Early secreted antigen ESAT-6 of Mycobacterium Tuberculosis promotes apoptosis of macrophages via targeting the microRNA155-SOCS1 interaction.

BACKGROUND: The early secreted antigenic target 6-kDa protein (ESAT-6) of Mycobacterium tuberculosis (Mtb) not only acts as a key player for virulence but also exhibits a strong immunotherapeutic potential against Mtb. However, little is known about the molecular basis for its potential in immunotherapy. The present study was designed to unravel the role of miRNA-155 in ESAT-6-mediated enhancement of host immunity and apoptosis in macrophages. METHODS: Lentivirus-mediated miR-155 sponge and miR-155 and SOCS1 overexpression vectors were developed in macrophages. TLR2- or p65-specific siRNA knockdown was employed to silence TLR2 or p65. Quantitative polymerase chain reaction and western blotting analyses were performed to determine mRNA and protein expression levels, respectively. Macrophage apoptosis was analyzed by flow cytometry. RESULTS: ESAT-6 significantly increased miR-155 expression, which was dependent on TLR2/NF-κB activation in macrophages. Induced expression of miRNA-155 was required for the ESAT-6-mediated protective immune response and macrophage apoptosis. ESAT-6 promoted macrophage apoptosis by targeting the miR-155-SOCS1 pathway. The differential expression levels of TLR2, BIC, and SOCS1 were involved in regulating the immune response in human peripheral blood mononuclear cells of patients with active tuberculosis (TB) and latent TB (LTB). CONCLUSION: ESAT-6 promotes apoptosis of macrophages via targeting the miRNA155-SOCS1 interaction.

Truncated hemoglobin GlbO from Mycobacterium leprae alleviates nitric oxide toxicity.

As a consequence of reductive genome evolution, the obligate intracellular pathogen Mycobacterium leprae has minimized the repertoire of genes implicated in defense against reactive oxygen and nitrogen species. Genes for multiple hemoglobin types coexist in mycobacterial genomes, but M. leprae has retained only glbO, encoding a group-II truncated hemoglobin. Mycobacterium tuberculosis GlbO has been involved in oxygen transfer and respiration during hypoxia, but a role in protection from nitric oxide (NO) has not been documented yet. Here, we report that the in vitro reaction of oxygenated recombinant M. leprae GlbO with NO results in an immediate stoichiometric formation of nitrate, concomitant with heme-protein oxidation. Overexpression of GlbO alleviates the growth inhibition of Escherichia colihmp (flavohemoglobin gene) mutants in the presence of NO-donors, partly complementing the defect in Hmp synthesis. A promoter element upstream of glbO was predicted in silico, and confirmed by using a glbO::lacZ transcriptional fusion in the heterologous Mycobacterium smegmatis system. The glbO::lacZ fusion was expressed through the whole growth cycle of M. smegmatis, and moderately induced by NO. We propose that M. leprae, by retaining the unique truncated hemoglobin GlbO, may have coupled O2 delivery to the terminal oxidase with a defensive mechanism to scavenge NO from respiratory enzymes. These activities would help to sustain the obligate aerobic metabolism required for intracellular survival of leprosy bacilli.

Identification and characterization of an immunogenic 22 kDa exported protein of Mycobacterium avium subspecies paratuberculosis.

An exported 22 kDa putative lipoprotein was identified in an alkaline phosphatase gene fusion library of Mycobacterium avium subsp. paratuberculosis and expressed in Mycobacterium smegmatis. The full nucleic acid sequence of the gene encoding P22 was determined and the ORF was cloned into a mycobacterial expression vector, enabling full-length P22 to be produced as a C-terminal polyhistidine-tagged protein in M. smegmatis. N-terminal sequencing of the recombinant protein confirmed cleavage of a signal sequence. Native P22 was detected in culture supernatants and cell sonicates of M. avium subsp. paratuberculosis strain 316F using rabbit antibody raised to recombinant P22. Investigation of the presence of similar genes in other mycobacterial species revealed that the gene was present in Mycobacterium avium subsp. avium and similar genes existed in Mycobacterium intracellulare and Mycobacterium scrofulaceum. Database searches showed that P22 belonged to the LppX/LprAFG family of mycobacterial lipoproteins also found in Mycobacterium leprae and in members of the Mycobacterium tuberculosis complex. P22 shared less than 75% identity to these proteins. Recombinant P22 was able to elicit interferon-gamma secretion in blood from eight of a group of nine sheep vaccinated with a live attenuated strain of M. avium subsp. paratuberculosis (strain 316F) compared to none from a group of five unvaccinated sheep. Antibody to P22 was detected by Western blot analysis in 10 out of 11 vaccinated sheep, in two out of two clinically affected cows and in 11 out of 13 subclinically infected cows.

The mycelium-associated Streptomyces reticuli catalase-peroxidase, its gene and regulation by FurS.

During early stages of growth, Streptomyces reticuli synthesizes a hyphae-associated, haem-containing enzyme which exhibits catalase and peroxidase activities with broad substrate specificity (CpeB). The purified dimeric enzyme (160 kDa) consists of two identical subunits. Using anti-CpeB antibodies and an expression- as well as a mini-library, the corresponding cpeB gene was identified and sequenced. It encodes a protein of 740 aa with a molecular mass of 81.3 kDa. The deduced protein shares the highest level of amino acid identity with KatG from Caulobacter crescentus and Mycobacterium tuberculosis, and PerA from Bacillus stearothermophilus. Streptomyces lividans transformants carrying cpeB and the upstream-located furS gene with its regulatory region on the bifunctional vector pWHM3 produced low or enhanced levels of CpeB in the presence or absence of Fe ions, respectively. An in-frame deletion of the major part of furS induces increased CpeB synthesis. The data imply that FurS regulates the transcription of cpeB. The deduced FurS protein is rich in histidine residues, contains a putative N-terminally situated helix-turn-helix motif and has a molecular mass of 15.1 kDa. It shares only 29% amino acid identity with the Escherichia coli ferric uptake regulator (Fur) protein, but about 64% with FurA deduced from the genomic sequences of several mycobacteria. The predicted secondary structures of FurS and FurA are highly similar and considerably divergent from those of the E. coli Fur. In contrast to some Gram-negative bacteria, within several mycobacteria an intact furA gene or a furA pseudogene is upstream of a catalase-peroxidase (katG) gene predicted to encode a functional or a non-functional (Mycobacterium leprae) enzyme. Thus the data obtained for Streptomyces reticuli are expected to serve as an additional model to elucidate the regulation of mycobacterial catalase-peroxidase genes.

Engineering a change in metal-ion specificity of the iron-dependent superoxide dismutase from Mycobacterium tuberculosis-- X-ray structure analysis of site-directed mutants.

We have refined the X-ray structures of two site-directed mutants of the iron-dependent superoxide dismutase (SOD) from Mycobacterium tuberculosis. These mutations which affect residue 145 in the enzyme (H145Q and H145E) were designed to alter its metal-ion specificity. This residue is either Gln or His in homologous SOD enzymes and has previously been shown to play a role in active-site interactions since its side-chain helps to coordinate the metal ion via a solvent molecule which is thought to be a hydroxide ion. The mutations were based on the observation that in the closely homologous manganese dependent SOD from Mycobacterium leprae, the only significant difference from the M. tuberculosis SOD within 10 A of the metal-binding site is the substitution of Gln for His at position 145. Hence an H145Q mutant of the M. tuberculosis (TB) SOD was engineered to investigate this residue's role in metal ion dependence and an isosteric H145E mutant was also expressed. The X-ray structures of the H145Q and H145E mutants have been solved at resolutions of 4.0 A and 2.5 A, respectively, confirming that neither mutation has any gross effects on the conformation of the enzyme or the structure of the active site. The residue substitutions are accommodated in the enzyme's three-dimensional structure by small local conformational changes. Peroxide inhibition experiments and atomic absorption spectroscopy establish surprisingly the H145E mutant SOD has manganese bound to it whereas the H145Q mutant SOD retains iron as the active-site metal. This alteration in metal specificity may reflect on the preference of manganese ions for anionic ligands.

A mycobacterial extracytoplasmic function sigma factor involved in survival following stress.

The extracytoplasmic function (ECF) sigma factors constitute a diverse group of alternative sigma factors that have been demonstrated to regulate gene expression in response to environmental conditions in several bacterial species. Genes encoding an ECF sigma factor of Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium smegmatis, designated sigE, were cloned and analyzed. Southern blot analysis demonstrated the presence of a single copy of this gene in these species and in Mycobacterium bovis BCG, Mycobacterium leprae, and Mycobacterium fortuitum. Sequence analysis showed the sigE gene to be highly conserved among M. tuberculosis, M. avium, M. smegmatis, and M. leprae. Recombinant M. tuberculosis SigE, when combined with core RNA polymerase from M. smegmatis, reconstituted specific RNA polymerase activity on sigE in vitro, demonstrating that this gene encodes a functional sigma factor. Two in vivo transcription start sites for sigE were also identified in M. smegmatis and M. bovis BCG. Comparison of wild-type M. smegmatis with a sigE mutant strain demonstrated decreased survival of the mutant under conditions of high-temperature heat shock, acidic pH, exposure to detergent, and oxidative stress. An inducible protective response to oxidative stress present in the wild type was absent in the mutant. The mycobacterial SigE protein, although nonessential for viability in vitro, appears to play a role in the ability of these organisms to withstand a variety of stresses.

Characterization of the gene encoding the immunodominant 35 kDa protein of Mycobacterium leprae.

Analysis of the interaction between the host immune system and the intracellular parasite Mycobacterium leprae has identified a 35 kDa protein as a dominant antigen. The native 35 kDa protein was purified from the membrane fraction of M. leprae and termed MMPI (major membrane protein I). As the purified protein was not amenable to N-terminal sequencing, partial proteolysis was used to establish the sequences of 21 peptides. A fragment of the 35 kDa protein-encoding gene was amplified by the polymerase chain reaction from M. leprae chromosomal DNA with oligonucleotide primers derived from internal peptide sequences and the whole gene was subsequently isolated from a M. leprae cosmid library. The nucleotide sequence of the gene revealed an open reading frame of 307 amino acids containing most of the peptide sequences derived from the native 35 kDa protein. The calculated subunit mass was 33.7 kDa, but the native protein exists as a multimer of 950 kDa. Database searches revealed no identity between the 35 kDa antigen and known protein sequences. The gene was expressed in Mycobacterium smegmatis under the control of its own promoter or at a higher level using an 'up-regulated' promoter derived from Mycobacterium fortuitum. The gene product reacted with monoclonal antibodies raised to the native protein. Using the bacterial alkaline phosphatase reporter system, we observed that the 35 kDa protein was unable to be exported across the membrane of recombinant M. smegmatis. The 35 kDa protein-encoding gene is absent from members of the Mycobacterium tuberculosis complex, but homologous sequences were detected in Mycobacterium avium, Mycobacterium haemophilum and M. smegmatis. The availability of the recombinant 35 kDa protein will permit dissection of both antibody- and T-cell-mediated immune responses in leprosy patients.

Molecular and immunological characterization of the highly conserved antigen 84 from Mycobacterium tuberculosis and Mycobacterium leprae.

Crossed immunoelectrophoresis (CIE) has been used to develop a reference system for classifying mycobacterial antigens. The subsequent use of specific antibodies allowed further determination of antigens by molecular weight. The monoclonal antibody F126-2, originally raised against a 34-kDa antigen of Mycobacterium kansasii, reacted with antigen 84 (Ag84) in the CIE reference system for Mycobacterium bovis BCG and Mycobacterium tuberculosis. To characterize Ag84, we screened a lambda gt11 gene library from M. tuberculosis with antibody F126-2 and identified the encoding gene. The corresponding Mycobacterium leprae Ag84 gene was subsequently selected from a cosmid library, using the M. tuberculosis gene as a probe. Both genes were expressed as 34-kDa proteins in Escherichia coli, and the recombinant proteins indeed corresponded to Ag84 in the CIE reference system. The derived amino acid sequences of the M. tuberculosis and M. leprae proteins showed 85% identity, which indicates that Ag84 constitutes a group of highly conserved mycobacterial antigens. Antibodies of almost 60% of lepromatous leprosy patients responded to Ag84, indicating that the protein is highly immunogenic following infection in multibacillary leprosy.

Cloning, sequencing and expression in Escherichia coli of the gene for alpha antigen from Mycobacterium scrofulaceum.

The gene for the extracellular alpha antigen of Mycobacterium scrofulaceum (S-alpha) was cloned by using the alpha antigen gene fragments of Mycobacterium bovis BCG as probes. The complete nucleotide sequence was determined. The gene was expressed in Escherichia coli. The gene encodes 330 amino acids, including 40 amino acids for the signal peptide, followed by 290 amino acids for the mature protein. The deduced amino acid sequences were highly homologous to the alpha antigen of the species of other mycobacteria. Interestingly, the alpha antigens of MAIS complex (Mycobacterium avium-Mycobacterium intracellulare-M. scrofulaceum) were closely homologous even at the C-terminal regions which were variable among those of M. bovis BCG, Mycobacterium leprae and Mycobacterium kansasii.

A fully modular vector system for the optimization of gene expression in Escherichia coli.

The new expression vector system CYTEXP is designed to facilitate the optimization of both transcription and translation in Escherichia coli, while at the same time allowing the exchange of its major components using unique restriction sites. In vitro mutagenesis can be performed in situ using single-stranded DNA generated from the bacteriophage f1 ORI sequence. The basic vector pCYTEXP1 bears a synthetic copy of the intercistronic sequence that enhances the translation of the E. coli atpE gene. Reading frames can be inserted directly downstream of this sequence. The bacteriophage lambda promoters, the atpE sequence, the bacteriophage fd transcriptional terminator, the f1 ORI, and the amp antibiotic resistance gene are all borne on exchangeable "modules." Thus, both the efficiency and the conditions of expression of cloned genes can be readily optimized.

Development of monoclonal antibodies reacting against mycobacterial 65 kDa heat shock protein by using recombinant truncated products.

A mycobacterial 65 kDa molecule is a member of the GroEL heat shock protein family. We developed mAbs reacting against recombinant 65 kDa protein by using a gene (pTB12) which encodes this protein. Three mAbs (B20, B97 and B167) reacted selectively with 65 kDa proteins of Mycobacterium tuberculosis, BCG and Mycobacterium leprae, although B20 and B167 may weakly react with a 15 kDa molecule of mammalian cells. One (B108) was obviously cross-reactive between mycobacterial 65 kDa and the mammalian intracytoplasmic protein. We also developed deletion mutants of pTB12. The localization of these mAb-defined epitopes was determined by using truncated proteins of the Mycobacterium tuberculosis 65 kDa molecule produced in E. coli. Immunohistochemical analysis showed that B20, B97 and B167 mAbs could detect this antigen in experimental granulomas induced by injection of BCG in the subcutaneous tissue of rats. These mAbs should be useful for analyzing the immunobiologic roles of mycobacterial 65 kDa molecules.

Glucose oxidase from Aspergillus niger. Cloning, gene sequence, secretion from Saccharomyces cerevisiae and kinetic analysis of a yeast-derived enzyme.

The gene for Aspergillus niger glucose oxidase (EC 1.1.3.4) has been cloned from both cDNA and genomic libraries using oligonucleotide probes derived from the amino acid sequences of peptide fragments of the enzyme. The mature enzyme consists of 583 amino acids and is preceded by a 22-amino acid presequence. No intervening sequences are found within the coding region. The enzyme contains 3 cysteine residues and 8 potential sites for N-linked glycosylation. The protein shows 26% identity with alcohol oxidase of Hansenuela polymorpha, and the N terminus has a sequence homologous with the AMP-binding region of other flavoenzymes such as p-hydroxybenzoate hydroxylase and glutathione reductase. Recombinant yeast expression plasmids have been constructed containing a hybrid yeast alcohol dehydrogenase II-glyceraldehyde-3-phosphate dehydrogenase promoter, either the yeast alpha-factor pheromone leader or the glucose oxidase presequence, and the mature glucose oxidase coding sequence. When transformed into yeast, these plasmids direct the synthesis and secretion of between 75 and 400 micrograms/ml of active glucose oxidase. Analysis of the yeast-derived enzymes shows that they are of comparable specific activity and have more extensive N-linked glycosylation than the A. niger protein.

High level expression of genes cloned in phage lambda gt11.

Plasmid cloning vectors have been constructed which allow genes originally cloned in lambda gt11 to be expressed at a high level in Escherichia coli. They are based on the pEMBL and pUC vectors, with the genes transcribed from the lac promoter. The EcoRI site in the vector has been altered to be in the same reading frame as the site used for cloning in lambda gt11. Cloned proteins are expressed fused to a 2-kDa leader sequence containing a run of six Aparagine residues which considerably improves the stability of the recombinant proteins, but does not interfere with immunological assays. Using these vectors, the Mycobacterium leprae 18-kDa protein was expressed at 20 mg per litre of culture and constituted 15% of total cell protein.

Expression of Mycobacterium leprae genes from a Streptococcus mutans promoter in Escherichia coli K-12.

Genomic libraries of Mycobacterium leprae DNA partially digested with Pst I were constructed in the expression vector pYA626, which contains the promoter region from the Streptococcus mutans gene encoding aspartate beta-semialdehyde dehydrogenase, which is very efficiently expressed in Escherichia coli. We have detected several clones that complement a mutation in the citrate synthase gene of E. coli. Southern blot analysis demonstrated that the complementing DNA was M. leprae DNA. Sodium dodecyl sulfate/polyacrylamide gel analysis of polypeptides produced by minicells containing the citrate synthase-complementing recombinant molecules demonstrated the production of a 46-kDa polypeptide. When the citrate synthase-complementing fragment was cloned in pYA626 in the reverse orientation, the recombinant molecule was no longer able to complement the mutation in the citrate synthase gene and no longer produced the 46-kDa polypeptide. When the DNA fragment was cloned in the Pst I site of pHC79, so as to allow expression from the beta-lactamase promoter, the resulting recombinant failed to complement the mutation in the E. coli citrate synthase gene yet still produced the 46-kDa polypeptide, but in one-fourth the amount than when expressed from the S. mutans asd promoters. This demonstrates that M. leprae translational sequences can be recognized by E. coli translational machinery. Promoter expression vectors can be used to obtain expression of protein antigens to be used for early diagnosis of leprosy or components of a vaccine and proteins that are targets of potential antileprosy drugs.