium sp. strain JC1 cutI ^- mutant substantially has resistance against NO. As a result of SNP test, the survival rate of Mycobacterium sp. strain JC1 cutI ^- mutant against NO was reduced by about 80% compared to that of Mycobacterium sp. strain JC1 wild type(FIG. 12).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the following claims.

<110> Industry-Academic Cooperation Foundation of Yonsei University

<120> Polypeptide and Polynucleotide Derived from Mycobacteria and

Screening Method for Anti-Mycobacterial Agent

<160> 2

<170> KopatentIn 1.71

<210> 1

<211> 1209

<212> DNA

<213> Mycobacterium sp. strain JC1

<400> 1

gtggcttcgc cgtttctgct gcgcggcgtc gatctcgcag ccttcgccgc cgccctggta 60

gcacgcttgc gtggtgccgg gttgctggtg tccgccagca gtgcagcagg actcgtcgag 120

gcactgcgcc ggttctggcc gactgaccgg gaacagctgt actggaccgc ccggctgaca 180

cttgttagcc gggcggagga cctgattggc ttcgacgccg cgttcgacgc tctgttcgcc 240

gacgccgtcc tgggtttgga cccgcccggc ctcaaacaaa gtctcggcac cacgacggta 300

cccgagcccg gcgtgcgggg ccgacagcac gcacaggctg agggcggttt gccgtgggcg 360

acccggccag cgtcgatcac agcagcgagc gaggacaacg acagcgacat cggtattccc 420

gagatgctgc ccagtcggct cgtcgcgcgc gccgaggagc cgttcgaacg ttttgacgcg 480

gccgatctac gcctgatcgg cgcctggctg gaacaggcgg tggctcgctg gccgcgtcgt 540

cggagtctgc gccgcgaacc gcatccgcat ggaaagcgca tcgacttgcg gcgcaccatg 600

aaagcgtcgc gggccaccgg ctgggagccg gtcgtactcg cgcggacccg gccgcgccag 660

cactcccggc gcatggtgct gatgtgcgac gtcagcgggt cgatgcaggc ctatgcatcc 720

gtctatctgc acttgatgcg tgcggcggcg ttgcagcaga aggggatgcg gccggaggtt 780

ttcgccttct cgacatcgct gacccgactg actccggtgc tgtcacatcg gtccgcggag 840

gtggcgctgg cgcgcgccaa cgccaaggtc gccgaccgct acggcggtac ccacctcggg 900

cggagtgtca ccgaattgct ggccacgtcg cacggtaatg cgctgcgcgg cgcggtggtg 960

atcatcgctt ccgacggttg ggacagcgat ccgccggagc tgctggcgcg agcggtcgcc 1020

cgggttcgcc ggcgtgcgca tcagctggtg tggctcaacc cgcgggccgc gcgcccggga 1080

tttcagccgc tggccggggc gatggcggcc gcgttgccgt actgcgacgc cgtgctgccg 1140

gcgcattcgc tttctgggtt gcaggagctg ttcgcggtgc tggccgaggg atcgggctac 1200

cgagcatga 1209

<210> 2

<211> 402

<212> PRT

<213> Mycobacterium sp. strain JC1

<400> 2

Met Ala Ser Pro Phe Leu Leu Arg Gly Val Asp Leu Ala Ala Phe Ala

1 5 10 15

Ala Ala Leu Val Ala Arg Leu Arg Gly Ala Gly Leu Leu Val Ser Ala

20 25 30

Ser Ser Ala Ala Gly Leu Val Glu Ala Leu Arg Arg Phe Trp Pro Thr

35 40 45

Asp Arg Glu Gln Leu Tyr Trp Thr Ala Arg Leu Thr Leu Val Ser Arg

50 55 60

Ala Glu Asp Leu Ile Gly Phe Asp Ala Ala Phe Asp Ala Leu Phe Ala

65 70 75 80

Asp Ala Val Leu Gly Leu Asp Pro Pro Gly Leu Lys Gln Ser Leu Gly

85 90 95

Thr Thr Thr Val Pro Glu Pro Gly Val Arg Gly Arg Gln His Ala Gln

100 105 110

Ala Glu Gly Gly Leu Pro Trp Ala Thr Arg Pro Ala Ser Ile Thr Ala

115 120 125

Ala Ser Glu Asp Asn Asp Ser Asp Ile Gly Ile Pro Glu Met Leu Pro

130 135 140

Ser Arg Leu Val Ala Arg Ala Glu Glu Pro Phe Glu Arg Phe Asp Ala

145 150 155 160

Ala Asp Leu Arg Leu Ile Gly Ala Trp Leu Glu Gln Ala Val Ala Arg

165 170 175

Trp Pro Arg Arg Arg Ser Leu Arg Arg Glu Pro His Pro His Gly Lys

180 185 190

Arg Ile Asp Leu Arg Arg Thr Met Lys Ala Ser Arg Ala Thr Gly Trp

195 200 205

Glu Pro Val Val Leu Ala Arg Thr Arg Pro Arg Gln His Ser Arg Arg

210 215 220

Met Val Leu Met Cys Asp Val Ser Gly Ser Met Gln Ala Tyr Ala Ser

225 230 235 240

Val Tyr Leu His Leu Met Arg Ala Ala Ala Leu Gln Gln Lys Gly Met

245 250 255

Arg Pro Glu Val Phe Ala Phe Ser Thr Ser Leu Thr Arg Leu Thr Pro

260 265 270

Val Leu Ser His Arg Ser Ala Glu Val Ala Leu Ala Arg Ala Asn Ala

275 280 285

Lys Val Ala Asp Arg Tyr Gly Gly Thr His Leu Gly Arg Ser Val Thr

290 295 300

Glu Leu Leu Ala Thr Ser His Gly Asn Ala Leu Arg Gly Ala Val Val

305 310 315 320

Ile Ile Ala Ser Asp Gly Trp Asp Ser Asp Pro Pro Glu Leu Leu Ala

325 330 335

Arg Ala Val Ala Arg Val Arg Arg Arg Ala His Gln Leu Val Trp Leu

340 345 350

Asn Pro Arg Ala Ala Arg Pro Gly Phe Gln Pro Leu Ala Gly Ala Met

355 360 365

Ala Ala Ala Leu Pro Tyr Cys Asp Ala Val Leu Pro Ala His Ser Leu

370 375 380

Ser Gly Leu Gln Glu Leu Phe Ala Val Leu Ala Glu Gly Ser Gly Tyr

385 390 395 400

Arg Ala

Claims (11)

1. A mycobacteria-derived polypeptide having an amino acid sequence of SEQ ID NO: 2.
2. The mycobacteria-derived polypeptide of claim 1, wherein the polypeptide activates the transcription of carbon monoxide dehydrogenase.
3. The mycobacteria-derived polypeptide of claim 1, wherein the polypeptide activates the expression of carbon monoxide dehydrogenase.
4. A mycobacteria-derived polynucleotide having: (a) a nucleotide sequence coding the amino acid sequence of SEQ ID NO: 2 of claim 1; (b) a nucleotide sequence complementary to the nucleotide sequence of (a); or (c) a nucleotide sequence obtained by hybridizing the nucleotides of (a) and (b) under stringent conditions.
5. The mycobacteria-derived polynucleotide of claim 4, wherein the nucleotide sequence has SEQ ID NO: 1.
6. A primer or probe specifically hybridized to the mycobacteria-derived polynucleotide of claim 4.
7. An antibody specifically binding to a mycobacteria-derived polypeptide according to any one of claims 1 to 3.
8. A recombinant vector comprising the mycobacteria-derived polynucleotide of claim 4 and a promoter operatively linked to the mycobacteria-derived polynucleotide.
9. A transformant by the recombinant vector of claim 8.
10. A method of screening an anti-mycobacterial agent, the method comprising:

    a) contacting a sample to be assayed to a cell comprising the mycobacteria-derived polypeptide according to any one of claims 1 to 3 or the mycobacteria-derived polynucleotide according to claim 4 or 5; and

    (b) measuring the activity of the polypeptide or the amount of transcribed or expressed polynucleotide, and if the activity of the polypeptide or the amount of transcribed or expressed polynucleotide is down-regulated, the sample is determined to be an anti-mycobacterial agent.
11. The method of claim 10, wherein the anti-mycobacterial agent is a substance for preventing or treating Buruli ulcer, lymphadenitis, or tuberculosis.

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