Detection and identification of mycobacteria by amplification of mycobacterial DNA

A 383bp segment of the gene coding for the 65kD mycobacterial antigens from Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium, Mycobacterium paratuberculosis, and Mycobacterium fortuitum was amplified using Taq polymerase and synthetic oligonucleotide primers and the amplified DNAs from four of these species were compared by nucleotide sequencing. Although the gene segments from these species showed considerable similarity, oligonucleotide probes which could distinguish M. tuberculosis/M. bovis, M. avium/M. paratuberculosis and M. fortuitum could be identified. Samples containing 10(6) human cells and serial dilutions of a suspension of intact mycobacteria were prepared, DNA was extracted, the segment of the mycobacterial DNA sequence amplified, and the amplified DNA hybridized with oligonucleotide probes. In two independent experiments, this procedure permitted the detection and identification of less than 100 mycobacteria in the original sample. These results suggest that this approach may prove useful in the early diagnosis of mycobacterial infection.     

Nucleotide sequence analysis and serologic characterization of the Mycobacterium intracellulare homologue of the Mycobacterium tuberculosis 19 kDa antigen

Disseminated Mycobacterium avium/Mycobacterium intracellulare complex (MAC) disease is a frequent complication in patients with the acquired immune deficiency syndrome (AIDS). In this report, we present the nucleotide sequence of the M. intracellulare MI22 gene. Computer sequence comparisons reveal that the MI22 gene, which encodes a serologically active protein, has 78% DNA sequence identity and 77% protein sequence identity with the seroreactive 19 kDa Mycobacterium tuberculosis lipoprotein antigen. Southern blot hybridizations indicate that an MI22 gene probe binds similar-sized restriction fragments in M. tuberculosis and M. intracellular genomic DNA. In addition, immunoblot analyses demonstrate that MI22 is recognized by sera from tuberculosis patients. These data further support the existence of 19 kDa MAC and M. tuberculosis protein homologues. Phase partitioning experiments and the presence of a consensus lipid modification site in the deduced MI22 protein sequence strongly suggest that M122 is also a lipoprotein. Comparative analyses of these mycobacterial antigenic homologues may provide the basis for the design of species-specific diagnostic reagents.     

Mycobacterium smegmatis laminin-binding glycoprotein shares epitopes with Mycobacterium tuberculosis heparin-binding haemagglutinin

Mycobacterium tuberculosis, the causative agent of tuberculosis, produces a heparin-binding haemagglutinin adhesin (HBHA), which is involved in its epithelial adherence. To ascertain whether HBHA is also present in fast-growing mycobacteria, Mycobacterium smegmatis was studied using anti-HBHA monoclonal antibodies (mAbs). A cross-reactive protein was detected by immunoblotting of M. smegmatis whole-cell lysates. However, the M. tuberculosis HBHA-encoding gene failed to hybridize with M. smegmatis chromosomal DNA in Southern blot analyses. The M. smegmatis protein recognized by the anti-HBHA mAbs was purified by heparin-Sepharose chromatography, and its amino-terminal sequence was found to be identical to that of the previously described histone-like protein, indicating that M. smegmatis does not produce HBHA. Biochemical analysis of the M. smegmatis histone-like protein shows that it is glycosylated like HBHA. Immunoelectron microscopy demonstrated that the M. smegmatis protein is present on the mycobacterial surface, a cellular localization inconsistent with a histone-like function, but compatible with an adhesin activity. In vitro protein interaction assays showed that this glycoprotein binds to laminin, a major component of basement membranes. Therefore, the protein was called M. smegmatis laminin-binding protein (MS-LBP). MS-LBP does not appear to be involved in adherence in the absence of laminin but is responsible for the laminin-mediated mycobacterial adherence to human pneumocytes and macrophages. Homologous laminin-binding adhesins are also produced by virulent mycobacteria such as M. tuberculosis and Mycobacterium leprae, suggesting that this adherence mechanism may contribute to the pathogenesis of mycobacterial diseases.     

Compiling a molecular inventory for Mycobacterium bovis BCG at two growth rates: evidence for growth rate-mediated regulation of ribosome biosynthesis and lipid metabolism

An experimental system of Mycobacterium tuberculosis growth in a carbon-limited chemostat has been established by the use of Mycobacterium bovis BCG as a model organism. For this model, carbon-limited chemostats with low concentrations of glycerol were used to simulate possible growth rates during different stages of tuberculosis. A doubling time of 23 h (D = 0.03 h(-1)) was adopted to represent cells during the acute phase of infection, whereas a lower dilution rate equivalent to a doubling time of 69 h (D = 0.01 h(-1)) was used to model mycobacterial persistence. This chemostat model allowed the specific response of the mycobacterial cell to carbon limitation at different growth rates to be elucidated. The macromolecular (RNA, DNA, carbohydrate, and lipid) and elemental (C, H, and N) compositions of the biomass were determined for steady-state cultures, revealing that carbohydrates and lipids comprised more than half of the dry mass of the BCG cell, with only a quarter of the dry weight consisting of protein and RNA. Consistent with studies of other bacteria, the specific growth rate impacts on the macromolecular content of BCG and the proportions of lipid, RNA, and protein increased significantly with the growth rate. The correlation of RNA content with the growth rate indicates that ribosome production in carbon-limited M. bovis BCG cells is subject to growth rate-dependent control. The results also clearly show that the proportion of lipids in the mycobacterial cell is very sensitive to changes in the growth rate, probably reflecting changes in the amounts of storage lipids. Finally, this study demonstrates the utility of the chemostat model of mycobacterial growth for functional genomic, physiology, and systems biology studies.     

The human CD1-restricted T cell repertoire is limited to cross-reactive antigens: implications for host responses against immunologically related pathogens

The repertoires of CD1- and MHC-restricted T cells are complementary, permitting the immune recognition of both lipid and peptide Ags, respectively. To compare the breadth of the CD1-restricted and MHC-restricted T cell repertoires, we evaluated T cell responses against lipid and peptide Ags of mycobacteria in leprosy, comparing tuberculoid patients, who are able to restrict the pathogen, and lepromatous patients, who have disseminated infection. The striking finding was that in lepromatous leprosy, T cells did not efficiently recognize lipid Ags from the leprosy pathogen, Mycobacterium leprae, or the related species, Mycobacterium tuberculosis, yet were able to efficiently recognize peptide Ags from M. tuberculosis, but not M. leprae. To identify a mechanism for T cell unresponsiveness against mycobacterial lipid Ags in lepromatous patients, we used T cell clones to probe the species specificity of the Ags recognized. We found that the majority of M. leprae-reactive CD1-restricted T cell clones (92%) were cross-reactive for multiple mycobacterial species, whereas the majority of M. leprae-reactive MHC-restricted T cells were species specific (66%), with a limited number of T cell clones cross-reactive (34%) with M. tuberculosis. In comparison with the MHC class II-restricted T cell repertoire, the CD1-restricted T cell repertoire is limited to recognition of cross-reactive Ags, imparting a distinct role in the host response to immunologically related pathogens.     

Structural elucidation and antigenicity of a novel phenolic glycolipid antigen from Mycobacterium haemophilum

The structure of a novel antigenic glycolipid that distinguishes the opportunistic pathogen Mycobacterium haemophilum from all other mycobacteria was established by a series of degradation reactions leading to products that were analyzed by gas/liquid chromatography-mass spectrometry. The complete structure of the oligosaccharide unit was determined as 2,3-di-O-CH3-alpha-L-Rhap(1----2)3-O-CH3-alpha-L-Rhap(1----4 )-2,3-di-O-CH3-alpha-L-Rhap(1----. The lipid portion of the phenolic glycolipid was composed of two component phenolphthiocerols differing by two methylene groups, as determined by analysis of their per-O-trideuteriomethylated derivatives. The diol unit of the phenolphthiocerols has a threo relative configuration. The absolute stereochemistry of the asymmetric centers of the phenolphthiocerols is uncertain, but the centers are probably 3R, 4S, 9R, and 11R as found for phthiocerol A from Mycobacterium tuberculosis. The hydroxyl functions of the branched glycolic chain are esterified to a complex mixture of multi-methyl branched mycocerosic acids, C27, C30, C32, C34, and C37 with molecular weights (as methyl esters) of 424, 466, 494, 522, and 564, respectively. The stereochemistry of the methyl branches of the mycocerosates have R absolute configuration. The glycolipid is highly antigenic and appears to be specific for M. haemophilum. There are intriguing similarities between the product from M. haemophilum and the well-known phenolic glycolipid I of Mycobacterium leprae, a matter that is discussed.     

Monoclonal antibodies to mycobacterial DNA gyrase A inhibit DNA supercoiling activity.

DNA gyrase is an essential type II topoisomerase found in bacteria. We have previously characterized DNA gyrase from Mycobacterium tuberculosis and Mycobacterium smegmatis. In this study, several monoclonal antibodies were generated against the gyrase A subunit (GyrA) of M. smegmatis. Three, MsGyrA:C3, MsGyrA:H11 and MsGyrA:E9, were further analyzed for their interaction with the enzyme. The monoclonal antibodies showed high degree of cross-reactivity with both fast-growing and slow-growing mycobacteria. In contrast, none recognized Escherichia coli GyrA. All the three monoclonal antibodies were of IgG1 isotype falling into two distinct types with respect to epitope recognition and interaction with the enzyme. MsGyrA:C3 and MsGyrA:H11 IgG, and their respective Fab fragments, inhibited the DNA supercoiling activity catalyzed by mycobacterial DNA gyrase. The epitope for the neutralizing monoclonal antibodies appeared to involve the region towards the N-terminus (residues 351-415) of the enzyme in a conformation-dependent manner. These monoclonal antibodies would serve as valuable tools for structure-function analysis and immunocytological studies of mycobacterial DNA gyrase. In addition, they would be useful for designing peptide inhibitors against DNA gyrase.     

Effect of silver-carrying photocatalyst "Hikari-Gintech" on mycobacterial growth in vitro

The antimycobacterial activity of "Hikari-Gintech" powder, which has photocatalytic activity, was examined in vitro. Both powder dissolved in liquid and Hikari-Gintech-coated cloths showed strong antimycobacterial activity against Mycobacterium tuberculosis H37Rv, M. bovis BCG Pasteur, multi-drug-resistant M. tuberculosis (a clinical isolate) and M. avium. Hikari-Gintech powder appeared to affect mycobacterial cell wall metabolism rather than mycobacterial DNA because no damage to mycobacterial DNA was detected after spraying with Hikari-Gintech solution.     

Inactivation of polyketide synthase and related genes results in the loss of complex lipids in Mycobacterium tuberculosis H37Rv

AIMS: Phthiocerol dimycocerosate (PDIM) waxes and other lipids are necessary for successful Mycobacterium tuberculosis infection, although the exact role of PDIM in host-pathogen interactions remains unclear. In this study, we investigated the contribution of tesA, drrB, pks6 and pks11 genes in complex lipid biosynthesis in M. tuberculosis. METHODS AND RESULTS: Four mutants were selected from M. tuberculosis H37Rv transposon mutant library. The transposon insertion sites were confirmed to be within the M. tuberculosis open reading frames for tesA (a probable thioesterase), drrB (predicted ABC transporter), pks11 (putative chalcone synthase) and pks6 (polyketide synthase). The first three of these transposon mutants were unable to generate PDIM and the fourth lacked novel polar lipids. CONCLUSIONS: Mycobacterium tuberculosis can be cultivated in vitro without the involvement of certain lipid synthesis genes, which may be necessary for in vivo pathogenicity. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of transposon mutants is a new functional genomic approach for the eventual definition of the mycobacterial 'lipidome'.     

Validation of qPCR Methods for the Detection of Mycobacterium in New World Animal Reservoirs

Zoonotic pathogens that cause leprosy (Mycobacterium leprae) and tuberculosis (Mycobacterium tuberculosis complex, MTBC) continue to impact modern human populations. Therefore, methods able to survey mycobacterial infection in potential animal hosts are necessary for proper evaluation of human exposure threats. Here we tested for mycobacterial-specific single- and multi-copy loci using qPCR. In a trial study in which armadillos were artificially infected with M. leprae, these techniques were specific and sensitive to pathogen detection, while more traditional ELISAs were only specific. These assays were then employed in a case study to detect M. leprae as well as MTBC in wild marmosets. All marmosets were negative for M. leprae DNA, but 14 were positive for the mycobacterial rpoB gene assay. Targeted capture and sequencing of rpoB and other MTBC genes validated the presence of mycobacterial DNA in these samples and revealed that qPCR is useful for identifying mycobacterial-infected animal hosts.     

Distribution of environmental mycobacteria in Karonga District, northern Malawi

The genus Mycobacterium includes many species that are commonly found in the environment (in soil and water or associated with plants and animals), as well as species that are responsible for two major human diseases, tuberculosis (Mycobacterium tuberculosis) and leprosy (Mycobacterium leprae). The distribution of environmental mycobacteria was investigated in the context of a long-term study of leprosy, tuberculosis, Mycobacterium bovis BCG vaccination, and the responses of individuals to various mycobacterial antigens in Karonga District, northern Malawi, where epidemiological studies had indicated previously that people may be exposed to different mycobacterial species in the northern and southern parts of the district. A total of 148 soil samples and 24 water samples were collected from various locations and examined to determine the presence of mycobacteria. The detection method involved semiselective culturing and acid-fast staining, following decontamination of samples to enrich mycobacteria and reduce the numbers of other microorganisms, or PCR with primers specific for the mycobacterial 16S rRNA gene, using DNA extracted directly from soil and water samples. Mycobacteria were detected in the majority of the samples, and subsequent sequence analysis of PCR products amplified directly from soil DNA indicated that most of the products were related to known environmental mycobacteria. For both methods the rates of recovery were consistently higher for dry season samples than for wet season samples. All isolates cultured from soil appeared to be strains of Mycobacterium fortuitum. This study revealed a complex pattern for the environmental mycobacterial flora but identified no clear differences between the northern and southern parts of Karonga District.     

Lipid domains of mycobacteria studied with fluorescent molecular probes

The complex mycobacterial cell envelope is recognized as a critical factor in our failure to control tuberculosis, leprosy and other non-tuberculous pathogens. Although its composition has been extensively determined, many details regarding the organization of the envelope remain uncertain. This is particularly so for the non-covalently bound lipids, whose natural distribution may be disrupted by conventional biochemical or cytological techniques. In order to study the native organization of lipid domains in the mycobacterial envelope, we have applied a range of fluorescent lipophilic probes to live mycobacteria, including Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium gadium and Mycobacterium aurum, and analysed the resultant signals by fluorescence microscopy and digital image processing. Five key features were observed: (i) the presence of both envelope and intracellular lipid domains; (ii) differential localization of probes into these domains influenced predominantly by their hydrophobicity, as modelled by their calculated octanol:water partition coefficients and by their amphiphilicities; (iii) uneven distribution of lipophilic material in the envelope; (iv) selective labelling of septal regions of the envelope; and (v) modification of labelling patterns by additional treatments such as fluorescence quenching antibodies, detergents and solvents. Using this last approach, a coherent cell envelope lipid domain was demonstrated outside the cytoplasmic membrane and, for the first time, the proposed covalently linked mycolyl-arabinogalactan-peptidoglycan macromolecular complex was imaged directly. The use of fluorescent probes and high-resolution fluorescence microscopy has enabled us to obtain a coherent view of distinct lipid domains in mycobacteria. Further application of this approach will facilitate understanding of the role of lipids in the physiology of these organisms.     

Ornithine lipid of Mycobacterium tuberculosis: its distribution in some slow- and fast-growing mycobacteria

An ornithine-amide lipid is present in Mycobacterium tuberculosis. Its structure was established by a combination of chemical analysis and mass spectrometry. 3-Hydroxyoctadecanoic and 3-hydroxyeicosanoic acids (and homologues) were found to be linked through an amide bond to the alpha-amino group of L-ornithine, the hydroxyl group of the fatty acid being esterified mainly by tuberculostearic acid (10-methyloctadecanoic acid). This ornithine-amide lipid was detected in several other slow-growing pathogenic mycobacteria by thin layer chromatography, but not in an avirulent strain (H37 Ra) of M. tuberculosis. In each case mass spectrometry showed that all the structures were identical, thus revising an earlier reported structure for the lipid from M. bovis.     

Characterization of mycobacterial protein glycosyltransferase activity using synthetic peptide acceptors in a cell-free assay

Synthetic peptides derived from a 45-kDa glycoprotein antigen of Mycobacterium tuberculosis were shown to function as glycosyltransferase acceptors for mannose residues in a mannosyltransferase cell-free assay. The mannosyltransferase activity was localized within both isolated membranes and a P60 cell wall fraction prepared from the rapidly growing mycobacterial strain, Mycobacterium smegmatis. Incorporation of radiolabel from GDP-[(14)C]mannose was inhibited by the addition of amphomycin, indicating that the glycosyl donor for the peptide acceptors was a member of the mycobacterial polyprenol-P-mannose (PPM) family of activated glycosyl donors. Furthermore, a direct demonstration of transfer from the in situ generated PP[(14)C]Ms was also demonstrated. It was also found that the enzyme activity was sensitive to changes in overall peptide length and amino acid composition. Because glycoproteins are present on the mycobacterial cell surface and are available for interaction with host cells during infection, protein glycosyltransferases may provide novel drug targets. The development of a cell-free mannosyltransferase assay will now facilitate the cloning and biochemical characterisation of the relevant enzymes from M. tuberculosis.     

PCR法快速检测临床标本中结核杆菌DNA

应用聚合酶链反应（ＰＣＲ）快速检测临床标本（脑脊液、胸水、腹水、血、痰液）中的结核杆菌ＤＮＡ,特异性扩增片段１２３ｂｐ,为结核杆菌的特异性重复序列ＩＳ６１１０部分基因。ＰＣＲ检测人型结核杆菌的敏感性达１０ｆｇＤＮＡ。临床标本的ＰＣＲ检测阳性率（２３．３％）明显高于抗酸染色涂片（２．９％）和细菌培养（５．７％）的阳性率（Ｐ〈０．０５）。通过设立对照系统及对扩增产物酶切分析,表明该法无假阴性结果（特异     

Characterization of the functional replication origin of Mycobacterium tuberculosis.

The gene order in the 5kb Mycobacterium tuberculosis dnaA region is rnpA, rpmH, dnaA, dnaN and recF. We show that M. tuberculosis DNA fragment containing the dnaA-dnaN intergenic region functioned as oriC, i.e., allowed autonomous replication to otherwise nonreplicative plasmids, in M. tuberculosis H37Ra (H37Ra), avirulent strain of M. tuberculosis, and in Mycobacterium bovis BCG (BCG), a closely related, slowly growing mycobacterial strain. Removal of Escherichia coli plasmid replication origin (ColE1) from the M. tuberculosis oriC plasmids did not abolish their ability to function as oriC, confirming that the autonomous replication activity of these plasmids is due to the presence of the DNA fragment containing the dnaA-dnaN intergenic region. Deletion analyses revealed that the minimal oriC DNA fragment is 814bp. The copy number of M. tuberculosis oriC plasmids containing ColE1 ori relative to chromosomal oriC is one and the 5' flanking region of minimal oriC contains features that support stable autonomous replication. The M. tuberculosis oriC did not function in rapidly growing mycobacterial species such as M. smegmatis. M. smegmatis oriC functioned only in M. fortuitum, but not in any of the slowly growing mycobacterial species such as M. tuberculosis and BCG. Together these data suggest that the replication initiation mechanisms in the slowly growing Mycobacteria are similar and probably different from those in the rapidly growing Mycobacteria and vice versa.     

Studies on alpha(1-->5) linked octyl arabinofuranosyl disaccharides for mycobacterial arabinosyl transferase activity

The appearance multi-drug resistant Mycobacterium tuberculosis (MTB) throughout the world has prompted a search for new, safer and more active agents against tuberculosis. Based on studies of the biosynthesis of mycobacterial cell wall polysaccharides, octyl 5-O-(alpha-D-arabinofuranosyl)-alpha-D-arabinofuranoside analogues were synthesized and evaluated as inhibitors for M. tuberculosis and Mycobacterium avium. A cell free assay system has been used for the evaluation of these disaccharides as substrates for mycobacterial arabinosyltransferase activity.     

The thymus as a target for mycobacterial infections

Mycobacterial infections are among the major health threats worldwide. Ability to fight these infections depends on the host's immune response, particularly on macrophages and T lymphocytes produced by the thymus. Using the mouse as a model, and two different routes of infection (aerogenic or intravenous), we show that the thymus is consistently colonized by Mycobacterium tuberculosis, Mycobacterium avium or Mycobacterium bovis BCG. When compared to organs such as the liver and spleen, the bacterial load reaches a plateau at later time-points after infection. Moreover, in contrast with organs such as the spleen and the lung no granuloma were found in the thymus of mice infected with M. tuberculosis or M. avium. Since T cell differentiation depends, to a large extent, on the antigens encountered within the thymus, infection of this organ might alter the host's immune response to infection. Therefore, from now on, the thymus should be considered in studies addressing the immune response to mycobacterial infection.