Characterization of a specific arabinosyltransferase activity involved in mycobacterial arabinan biosynthesis

Mycobacterium smegmatis strains that contain inactivated EmbA or EmbB proteins are unable to synthesize terminal Arabeta1-->2Araalpha1-->5(Arabeta1-->2Araalpha1-->3)Araalpha1-->5Araalpha1-->(Ara(6)) motif in the cell wall polysaccharide arabinogalactan. Instead, the mutants contain a linear Arabeta1-->2Araalpha1-->5Araalpha1-->5Araalpha1-->(Ara(4)) motif, suggesting that these proteins are involved in the synthesis or transfer of the disaccharide Arabeta1-->2Araalpha1--> to an internal 5-linked Ara. Therefore, we synthesized a linear Arabeta1-->2Araalpha1-->5Araalpha1-->5Araalpha1-->5Araalpha1--> with an octyl aglycon as an arabinosyl acceptor in cell-free assays. A facile assay was developed using the chemically synthesized glycan, membrane, and particulate cell wall as the enzyme source, and 5-phosphoribose diphosphate pR[(14)C]pp as the arabinose donor. The results unequivocally show that two arabinofuranosyl residues were added at the tertiary -->5Araalpha1--> of the synthetic glycan. This activity was undetectable in strains of M. smegmatis where embB or embA had been genetically disrupted. Normal activity could be restored only in the presence of both EmbA and EmbB proteins.     

Structural analysis of mycobacterial lipoglycans

Mycobacterium tuberculosis, the causative agent of tuberculosis, is one of the most effective human pathogens. The mycobacterial cell envelope contains lipoglycans, and of particular interest is lipoarabinomannan (LAM), one of the most potent mycobacterial immunomodulatory molecules. The importance of lipoarabinomannan (LAM) in the immunopathogenesis of tuberculosis has incited structural studies on this molecule to (1) establish a precise structural model of the molecule and (2) decipher the structure/function relationships. In recent years, we have focused on the two domains essential for LAM biologic activities: the mannosyl-phosphatidyl-myo-inositol anchor and the caps. We review here the recent procedures developed for the structural analysis of these domains.     

Characterization of a distinct arabinofuranosyltransferase in Mycobacterium smegmatis

The D-arabinans in Mycobacterium are essential, extraordinarily complex entity comprised of d-arabinofuranose residues which are rarely found in nature. Despite the well-recognized importance of the mycobacterial arabinan, delineation of the arabinosylation process has been severely hampered due to lack of positively identified arabinosyltransferases. Identification of genes involved in arabinan biosynthesis entailed the use of ethambutol (EMB), a first-line antituberculosis agent that is known to inhibit cell wall arabinan synthesis. The three genes (embA, embB, and embC) encode novel membrane proteins, implicated as the only known mycobacterial arabinosyltransferases to this date. We have now adapted a multifaceted approach involving development of convenient arabinosyltransferase assay using novel synthetic acceptors to identify arabinosyltransferase/s that will be distinct from the Emb proteins. In our present work, Mycobacterium smegmatis mc(2) 155 (WTMsm) was used as a model to study the biosynthesis of cell wall arabinan. In an in vitro assay, we demonstrate that transfer of only alpha-Araf had occurred from decaprenylphosphoryl-D-arabinofuranose (DPA) on a newly synthesized branched acceptor [alpha-D-Araf](2)-3,5-alpha-D-Araf-(1-->5)-alpha-d-Araf-(1-->5)-alpha-D-Araf with an octyl aglycon. Higher molecular weight (up to Ara(10)) oligomers were also detected in a parallel reaction using cold phosphoribosepyrophosphate (pRpp). Matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS) analysis of these products revealed that isomeric products were formed and initiation and elongation of arabinan can occur either on the 5-arm or 3-arm of the branched 3,5-alpha-D-Araf. Individual embA, embB, and embC knockout strains retained this alpha-1,5 arabinosyltransferase activity, and the activity was partially inhibited by ethambutol. This particular enzyme function is distinct from the function of the Emb proteins.     

Identification of proteins from Mycobacterium tuberculosis missing in attenuated Mycobacterium bovis BCG strains

A proteome approach, combining high-resolution two-dimensional electrophoresis (2-DE) with mass spectrometry, was used to compare the cellular protein composition of two virulent strains of Mycobacterium tuberculosis with two attenuated strains of Mycobacterium bovis Bacillus Calmette-Guerin (BCG), in order to identify unique proteins of these strains. Emphasis was given to the identification of M. tuberculosis specific proteins, because we consider these proteins to represent putative virulence factors and interesting candidates for vaccination and diagnosis of tuberculosis. The genome of M. tuberculosis strain H37Rv comprises nearly 4000 predicted open reading frames. In contrast, the separation of proteins from whole mycobacterial cells by 2-DE resulted in silver-stained patterns comprising about 1800 distinct protein spots. Amongst these, 96 spots were exclusively detected either in the virulent (56 spots) or in the attenuated (40 spots) mycobacterial strains. Fifty-three of these spots were analyzed by mass spectrometry, of which 41 were identified, including 32 M. tuberculosis specific spots. Twelve M. tuberculosis specific spots were identified as proteins, encoded by genes previously reported to be deleted in M. bovis BCG. The remaining 20 spots unique for M. tuberculosis were identified as proteins encoded by genes that are not known to be missing in M. bovis BCG.     

Specific electrochemical phage sensing for Bacillus cereus and Mycobacterium smegmatis

The rapid and reliable detection of pathogenic microorganisms is an important issue for the safety and security of our society. Here we describe the use of a sensitive, inexpensive, amperometric, phage-based biosensor for the detection of extremely low concentrations of Bacillus cereus and Mycobacterium smegmatis as models for Bacillus anthracis (the causative agent of anthrax) and for Mycobacterium tuberculosis (the causative agent of tuberculosis), respectively. The detection procedure developed here enabled the determination of bacteria at a low concentration of 10 viable cells/mL within 8 h. This experimental setup allows the simultaneous analysis of up to eight independent samples, using disposable screen-printed electrodes.     

The Discovery of 2‐Aminobenzimidazoles That Sensitize Mycobacterium smegmatis and M. tuberculosis to β‐Lactam Antibiotics in a Pattern Distinct from β‐Lactamase Inhibitors

A library of 2‐aminobenzimidazole derivatives was screened for the ability to suppress β‐lactam resistance in Mycobacterium smegmatis. Several non‐bactericidal compounds were identified that reversed intrinsic resistance to β‐lactam antibiotics in a manner distinct from β‐lactamase inhibitors. Activity also translates to M. tuberculosis, with a lead compound from this study potently suppressing carbenicillin resistance in multiple M. tuberculosis strains (including multidrug‐resistant strains). Preliminary mechanistic studies revealed that the lead compounds act through a mechanism distinct from that of traditional β‐lactamase inhibitors.     

Synthesis of a core arabinomannan oligosaccharide of Mycobacterium tuberculosis

The synthesis of a core arabinomannan (AM) oligosaccharide from Mycobacterium tuberculosis has been achieved using a convergent [6 + 6] glycosylation strategy and a defined set of building blocks. Dodecasaccharide 1, containing the key AM structural features of lipoarabinomannan (LAM), was obtained in excellent yield and selectivity from hexamannan 3 and hexaarabinan 5. This flexible synthetic strategy involves late-stage couplings and modifications, thus providing ready access to several different LAM fragments. The incorporation of a thiol linker at the reducing end of the oligosaccharide allows for the attachment of these compounds to microarrays and protein carriers.     

Towards the proteome of Mycobacterium tuberculosis

Human tuberculosis is caused by the intracellular pathogen Mycobacterium tuberculosis. Sequencing of the genome of M. tuberculosis strain H37Rv has predicted 3924 open reading frames, and enabled identification of proteins from this bacterium by peptide mass fingerprinting. Extracellular proteins from the culture medium and proteins in cellular extracts were examined by two-dimensional gel electrophoresis using immobilized pH gradient technology. By mass spectrometry and immunodetection, 49 culture filtrate proteins and 118 lysate proteins were identified, 83 of which were novel. To date, 288 proteins have been identified in M. tuberculosis proteome studies, and a list is presented which includes all identified proteins (available at http://www.ssi.dk/publichealth/tbimmun). The information obtained from the M. tuberculosis proteome so far is discussed in relation to the information obtained from the complete genome sequence.     

Top down characterization of secreted proteins from Mycobacterium tuberculosis by electron capture dissociation mass spectrometry

Secreted proteins of Mycobacterium tuberculosis are implicated in its disease pathogenesis and so are considered as potential diagnostic and vaccine candidates. The search for these has been slow, even though the entire genome sequence of M. tuberculosis is now available; of the 620 protein spots resolved by 2-D gel electrophoresis, 114 secreted proteins have been identified, but for only 13 has the primary structure been partly characterized. For comparison, in this top down mass spectrometry (MS) approach the secreted proteins were precipitated from cell culture filtrate, resuspended, and examined directly by electrospray ionization (ESI) Fourier transform MS. The ESI spectra of three precipitates showed 93, 535, and 369 molecular weight (M(r)) values, for a total of 689 different values. However, only approximately 10% of these values matched (+/-1 Da) the DNA predicted M(r) values, but these identifications were unreliable. Of nine molecular ions characterized by MS/MS, only one protein match was confirmed, and its isotopic molecular ions were overlapped by those of another protein. MS/MS identified a total of ten proteins by sequence tag search, of which three were unidentified previously. The low success of M(r) matching was due to unusually extensive posttranslational modifications, including loss of a signal sequence, loss of the N-terminal residue, proteolytic degradation, oxidation, and glycosylation. Although in eubacteria the latter is relatively rare, a 9 kDa protein showed 7 hexose attachments and two 20 kDa proteins each had 20 attachments. For MS/MS, electron capture dissociation was especially effective.     

Dual inhibition of mycobacterial fatty acid biosynthesis and degradation by 2-alkynoic acids

2-Hexadecynoic acid and 2-octadecynoic acid have cidal activity against Mycobacterium smegmatis and Mycobacterium bovis BCG. At subinhibitory concentrations, M. smegmatis rapidly transformed [1-(14)C]-2-hexadecynoic acid into endogenous fatty acids and elongated them into mycolic acids. Toxic concentrations of 2-hexadecynoic acid resulted in accumulation of 3-ketohexadecanoic acid, which blocked fatty acid biosynthesis, and 3-hexadecynoic acid, an inhibitor of fatty acid degradation. The combination of these two metabolites is necessary to achieve the inhibition of M. smegmatis. We conclude that 2- and 3-hexa/octadecynoic acids inhibit mycolic acid biosynthesis, fatty acid biosynthesis, and fatty acid degradation, pathways of significant importance for mycobacteria.     

Loss of a mycobacterial gene encoding a reductase leads to an altered cell wall containing beta-oxo-mycolic acid analogs and accumulation of ketones

Mycolic acids are essential components of the mycobacterial cell wall. In this study, we show that a gene encoding a reductase involved in the final step of mycolic acid biosynthesis can be deleted in Mycobacterium smegmatis without affecting cell viability. Deletion of MSMEG4722 (ortholog of Mycobacterium tuberculosis Rv2509) altered culture characteristics and antibiotic sensitivity. The DeltaMSMEG4722 strain synthesized alpha-alkyl, beta-oxo intermediates of mycolic acids, which were found esterified to cell wall arabinogalactan. While the precursors could not be isolated directly due to their inherent instability during base treatment, their presence was established by prior reduction of the beta-oxo group by sodium borohydride. Interestingly, the mutant also accumulated unsaturated ketones, similar to tuberculenone from M. tuberculosis, which were shunt products derived from spontaneous decarboxylation of alpha-alkyl, beta-oxo fatty acid precursors of mycolic acids.     

Beiträge zur Chemie von Phosphorverbindungen mit Adamantanstruktur. X. Zur Solvolyse von Phosphor(III/V)-oxiden

Contributions to the Chemistry of Phosphorus Compounds with Adamantane Structure. X. Solvolysis of Phosphorus(III/V) Oxides The phosphorus(III/V) oxides of adamantan-like structure have been prepared by oxydation of phosphorus or of phosphorus trioxide with oxygen and characterized by analysis in water solution and by ³¹P n.m.r. spectroscopy. Paper chromatographic and n.m.r. studies show that the alcoholysis of the phosphorus(III/V) oxides leads to tri-, di-, and monoesters of phosphorous acid and also to mono-, di-, and cyclotriphosphoric acid, in dependence on their P^III and P^V contents. Nucleophilic attack by the alcohol takes place not statistically but in principle on the P^III atoms of the phosphorus(III/V) oxide molecule and their primary degradation products. In analogy to the alcoholysis the hydrolysis of the phosphorus(III/V) oxides yields phosphorous acid, mono-, di-, and cyclotriphosphoric acid. The mechanism of degradation is discussed.     

Rapid, iterative assembly of octyl alpha-1,6-oligomannosides and their 6-deoxy equivalents

Mycobacterium tuberculosis is the cause of the deadly human disease tuberculosis. In studies over the last 40 years it has been revealed that this organism possesses a complex cell wall including glycophospholipids such as the phosphatidylinositiol mannosides (PIMs), lipomannan (LM) and lipoarabinomannan (LAM). These glycolipids all contain a common alpha-1,6-linked mannoside core, and the higher PIMs and LAM possess alpha-1,2-linked mannosyl residues. It has been shown that simple alpha-1,6-linked oligomannosides can act as substrates for alpha-1,6-mannosyltransferases in mycobacteria. Here we report a simple iterative synthesis of a series of hydrophobic octyl alpha-1,6-linked oligomannosides from mono- through to tetrasaccharides. We have utilized a single thioglycoside donor and alcohol acceptor. Further, we have developed conditions for the conversion of each of these compounds to the 6-deoxy congeners. Deoxygenation of the 6-position of the terminal mannosyl residue should prevent these compounds acting as substrates for the abundant alpha-1,6-mannosyltransferases in mycobacteria and should permit detection of the elusive alpha-1,2-mannosyltransferase activity responsible for elaboration of LM to mature LAM and the biosynthesis of the higher PIMs.     

Comparative proteome analysis of culture supernatant proteins from virulent Mycobacterium tuberculosis H37Rv and attenuated M. bovis BCG Copenhagen

A comprehensive analysis of culture supernatant (CSN) proteins of Mycobacterium tuberculosis H37Rv was accomplished by combination of two-dimensional electrophoresis (2-DE), mass spectrometry, and N-terminal sequencing by Edman degradation. Analytical 2-DE gels resolved approximately 1250 protein spots from CSN of M. tuberculosis H37Rv, 381 of which were identified by mass spectrometry and/or Edman degradation. This study revealed 137 different proteins, 42 of which had previously been described as secreted. Comparative proteome analysis of CSN from virulent M. tuberculosis H37Rv and attenuated Mycobacterium bovis BCG Copenhagen identified 39 M. tuberculosis-specific spots containing 27 different proteins, representing candidate antigens for novel vaccines and diagnostics in tuberculosis. These included five proteins encoded by open reading frames absent from M. bovis BCG, e.g., early secretory antigen target (Esat6), as well as 22 novel differential proteins, such as acetyl-CoA C-acetyltransferase (Rv0243) and two putative Esat6-like proteins (Rv1198, Rv1793).     

Effects of ion-pairing reagents on the electrospray signal suppression of sulphonated dyes and intermediates

High-performance liquid chromatography/mass spectrometry (HPLC/MS) analysis of anionic species such as sulphonic acid dyes and intermediates requires volatile ion-pairing mobile phase additives. Six di- and trialkylammonium acetates were compared with tetraalkylammonium salts and ammonium acetate in the concentration range 0-20 mmol l(-1) as mobile phase additives for HPLC/MS of polysulphonated compounds. The effects of the structure and concentration of the ion-pairing reagents on the electrospray response of mono-, di- and tetrasulphonic aromatic acids and acid dyes were studied in detail. Further, five different mass analysers and instrument geometries were compared. A higher signal decrease is observed with linear geometry instruments in comparison to orthogonal or even Z-spray geometry mass spectrometers. The concentration of mobile phase additives has a significant influence on the abundance ratios of multiply charged ions in the mass spectra of polysulphonated compounds. The competing ions of sulphonic acids may also cause significant signal suppression.     

A fast method for the identification of Mycobacterium tuberculosis in sputum and cultures based on thermally assisted hydrolysis and methylation followed by gas chromatography–mass spectrometry

A fast gas chromatography–mass spectrometry (GC–MS) method with minimum sample preparation is described for early diagnosis of tuberculosis (TB). The automated procedure is based on the injection of sputum samples which are then methylated inside the GC injector using thermally assisted hydrolysis and methylation (THM). The THM–GC–MS procedure was optimized for the injection of sputum samples. For the identification of Mycobacterium tuberculosis the known marker tuberculostearic acid (TBSA) and other potential markers were evaluated. Hexacosanoic acid in combination with TBSA was found to be specific for the presence of M. tuberculosis. For validation of the method several sputum samples with different viscosities spiked with bacterial cultures were analyzed. Finally, 18 stored sputum samples collected in Vietnam from patients suspected to suffer from TB were re-analyzed in Amsterdam by microscopy after decontamination/concentration and using the new THM–GC–MS method. No false positives were found by THM–GC–MS and all patients who were diagnosed with TB were also found positive using our newly developed THM–GC–MS method. These results show that the new fast and sensitive THM–GC–MS method holds great potential for the diagnosis of TB.     

Tape measure protein having MT3 motif facilitates phage entry into stationary phase cells of Mycobacterium tuberculosis

Tape measure protein (TMP) having MT3 motif in mycobacteriophage TM4 genome has been reported to enable the phage infection of Mycobacterium smegmatis during stationary phase. In the present work looking at eight additional mycobacteriophage genomes by in silico analysis, six of them have been found to possess MT3 motif in TMP. The absence of MT3 motif in Che12 and D29 probably makes them incapable of infecting stationary phase cells of Mycobacterium tuberculosis which was experimentally evaluated by the performance of respective luciferase reporter phage constructs developed from the parental phages Che12, D29 and TM4.     

Comprehensive synthesis of ER related high-mannose-type sugar chains by convergent strategy

Systematic synthesis of high-mannose-type sugar chains of asparagine-linked glycoproteins is described. To construct the target sugar chains, we employed the convergent route, using three oligosaccharide components, the common hexasaccharide, branched tri-, tetra- and pentasaccharides, and mono-, di-, and triglucosyl fragments. Construction of the β-mannoside linkage was performed using p-methoxybenzyl-assisted intramolecular aglycon delivery. The hexasaccharide fragment was coupled with the branched mannooligosaccharide donors such as M5, M4B, M4C, and M3 to give undecasaccharide (M9), decasaccharide (M8B and M8C), and nonasaccharide (M7), respectively. Incorporation of mono-, di-, and triglucosyl fragments toward them gave tetradecasaccharide (G3M9), tridecasaccharide (G2M9), dodecasaccharide (G1M9), undecasaccharide (G1M8B and G1M8C), and decasaccharide (G1M7), respectively.     

Farnesol, a potential efflux pump inhibitor in Mycobacterium smegmatis

The active multidrug efflux pump (EP) has been described as one of the mechanisms involved in the natural drug resistance of bacteria, such as mycobacteria. As a result, the development of efflux pumps inhibitors (EPIs) is an important topic. In this study, a checkerboard synergy assay indicated that farnesol both decreased the minimum inhibitory concentration (MIC) of ethidium bromide (EtBr) 8-fold against Mycobacterium smegmatis (M. smegmatis) mc2155 ATCC 700084 when incorporated at a concentration of 32 μg/mL (FICI = 0.625) and decreased MIC 4-fold at 16 μg/mL (FICI = 0.375). Farnesol also showed synergism when combined with rifampicin. A real-time 96-well plate fluorometric method was used to assess the ability of farnesol to inhibit EPs in comparison with four positive EPIs: chlorpromazine, reserpine, verapamil, and carbonyl cyanide m-chlorophenylhydrazone (CCCP). Farnesol significantly enhanced the accumulation of EtBr and decreased the efflux of EtBr in M. smegmatis; these results suggest that farnesol acts as an inhibitor of mycobacterial efflux pumps.