Gene Expression Profiling ; Humans ; Macrophages ; metabolism ; MicroRNAs ; genetics ; metabolism ; Mycobacterium tuberculosis ; genetics ; metabolism

Treatment of latent tuberculosis infection remains an important goal of global TB eradication. To this end, targets that are essential for intracellular survival of Mycobacterium tuberculosis are particularly attractive. Arylamine N-acetyltransferase (NAT) represents such a target as it is, along with the enzymes encoded by the associated gene cluster, essential for mycobacterial survival inside macrophages and involved in cholesterol degradation. Cholesterol is likely to be the fuel for M. tuberculosis inside macrophages. Deleting the nat gene and inhibiting the NAT enzyme prevents survival of the microorganism in macrophages and induces cell wall alterations, rendering the mycobacterium sensitive to antibiotics to which it is normally resistant. To date, NAT from M. marinum (MMNAT) is considered the best available model for NAT from M. tuberculosis (TBNAT). The enzyme catalyses the acetylation and propionylation of arylamines and hydrazines. Hydralazine is a good acetyl and propionyl acceptor for both MMNAT and TBNAT. The MMNAT structure has been solved to 2.1 Å resolution following crystallisation in the presence of hydralazine and is compared to available NAT structures. From the mode of ligand binding, features of the binding pocket can be identified, which point to a novel mechanism for the acetylation reaction that results in a 3-methyltriazolo[3,4-a]phthalazine ring compound as product.
Acetyltransferases ; metabolism ; Arylamine N-Acetyltransferase ; chemistry ; genetics ; metabolism ; Catalysis ; Catalytic Domain ; Crystallization ; Mycobacterium ; enzymology ; metabolism ; Mycobacterium marinum ; enzymology ; Mycobacterium tuberculosis ; enzymology ; genetics ; metabolism ; Protein Binding

This study was conducted to amplify the cfpl0-esat6 fusion gene by SOE and insert into the integrating shuttle plasmid pMV361 to form the recombinant plasmid. Then another recombinant plasmid was constructed by insertinga-A g signal sequence of BCG. The two recombinant plasmids were introduced into BCG and the induced products from recombinant BCG were analyzed. In conclusion,the successful construction of rBCG expressing the fusion protein CFP10-ESAT6 will be the base of the development of novel Mycobacterium tuberclosis vaccines.
Antigens, Bacterial ; biosynthesis ; genetics ; Bacterial Proteins ; biosynthesis ; genetics ; Mycobacterium bovis ; genetics ; metabolism ; Mycobacterium tuberculosis ; genetics ; Plasmids ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Tuberculosis Vaccines ; biosynthesis

OBJECTIVETo clone and express the Rv3871 gene related to the virulent protein secretion of Mycobacterium tuberculosis and analyze its molecular structure, function and homology using bioinformatic approach.

METHODSA pair of primers was designed to amplify the Rv3871 gene, which was subcloned into the prokaryotic plasmid pET32a(+). The recombinant plasmid was identified by sequence analysis and the expressed recombinant protein by SDS-PAGE. The structure, function and homology alignment of Rv3871 were analyzed comparatively against other mycobacteria.

RESULTSThe restriction fragments through molecular cloning matched perfectly in size with our prediction. The gene sequence was consistent with the corresponding sequence in GenBank. The expression protein was detected by SDS-PAGE with a molecular weight of 84 kD. Two FtsK/SpoE III domains were found by bioinformatic analysis. The homology results showed distinct differences between Rv3871 of the pathogenic M. tuberculosis and its counterparts in non-pathogenic mycobacteria.

CONCLUSIONMolecular cloning, expression and sequencing identify the structural and functional characteristics of Rv3871. The structural and functional differences of the gene between pathogenic and non-pathogenic mycobacteria identified by bioinformatics provide some evidence for the pathogenesis and drug targets of tuberculosis.

Bacterial Proteins ; genetics ; metabolism ; Cloning, Molecular ; Computational Biology ; Mycobacterium tuberculosis ; genetics ; metabolism ; pathogenicity ; Recombinant Proteins ; genetics ; metabolism ; Virulence ; genetics

OBJECTIVETo obtain the recombinant rv1837c and rv3803c of Mycobacterium tuberculosis using gene engineering technology and explore their prokaryotic expression, purification, and immunogenicity.

METHODSThe Mycobacterium tuberculosis rv1837c and rv3803c genes were amplified by polymerase chain reaction, and then cloned into the vector pTA2, followed by the subclone into the expression vector pET30a (+). The resulting plasmids, named pET30a (+): rv1837c and pET30a (+): rv3803c, encode recombinant protein containing a hexa-histidine tag on its N-terminus. pET30a (+): rv1837c and pET30a (+): rv3803c were introduced into E. coli BL21 (DE3) by transformation respectively, and the recombinant gene was induced with 0.4 mmol/L isopropyl-D-thiogalactopyranoside. The expressed products were identified by Western blot with hexa-histidine tag antibody and serum from tuberculotic patients. The histidine tagged protein was purified by nickel nitrilotriacetic acid His-Bind resin. Rabbits were immunized with purified recombinant Rv1837c and Rv3803c proteins. Then the purified recombinant Rv1837c and Rv3803c proteins were used to detect antibody in rabbit serum, which had been immunized by Western blot.

RESULTSAfter transformation of the E. coli and induction with 0.4 mmol/L of isopropyl-D-thiogalactopyranoside, recombinant target proteins Rv1837c (relative molecular mass: 92000) and Rv3803c (relative molecular mass: 38 000) were expressed in pET30a (+): rv1837c and pET30a (+): rv3803c system. The expressed protein existed in cytoplasm in an unsoluble form and amounted to 30% and 50% of the total proteins of E. coli. The purity of the purified protein reached 90%. The immunogenicity of the recombinant proteins Rv1837c and Rv3803c was strong, as identified by Western blot.

CONCLUSIONThe prokaryotic expression recombinant plasmids pET30a (+): rv1837c and pET30a (+): rv3803c was successfully constructed and the recombinant proteins Rv1837c and Rv3803c were obtained, which laid a basis for the optimized diagnosis of active tuberculosis.

Antibodies ; metabolism ; Bacterial Proteins ; genetics ; immunology ; metabolism ; Blotting, Western ; Escherichia coli ; metabolism ; Genetic Vectors ; Mycobacterium tuberculosis ; genetics ; immunology ; metabolism ; Plasmids ; metabolism ; Polymerase Chain Reaction ; Recombinant Proteins ; genetics ; metabolism

The AdvanSure tuberculosis/non-tuberculous mycobacterium (TB/NTM) PCR (LG Life Science, Korea) and COBAS TaqMan Mycobacterium tuberculosis (MTB) PCR (Roche Diagnostics, USA) are commonly used in clinical microbiology laboratories. We aimed to evaluate these two commercial real-time PCR assays for detection of MTB in a large set of clinical samples over a two-year period. AdvanSure TB/NTM PCR and COBAS TaqMan MTB PCR were performed on 9,119 (75.2%) and 3,010 (24.8%) of 12,129 (9,728 respiratory and 2,401 non-respiratory) MTB specimens, with 361 (4.0%) and 102 (3.4%) acid-fast bacilli (AFB)-positive results, respectively. In MTB culture, 788 (6.5%) MTB and 514 (4.2%) NTM were identified. The total sensitivity and specificity of the AdvanSure assay were 67.8% (95% confidence interval [CI], 63.9-71.6) and 98.3% (95% CI, 98.0-98.6), while those of the COBAS TaqMan assay were 67.2% (95% CI, 60.0-73.8) and 98.4% (95% CI, 97.9-98.9), respectively. The sensitivities and specificities of the AdvanSure and COBAS TaqMan assays for AFB-positive and AFB-negative samples were comparable. Furthermore, the AdvanSure assay showed fewer invalid results compared with the COBAS TaqMan assay (5.0 vs. 20.4 invalid results/1,000 tests, P<0.001). AdvanSure assay represents a comparable yet more reliable method than COBAS TaqMan for the identification of mycobacteria in routine clinical microbiology.
DNA, Bacterial/genetics/metabolism ; Humans ; Mycobacterium tuberculosis/*genetics/isolation & purification ; Reagent Kits, Diagnostic ; Real-Time Polymerase Chain Reaction ; Republic of Korea ; Sensitivity and Specificity ; Tuberculosis, Pulmonary/diagnosis

Differences in ability to produce the specific phenolic glycolipid-Tb (PGL-Tb) antigen among Mycobacterium tuberculosis strains have been reported. One of the explanations would be the genotypic variation between the strains. In this study, we compared the DNA fragment patterns after digestion of DNA with various restriction enzymes between the PGL-Tb producing and non-producing strains of M. tuberculosis. Three clinical isolates of M. tuberculosis producing the PGL-Tb antigen detectable by thin-layer chromatography, and M. tuberculosis H37Rv and M. bovis BCG not producing the antigen were grown in Sauton medium. The chromosomal DNA was digested with the restriction endonucleases, Eco RI, Sau3A I, BamH I, Xho I, Sma I, Pst I, Hinc II, and Bst EII. Most of the restriction enzymes used gave no clear DNA bands or no DNA fragment common just to the PGL-Tb producing strains. When DNAs were digested with Bst EII, however, there was a 13 kb DNA fragment common to the PGL-Tb producing isolates of M. tuberculosis and not present in the H37Rv strain and M. bovis BCG. This study thus suggests that there might be differences in DNA fragment patterns between the PLG-Tb producing and non-producing strains of M. tuberculosis.
Base Sequence ; Comparative Study ; DNA Restriction Enzymes ; DNA, Bacterial/*metabolism ; Glycolipids/*biosynthesis ; Molecular Sequence Data ; Mycobacterium tuberculosis/*genetics/metabolism ; Support, Non-U.S. Gov't ; Tuberculosis/microbiology

To begin with, we constructed cfp10-esat6 fusion gene and its prokaryotic expression vector and had it express in E. coli. By GeneSOEing techniques, a fusion gene was constructed by splicing cfpl0 gene and esat6 gene, and then was cloned into pGEX-4T-1 plasmid. Secondly, we constructed the prokaryotic expression recombinant plasmid pGcfp10-esat6. After identification with restriction enzyme analysis, PCR and nucleotide sequencing analysis, The E. coli BL21 containing the recombinant plasmid was induced by IPTG (Isopropy-beta-D-thiogalatoside). The fusion protein CFP10-ESAT6 with GST-tag about 42 kDa was expressed and purified with GST-fusion protein purification kit,The expression of cfp10-esat6 fusion gene was subsequently detected by SDS-polyacrylamine gel electrophoresis and Western-blot analysis. The sequence of cfp10 and esat6 in recombinant plasmid was consistent with that of GenBank report. The fusion protein existed in cytoplasm in soluble form and represented about 40% total bacterial protein of E. coil. The fusion protein was purified and the purity reached 90%. Its antigenicity was confirmed by Western-blotting. The prokaryotic expression vector (pGcfp1o-esat6) was constructed successfully, and the fusion protein CFP10-ESAT6 was obtained. This study provided an experimental basis for potential application of the recombinant CFP10-ESAT6 in the diagnosis of tuberculosis.
Antigens, Bacterial ; biosynthesis ; genetics ; Bacterial Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Mycobacterium tuberculosis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

OBJECTIVETuberculosis remains one of the most serious infectious diseases in the world. In this study, a scheme of Mycobacterium tuberculosis (M. tuberculosis) multilocus sequence analysis (MLSA) was established for the phylogenetic and epidemiology analysis.

METHODSTo establish the scheme of M. tuberculosis MLSA, the genome of H37Rv, CCDC5079 and CCDC5180 were compared, and some variable genes were chosen to be the MLSA typing scheme. 44 M. tuberculosis clinical isolates were typed by MLSA, IS6110-RFLP, and soligotyping, to evaluate the MLSA methods.

RESULTSAfter comparison of the genome, seven high discrimination gene loci (recX, rpsL, rmlC, rpmG1, mprA, gcvH, ideR) were chosen to be the MLSA typing scheme finally. 11 variable SNP sites of those seven genes were found among the 44 M. tuberculosis isolate strains and 11 sequence types (STs) were identified. Based on the Hunter-Gaston Index (HGI), MLSA typing was not as good for discrimination at the strain level as IS6110-RFLP, but the HGI was much better than that of spoligotyping. In addition, the MEGA analysis result of MLSA data was similar to spoligotyping/PGG lineage, showing a strong phylogenetic signal in the modern strains of M. tuberculosis. The MLSA data analysis by eBURST revealed that 4 sequence types (ST) came into a main cluster, showing the major clonal complexes in those 44 strains.

CONCLUSIONMLSA genotyping not only can be used for molecular typing, but also is an ideal method for the phylogenetic analysis for M. tuberculosis.

Chromosome Mapping ; Chromosomes, Bacterial ; Gene Expression Regulation, Bacterial ; Genome, Bacterial ; Genotype ; Multilocus Sequence Typing ; methods ; Mycobacterium tuberculosis ; genetics ; metabolism

LysinB (LysB) in mycobacteriophage D29 was cloned and expressed and its enzymatic properties were analysed. The lysB gene was amplified by PCR from mycobacteriophage D29 genomic DNA and inserted into pET22b vector. The constructed recombinant plasmid was transformed into Escherichia coli BL21(DE3) to express fusion protein, which was purified by Ni-NTA column and enzymatic activity detected. The results showed that expression plasmid pET22b-lysB was constructed successfully. Highly purified recombination protein (His-LysB) was obtained 33.2 mg from 1 L LB culture medium. A screening for His-LysB activity on esterase and lipase substrates confirmed the lipolytic activity. With p-nitrophenyl butyrate as substrate, the thermal stability of the enzyme was poor when the temperature was above 30 degrees C. The enzyme exhibited higher stability at pH 5.0-9.5. The optimum temperature and pH for the lipolytic activity of His-LysB were 23 degrees C and 7.5 respectively. Under the optimum conditions, the specific activity of His-LysB was 1.3 U/mg. Zn2+, CU2+, Mg2+, Mn2+ and phenylmethane sulfonyl fruoride severely inhibited the lipolytic activity of His-LysB. The result provides a new option for tuberculosis drug research and development.
Cloning, Molecular ; Enzyme Stability ; Escherichia coli ; genetics ; metabolism ; Mycobacteriophages ; enzymology ; Mycobacterium tuberculosis ; drug effects ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; metabolism ; Viral Proteins ; biosynthesis ; genetics