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.     

Mycobacterium tuberculosis FurA autoregulates its own expression

The furA-katG region of Mycobacterium tuberculosis, encoding a Fur-like protein and the catalase-peroxidase, is highly conserved among mycobacteria. Both genes are induced upon oxidative stress. In this work we analyzed the M. tuberculosis furA promoter region. DNA fragments were cloned upstream of the luciferase reporter gene, and promoter activity in Mycobacterium smegmatis was measured in both the presence and absence of oxidative stress. The shortest fragment containing an inducible promoter extends 45 bp upstream of furA. In this region, -35 and -10 promoter consensus sequences can be identified, as well as a 23-bp AT-rich sequence that is conserved in the nonpathogenic but closely related M. smegmatis. M. tuberculosis FurA was purified and found to bind upstream of furA by gel shift analysis. A ca. 30-bp DNA sequence, centered on the AT-rich region, was essential for FurA binding and protected by FurA in footprinting analysis. Peroxide treatment of FurA abolished DNA binding. Three different AT-rich sequences mutagenized by site-directed mutagenesis were constructed. In each mutant, both M. tuberculosis FurA binding in vitro and pfurA regulation upon oxidative-stress in M. smegmatis were abolished. Thus, pfurA is an oxidative stress-responsive promoter controlled by the FurA protein.     

Purification and characterization of three immunodominant proteins (38, 30, and 16 kDa) of Mycobacterium tuberculosis

Specific mycobacterial antigens are an important prerequisite in the serodiagnosis of tuberculosis. Many studies have reported the use of both native and recombinant proteins. Even though recombinant proteins can form standardized reagents with unlimited supply, their diagnostic test characteristics were not satisfactory in some cases. In this study we have purified the 38-, 30- (antigen 85B), and 16-kDa native antigens of Mycobacterium tuberculosis by procedures with limited number of steps. Starting with the secreted antigens of M. tuberculosis H37Rv, the 38-kDa form was purified by preparative isoelectric focusing, followed by preparative electrophoresis. Separation of antigen 85 components was achieved by anion-exchange chromatography, followed by hydrophobic interaction chromatography. Gel-permeation chromatography was employed for the isolation of the 16-kDa form, from the cytosol fraction of M. tuberculosis H37Rv. By using a minimal number of steps, considerable yields of these proteins were obtained without loss of immunological activity. The native proteins purified were characterized by analytical two-dimensional electrophoresis, HPLC, and circular dichroism studies. Conformation of the native 38-kDa form purified in our laboratory was different from that of the recombinant 38-kDa form from the WHO Bank. The identities of these native antigens were established by immunoblotting with known monoclonal antibodies from the WHO Bank.     

Identification and characterization of a gene encoding a 35-kDa protein from Mycobacterium avium subspecies paratuberculosis

Mycobacterium avium subspecies paratuberculosis is the causative agent of Johne's disease, a chronic enteritis in ruminants. A gene homologous to that of 35-kDa antigen of Mycobacterium leprae was cloned and sequenced from Mycobacterium paratuberculosis. The database searches revealed 82.79% and 95.67% similarities of its nucleotide sequence, with those of immunodominant 35-kDa protein of M. leprae and M. avium, respectively.     

Decreased capacity of recombinant 45/47-kDa molecules (Apa) of Mycobacterium tuberculosis to stimulate T lymphocyte responses related to changes in their mannosylation pattern.

The Apa molecules secreted by Mycobacterium tuberculosis, Mycobacterium bovis, or BCG have been identified as major immunodominant antigens. Mass spectrometry analysis indicated similar mannosylation, a complete pattern from 1 up to 9 hexose residues/mole of protein, of the native species from the 3 reference strains. The recombinant antigen expressed in M. smegmatis revealed a different mannosylation pattern: species containing 7 to 9 sugar residues/mole of protein were in the highest proportion, whereas species bearing a low number of sugar residues were almost absent. The 45/47-kDa recombinant antigen expressed in E. coli was devoid of sugar residues. The proteins purified from M. tuberculosis, M. bovis, or BCG have a high capacity to elicit in vivo potent delayed-type hypersensitivity (DTH) reactions and to stimulate in vitro sensitized T lymphocytes of guinea pigs immunized with living BCG. The recombinant Apa expressed in Mycobacterium smegmatis was 4-fold less potent in vivo in the DTH assay and 10-fold less active in vitro to stimulate sensitized T lymphocytes than the native proteins. The recombinant protein expressed in Escherichia coli was nearly unable to elicit DTH reactions in vivo or to stimulate T lymphocytes in vitro. Thus the observed biological effects were related to the extent of glycosylation of the antigen.     

Staphylococcus aureus nuclease is a useful secretion reporter for mycobacteria.

A secretion reporter system based on Staphylococcus aureus nuclease (nuc) was developed for use in mycobacteria. Fusion of secretion signals to the reporter cloned in a shuttle vector, pBPnuc1, resulted in halo formation around colonies of Mycobacterium smegmatis and Mycobacterium tuberculosis grown on DNase agar plates containing Methyl Green indicator dye. This in-situ detection system was used to identify secreted proteins by screening a pBPnuc1::H37Rv nuc gene fusion library in M. smegmatis. The clones identified in this screen all formed colony halos when present in M. tuberculosis grown on indicator media. The proteins corresponded to DesA2, a stearoyl-acyl carrier protein desaturase, PepA, a putative serine protease and the Apa antigen, which is the ATP-binding subunit of an ABC transport system. Of these proteins, only PepA and Apa contained recognizable leader peptides.     

Identification of mycobacterium tuberculosis DNA sequences encoding exported proteins by using phoA gene fusions.

The activity of bacterial alkaline phosphatase (PhoA) is dependent on it being exported across the plasma membrane. A plasmid vector (pJEM11) allowing fusions between phoA and genes encoding exported proteins was constructed to study protein export in mycobacteria. Introduction of the Mycobacterium fortuitum beta-lactamase gene (blaF*) into this vector led to the production in M. smegmatis of protein fusions with PhoA activity. A genomic library from M. tuberculosis was constructed in pJEM11 and screened in M. smegmatis for clones with PhoA activity. Sequences of the M. tuberculosis inserts directing the production of protein fusions in these PhoA-positive clones were determined. They include part of the already-known exported 19-kDa lipoprotein, a sequence with similarities to the exported 28-kDa antigen from M. leprae, a sequence encoding a protein sharing conserved amino acid motifs with stearoyl-acyl-carrier-protein desaturases, and unknown sequences. This approach thus appears to identify sequences directing protein export, and we expect that more extensive screening of such libraries will lead to a better understanding of protein export in M. tuberculosis.     

The 65-kilodalton antigen of Mycobacterium tuberculosis.

The immune response of the host to the antigens of Mycobacterium tuberculosis plays the key role in determining immunity from infection with as well as the pathogenicity of this organism. A 65-kilodalton (kDa) protein has been identified as one of the medically important antigens of M. tuberculosis. The gene encoding this antigen was isolated from a lambda gt11-M. tuberculosis recombinant DNA library using monoclonal antibodies directed against the 65-kDa antigen as the specific probes. The nucleotide sequence of this gene was determined, and a 540-amino-acid sequence was deduced. This sequence was shown to correspond to that of the 65-kDa antigen by constructing a plasmid in which this open reading frame was fused to the lacZ gene. The resulting fusion protein reacted specifically with the anti-65-kDa protein antibodies. A second long open reading frame was found downstream of the 65-kDa antigen gene which could encode a protein of 517 amino acids. This putative protein contained 29 tandemly arranged partial or complete matches to a pentapeptide sequence.     

Identification and characterization of a regulatory sequence recognized by Mycobacterium tuberculosis persistence regulator MprA

Establishment and maintenance of persistent, latent infection by Mycobacterium tuberculosis are dependent on expression of the mprA-mprB regulatory system. Previously, MprA and MprB were shown to participate in phosphotransfer reactions characteristic of two-component signaling systems. To begin identifying downstream effector genes regulated by mprA-mprB during persistent stages of infection, a search for the regulatory sequence(s) recognized by response regulator MprA was carried out. Here, evidence is presented demonstrating that MprA recognizes a 19-bp sequence comprising two loosely conserved 8-bp direct repeat subunits separated by 3 nucleotides. This motif, termed the MprA box, is found upstream of the mprA coding sequence and that of downstream gene pepD (Rv0983). Protein phosphorylation was not required for binding to this DNA sequence by MprA in vitro; however, phosphorylation enhanced DNA binding by MprA and was required for the regulation of mprA and pepD by MprA in vivo. Binding of MprA to the MprA box was dependent on conserved nucleotides contained within repeat subunits and on the spacer length separating these repeats. In addition, recognition of this sequence proceeded via tandem binding of two monomers of MprA. Identification of the genetic determinants regulated by MprA will ultimately enhance our understanding of the mechanisms utilized by M. tuberculosis to undergo latency.     

The MprB Extracytoplasmic Domain Negatively Regulates Activation of the Mycobacterium tuberculosis MprAB Two-Component System

Mycobacterium tuberculosis is an acid-fast pathogen of humans and the etiological agent of tuberculosis (TB). It is estimated that one-third of the world''s population is latently (persistently) infected with M. tuberculosis. M. tuberculosis persistence is regulated, in part, by the MprAB two-component signal transduction system, which is activated by and mediates resistance to cell envelope stress. Here we identify MprAB as part of an evolutionarily conserved cell envelope stress response network and demonstrate that MprAB-mediated signal transduction is negatively regulated by the MprB extracytoplasmic domain (ECD). In particular, we report that deregulated production of the MprB sensor kinase, or of derivatives of this protein, negatively impacts M. tuberculosis growth. The observed growth attenuation is dependent on MprAB-mediated signal transduction and is exacerbated in strains of M. tuberculosis producing an MprB variant lacking its ECD. Interestingly, full-length MprB, and the ECD of MprB specifically, immunoprecipitates the Hsp70 chaperone DnaK in vivo, while overexpression of dnaK inhibits MprAB-mediated signal transduction in M. tuberculosis grown in the absence or presence of cell envelope stress. We propose that under nonstress conditions, or under conditions in which proteins present in the extracytoplasmic space are properly folded, signaling through the MprAB system is inhibited by the MprB ECD. Following exposure to cell envelope stress, proteins present in the extracytoplasmic space become unfolded or misfolded, leading to removal of the ECD-mediated negative regulation of MprB and subsequent activation of MprAB.     

The twin-arginine translocation pathway of Mycobacterium smegmatis is functional and required for the export of mycobacterial beta-lactamases

The twin-arginine translocation (Tat) pathway exports folded proteins across the bacterial cytoplasmic membrane and is responsible for the proper extracytoplasmic localization of proteins involved in a variety of cellular functions, including pathogenesis. The Mycobacterium tuberculosis and Mycobacterium smegmatis genomes contain open reading frames with homology to components of the Tat export system (TatABC) as well as potential Tat-exported proteins possessing N-terminal signal sequences with the characteristic twin-arginine motif. Due to the importance of exported virulence factors in the pathogenesis of M. tuberculosis and the limited understanding of mycobacterial protein export systems, we sought to determine the functional nature of the Tat export pathway in mycobacteria. Here we describe phenotypic analyses of DeltatatA and DeltatatC deletion mutants of M. smegmatis, which demonstrated that tatA and tatC encode components of a functional Tat system capable of exporting characteristic Tat substrates. Both mutants displayed a growth defect on agar medium and hypersensitivity to sodium dodecyl sulfate. The mutants were also defective in the export of active beta-lactamases of M. smegmatis (BlaS) and M. tuberculosis (BlaC), both of which possess twin-arginine signal sequences. The Tat-dependent nature of BlaC was further revealed by mutation of the twin-arginine motif. Finally, we demonstrated that replacement of the native signal sequence of BlaC with the predicted Tat signal sequences of M. tuberculosis phospholipase C proteins (PlcA and PlcB) resulted in the Tat-dependent export of an enzymatically active 'BlaC. Thus, 'BlaC can be used as a genetic reporter for Tat-dependent export in mycobacteria.     

Characterization of T cell antigens associated with the cell wall protein-peptidoglycan complex of Mycobacterium tuberculosis

Mycobacterium tuberculosis cell walls are likely to contain critical T cell Ag capable of inducing protective immunity against the development of tuberculosis in animal models. Therefore, we characterized cell wall-associated Ag that stimulate T lymphocytes in tuberculosis patients and clinically well tuberculin-positive individuals. A protein-peptidoglycan complex isolated from the M. tuberculosis cell wall had potent immunologic activity, evoking PBMC proliferative responses similar to those induced by sonicated whole M. tuberculosis. In order to characterize the immunoreactive protein determinants associated with the protein-peptidoglycan complex, T cell lines were established to cell wall Ag and used to probe M. tuberculosis proteins separated by SDS-PAGE. These T cell lines proliferated primarily to protein Ag of 10, 19, 23, 28, 30, 40 to 50, and 65 kDa. Cell wall-reactive T cell clones that recognized the 10-, 23-, 28-, and 30-kDa proteins as single bands on SDS-PAGE did so under reducing and nonreducing conditions, suggesting that these are not proteolytic fragments or subunits of larger protein aggregates. We propose that these protein monomers, when post-translationally complexed with peptidoglycan, are the key ingredients of the immunogenic protein-peptidoglycan complex. In order to assess the relationship of the cell wall-associated Ag to those secreted proteins from "early culture filtrates" of actively growing M. tuberculosis recently implicated in eliciting protective immunity, cell wall-reactive T cell clones were tested for their ability to recognize early culture filtrates. Results revealed that at least three proteins shared with the cell wall complex are contained within early culture filtrates. Our data indicate that antigenic determinants associated with the protein-peptidoglycan complex of the M. tuberculosis cell wall may be involved in protective immunity and hence are potential candidates for inclusion in an effective antituberculosis vaccine.     

Identification of the new T-cell-stimulating antigens from Mycobacterium tuberculosis culture filtrate

The proteins secreted by Mycobacterium tuberculosis are an important target for vaccine development. To identify the antigens from M. tuberculosis culture filtrate (CF) that strongly stimulate T-cells, the CF was fractionated by ion-exchange chromatography and then non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis with mini-whole gel elution. Each fraction was screened for its ability to induce interferon-gamma (IFN-gamma) production in peripheral blood mononuclear cells isolated from healthy tuberculin reactors. The protein bands that strongly induced IFN-gamma production were subjected to N-terminal sequencing. Two new proteins, a 17-kDa protein (Rv0164, MTSP17) and an 11-kDa (Rv3204, MTSP11) protein, were identified. The recombinant MTSP17 (rMTSP17) and rMTSP11 induced significant production of IFN-gamma and interleukin (IL)-12p40 in peripheral blood mononuclear cells from healthy tuberculin reactors. Interestingly, IL-12p40 production in response to rMTSP11 was significantly higher than that in response to rMTSP17 or the three components of the antigen 85 complex. These results suggest that MTSP11 antigen should be further evaluated as a component of a subunit vaccine.     

High-level heterologous expression and secretion in Streptomyces lividans of two major antigenic proteins from Mycobacterium tuberculosis

Two major antigens from Mycobacterium tuberculosis were produced by Streptomyces lividans as secreted extracellular proteins. An expression-secretion vector had been constructed that contained the promoter of xylanase A and the signal sequence of cellulase A. The latter contained two initiation codons preceded by a Shine-Dalgarno sequence plus eight nucleotides complementary to the 16S rRNA. The genes encoding the 38-kDa (Rv0934) and 19-kDa (Rv3763) proteins, respectively, were amplified by polymerase chain reaction and cloned into that vector. The recombinant proteins were then purified from the culture supernatants of the clones. The yields after purification were 80 mg/L for the 38-kDa protein and 200 mg/L for the 19-kDa protein. Sequence analysis of the N-terminal sequences showed a deletion of seven or eight amino acids for the 38-kDa protein, while in the 19-kDa protein 22 or 23 amino acids were lost, as compared with the respective wild-type proteins. However, the 19 kDa recombinant protein had the same N-terminal sequence as the one recovered from the M. tuberculosis culture supernatant. The high yields obtained for these two proteins demonstrated the potential of S. lividans as an alternative host for the production of recombinant proteins from M. tuberculosis. The culture conditions have yet to be worked out to minimize proteolytic degradation and to recover intact products.     

Stationary phase-associated protein expression in Mycobacterium tuberculosis: function of the mycobacterial alpha-crystallin homolog.

The majority of active tuberculosis cases arise as a result of reactivation of latent organisms which are quiescent within the host. The ability of mycobacteria to survive extended periods without active replication is a complex process whose details await elucidation. We used two-dimensional gel electrophoresis to examine both steady-state protein composition and time-dependent protein synthetic profiles in aging cultures of virulent Mycobacterium tuberculosis. At least seven proteins were maximally synthesized 1 to 2 weeks following the end of log-phase growth. One of these proteins accumulated to become a predominant stationary-phase protein. N-terminal amino acid sequencing and immunoreactivity identified this protein as the 16-kDa alpha-crystallin-like small heat shock protein. The gene for this protein was shown to be limited to the slowly growing M. tuberculosis complex of organisms as assessed by Southern blotting. Overexpression of this protein in wild-type M. tuberculosis resulted in a slower decline in viability following the end of log-phase growth. Accumulation of this protein was observed in log-phase cultures following a shift to oxygen-limiting conditions but not by other external stimuli. The protein was purified to homogeneity from overexpressing M. smegmatis in two steps and shown to have a significant ability to suppress the thermal denaturation of alcohol dehydrogenase. Collectively, these results suggest that the mycobacterial alpha-crystallin protein may play a role in enhancing long-term protein stability and therefore long-term survival of M. tuberculosis.     

Mycobacterium tuberculosis Rv2536 protein implicated in specific binding to human cell lines

The gene encoding the Mycobacterium tuberculosis Rv2536 protein is present in the Mycobacterium tuberculosis complex (as assayed by PCR) and transcribed (as determined by RT-PCR) in M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. bovis BCG, and M. africanum strains. Rabbits immunized with synthetic polymer peptides from this protein produced antibodies specifically recognizing a 25-kDa band in mycobacterial sonicate. U937 and A549 cells were used in binding assays involving 20-amino-acid-long synthetic peptides covering the whole Rv2536 protein sequence. Peptide 11207 (161DVFSAVRADDSPTGEMQVAQY180) presented high specific binding to both types of cells; the binding was saturable and presented nanomolar affinity constants. Cross-linking assays revealed that this peptide specifically binds to 50 kDa U937 cell membrane and 45 kDa A549 cell membrane proteins.