The solution structure of antigen MPT64 from Mycobacterium tuberculosis defines a new family of beta-grasp proteins

The MPT64 protein and its homologs form a highly conserved family of secreted proteins with unknown function that are found within the pathogenic Mycobacteria genus. The founding member of this family from Mycobacterium tuberculosis (MPT64 or protein Rv1980c) is expressed only when Mycobacteria cells are actively dividing. By virtue of this relatively unique expression profile, Rv1980c is currently under phase III clinical trials to evaluate its potential to replace tuberculin, or purified protein derivative, as the rapid diagnostic of choice for detection of active tuberculosis infection. We describe here the NMR solution structure of Rv1980c. This structure reveals a previously undescribed fold that is based upon a variation of a beta-grasp motif most commonly found in protein-protein interaction domains. Examination of this structure in conjunction with multiple sequence alignments of MPT64 homologs identifies a candidate ligand-binding site, which may help guide future studies of Rv1980c function. The work presented here also suggests structure-based approaches for increasing the antigenic potency of a Rv1980c-based diagnostic.     

Immunohistochemical diagnosis of abdominal and lymph node tuberculosis by detecting Mycobacterium tuberculosis complex specific antigen MPT64

Background

Epstein-Barr virus has been proved to be associated with many of the human malignancy including gastric carcinoma, one of the most important human malignancies in the world. There has been no study about the presence of EBV in gastric adenocarcinoma in Iran.

Methods

We examined the presence of EBV in 273 formalin fixed paraffin-embedded cases of gastric carcinoma from Cancer institute of Tehran University, from 1969 to 2004. In situ hybridization of EBV-encoded small RNA-1 (EBER-1) was conducted. The strain of positive cases was examined by means of polymerase chain reaction and/or restriction fragment length polymorphism analysis.

Results

We found 9 (3%; 95% CI = 1–5%) EBV positive cases. The gender difference was not statisticaly significant. The proportion of EBV-GC cases in diffuse type was higher than intestinal type (OR = 0.08; 95% CI = 0.002–0.64). EBV-GC cases had no relation with age, location and invasion. Six out of 9 EBV-GC cases were born during the period between 1928 and 1930. All 9 cases were Type A. Prototype F was seen in 6 out of 8 cases. Type "i" was found in 8 cases and type I in 1 case. XhoI+ and XhoI- polymorphism accounted 6 and 3 of the cases, respectively.

Conclusion

Our study is the first to describe the frequency of EBV-GC in Iran and the Middle East, highlighting a very low prevalence with specific clinicopathologic features. The predominance of EBV-GC birth year in a fixed period, suggests that EBV infection or other events at early childhood may be related to the development of EBV-GC later in the life. The predominance of the type "i" and XhoI+ cases are contradictory to other studies in Asia and is similar to what is reported from Latin American countries.     

Expression, purification, and characterization of protective MPT64 antigen protein and identification of its multimers isolated from nontoxic Mycobacterium tuberculosis H37Ra

MPT64, a secreted protein of Mycobacterium tuberculosis (MTB), stimulates the immune reactions within cells and is a protective antigen that is lost by the bacilli Calmette-Guérin (BCG) vaccine during propagation. To minimize the toxicity caused by MTB, we used the MPT64 gene encoded by nontoxic H37Ra MTB to carry out genetic expansion via polymerase chain reaction and gene clone MPT64. The plasmid DNA encoded MPT64 was expressed at 20°C for 22 H, and a large quantity of MPT64 was obtained. In the absence of urea, MPT64 multimers with subunits being covalently connected via disulfide bonds were detected by Western blot showing strong protein-protein interactions, as evidenced by the formation of MPT64 tetramers. Finally, with urea of decreasing concentrations, we refolded MPT64 purified in the presence of urea and determined its secondary structures using circular dichroism. MPT64 was found to contain 2.2% α-helix, 50.9% β-sheet, 19.5% turn, and 27.4% random coil. The molecular weight of MPT64 was determined by a matrix-assisted laser desorption ionization-time of flight mass spectrometer and found to be 23,497 Da, very close to the theoretical molecular weight of MPT64. The results presented here provide a sound basis for future biochemical and biophysical studies of MPT64 or any other proteins encoded by nontoxic H37Ra MTB.     

Diagnosis of active tuberculosis using MPB64, a specific antigen of Mycobacterium bovis

Because the incidence of tuberculosis (TB) is still high in developing countries, an inexpensive and rapid diagnostic test for this infection is needed. To develop a screening test for TB, MPB64 antigen was produced by recombinant technology and purified with a polyhistidine tag. Next, serum and urine samples from patients with TB and uninfected individuals were examined by the dot‐blot assay method using this purified antigen. Serum samples from patients with TB reacted more strongly with MPB64 antigen than did those from uninfected individuals. In addition, serum samples from TB patients with active infection reacted more strongly with the antigen than did samples from patients with inactive TB. When urine samples were assessed using this assay, similar results were obtained. Correlations between the data obtained from serum and urine samples were analyzed for all subjects, including uninfected individuals, and a strong positive correlation between the results of serum and urine tests (n = 36, r = 0.672) was found. The sensitivity and specificity of this assay for serum samples was 85.7 % and 85.0 %, and for urine samples 75.0 % and 85.0 %, respectively. These results suggest that dot‐blot assay with MPB64 antigen could be a useful screening test for active TB. Because urine samples can be obtained more easily than serum samples and because urine is less contagious, urine testing should probably be employed for screening purposes.     

Recombinant MPT83 derived from Mycobacterium tuberculosis induces cytokine production and upregulates the function of mouse macrophages through TLR2

TLR2 recognizes components of Mycobacterium tuberculosis and initiates APC activities that influence both innate and adaptive immunity. M. tuberculosis lipoproteins are an important class of TLR2 ligands. In this study, we focused on recombinant MPT83 (rMPT83) to determine its effects on mouse macrophages. We demonstrated that rMPT83 induced the production of TNF-α, IL-6, and IL-12 p40 and that cytokine induction depended on activated MAPKs, because we observed the rapid phosphorylation of ERK1/2, p38, and JNK in macrophages. Additionally, neutralizing Abs against TLR2 significantly inhibited cytokine secretion and reduced or attenuated the rMPT83-induced activation of p38 and JNK in RAW264.7 cells, a mouse macrophage cell line. Furthermore, rMPT83-induced cytokine production was significantly lower in macrophages from TLR2(-/-) mice than in macrophages from wild-type mice. We further found that prolonged exposure (>24 h) of RAW264.7 cells or macrophages from wild-type and TLR2(-/-) mice to rMPT83 resulted in a significant enhancement of IFN-γ-induced MHC class II expression and an enhanced ability of macrophages to present the rMPT83 peptide to CD4(+) T cells. These results indicated that rMPT83 is a TLR2 agonist that induces the production of cytokines by macrophages and upregulates macrophage function.     

Expression and purification of Mycobacterium tuberculosis ESAT-6 and MPT64 fusion protein and its immunoprophylactic potential in mouse model

The completion of Mycobacterium tuberculosis genome sequence has opened a new way for the identification and characterization of bacterial antigens, such as ESAT-6, CFP10, MPT64, and Ag85 complex, which are helpful for tuberculosis control. In this work, genes of ESAT-6 and MPT64 were fused and expressed in Escherichia coli in form of inclusion bodies with a histidine tag. The expressed fusion protein was purified by nitrilotriacetic acid (Ni-NTA) affinity chromatography under denaturing conditions, and the yield was 18mg/L of culture. In mice, the purified ESAT-6-MPT64 fusion protein elicited stronger humoral response, greater splenic lymphocyte stimulated index, and higher levels of IFN-gamma and IL-12 production than that of the single MPT64 inoculation group, and rendered modest protection on the experimental tuberculosis mouse models. In short, the ESAT-6-MPT64 fusion protein might be a potential candidate vaccine for tuberculosis.     

Process optimization of large-scale production of recombinant adeno-associated vectors using dielectric spectroscopy

A well-characterized manufacturing process for the large-scale production of recombinant adeno-associated vectors (rAAV) for gene therapy applications is required to meet current and future demands for pre-clinical and clinical studies and potential commercialization. Economic considerations argue in favor of suspension culture-based production. Currently, the only feasible method for large-scale rAAV production utilizes baculovirus expression vectors and insect cells in suspension cultures. To maximize yields and achieve reproducibility between batches, online monitoring of various metabolic and physical parameters is useful for characterizing early stages of baculovirus-infected insect cells. In this study, rAAVs were produced at 40-l scale yielding ~1 × 10¹⁵ particles. During the process, dielectric spectroscopy was performed by real time scanning in radio frequencies between 300 kHz and 10 MHz. The corresponding permittivity values were correlated with the rAAV production. Both infected and uninfected reached a maximum value; however, only infected cell cultures permittivity profile reached a second maximum value. This effect was correlated with the optimal harvest time for rAAV production. Analysis of rAAV indicated the harvesting time around 48 h post-infection (hpi), and 72 hpi produced similar quantities of biologically active rAAV. Thus, if operated continuously, the 24-h reduction in the production process of rAAV gives sufficient time for additional 18 runs a year corresponding to an extra production of ~2 × 10¹⁶ particles. As part of large-scale optimization studies, this new finding will facilitate the bioprocessing scale-up of rAAV and other bioproducts.     

Protection of mice with a divalent tuberculosis DNA vaccine encoding antigens Ag85B and MPT64

Tuberculosis (TB) remains to be a major challenge tothe public health in the world. It is estimated that, through-out the world, 15 individuals are affected by TB and 6 ofthem die from it every minute [1]. Drug resistance andcoinfection with HIV, which ut…     

Isolation and structural characteristics of a monoclonal antibody-defined cross-reactive phospholipid antigen from Mycobacterium tuberculosis and Mycobacterium leprae.

A low molecular weight antigen of Mycobacterium leprae and other mycobacteria was previously defined in our laboratory by means of IgG2a monoclonal antibody termed L4. The antigen had an apparent molecular mass of 4.5-6 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was assumed to be a glycoprotein on the basis of its staining with periodic acid Schiff and sensitivity to periodate treatment. In the present work, the cross-reactive and phospholipidic nature of the antigen, present in Mycobacterium tuberculosis as well as in M. leprae sonicates, was demonstrated and this enabled us to undertake its purification from crude M. tuberculosis phospholipidic extracts. The L4-reactive antigen from M. tuberculosis called L4-PIM, was purified by means of silicic acid high pressure liquid chromatography. Its characterization by gas chromatography and FAB-MS showed the antigen to be the common mycobacterial dimannosylated phosphatidylinositol (PIM2), the structure of which had been previously established by others (Lee, Y. C., and Ballou, C. E. (1964) J. Biol. Chem. 239, 1316-1327; Lee, Y. C., and Ballou, C. E. (1965) J. Biochem. (Tokyo) 4, 1395-1404). Delineation of the L4 epitope on M. tuberculosis L4-PIM revealed the involvement of the axial 2-hydroxyl of the alpha-D-mannosyl residues, without any detectable contribution from the myo-inositol. Consequently, L4 was shown to react with PIM5, the structure of which contains twice the number of epitopes as does PIM2. By using both immunostained thin layer chromatography and indirect enzyme-linked immunosorbent assay, similar L4-PIM epitopes were demonstrated in M. leprae sonicate, thereby explaining the cross-reactive nature of the L4-monoclonal antibody. Antibodies of IgG class directed against M. tuberculosis L4-PIM were detectable in sera from patients with leprosy, but no evidence of T cell reactivity to L4-PIM was obtained. The demonstration of a correlation of anti-L4-PIM IgG and anti-disacharide-conjugated bovine serum albumin IgM antibody titers in the sera of leprosy patients indicates that measurement of antibodies directed against L4-PIM may have the potential to be used as a complementary assay to the disaccharide-conjugated bovine serum albumin test for diagnosis and monitoring of patients undergoing leprosy therapy.     

A semi-automated large-scale process for the production of recombinant tagged proteins in the Baculovirus expression system

The efficient preparation of recombinant proteins at the lab-scale level is essential for drug discovery, in particular for structural biology, protein interaction studies and drug screening. The Baculovirus insect-cell expression system is one of the most widely applied and highly successful systems for production of recombinant functional proteins. However, the use of eukaryotic cells as host organisms and the multi-step protocol required for the generation of sufficient virus and protein has limited its adaptation to industrialized high-throughput operation. We have developed an integrated large-scale process for continuous and partially automated protein production in the Baculovirus system. The instrumental platform includes parallel insect-cell fermentation in 10L BioWave reactors, cell harvesting and lysis by tangential flow filtration (TFF) using two custom-made filtration units and automated purification by multi-dimensional chromatography. The use of disposable materials (bags, filters and tubing), automated cleaning cycles and column regeneration, prevent any cross-contamination between runs. The preparation of the clear cell lysate by sequential TFF takes less than 2 h and represents considerable time saving compared to standard cell harvesting and lysis by sonication and ultra-centrifugation. The process has been validated with 41 His-tagged proteins with molecular weights ranging from 20 to 160 kDa. These proteins represented several families, and included 23 members of the deubiquitinating enzyme (DUB) family. Each down-stream unit can process four proteins in less than 24 h with final yields between 1 and 100 mg, and purities between 50 and 95%.     

6,6'-Dimycoloyl trehalose from a rapidly growing Mycobacterium: an alternative antigen for tuberculosis serodiagnosis

Mycobacterial O-acyltrehaloses have been described as highly specific and sensitive reagents for tuberculosis immunodiagnosis. An O-acyltrehalose-containing lipid fraction from the rapidly growing Mycobacterium fortuitum was found to include additional antigens, which presented high cross-reactivity with sera from tuberculosis-infected patients. Based on a combination of selective chemical degradations, thin-layer-chromatography analyses and (1)H-nuclear magnetic resonance spectroscopy, the antigenic by-product was identified as 6,6'-dimycoloyl trehalose, the so-called cord factor. The lipid was purified and tested in ELISA for pulmonary tuberculosis serodiagnosis. Sensitivity and specificity of the test were found to be 66.6-74.1% and 95.2-99.0%, respectively, showing a slightly higher efficiency as compared to the ELISA performed using 6,6'-dimycoloyl trehalose from Mycobacterium tuberculosis. No cross-reactivity was found with sera from Nocardia-infected individuals.     

Use of a tetracycline-inducible system for conditional expression in Mycobacterium tuberculosis and Mycobacterium smegmatis

A number of essential genes have been identified in mycobacteria, but methods to study these genes have not been developed, leaving us unable to determine the function or biology of the genes. We investigated the use of a tetracycline-inducible expression system in Mycobacterium tuberculosis and Mycobacterium smegmatis. Using a reporter gene which encodes an unstable variant of GFP, we showed that tetracycline-inducible expression occurred in M. smegmatis and that expression levels were titratable to some extent by varying the concentration of tetracycline. The removal of tetracycline led to cessation of GFP expression, and we showed that this was a controllable on/off switch for fluorescence upon addition and removal of the antibiotic inducer. The system also functioned in M. tuberculosis, giving inducible expression of the reporter gene. We used homologous recombination to construct a strain of M. tuberculosis that expressed the only copy of the tryptophan biosynthetic enzyme, TrpD, from the tetracycline-inducible promoter. This strain was conditionally auxotrophic, showing auxotrophy only in the absence of tetracycline, confirming that trpD was tightly controlled by the foreign promoter. This is the first demonstration of the use of an inducible promoter to generate a conditional auxotroph of M. tuberculosis. The ability to tightly regulate genes now gives us the possibility to define the functions of essential genes by switching them off under defined conditions and paves the way for in vivo studies.     

Characterization of serologic and cell-mediated reactivity of a 38-kDa antigen isolated from Mycobacterium tuberculosis

An antigen of Mycobacterium tuberculosis with an m.w. of 38,000 has been isolated by affinity chromatography using a monoclonal antibody. This antibody bound only to an antigen found in M. tuberculosis and Mycobacterium bovis BCG. The specificity of the antigen was tested in a vertical study by immunodetection on western blots reacted with hyperimmune sera against M. tuberculosis, M. bovis, and 10 other Mycobacterium species. The antigen was detected only by antisera to M. tuberculosis and M. bovis. Specificity in cell-mediated immunity was tested by skin tests in guinea pigs sensitized with M. tuberculosis, Mycobacterium intracellulare, and Mycobacterium kansasii and by lymphocyte proliferation tests. The 38-kDa antigen induced positive skin test reactions regardless of the Mycobacterium species used to sensitize the animal. The ability of the 38-kDa antigen to sensitize for cell-mediated immunity was tested by injecting mice with the 38-kDa antigen and challenging their lymphocytes in vitro with various mycobacterial antigens. Lymphocyte proliferation was observed in the presence of 38-kDa antigen, M. tuberculosis sonicate antigen, and tuberculin purified protein derivative and to M. kansasii and M. intracellulare. The 38-kDa antigen may contain a specific epitope detected by serology, but also contains epitopes that are cross-reactive for cellular immunity.     

Immunoreactivity of a 10-kDa antigen of Mycobacterium tuberculosis.

Identification of Ag of Mycobacterium tuberculosis recognized by T cells is essential to understanding the pathogenesis of tuberculosis and mechanism(s) of resistance to infection. Previous studies evaluating the immunoreactivity of nitrocellulose transfers of M. tuberculosis Ag separated by SDS-PAGE indicated that a high proportion of M. tuberculosis-reactive T cell lines proliferate in response to a 10-kDa Ag. We therefore purified this Ag from M. tuberculosis culture filtrates and evaluated its immunoreactivity in patients with tuberculous infection. Proliferative responses of PBMC to the 10-kDa Ag were similar to those induced by whole M. tuberculosis and greater than those elicited by other proteins isolated from culture filtrate. Furthermore, in patients with tuberculous pleuritis, proliferative responses to the 10-kDa Ag were higher in pleural fluid mononuclear cells than in PBMC, indicating that T cell reactivity to this Ag is enhanced at the site of disease. The first 15 amino acids of the 10-kDa Ag were identical to those defined previously for Bacillus Calmette-Guérin-a (BCG-a), and a T cell clone recognized the 10-kDa Ag and a peptide of BCG-a, indicating that the 10-kDa Ag corresponds to BCG-a. This Ag elicited IFN-gamma production by pleural fluid mononuclear cells and by PBMC from healthy tuberculin reactors, suggesting that the 10-kDa Ag can enhance macrophage activation and resistance to mycobacterial infection. Our findings indicate that the 10-kDa Ag of M. tuberculosis is highly immunoreactive and should be evaluated for its capacity to elicit protective immunity.     

Expression and purification of immunologically reactive DPPD, a recombinant Mycobacterium tuberculosis skin test antigen, using Mycobacterium smegmatis and Escherichia coli host cells

DPPD is a Mycobacterium tuberculosis recombinant antigen that elicits specific delayed type hypersensitivity reactions similar in size and morphological aspects to that elicited by purified protein derivative, in both guinea pigs and humans infected with M. tuberculosis. In addition, earlier clinical studies with DPPD suggested that this molecule could improve the specificity of the tuberculin skin test, which is used as an important aid for the diagnosis of tuberculosis. However, these studies could only be performed with DPPD engineered as a fusion molecule with another Mycobacterium spp. protein because no expression of DPPD could be achieved as a single molecule or as a conventional fusion protein in any commercial system. Although recombinant fusion proteins are in general suitable for several biological studies, they are by definition not ideal for studies involving highly purified and defined polypeptide sequences. Here, we report two alternative approaches for the expression of immunologically reactive recombinant genuine DPPD. The first approach used the rapidly growing, nonpathogenic Mycobacterium smegmatis as host cells transformed with the pSMT3 plasmid vector containing the full-length DPPD gene. The second approach used Escherichia coli transformed with the pET-17b plasmid vector containing the DPPD gene engineered in a three-copy fusion manner in tandem with itself. Though at low levels, expression and purification of immunologically reactive DPPD in M. smegmatis could be achieved. More abundant expression and purification of DPPD as a homo-trimer molecule was achieved in E. coli (> or =2 mg/L of bacterial broth cultures). Interestingly, expression could only be achieved in host cells transformed with the DPPD gene containing its leader peptide. However, the expressed proteins lacked the leader sequence, which indicates that processing of the M. tuberculosis DPPD gene was accurately achieved and necessary in both M. smegmatis and E. coli. More importantly, the delayed type hypersensitivity reactions elicited by purified molecules in guinea pigs infected with M. tuberculosis were indistinguishable from that elicited by purified protein derivative. Because the DPPD gene is present only in the tuberculosis-complex organisms of the Mycobacterium genus, these highly purified molecules should be helpful in identifying individuals sensitized with tubercle bacilli.     

Cloning and expression of MPT83 gene from Mycobacterium tuberculosis in E. coli BL21 as vaccine candidate of tuberculosis: A preliminary study

The appearance of Mycobacterium tuberculosis strains leading to drug resistance has caused new problems in TB treatment in various parts of the world and forces WHO to declare TB as a global emergency. With the increase of TB drug resistance, it is convinced that a more effective vaccine development will stop the epidemic of TB. Some M. tuberculosis antigens, one of which is MPT83, have been examined as TB vaccine candidate. MPT83 antigen, which is very immunogenic in lipoprotein micro bacteria, is identified as surface cell interrelated to antigen with cytometry circulation. Having TB resistance from BCG vaccine, MPT83 is considered TB vaccine candidate that can protect people against TB at adult age. The purpose of this research is to conduct amplification of MPT83 antigen cloning, and expression of its antigen on E. coli bacteria. From the result of the research, it is expected that raw material to produce TB vaccine as well as a high-quality antigen can be obtained. The band of DNA in PCR product is 660?bp, while the one in pGEMT-Easy-Mpt83 recombinant plasmid is 3678?bp. This is expressed in E. coli BL21 strain and produces 48?kDa protein as well as GST-MPT83 fusion protein.