Trehalose-containing lipooligosaccharides. A new class of species-specific antigens from Mycobacterium

Mycobacterium kansasii is characterized by the presence of seven species-specific neutral lipooligosaccharide antigens. All react with hyperimmune anti-M. kansasii serum in enzyme-linked immunosorbent assays, and the more glycosylated members also react by gel diffusion. Both the native glycolipids and their inherent oligosaccharides were purified and the major features of their unique structures determined by acetolysis, partial acid cleavage, 1H-NMR and 13C-NMR, and chemical ionization and electron impact mass spectrometry of the permethylated products. They have in common a tetraglucose "core," beta-D-Glcp-(1 leads to 3)-beta-D-Glcp-(1 leads to 4)- alpha-D-Glcp(1 leads to 1)-alpha-D-Glcp where Glcp is glucopyranose, distinguished by the presence of an alpha,alpha-trehalose substituent. Variable residues of xylose, 3-O-methylrhamnose, fucose, and a novel N-acyl aminosugar, (4,6-dideoxy-2-O-methyl-3-C-methyl-4-(2'-methoxypropionamido)hexose; proof of this structure is not given) are linked to the core and the resulting oligosaccharides are acylated with 2,4-dimethyltetradecanoyl and acetyl functions to present a familial arrangement. Related but species-specific lipooligosaccharides typify Mycobacterium szulgai and apparently a host of other atypical mycobacteria. Thus, the antigenicity, and perhaps other features such as pathogenesis and drug resistance, of most atypical mycobacteria may be rationalized in terms of these new found cell wall lipooligosaccharides and the previously described glycopeptidolipids.     

Novel drug target strategies against Mycobacterium tuberculosis

The resurgence of drug resistant tuberculosis (TB) is a significant global healthcare challenge. Mycobacterium tuberculosis (MTB), TB's causative agent, evades the host immune system and drug regimes by entering prolonged periods of non-proliferation or dormancy. In infected individuals, the immune system sequesters MTB into structures called granulomas where the bacterium survives by shifting into a non-replicative state. Although still not well understood, progress has been made in characterizing the genetic program of MTB, activated by DosR (DevR) signal transduction that allows adaptation to the hypoxic, nutrient limiting granuloma microenvironment. Recent work, especially the identification genes involved in regulatory networks and the Enduring Hypoxic Response (EHR), hold promise for developing new drugs targeting dormancy phase MTB.     

Lessons from experimental Mycobacterium tuberculosis infections

Mycobacterium tuberculosis is the cause of enormous human morbidity and mortality each year. Although this bacterium can infect and cause disease in many animals, humans are the natural host. For the purposes of studying the pathogenesis of M. tuberculosis, as well as the protective and immunopathologic host responses against this pathogen, suitable animal models must be used. However, modeling the human infection and disease in animals can be difficult, and interpreting the data from animal models must be done carefully. In this paper, the animal models of tuberculosis are discussed, as well as the limitations and advantages of various models. In particular, the lessons we have learned about tuberculosis from the mouse models are highlighted. The careful and thoughtful use of animal models is essential to furthering our understanding of M. tuberculosis, and this knowledge will enhance the discovery of improved treatment and prevention strategies.     

Novel substrates of Mycobacterium tuberculosis PknH Ser/Thr kinase

PknH Ser/Thr protein kinase of Mycobacterium tuberculosis controls the expression of a variety of cell wall related enzymes and regulates the in vivo growth in mice. Therefore, we predicted that the PknH kinase could phosphorylate several substrates controlling different metabolic and physiological pathways. Using a bioinformatic approach, we identified 40 potential substrates. Two substrates were shown to be phosphorylated by recombinant PknH kinase in vitro. Point mutation studies verified that substrates are phosphorylated at the in silico-predicted sites. Kinetic studies revealed a similar relative-phosphorylation rate (V(max)) of PknH towards two new substrates and the only previously known substrate, EmbR. Unlike the EmbR protein, the Rv0681 and DacB1 proteins do not contain an FHA domain and are possible participants of new signaling pathways mediated by the PknH kinase in M. tuberculosis.     

Novel use of guanidinium isothiocyanate in the isolation of Mycobacterium tuberculosis DNA from clinical material

Nucleic acid amplification technologies offer great promise for the rapid, sensitive and specific diagnosis of tuberculosis. However, the isolation of inhibitor-free DNA from biological specimens is a bottleneck of the PCR assay. Here we describe a simple method for the isolation of PCR-amplifiable DNA of Mycobacterium tuberculosis from all types of samples of pulmonary and extrapulmonary origin tested. Briefly, it involves concentration of the bacilli by high-speed centrifugation, removal of PCR inhibitors by a wash solution containing guanidinium isothiocyanate and the release of bacterial DNA by heating in the presence of detergents and Chelex-100 resin. The entire process is accomplished within approximately 3 h. The method has been validated on 780 samples of human, bovine and guinea pig origin including sputum, cerebrospinal fluid, pulmonary fluids, pus, fine needle aspirate, tissue, blood and milk.     

Identification of a glycosyltransferase from Mycobacterium marinum involved in addition of a caryophyllose moiety in lipooligosaccharides

Deletion of Mycobacterium marinum MMAR2333 resulted in the loss of three of four subclasses of lipooligosaccharides (LOSs). The mutant was unable to extend an intermediate (LOS-II*) by addition of caryophyllose. These data and the predicted domain structure suggest that MMAR2333 is a glycosyltransferase involved in the generation of a lipid-linked caryophyllose donor.     

An unconventional hexacoordinated flavohemoglobin from Mycobacterium tuberculosis

Being an obligate aerobe, Mycobacterium tuberculosis faces a number of energetic challenges when it encounters hypoxia and environmental stress during intracellular infection. Consequently, it has evolved innovative strategies to cope with these unfavorable conditions. Here, we report a novel flavohemoglobin (MtbFHb) from M. tuberculosis that exhibits unique features within its heme and reductase domains distinct from conventional FHbs, including the absence of the characteristic hydrogen bonding interactions within the proximal heme pocket and mutations in the FAD and NADH binding regions of the reductase domain. In contrast to conventional FHbs, it has a hexacoordinate low-spin heme with a proximal histidine ligand lacking imidazolate character and a distal heme pocket with a relatively low electrostatic potential. Additionally, MtbFHb carries a new FAD binding site in its reductase domain similar to that of D-lactate dehydrogenase (D-LDH). When overexpressed in Escherichia coli or Mycobacterium smegmatis, MtbFHb remained associated with the cell membrane and exhibited D-lactate:phenazine methosulfate reductase activity and oxidized D-lactate into pyruvate by converting the heme iron from Fe(3+) to Fe(2+) in a FAD-dependent manner, indicating electron transfer from D-lactate to the heme via FAD cofactor. Under oxidative stress, MtbFHb-expressing cells exhibited growth advantage with reduced levels of lipid peroxidation. Given the fact that D-lactate is a byproduct of lipid peroxidation and that M. tuberculosis lacks the gene encoding D-LDH, we propose that the novel D-lactate metabolizing activity of MtbFHb uniquely equips M. tuberculosis to balance the stress level by protecting the cell membrane from oxidative damage via cycling between the Fe(3+)/Fe(2+) redox states.     

Structure of chorismate synthase from Mycobacterium tuberculosis

In bacteria, fungi, plants, and apicomplexan parasites, the aromatics compounds, such as aromatics amino acids, are synthesized through seven enzymes from the shikimate pathway, which are absent in mammals. The absence of this pathway in mammals make them potential targets for development of new therapy against infectious diseases, such as tuberculosis, which is the world's second commonest cause of death from infectious disease. The last enzyme of shikimate pathway is the chorismate synthase (CS), which is responsible for conversion of the 5-enolpyruvylshikimate-3-phosphate to chorismate. Here, we report the crystallographic structure of CS from Mycobacterium tuberculosis (MtCS) at 2.65 A resolution. The MtCS structure is similar to other CS structures, presenting beta-alpha-beta sandwich structural topology, in which each monomer of MtCS consists of a central helical core. The MtCS can be described as a tetramer formed by a dimer of dimers. However, analytical ultracentrifugation studies suggest the MtCS is a dimer with a more asymmetric shape than observed on the crystallographic dimer and the existence of a low equilibrium between dimer and tetramer. Our results suggest that the MtCS oligomerization is concentration dependent and some conformational changes must be involved on that event.     

Characterization of mRNA interferases from Mycobacterium tuberculosis

mRNA interferases are sequence-specific endoribonucleases encoded by the toxin-antitoxin systems in the bacterial genomes. MazF from Escherichia coli has been shown to be an mRNA interferase that specifically cleaves at ACA sequences in single-stranded RNAs. It has been shown that MazF induction in E. coli effectively inhibits protein synthesis leading to cell growth arrest in the quasidormant state. Here we have demonstrated that Mycobacterium tuberculosis contains at least seven genes encoding MazF homologues (MazF-mt1 to -mt7), four of which (MazF-mt1, -mt3, -mt4, and -mt6) caused cell growth arrest when induced in E. coli. MazF-mt1 and MazF-mt6 were purified and characterized for their mRNA interferase specificities. We showed that MazF-mt1 preferentially cleaves the era mRNA between U and A in UAC triplet sequences, whereas MazF-mt6 preferentially cleaves U-rich regions in the era mRNA both in vivo and in vitro. These results indicate that M. tuberculosis contains sequence-specific mRNA interferases, which may play a role in the persistent dormancy of this devastating pathogen in human tissues.     

Mycobacterium tuberculosis phagosome

The arrest of Mycobacterium tuberculosis phagosome maturation in infected macrophages is a phenomenon of dual significance both for the pathogenesis of tuberculosis and as a model system to study interference of microbes with membrane trafficking and organelle biogenesis in host cells. Among other factors, compartment-specialized regulators of vesicular trafficking and other parts of membrane fusion machinery are likely to play a role in these processes. Here we summarize the emerging view of mycobacterial phagosome maturation arrest in the context of the dynamic processes of intracellular membrane trafficking.     

Novel Mycobacterium tuberculosis anti-sigma factor antagonists control sigmaF activity by distinct mechanisms

The aetiological agent of tuberculosis, Mycobacterium tuberculosis, encodes 13 sigma factors, as well as several putative anti-, and anti-anti- sigma factors. Here we show that a sigma factor that has been previously shown to be involved in virulence and persistence processes, sigmaF, can be specifically inhibited by the anti-sigma factor UsfX. Importantly, the inhibitory activity of UsfX, in turn, can be negatively regulated by two novel anti-anti-sigma factors. The first anti-anti-sigma factor seems to be regulated by redox potential, and the second may be regulated by phosphorylation as it is rendered non-functional by the introduction of a mutation that is believed to mimic phosphorylation of the anti-anti-sigma factor. These results suggest that sigmaF activity might be post-translationally modulated by at least two distinct pathways in response to different possible physiological cues, the outcome being consistent with the bacteria's ability to adapt to diverse host environments during disease progression, latency and reactivation.     

Molecular model of shikimate kinase from Mycobacterium tuberculosis

Tuberculosis (TB) resurged in the late 1980s and now kills approximately 3 million people a year. The reemergence of tuberculosis as a public health threat has created a need to develop new anti-mycobacterial agents. The shikimate pathway is an attractive target for herbicides and anti-microbial agents development because it is essential in algae, higher plants, bacteria, and fungi, but absent from mammals. Homologs to enzymes in the shikimate pathway have been identified in the genome sequence of Mycobacterium tuberculosis. Among them, the shikimate kinase I encoding gene (aroK) was proposed to be present by sequence homology. Accordingly, to pave the way for structural and functional efforts towards anti-mycobacterial agents development, here we describe the molecular modeling of M. tuberculosis shikimate kinase that should provide a structural framework on which the design of specific inhibitors may be based.     

Kinetics and inhibition of nicotinamidase from Mycobacterium tuberculosis

Nicotinamidase/pyrazinamidase (PncA) is involved in the NAD+ salvage pathway of Mycobacterium tuberculosis and other bacteria. In addition to hydrolyzing nicotinamide into nicotinic acid, PncA also hydrolyzes the prodrug pyrazinamide to generate the active form of the drug, pyrazinoic acid, which is an essential component of the multidrug treatment of TB. A coupled enzymatic activity assay has been developed for PncA that allows for the spectroscopic observation of enzyme activity. The enzyme activity was essentially pH-independent under the conditions tested; however, the measurement of the pH dependence of iodoacetamide alkylation revealed a pK value of 6.6 for the active site cysteine. Solvent deuterium kinetic isotope effects revealed an inverse value for kcat of 0.64, reconfirming the involvement of a thiol group in the mechanism. A mechanism is proposed for PncA catalysis that is similar to the mechanisms proposed for members of the nitrilase superfamily, in which nucleophilic attack by the active site cysteine generates a tetrahedral intermediate that collapses with the loss of ammonia and subsequent hydrolysis of the thioester bond by water completes the cycle. An inhibitor screen identified the competitive inhibitor 3-pyridine carboxaldehyde with a Ki of 290 nM. Additionally, pyrazinecarbonitrile was found to be an irreversible inactivator of PncA, with a kinact/KI of 975 M(?1) s(?1).     

Structure-Activity relationship in mutated pyrazinamidases from Mycobacterium tuberculosis

The pncA gene codes the pyrazinamidase of Mycobacterium tuberculosis, which converts pyrazinamide to ammonia and pyrazinoic-acid, the active antituberculous compound. Pyrazinamidase mutations are associated to pyrazinamide-resistant phenotype, however how mutations affect the structure of the pyrazinamidase, and how structural changes affect the enzymatic function and the level of pyrazinamide-resistance is unknown. The structures of mutated pyrazinamidases from twelve Mycobacterium tuberculosis strains and the pyrazinamide-susceptible H37Rv reference strain were modelled using homology modelling and single amino acid replacement. Physical-chemical and structural parameters of each pyrazinamidase were calculated. These parameters were: The change of electrical charge of the mutated amino acid, the change of volume of the mutated amino acid, the change of a special amino acid, the distance of the mutated amino acid to the active site, the distance of the mutated amino acid to the metal-coordination site, and the orientation of the side-chain of the mutated amino acid. The variability of the enzymatic activity of the recombinant pyrazinamidases, and the microbiological susceptibility to pyrazinamide determined by BACTEC 460TB, were modelled in multiple linear regressions. Physical-chemical and structural parameters of the mutated pyrazinamidases were tested as predictors. Structural and physical-chemical variations of the pyrazinamidase explained 75% of the variability of the enzymatic activity, 87% of the variability of the kinetic constant and 40% of the variability of the pyrazinamide-resistance level. Based on computer models of mutated pyrazinamidases, the structural parameters explained a high variability of the enzymatic function, and to a lesser extent the resistance level.