Glycolipids of Mycobacterium tuberculosis strain H37Rv are potential serological markers for diagnosis of active tuberculosis

A simple and cost-effective diagnostic tool (TB Screen Test) for the screening of patients with pulmonary and extrapulmonary tuberculosis and for differentiation of those individuals from individuals without tuberculosis, other common infections, and healthy controls has been developed. The serological responses of purified mycobacterial glycolipid antigens were examined by a liposome agglutination assay. The assay was able to detect very low antiglycolipid antibody concentrations in the infected individuals. The sera from the tuberculosis patient group had significantly higher concentrations of antiglycolipid antibody than the sera from uninfected control subjects, with 94% sensitivity and 98.3% specificity. Glycolipids of Mycobacterium tuberculosis H37Rv antigens were isolated, purified, and characterized. After interchelation with liposome particles, these purified antigens specifically bound to the antiglycolipid antibodies present in the sera of patients with tuberculosis, resulting in the formation of a blue agglutination. This protocol clearly differentiates healthy controls and M. bovis BCG-vaccinated subjects from those with active tuberculosis. The resultant diagnostic tool, the TB Screen Test, is more economical and rapid (4 min) than other currently available products and can be used for the mass screening of a heavily afflicted population.     

Genomic Analysis Reveals Variation between Mycobacterium tuberculosis H37Rv and the Attenuated M. tuberculosis H37Ra Strain

Mycobacterium tuberculosis H37Ra is an attenuated tubercle bacillus closely related to the virulent type strain M. tuberculosis H37Rv. Despite extensive study, the reason for the decreased virulence of M. tuberculosis H37Ra has not been determined. A genomic approach was therefore initiated to identify genetic differences between M. tuberculosis H37Rv and M. tuberculosis H37Ra as a means of pinpointing the attenuating mutation(s). Digestion with the rare-cutting restriction endonuclease DraI revealed two polymorphisms between the strains: a 480-kb fragment in M. tuberculosis H37Rv was replaced by two fragments of 220 and 260 kb in M. tuberculosis H37Ra, while there was a approximately 7.9-kb DraI fragment in M. tuberculosis H37Ra that had no counterpart in M. tuberculosis H37Rv. As the M. tuberculosis insertion sequence IS6110 contains a single DraI restriction site, it was considered possible that these polymorphisms were the result of IS6110 transposition events in M. tuberculosis H37Ra, events that may have inactivated virulence genes. The 7.9-kb polymorphism was found to be due to the presence of the previously described H37Rv RvD2 deletion in M. tuberculosis H37Ra, with sequence analysis suggesting an IS6110-mediated deletion mechanism for loss of RvD2. Three other IS6110-catalyzed deletions from the M. tuberculosis H37Rv chromosome (RvD3 to RvD5) were also identified, suggesting that this mechanism plays an important role in genome plasticity in the tubercle bacilli. Comparative mapping and sequencing revealed that the 480-kb polymorphism was due to an IS6110 insertion in M. tuberculosis H37Ra near oriC. Complementation of M. tuberculosis H37Ra with a 2.9-kb restriction fragment from M. tuberculosis H37Rv that encompassed the IS6110 insertion did not increase the survival of recombinant M. tuberculosis H37Ra in mice. In conclusion, this study describes the presence and mechanisms of genomic variation between M. tuberculosis H37Ra and M. tuberculosis H37Rv, although the role that they play in the attenuation of M. tuberculosis H37Ra is unclear.     

Antigen analysis of Mycobacterium tuberculosis H37Rv culture filtrate proteins

Culture filtrates of Mycobacterium tuberculosis H37Rv highly enriched with secreted proteins were used to identify antigens recognized by a serum pool from tuberculosis patients. Two different approaches were used to separate the culture filtrate protein mixture: (i) proteins were fractionated according to their hydrophobicity using an HPLC-C18 chromatography column followed by separation based on their molecular mass by SDS-PAGE and subsequent immunoblotting or (ii) proteins were separated by two-dimensional gel electrophoresis, based on their isoelectric point and their molecular mass. Twenty serologically reactive proteins were ultimately identified by both methods, including four novel antigens. Further, to estimate the immunogenicity of the identified culture filtrate proteins, the relative antibody quantities were measured using I mage master software. Our results show that the antibodies against proteins belonging to the antigen 85 complex were the most abundant in the serum of patients with active tuberculosis. The most immunogenic proteins in terms of high antibody-to-protein-ratio were Rv3881c and three lipoproteins Rv0934 (the 38 kDa antigen), Rv0932c (pstS2), and Rv3006 (LppZ). Rv3881c is located in the region of difference 1 (RD1) which is deleted from Mycobacterium bovis BCG, and is therefore a particularly promising candidate for development of serodiagnostic assays to detect active tuberculosis. The proteins from the M. tuberculosis H37Rv culture filtrate are strong candidates to be evaluated for improvement of the serodiagnostic tests of tuberculosis.     

Human alveolar macrophage gene responses to Mycobacterium tuberculosis strains H37Ra and H37Rv

H37Rv and H37Ra have been widely used as models of virulent and avirulent strains, respectively, of Mycobacterium tuberculosis. Since the sequencing of H37Rv, microarrays have been used to investigate gene expression of M. tuberculosis strains under various conditions, and to compare gene expression of specific isolates of the organism. Because differences in the virulence of these organisms could also be manifest via their differential induction of host genes, we used Affymetrix Human Genome Arrays U133A and U133B to evaluate human alveolar macrophage (AM) responses to infection with H37Rv and H37Ra. H37Rv altered expression of far more genes than did H37Ra. Moreover, the genes induced by H37Rv to a greater extent than by H37Ra were predominantly associated with the development of effective immunity. H37Rv markedly increased expression of IL-23 p19, whereas neither organism significantly induced IL-12 p35 expression. Quantitative PCR confirmed that H37Rv induced significantly more AM p19 expression than did H37Ra. After low-level infection of both AM and peripheral blood monocytes (MN) with H37Rv, neither cell type produced IL-12 (by ELISA). In contrast, AM displayed significant IL-23 production in response to H37Rv, whereas MN did not. Our findings thus suggest an important role for IL-23 in human host responses to pulmonary infection with M. tuberculosis, and are consistent with epidemiologic and genetic studies that imply that H37Rv may not have unusual capacity to cause human disease.     

Serodiagnostic efficiency of phospholipid associated protein of Mycobacterium tuberculosis H37Rv

The phospholipid-associated protein (55–67 kDa) fraction of Mycobacterium tuberculosis H₃₇Rv was purified as the DE-V protein fraction. This DE-V fraction was used for diagnosis of tuberculosis by enzyme-linked immunosorbent assay (ELISA), detecting IgG antibody in sera collected from different categories of tuberculosis patients, i.e. with acid fast bacilli (AFB) culture-positive pulmonary tuberculosis, with AFB culture-negative, but radiologically suspected, pulmonary tuberculosis, extrapulmonary tuberculosis, and control groups of patients suffering from diseases other than tuberculosis (asthma and/or rhinitis, lepromatous leprosy) as well as from healthy volunteers. Encouraging operational ELISA validity could be achieved with 93% sensitivity, 100% specificity, 97% efficiency, 100% positive predictivity and 95% negative predictability even among the extrapulmonary and suspected pulmonary tuberculosis patients. The above assay was insensitive but with 100% specificity among control group of patients suffering from diseases other than tuberculosis. The DE-V protein fraction was associated with phosphatidyl inositol and phosphatidyl inositol mannosides. The dissociation of phospholipid-protein complex decreased ELISA specificity. ELISA reactivity of the DE-V fraction appeared to be thermostable; thus, it may have serodiagnostic utility in developing countries.     

Isolation of a 33-kDa protein antigen from delipidified Mycobacterium tuberculosis H37Rv

A 33-kDa protein (TB33) was isolated from a delipidated cell sonicate (CS) of Mycobacterium tuberculosis H₃₇Rv (grown in Middlebrook 7H9 broth supplemented with glucose) using immobilized metal affinity chromatography (IMAC) on a nickel-nitrilotriacetic acid (Ni-NTA) column. TB33 could not be isolated from the culture filtrate (CF) of M. tuberculosis H₃₇Rv using Ni-NTA. TB33 was recognized by monoclonal antibodies (mAb) known to react with proteins of M. tuberculosis with a molecular mass of 33/34 kDa; namely, mAb F126-5, F67-1 and F126-2. The N-terminal amino acid sequence of TB33 was found to be Xaa-Xaa-Thr-Pro-Ala-Asp-Val-Ser/Cys-Asn-Val-Ala-Ile and thus, shows identity with the N-terminal of antigen 84 of M. tuberculosis except for two mismatches. Antibodies to TB33 could be raised in mice by administering four injections of TB33 (40 μg total protein). Sera from tuberculosis patients reacted with TB33, while those from normal healthy individuals did not. Received: 17 April 1996     

Cloning and B-cell-epitope mapping of MPT64 from Mycobacterium tuberculosis H37Rv.

The gene of the immunogenic protein MPT64 found in culture filtrates of Mycobacterium tuberculosis H37Rv was cloned and sequenced. A comparison showed mpt64 and the gene encoding MPB64 from Mycobacterium bovis BCG Tokyo to be identical except for one silent mutation. The regions encoding the promoter and the signal peptide were also well conserved for the two sequences. Southern blot experiments on genomic mycobacterial DNA showed the presence of mpt64 in the M. tuberculosis substrains H37Rv, H37Ra, and Erdman and in the M. bovis BCG substrains Tokyo, Moreau, and Russian, whereas the M. bovis BCG substrains Glaxo, Pasteur, Canadian, Tice, and Danish 1331 and Mycobacterium leprae lack the gene. Southern blot analyses revealed differences in the restriction enzyme patterns within the M. tuberculosis substrains as well as within the M. bovis BCG substrains, indicating either different chromosomal localization of mpt64 or that mutations have occurred at different locations on the chromosomes. N-terminal and C-terminal deletion mutants were constructed for the mapping of B-cell epitopes on MPT64 with five monoclonal antibodies, C24b1, C24b2, C24b3, L24b4, and L24b5. Western blot (immunoblot) analysis revealed that the murine antibodies bind to one linear and three conformational epitopes.     

Rv1813c在结核分枝杆菌潜伏感染诊断中的价值

目的 研究重组结核分枝杆菌潜伏感染蛋白Rv1813c在诊断结核分枝杆菌潜伏感染方面的价值.方法 20例结核潜伏感染者和79例健康志愿者均行胸部X线检查、PPD皮肤试验、结核抗体检测,同时应用ELISA联合重组融合蛋白CFP10-ESAT6和潜伏感染蛋白Rv1813c进行IGRA检测.结果 所有受试者中,PPD皮肤试验和抗体检测的阳性率分别为50％和28％.Rv1813c刺激潜伏感染者后产生IFN-γ的水平显著高于健康对照(P＜0.05),其刺激PPD强阳性组(皮试直径≥15mm)产生的IFN-γ值低于弱阳性组(5mm≤皮试直径＜15mm),但无统计学意义,而CFP10-ESAT6刺激PPD强阳性组后产生的IFN-γ值高于弱阳性组(P＜0.05).CFP10-ESAT6和Rv1813c刺激抗体阴性及阳性组后产生的IFN-γ水平没有显著性差异(P＞0.05).Rv1813c诊断结核感染的ROC曲线下面积为0.834,特异性80％时,灵敏度为85％;特异性为90％时,灵敏度为45％.结论 同时检测Rv1813c及CFP10-ESAT6抗原特异的IFN-γ值有可能筛选出潜伏感染者中的活动感染人群,对于结核分枝杆菌潜伏感染诊断有重要的应用价值.     

Immunological activity of a 14-kilodalton recombinant protein of Mycobacterium tuberculosis H37Rv.

A 14-kilodalton peptide antigen from Mycobacterium tuberculosis was isolated from an Escherichia coli lambda gt 11 recombinant DNA clone and was identified by Western blotting (immunoblotting) with monoclonal antibody TB68. Immunization of mice and guinea pigs with the recombinant peptide (rTB68) induced in vitro lymphoproliferative responses in draining lymph node lymphocyte cultures as well as in vivo delayed-type hypersensitivity reactions. Moreover, rTB68 was found both to induce and to cross-react with Mycobacterium leprae immune lymphocytes, but did not generate protective effects against live M. leprae challenge in mice. These findings showed that a 14-kilodalton peptide which has been characterized as specific for M. tuberculosis on the basis of B-cell recognition was capable of generating cell-mediated immune responses and moreover contained T-cell epitopes which were cross-reactive with M. leprae antigens.     

IS6110-based restriction fragment length polymorphism (RFLP) analysis of Mycobacterium tuberculosis H37Rv and H37Ra

IS6110-based restriction fragment length polymorphism (RFLP) analysis of Mycobacterium tuberculosis H37Rv and its avirulent mutant H37Ra was performed by a number of restriction enzymes, including Nru I, EcoN I, Pst I, and Pvu II. No differences were found in the IS 6110-fingerprints of the study strains by Nru I. One differential IS6110-positive restriction fragment was detected by EcoN I in strain H37Ra, while analysis by Pst I revealed that two fragments of the strain H37Rv were replaced by four novel IS6110-positive fragments in the strain H37Ra. By using Pvu II, a restriction enzyme that cleaves IS 6110 once, and by probing for an IS6110 specific target sequence located to the right of the Pvu II site, we found that the strains H37Rv and H37Ra share 13 IS6110-positive restriction fragments and that one IS6110-positive restriction fragment of H37Rv is replaced by four novel fragments in H37Ra; by probing for an IS6110-specific target sequence to the left of the Pvu II site, 13 shared restriction fragments and 2 differential bands in strain H37Ra were detected. These findings demonstrate that novel insertions of the IS6110 element exist in the avirulent strain H37Ra and raise the question of the role, if any, of IS6110-insertional mutagenesis in the establishment of the avirulent M. tuberculosis H37Ra phenotype.     

Proteomics reveals open reading frames in Mycobacterium tuberculosis H37Rv not predicted by genomics

Genomics revealed the sequence of 3924 genes of the H37Rv strain of Mycobacterium tuberculosis. Proteomics complements genomics in showing which genes are really expressed, and here we show the expression of six genes not predicted by genomics, as proved by two-dimensional electrophoresis and matrix-assisted laser desorption ionization and nano-electrospray mass spectrometry.     

人型结核杆菌H37Ra的抗原决定簇的筛选及其初步鉴定

本文报道一种筛选人型结核杆菌H37Ra的抗原决定簇的Western Blot分析技术。细菌培养滤液经15％SDS—PAGE电泳进行分离,然后电转移到硝酸纤维素薄膜上,应用鼠抗人结核杆菌单克隆抗体进行免疫反应。通过免疫印染,一条能与McAb起强阳性反应的蛋白带被识别。应用蛋白质洗脱技术,这个蛋白质片段被提纯。经SDS—PAGE电泳分析,这个含有抗原决定簇的蛋白质片段为分子量17,500道尔顿,斑点免疫试验证实该片段具有生物学活性。本文提示,Western Blot分析技术是对抗原决定簇进行分析的有用方法,值得推荐。     

Phagosomal membranes of Mycobacterium bovis BCG-immune alveolar macrophages are resistant to disruption by Mycobacterium tuberculosis H37Rv.

Data obtained in this study reaffirm that virulent Mycobacterium tuberculosis H37Rv has a potent phagosome-destroying capacity when ingested by normal alveolar macrophages. In contrast, Mycobacterium bovis BCG-immune alveolar macrophages are highly resistant to this virulence mechanism. BCG-immune sera incubated with BCG-immune alveolar macrophages did not increase resistance of BCG-immune alveolar macrophages as compared with the data obtained from experiments with normal sera. BCG-immune sera failed to confer resistance to normal alveolar macrophages against the phagosomal membrane-destroying H37Rv virulence mechanism.     

Identification and characterization of genomic variations between Mycobacterium bovis and M. tuberculosis H37Rv

Genetic differences between Mycobacterium bovis and M. tuberculosis were identified. We found (i) a deletion of Rv3479 specific to M. bovis, (ii) that the rpfA gene is shortened to various extents in M. bovis, and (iii) an insertion in Rv0648 and a duplication of lppA common in M. tuberculosis complex isolates.     

Human natural killer cells mediate killing of intracellular Mycobacterium tuberculosis H37Rv via granule-independent mechanisms

Despite the continued importance of tuberculosis as a world-wide threat to public health, little is known about the mechanisms used by human lymphocytes to contain and kill the intracellular pathogen Mycobacterium tuberculosis. We previously described an in vitro model of infection of human monocytes (MN) with virulent M. tuberculosis strain H37Rv in which the ability of peripheral blood lymphocytes to limit intracellular growth of the organism could be measured. In the current study, we determined that lymphocyte-mediated killing of intracellular M. tuberculosis occurs within the first 24 h of coculture with infected MN. Natural killer (NK) cells isolated from both purified protein derivative (PPD)-positive and PPD-negative subjects were capable of mediating this early killing of intracellular H37Rv. NK cell-mediated killing of intracellular M. tuberculosis was not associated with the production of gamma interferon. Transferred supernatants of cocultured NK cells and M. tuberculosis-infected MN could not mediate the killing of intracellular M. tuberculosis, and Transwell studies indicated that direct cell-to-cell contact was required for NK cells to mediate the killing of the organism. Killing was not dependent upon exocytosis of NK cell cytotoxic granules. NK cells induced apoptosis of mycobacterium-infected MN, but neither killing of intracellular M. tuberculosis by NK cells nor NK cell-induced apoptosis of infected MN was inhibited by blocking the interaction of FasL and Fas. Thus, human NK cells may mediate killing of intracellular M. tuberculosis via alternative apoptotic pathways.     

Induction of Cell Death in Human Macrophages by a Highly Virulent Korean Isolate of Mycobacterium tuberculosis and the Virulent Strain H37Rv

Recent studies have suggested that virulent strains of Mycobacterium tuberculosis induce apoptosis in macrophages less often than do attenuated strains. K-strain, which belongs to the Beijing family, is the most frequently isolated clinical strain of M. tuberculosis in Korea. In this study, we investigated the differential induction of cell death in human monocytic THP-1 cells by K-strain and H37Rv, a virulent but laboratory-adapted strain of M. tuberculosis . Although no significant difference in growth rate was observed between the cells exposed to K-strain and those exposed to H37Rv, the levels of protective cytokines such as tumour necrosis factor (TNF)-α, interleukin (IL)-6 and IL-12p40 were lower in K-strain-infected cells than in H37Rv-infected cells. Cell viability assays showed that both K-strain and H37Rv, but not heat- or streptomycin-killed bacteria, induced THP-1 cell death in a TNF-independent manner. In contrast, double staining with fluorochrome-labelled inhibitors of caspase and propidium iodide and lactate dehydrogenase release assays revealed that K-strain induced significantly higher levels of necrotic cell death, rather than apoptosis, in THP-1 cells than did H37Rv. Anti-apoptotic Bcl-2 , Mcl-1 , Bfl-1 and Bcl-xL in the cells were significantly upregulated following infection with K-strain compared with H37Rv, whereas Bax was slightly upregulated in response to infection with both H37Rv and K-strain. These results suggest that the highly virulent K-strain keeps cellular apoptosis as a host defense mechanism to a minimum and induces necrosis in macrophages.     

Mycobacterium tuberculosis H37Ra and H37Rv differential growth and cytokine/chemokine induction in murine macrophages in vitro.

The role of tumor necrosis factor-alpha (TNF-alpha) in controlling growth of Mycobacterium tuberculosis in murine peritoneal macrophages infected in vitro was studied. TNF-alpha was shown to be required but not sufficient, and the amount of TNF-alpha produced by the infected cells did not correlate with the extent of growth control. In this system, TNF-alpha-dependent control of growth of the avirulent strain H37Ra was independent of inducible nitric oxide synthase (iNOS) and interferon-gamma (IFN-gamma), as shown by the infection of macrophages from selected gene-disrupted mice. TNF-alpha-mediated bacteriostasis of H37Ra in the infected macrophages was associated with increased expression of selected Th1-type cytokines and chemokines. In contrast, growth of the virulent strain H37Rv in macrophages involved upregulation by infected cells of Th2-type cytokines, including interleukin-5 (IL-5), IL-10, and IL-13. Taken together, these results suggest that the particular nature of macrophage activation and the cytokine and chemokine response to infection with different M. tuberculosis strains determine the ability of the cells to control the growth of the intracellular bacilli.