System-level strategies for studying the metabolism of Mycobacterium tuberculosis

Despite decades of research many aspects of the biology of Mycobacterium tuberculosis remain unclear and this is reflected in the antiquated tools available to treat and prevent tuberculosis and consequently this disease remains a serious public health problem. Important discoveries linking M. tuberculosis's metabolism and pathogenesis have renewed interest in this area of research. Previous experimental studies were limited to the analysis of individual genes or enzymes whereas recent advances in computational systems biology and high throughput experimental technologies now allow metabolism to be studied on a genome scale. Here we discuss the progress being made in applying system level approaches to studying the metabolism of this important pathogen. The information from these studies will fundamentally change our approach to tuberculosis research and lead to new targets for therapeutic drugs and vaccines.     

Genetic basis for the biosynthesis of methylglucose lipopolysaccharides in Mycobacterium tuberculosis

Mycobacteria produce two unusual polymethylated polysaccharides, the 6-O-methylglucosyl-containing lipopolysaccharides (MGLP) and the 3-O-methylmannose polysaccharides, which have been shown to regulate fatty acid biosynthesis in vitro. A cluster of genes dedicated to the synthesis of MGLP was identified in Mycobacterium tuberculosis and Mycobacterium smegmatis. Overexpression of the putative glycosyltransferase gene Rv3032 in M. smegmatis greatly stimulated MGLP production, whereas the targeted disruption of Rv3032 in M. tuberculosis and that of the putative methyltransferase gene MSMEG2349 in M. smegmatis resulted in a dramatic reduction in the amounts of MGLP synthesized and in the accumulation of precursors of these molecules. Disruption of Rv3032 also led to a significant decrease in the glycogen content of the tubercle bacillus, indicating that the product of this gene is likely to be involved in the elongation of more than one alpha-(1-->4)-glucan in this bacterium. Results thus suggest that Rv3032 encodes the alpha-(1-->4)-glucosyltransferase responsible for the elongation of MGLP, whereas MSMEG2349 encodes the O-methyltransferase required for the 6-O-methylation of these compounds.     

Genetic diversity among Mycobacterium tuberculosis isolates from Mexican patients

Forty-six isolates of the Mycobacterium tuberculosis complex were typified by PCR of the IS6110 region and by Mycobacterium bovis specific primers JB21/JB22. Isolate MVG01 was typified as M. bovis, being the first record of a case of human tuberculosis caused by this species in Mexico. RAPD-PCR was used to describe the genetic diversity of the remaining 45 M. tuberculosis complex isolates. The corrected genotypic diversity value calculated for the analyzed population was 0.96, the estimated mean gene diversity was 0.235, and the corrected Shannon-Weiner index was 2.15. All allele-loci combinations generated showed significant linkage disequilibria. The distribution of genetic variation was analyzed both by the unweighted pair group method with arithmetic averages clustering and by principal coordinates analysis. Unweighted pair group method with arithmetic averages clustering resulted in a tree with four main clusters and one unclustered strain (MVG20), the principal coordinates analysis strain distribution pattern being consistent with this grouping. The obtained results suggest that the studied isolates belong to a clonal population having significant genetic diversity. Our genetic diversity results are comparable with those reported for other populations of M. tuberculosis, although only three RAPD primers were used.     

Genetic diversity of Iranian clinical isolates of Mycobacterium tuberculosis

In the current study we aimed to execute a rather less complicated molecular tying method, i.e. the random amplification of polymorphic DNA (RAPD) analysis to find the heterogeneity of Iranian strains of Mycobacterium tuberculosis. The isolates comprised a total of 96 strains of M. tuberculosis collected from clinical specimens of patients in Isfahan and Tehran. The isolates were assigned to the species M. tuberculosis by the key conventional and molecular methods. They were then subjected to RAPD analysis by four arbitrary primers, namely, the primers 27F, 1525R, MS- GF and INS-2. They were then evaluated for the number and intensity of the band patterns. The RAPD profiles of the Iranian isolates showed a degree of heterogeneity which varied based on the primer used. However, analysis of the isolates by primer INS-2 revealed the highest degree of diversity yielding 31 distinguishable RAPD types. RAPD analysis provides a rapid and easy means of identifying heterogeneity among the M. tuberculosis isolates. This typing system might be considered a valuable alternative molecular typing for countries with limited resources provided that the reproducibility and reliability of the method is carefully assured.     

Evidence for phylogenetic inheritance in pathogenicity of Mycobacterium

In this study, we attempt to highlight part of the adaptive and phylogenetic constraints in mycobacterial pathogenicity. For this purpose, we first provide a phylogeny of Mycobacteria based on cladistic analyses of 64 different taxa. We then performed a comparative analysis, taking into account both ecological factors and phylogenetic relationships. The GLIM modelling analysis showed that different ecological and phylogenetic factors might be invoked to explain the variation in pathogenicity levels. Interestingly, the most harmful species were shown to be connected with the most diversified habitats. However, the independent contrast analysis revealed that once phylogeny was taken into account, none of the relationships between ecological factors and pathogenicity remained significant, and the pathogenicity appeared to be phylogenetically inherited among mycobacteria. The most pathogen were found in the slow-growing/long helix 18 group, and within this group in the most derived taxa. This revised version was published online in June 2006 with corrections to the Cover Date.     

The physiology and pathogenicity of Mycobacterium tuberculosis grown under controlled conditions in a defined medium

A chemically-defined culture medium was developed which supported batch growth of Mycobacterium tuberculosis, strain H37Rv, at a minimum doubling time of 14.7 h. This medium also facilitated chemostat culture of M. tuberculosis at a constant doubling time of 24 h. Chemostat growth was optimized at a dissolved oxygen tension of 20% (v/v) and 0.2% (v/v) Tween-80. Chemostat cultures were dispersed suspensions of single bacilli (1.5-3 microm long), or small aggregates, at a mean density of log10 8.3 cfu ml-1. A limited number of amino acids was utilized (alanine, asparagine, aspartate and serine were depleted by >50%; glycine, arginine, isoleucine, leucine and phenylalanine, by approximately 40%). Chemostat-grown cells were pathogenic in aerosol-infected guinea pigs, producing disseminated infection similar to that caused by plate-grown cells. Cells from chemostat culture were significantly more invasive for J774A.1 mouse macrophages than agar- or batch-grown cells. This study demonstrates the suitability of chemostat culture for the growth of pathogenic mycobacteria in a defined physiological state with potential applications for the controlled production of mycobacterial components for therapeutic and vaccine applications.     

Genetic characterization of the Guinea-Bissau family of Mycobacterium tuberculosis complex strains

In a previous study of Mycobacterium tuberculosis complex isolates from Guinea-Bissau in West Africa, we identified a unique group of strains, designated here as the Guinea-Bissau family of strains, which, although genotypically closely related, phenotypically demonstrated a considerable heterogeneity. We conducted here a detailed genotypic analysis of a subset (n = 35) of these isolates. Based on the data obtained, and by comparison of known corresponding genes in mycobacteria outside the M. tuberculosis complex, we propose that the Guinea-Bissau strains belong to a unique branch of the M. tuberculosis complex tree in between classical M. tuberculosis and classical M. bovis. These observations are discussed in their significance in M. tuberculosis complex classification.     

Nitric oxide and Mycobacterium leprae pathogenicity

Leprosy is an old, still dreaded infectious disease caused by the obligate intracellular bacterium Mycobacterium leprae. During the infectious process, M. leprae is faced with the host macrophagic environment, where the oxidative stress and NO release, combined with low pH, low pO2, and high pCO2, contribute to limit the growth of the bacilli. Comparative genomics has unraveled massive gene decay in M. leprae, linking the strictly parasitic lifestyle with the reductive genome evolution. Compared with Mycobacterium tuberculosis and Mycobacterium bovis, the leprosy bacillus has lost most of the genes involved in the detoxification of reactive oxygen and nitrogen species. The very low reactivity of the unique truncated hemoglobin retained by M. leprae could account for the susceptibility of this exceptionally slow-growing microbe to NO.     

Genetic approaches for studying rhizosphere colonization

Most bacterial traits involved in colonization of plant roots are yet to be defined. Studies were initiated to identify genes in Pseudomonas which play significant roles in this process. The general approach is to use transposons to construct collections of insertion mutants, each of which is then screened for alterations in its interactions with the host plant. In one study a Tn5 derivative containing a constitutively expressed -galactosidase (lacZ) gene was used to generate a collection of insertion mutants which could be distinguished from the wild-type parent on X-gal plates. Each mutant was examined for its ability to colonize wheat seedlings in the presence of the wild-type parent. Mutants which gave wild-type:mutant ratio of 20:1 or greater were obtained. In a second study a Tn5 derivative which carries a promoterless lacZ gene located near one end of the transposon was constructed. Expression of the lacZ gene depends on the presence of an active promoter outside of the transposon in the correct orientation. Insertion mutants generated with this transposon were examined for changes in -galactosidase expression in the presence and absence of plant root exudate. A number of mutants which showed differential lacZ expression have been identified.     

Evidence for recombination in Mycobacterium tuberculosis

Due to its mostly isolated living environment, Mycobacterium tuberculosis is generally believed to be highly clonal, and thus recombination between different strains must be rare and is not critical for the survival of the species. To investigate the roles recombination could have possibly played in the evolution of M. tuberculosis, an analysis was conducted on previously determined genotypes of 36 synonymous single nucleotide polymorphisms (SNPs) in 3,320 M. tuberculosis isolates. The results confirmed the predominant clonal structure of the M. tuberculosis population. However, recombination between different strains was also suggested. To further resolve the issue, 175 intergenic SNPs and 234 synonymous SNPs were genotyped in 37 selected representative strains. A clear mosaic polymorphic pattern ahead of the MT0105 locus encoding a PPE (Pro-Pro-Glu) protein was obtained, which is most likely a result of recombination hot spot. Given that PPE proteins are thought to be critical in host-pathogen interactions, we hypothesize that recombination has been influential in the history of M. tuberculosis and possibly a major contributor to the diversity observed ahead of the MT0105 locus.     

Genetic basis for the synthesis of the immunomodulatory mannose caps of lipoarabinomannan in Mycobacterium tuberculosis

Lipoarabinomannan (LAM) is a high molecular weight, heterogenous lipoglycan present in abundant quantities in Mycobacterium tuberculosis and many other actinomycetes. In M. tuberculosis, the non-reducing arabinan termini of the LAM are capped with alpha1-->2 mannose residues; in some other species, the arabinan of LAM is not capped or is capped with inositol phosphate. The nature and extent of this capping plays an important role in disease pathogenesis. MT1671 in M. tuberculosis CDC1551 was identified as a glycosyltransferase that could be involved in LAM capping. To determine the function of this protein a mutant strain of M. tuberculosis CDC1551 was studied, in which MT1671 was disrupted by transposition. SDS-PAGE analysis showed that the LAM of the mutant strain migrated more rapidly than that of the wild type and did not react with concanavalin A as did wild-type LAM. Structural analysis using NMR, gas chromatography/mass spectrometry, endoarabinanase digestion, Dionex high pH anion exchange chromatography, and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry demonstrated that the LAM of the mutant strain was devoid of mannose capping. Since an ortholog of MT1671 is not present in Mycobacterium smegmatis mc(2)155, a recombinant strain was constructed that expressed this protein. Analysis revealed that the LAM of the recombinant strain was larger than that of the wild type, had gained concanavalin A reactivity, and that the arabinan termini were capped with a single mannose residue. Thus, MT1671 is the mannosyltransferase involved in deposition of the first of the mannose residues on the non-reducing arabinan termini and the basis of much of the interaction between the tubercle bacillus and the host cell.     

Genetic typing of Mycobacterium tuberculosis strains by spoligotyping and genome fingerprinting techniques

A total of 122 M. tuberculosis clinical drug-resistant strains isolated in Central Russia were studied by spoligotyping and genome fingerprinting techniques. According to spoligotyping results 77% of M. tuberculosis strains were distributed to 13 oligotypes, while 23% of these strains were found to form unique patterns. Most of them belonged to the families Beijing and Haarlem (43.4% and 13.9% respectively). The patterns of the strains of oligotype 12 (7F-7F-7E-0E-78-3E) were identical to those of the strains isolated in Brazil, France and the Netherlands. The strains of the spoligotype 22 (7F-1E-7F-7F-07-3E) had the patterns identical to those of the strains of group S13, also isolated in Brazil. According to genome fingerprinting 31.4% of the strains were found to belong to clusters with the similarity coefficient equal to 1. The strains belonging to genotypes W and A1 were found to prevail in the analyzed group.     

Genetic diversity of Mycobacterium tuberculosis isolates from Tibetans in Tibet, China

Background

Tuberculosis (TB) is a serious health problem in Tibet where Tibetans are the major ethnic group. Although genotyping of Mycobacterium tuberculosis (M. tuberculosis) isolates is a valuable tool for TB control, our knowledge of population structure of M. tuberculosis circulating in Tibet is limited.

Methodology/Principal Findings

In our study, a total of 576 M. tuberculosis isolates from Tibetans in Tibet, China, were analyzed via spoligotyping and 24-locus MIRU-VNTR. The Beijing genotype was the most prevalent family (90.63%, n = 522). Shared-type (ST) 1 was the most dominant genotype (88.89%, n = 512). We found that there was no association between the Beijing genotype and sex, age and treatment status. In this sample collection, 7 of the 24 MIRU-VNTR loci were highly or moderately discriminative according to their Hunter-Gaston discriminatory index. An informative set of 12 loci had similar discriminatory power with 24 loci set.

Conclusions/Significance

The population structure of M. tuberculosis isolates in Tibetans is homogeneous and dominated by Beijing genotype. The analysis of 24-locus MIRU-VNTR data might be useful to select appropriate VNTR loci for the genotyping of M. tuberculosis.     

Electrochemical-based biosensors for detection of Mycobacterium tuberculosis and tuberculosis biomarkers

Abstract

Early detection of tuberculosis (TB) reduces the interval between infection and the beginning of treatment. However, commercially available tests cannot discriminate between BCG-vaccinated healthy persons and patients. Also, they are not suitable to be used for immunocompromised persons. In recent years, biosensors have attracted great attention due to their simple utility, accessibility, and real-time outputs. These sensors are increasingly being considered as pioneering tools for point-of-care diagnostics in communities with a high burden of TB and limited accessibility to reference laboratories. Among other types of biosensors, the electrochemical sensors have the advantages of low-cost operation, fast processing, simultaneous multi-analyte analyzing, operating with turbid samples, comparable sensitivity and readily available miniaturization. Electrochemical biosensors are sub-divided into several categories including: amperometric, impedimetric, potentiometric, and conductometric biosensors. The biorecognition element in electrochemical biosensors is usually based on antibodies (immunosensors), DNAs or PNAs (genosensors), and aptamers (aptasensors). In either case, whether an interaction of the antigen–antibody/aptamer or the hybridization of probe with target mycobacterial DNA is detected, a change in the electrical current occurs that is recorded and displayed as a plot. Therefore, impedimetric-based methods evaluate resistance to electron transfer toward an electrode by a Nyquist plot and amperometric/voltammetric-based methods weigh the electrical current by means of cyclic voltammetry, square wave voltammetry, and differential pulse voltammetry. Electrochemical biosensors provide a promising scope for the new era of diagnostics. As a consequence, they can improve detection of Mycobacterium tuberculosis traces even in attomolar scales.     

Crystal structure of Mycobacterium tuberculosis zinc-dependent metalloprotease-1 (Zmp1), a metalloprotease involved in pathogenicity

Mycobacterium tuberculosis, the causative agent of tuberculosis, parasitizes host macrophages. The resistance of the tubercle bacilli to the macrophage hostile environment relates to their ability to impair phagosome maturation and its fusion with the lysosome, thus preventing the formation of the phago-lysosome and eventually arresting the process of phagocytosis. The M. tuberculosis zinc-dependent metalloprotease Zmp1 has been proposed to play a key role in the process of phagosome maturation inhibition and emerged as an important player in pathogenesis. Here, we report the crystal structure of wild-type Zmp1 at 2.6 Å resolution in complex with the generic zinc metalloprotease inhibitor phosphoramidon, which we demonstrated to inhibit the enzyme potently. Our data represent the first structural characterization of a bacterial member of the zinc-dependent M13 endopeptidase family and revealed a significant degree of conservation with eukaryotic enzymes. However, structural comparison of the Zmp1-phosphoramidon complex with homologous human proteins neprilysin and endothelin-converting enzyme-1 revealed unique features of the Zmp1 active site to be exploited for the rational design of specific inhibitors that may prove useful as a pharmacological tool for better understanding Zmp1 biological function.     

Genetic mosaic techniques for studying Drosophila development

Genetic screens for recessive mutations continue to provide the basis for much of the modern work on Drosophila developmental genetics. However, many of the mutations isolated in these screens cause embryonic or early larval lethality. Studying the effects of such mutations on later developmental events is still possible, however, using genetic mosaic techniques, which limit losses or gains of genetic function to specific tissues and cells, and to selected stages of development. A variety of genetic mosaic techniques have been developed, and these have led to key insights into developmental processes in the fly. Variations on these techniques can also be used to screen for novel genes that are involved in non-embryonic patterning and growth.