Cloning and sequence analysis of the 10 kDa antigen gene of Mycobacterium tuberculosis

The gene encoding a major protein antigen of Mycobacterium tuberculosis has been cloned and sequenced using oligonucleotide probes derived from the N-terminal sequence of the analogous protein from Mycobacterium bovis BCG. The gene analysis revealed a sequence encoding a protein of 99 amino acid residues, with a molecular mass of 10.7 kDa. Computer prediction suggests that the protein contains three T-cell-determined epitopes (of which one has been demonstrated experimentally) and three B-cell-determined epitopes. The protein sequence was homologous to two prokaryote heat-shock proteins and the gene possessed heat-shock-like promoter sequences upstream of the initiation codon. A hairpin loop identified in the upstream sequence may also be important in regulation of the gene.     

Duplication of genes encoding the immunodominant 38 kDa antigen in Mycobacterium intracellulare

Abstract Mycobacterium avium is a causative agent of mycobacterioses in systemically immunocompromised individuals, whereas Mycobacterium intracellulare is responsible for causing infections in relatively immunocompetent hosts. In an attempt to identify components that could be involved in virulence, we characterised the 38 kDa-encoding gene of M. intracellulare that is absent in M. avium . This antigen cross-reacts immunologically with a major 38 kDa antigen of M. tuberculosis , and both antigens are homologues of the phosphate transport subunit S (PstS) of the pst complex of Escherichia coli . Unlike the M. tuberculosis complex the M. intracellulare coding gene was found to be duplicated. We also identified and characterised other pst genes that may constitute an operon. Considering that multiple isoforms of PstS are present in mycobacteria the possible role of pstS1 genes for pathogenesis is discussed.     

Extensive sequence homology between the mycobacterium leprae LSR (12 kDa) antigen and its Mycobacterium tuberculosis counterpart

The Mycobacterium leprae LSR (12 kDa) protein antigen has been reported to mimic whole cell M. leprae in T cell responses across the leprosy spectrum. In addition, B cell responses to specific sequences within the LSR antigen have been shown to be associated with immunopathological responses in leprosy patients with erythema nodosum leprosum. We have in the present study applied the M. leprae LSR DNA sequence as query to search for the presence of homologous genes within the recently completed Mycobacterium tuberculosis genome database (Sanger Centre, UK). By using the BLASTN search tool, a homologous M. tuberculosis open reading frame (336 bp), encoding a protein antigen of 12.1 kDa, was identified within the cosmid MTCY07H7B.25. The gene is designated Rv3597c within the M. tuberculosis H37Rv genome. Sequence alignment revealed 93% identity between the M. leprae and M. tuberculosis antigens at the amino acid sequence level. The finding that some B and T cell epitopes were localized to regions with amino acid substitutions may account for the putative differential responsiveness to this antigen in tuberculosis and leprosy.     

Purification and characterization of a 36 kDa antigen of Mycobacterium leprae

A 36 kDa antigen of Mycobacterium leprae was purified by phenol biphasic partition followed by preparative SDS-PAGE. The purified antigen appeared as a single band in SDS-PAGE and eluted as a single peak in ion-exchange chromatography. The antigen comprised epitopes which were cross-reactive with M. tuberculosis, as well as a species-specific epitope (recognized by MAb F47-9). Different treatments of the 36 kDa antigen suggested it to be largely protein in nature; the amino acid composition of 81% of the antigen was determined. A majority of sera from leprosy patients contained antibodies recognizing the 36 kDa antigen.     

Bacterial glycoproteins: a link between glycosylation and proteolytic cleavage of a 19 kDa antigen from Mycobacterium tuberculosis.

Protein glycosylation has an important influence on a broad range of molecular interactions in eukaryotes, but is comparatively rare in bacteria. Several antigens from Mycobacterium tuberculosis, the causative agent of human tuberculosis, have been identified as glycoproteins on the basis of lectin binding, or by detailed structural analysis. By production of a set of alkaline phosphatase (PhoA) hybrid proteins in a mycobacterial expression system, the peptide region required for glycosylation of the 19 kDa lipoprotein antigen from M.tuberculosis was defined. Mutagenesis of two threonine clusters within this region abolished lectin binding by PhoA hybrids and by the 19 kDa protein itself. Substitution of the threonine residues also resulted in generation of a series of smaller forms of the protein as a result of proteolysis. In a working model to account for these observations, we propose that the role of glycosylation is to regulate cleavage of a proteolytically sensitive linker region close to the acylated N-terminus of the protein.     

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.     

Profile of a 24 kDa Mycobacterium tuberculosis antigen in the urine samples of tuberculosis patients under therapy regime

Secretion of a low molecular weight (24 kDa) Mycobacterium tuberculosis-specific antigen was analysed in the urine samples of tuberculosis patients under antimycobacterial therapy regime. The urine samples of sputum-positive and culture-positive tuberculosis patients under therapy regime were collected after 2, 3, 4, 5 and 6 months of antimycobacterial therapy. After concentration of the samples by ultrafiltration the proteins were resolved by SDS–PAGE. The antibodies raised in rabbits against M. tuberculosis strain H₃₇Ra were used to check specificity of the bands by Western blotting. It was found that the tuberculosis patients secreted a 24 kDa M. tuberculosis-specific antigen in their urine. This band was present in the samples taken upto 5 months of drug therapy but was absent in the samples taken after 6 months of antimycobacterial therapy. This study gives evidence in support of the continuation of chemotherapy of tuberculosis patients for at least 6 months. Also, the 24 kDa excreted/secreted antigen can be used as a marker for monitoring the drug therapy as well as for the diagnosis of tuberculosis patients. Moreover, the urine sample taken in the study is a non-invasive and safer sampling method as compared to sampling blood or sputum which is the usual procedure in these patients.     

Cloning and characterization of a bifunctional RelA/SpoT homologue from Mycobacterium tuberculosis.

A 2.2kb relA/spoT homologue was isolated from Mycobacterium tuberculosis (Mtb) genomic DNA by PCR-amplification. The Mtb gene encodes a protein of 738 amino acid residues, and is flanked upstream by an ORF that is highly similar to the apt gene, and downstream by an ORF that is highly similar to the cypH gene. This dual function Mtb homologue belongs to the relA/spoT family of genes that mediate the stringent response by regulating the synthesis and degradation of guanosine 3',5'-bis(diphosphate) (ppGpp) and pppGpp. In vitro biochemical data indicate that purified RelMtb is a ribosome- and tRNA-independent ATP:GTP/GDP/ITP 3'-pyrophosphoryltransferase. Additionally, purified RelMtb is an Mn2+-dependent, ribosome and tRNA-independent, (p)ppGpp 3'-pyrophosphohydrolase. These reactions were also assessed in vivo in E. coli deleted in both the relA and spoT genes, which generates a (p)ppGpp0 phenotype. RelMtb can suppress this phenotype and can generate more (p)ppGpp than relA in the wild type E. coli control.     

The catalase-peroxidase of Mycobacterium intracellulare: nucleotide sequence analysis and expression in Escherichia coli.

The activation of catalase genes in response to oxidative stress may contribute to the intracellular survival of mycobacteria. In this report, the nucleotide sequence of a mycobacterial catalase gene is described. The deduced protein sequence of this Mycobacterium intracellulare gene (MI85) was 60% identical to the Escherichia coli hydroperoxidase I (HPI) protein, 59% identical to the Salmonella typhimurium (HPI) catalase, and 47% identical to a Bacillus stearothermophilus peroxidase. The MI85 protein, expressed in E. coli, has also been shown to have peroxidase and catalase activities. Furthermore, Southern blot hybridizations, which demonstrated that a MI85 gene probe hybridizes with chromosomal DNA from thirteen different strains of mycobacteria, suggest that this catalase-peroxidase gene is prevalent in the mycobacterial genus. The availability of catalase gene probes should permit an evaluation, at the molecular level, of the role of catalase in mycobacterial pathogenesis.     

Complete genome sequence of Mycobacterium intracellulare clinical strain MOTT-02

Here, we report the first complete genome sequence of the Mycobacterium intracellulare clinical strain MOTT-02, which was previously grouped in the INT2 genotype of M. intracellulare. This genome sequence will serve as a valuable reference for improving the understanding of the disparity in the virulence and epidemiologic traits between M. intracellulare genotypes.     

结核分枝杆菌16kDa与GST融合表达及纯化

目的:构建融合表达GST和结核分枝杆菌16kDa蛋白的表达系统,摸索表达条件及制备方法.方法:根据16kDa蛋白的基因序列设计引物,从结核菌基因组DNA中扩增该基因,酶切后插入表达载体,测序证实后转入大肠杆菌JM109菌株,经IPTG诱导表达,产物经谷胱甘肽-琼脂糖凝胶法纯化.结果:16kDa基因产物大小、酶切片断大小和载体的大小与设计一致、16kDa基因测序的结果与目的基因完全符合.表达纯化的蛋白的大小与蛋白质分子量标准相比较一致.结论:构建的表达载体成功,纯化的蛋白即为目的蛋白.     

Characterization of a Mycobacterium tuberculosis proteasomal ATPase homologue

A screen for Mycobacterium tuberculosis (Mtb) mutants sensitive to reactive nitrogen intermediates identified transposon insertions in the presumptive proteasomal ATPase gene mpa (mycobacterium proteasome ATPase; Rv2115c). mpa mutants are attenuated in both wild type and nitric oxide synthase 2 deficient mice. In this work, we show that attenuation of mpa mutants is severe, and that Mpa is an ATPase associated with various cellular activities (AAA) ATPase that forms hexameric rings resembling the eukaryotic complex p97/valosin-containing protein (VCP). Point mutations in the conserved Walker box ATPase motifs of Mpa greatly reduced or abolished ATPase activity in vitro and abrogated protection of Mtb against acidified nitrite. A mutant Mpa protein missing only its last two amino acids retained ATPase activity, yet failed to protect Mtb against nitrite. The corresponding strain was attenuated in mice. Thus, Mpa is an ATPase whose enzymatic activity is necessary but not sufficient to protect against reactive nitrogen intermediates.     

The mapping of an antibody-binding region on the Mycobacterium tuberculosis 19 kilodalton antigen.

To localize the epitopes of four independently derived murine mAb IT-10, IT-12, IT-16, and IT-19 on the 19-kDa Ag protein of Mycobacterium tuberculosis, expression plasmids were constructed containing deletions of the gene encoding the 19-kDa protein. Reaction of the 4 mAb with Western blots of the truncated recombinant proteins revealed two epitope specificities in the recognition of the 19-kDa protein. IT-10 was found to be dependent only on the presence of amino acids surrounding the first cysteine residue, whereas IT-12, IT-16, and IT-19 all required the presence of both the first and third cysteine residues. These two cysteine residues are separated by 135 amino acids, and are considered to be brought together by tertiary folding of the protein to form an assembled epitope for IT-12, IT-16, and IT-19. These three mAb demonstrated differing sensitivities to the modification of reduced 19-kDa protein using iodoacetamide: a treatment that should have prevented the reformation of disulfide bonds within the protein. This result suggests that, although IT-12, IT-16, and IT-19 appear to be specific for the same epitope, there are probably fine-specificity differences in this recognition. IT-10 was not sensitive to the absence of disulfide bonds within the 19-kDa protein, suggesting that the epitope is not conformationally sensitive, and is likely to be linear in nature.     

Cloning and characterization of the gene for the '19 kDa' antigen of Mycobacterium bovis

Monoclonal antibody CMA134.1 reacted with a protein antigen of apparent molecular mass 22 kDa from Mycobacterium bovis and Mycobacterium tuberculosis, and with an apparently 24 kDa antigen of Mycobacterium kansasii, but not with other mycobacteria or related species. This antibody was used to screen a gene library of M. bovis in lambda gt11 and identified a recombinant clone that expressed a protein with an apparent molecular mass of 19-20 kDa. Gene expression occurred from the lac promoter in lambda gt11, but used an unidentified vector promoter, possibly that of the replication primer RNA, in the final plasmid construct. The sequence of an 840 bp fragment was determined and shown to code for a product of 15 kDa. This sequence is identical to that, independently determined, of a gene from M. tuberculosis, usually referred to as the 19 kDa antigen. The reasons for the apparent size discrepancies are discussed.     

Nucleotide sequence of the Mycobacterium leprae katG region.

Synthetic oligonucleotide primers based on the DNA sequence data of the Escherichia coli, Mycobacterium tuberculosis, and Mycobacterium intracellulare katG genes encoding the heme-containing enzyme catalase-peroxidase were used to amplify and analyze the Mycobacterium leprae katG region by PCR. A 1.6-kb DNA fragment, which hybridized to an M. tuberculosis katG probe, was obtained from an M. leprae DNA template. Southern hybridization analysis with a probe derived from the PCR-amplified fragment showed that the M. leprae chromosome contains only one copy of the putative katG sequence in a 3.4-kb EcoRI-BamHI DNA segment. Although the nucleotide sequence of the katG region of M. leprae was approximately 70% identical to that of the M. tuberculosis katG gene, no open reading frame encoding a catalase-peroxidase was detectable in the whole sequence. Moreover, two DNA deletions of approximately 100 and 110 bp were found in the M. leprae katG region, and they seemed to be present in all seven M. leprae isolates tested. These results strongly suggest that M. leprae lacks a functional katG gene and catalase-peroxidase activity.