LdtC Is a Key l,d-Transpeptidase for Peptidoglycan Assembly in Mycobacterium smegmatis.

The peptidoglycan of mycobacteria has two types of direct cross-links, classical 4-3 cross-links that occur between diaminopimelate (DAP) and alanine residues, and nonclassical 3-3 cross-links that occur between DAP residues on adjacent peptides. The 3-3 cross-links are synthesized by the concerted action of d,d-carboxypeptidases and l,d-transpeptidases (Ldts). Mycobacterial genomes encode several Ldt proteins that can be classified into six classes based upon sequence identity. As a group, the Ldt enzymes are resistant to most ß-lactam antibiotics but are susceptible to carbapenem antibiotics, with the exception of LdtC, a class 5 enzyme. In previous work, we showed that loss of LdtC has the greatest effect on the carbapenem susceptibility phenotype of Mycobacterium smegmatis (also known as Mycolicibacterium smegmatis) compared to other ldt deletion mutants. In this work, we show that a M. smegmatis mutant lacking the five ldt genes other than ldtC has a wild-type phenotype with the exception of increased susceptibility to rifampin. In contrast, a mutant lacking all six ldt genes has pleiotropic cell envelope defects, is temperature sensitive, and has increased susceptibility to a variety of antibiotics. These results indicate that LdtC is capable of functioning as the sole l,d-transpeptidase in M. smegmatis and suggest that it may represent a carbapenem-resistant pathway for peptidoglycan biosynthesis. IMPORTANCE Mycobacteria have several enzymes to catalyze nonclassical 3-3 linkages in the cell wall peptidoglycan. Understanding the biology of these cross-links is important for the development of antibiotic therapies to target peptidoglycan biosynthesis. Our work provides evidence that LdtC can function as the sole enzyme for 3-3 cross-link formation in M. smegmatis and suggests that LdtC may be part of a carbapenem-resistant l,d-transpeptidase pathway.

Interactions of Truncated Menaquinones in Lipid Monolayers and Bilayers.

Menaquinones (MK) are hydrophobic molecules that consist of a naphthoquinone headgroup and a repeating isoprenyl side chain and are cofactors used in bacterial electron transport systems to generate cellular energy. We have previously demonstrated that the folded conformation of truncated MK homologues, MK-1 and MK-2, in both solution and reverse micelle microemulsions depended on environment. There is little information on how MKs associate with phospholipids in a model membrane system and how MKs affect phospholipid organization. In this manuscript, we used a combination of Langmuir monolayer studies and molecular dynamics (MD) simulations to probe these questions on truncated MK homologues, MK-1 through MK-4 within a model membrane. We observed that truncated MKs reside farther away from the interfacial water than ubiquinones are are located closer to the phospholipid tails. We also observed that phospholipid packing does not change at physiological pressure in the presence of truncated MKs, though a difference in phospholipid packing has been observed in the presence of ubiquinones. We found through MD simulations that for truncated MKs, the folded conformation varied, but MKs location and association with the bilayer remained unchanged at physiological conditions regardless of side chain length. Combined, this manuscript provides fundamental information, both experimental and computational, on the location, association, and conformation of truncated MK homologues in model membrane environments relevant to bacterial energy production.

Stearoly-CoA desaturase 1 differentiates early and advanced dengue virus infections and determines virus particle infectivity.

Positive strand RNA viruses, such as dengue virus type 2 (DENV2) expand and structurally alter ER membranes to optimize cellular communication pathways that promote viral replicative needs. These complex rearrangements require significant protein scaffolding as well as changes to the ER chemical composition to support these structures. We have previously shown that the lipid abundance and repertoire of host cells are significantly altered during infection with these viruses. Specifically, enzymes in the lipid biosynthesis pathway such as fatty acid synthase (FAS) are recruited to viral replication sites by interaction with viral proteins and displayed enhanced activities during infection. We have now identified that events downstream of FAS (fatty acid desaturation) are critical for virus replication. In this study we screened enzymes in the unsaturated fatty acid (UFA) biosynthetic pathway and found that the rate-limiting enzyme in monounsaturated fatty acid biosynthesis, stearoyl-CoA desaturase 1 (SCD1), is indispensable for DENV2 replication. The enzymatic activity of SCD1, was required for viral genome replication and particle release, and it was regulated in a time-dependent manner with a stringent requirement early during viral infection. As infection progressed, SCD1 protein expression levels were inversely correlated with the concentration of viral dsRNA in the cell. This modulation of SCD1, coinciding with the stage of viral replication, highlighted its function as a trigger of early infection and an enzyme that controlled alternate lipid requirements during early versus advanced infections. Loss of function of this enzyme disrupted structural alterations of assembled viral particles rendering them non-infectious and immature and defective in viral entry. This study identifies the complex involvement of SCD1 in DENV2 infection and demonstrates that these viruses alter ER lipid composition to increase infectivity of the virus particles.

Investigating Substrate Analogues for Mycobacterial MenJ: Truncated and Partially Saturated Menaquinones.

Menaquinones (MKs) are essential for electron transport in prokaryotes, and importantly, partially saturated MKs represent a novel virulence factor. However, little is known regarding how the degree of saturation in the isoprenyl side chain influences conformation or quinone redox potential. MenJ is an enzyme that selectively reduces the second isoprene unit on MK-9 and is contextually essential for the survival of Mycobacterium tuberculosis in J774A.1 macrophage-like cells, suggesting that MenJ may be a conditional drug target for pathogenic mycobacteria. Therefore, fundamental information about the properties of this system is important, and we synthesized the simplest MKs, unsaturated MK-1 and the saturated analogue, MK-1(H2). Using two-dimensional nuclear magnetic resonance spectroscopy, we established that MK-1 and MK-1(H2) adopted similar folded-extended conformations (i.e., the isoprenyl side chain folds upward) in each solvent examined but the folded-extended conformations differed slightly between organic solvents. Saturation of the isoprenyl side chain slightly altered the MK-1 analogue conformation in each solvent. We used molecular mechanics to illustrate the MK-1 analogue conformations. The measured quinone redox potentials of MK-1 and MK-1(H2) differed between organic solvents (presumably due to differences in dielectric constants), and remarkably, an ∼20 mV semiquinone redox potential difference was observed between MK-1 and MK-1(H2) in pyridine, acetonitrile, and dimethyl sulfoxide, demonstrating that the degree of saturation in the isoprenyl side chain of MK-1 influences the quinone redox potential. Finally, MK-1 and MK-1(H2) interacted with Langmuir phospholipid monolayers and Aerosol-OT reverse micelle (RM) model membrane interfaces, where MK-1 adopted a slightly different folded conformation within the RM model membrane interface.

Structure Dependence of Pyridine and Benzene Derivatives on Interactions with Model Membranes.

Pyridine-based small-molecule drugs, vitamins, and cofactors are vital for many cellular processes, but little is known about their interactions with membrane interfaces. These specific membrane interactions of these small molecules or ions can assist in diffusion across membranes or reach a membrane-bound target. This study explores how minor differences in small molecules (isoniazid, benzhydrazide, isonicotinamide, nicotinamide, picolinamide, and benzamide) can affect their interactions with model membranes. Langmuir monolayer studies of dipalmitoylphosphatidylcholine (DPPC) or dipalmitoylphosphatidylethanolamine (DPPE), in the presence of the molecules listed, show that isoniazid and isonicotinamide affect the DPPE monolayer at lower concentrations than the DPPC monolayer, demonstrating a preference for one phospholipid over the other. The Langmuir monolayer studies also suggest that nitrogen content and stereochemistry of the small molecule can affect the phospholipid monolayers differently. To determine the molecular interactions of the simple N-containing aromatic pyridines with a membrane-like interface, 1H one-dimensional NMR and 1H-1H two-dimensional NMR techniques were utilized to obtain information about the position and orientation of the molecules of interest within aerosol-OT (AOT) reverse micelles. These studies show that all six of the molecules reside near the AOT sulfonate headgroups and ester linkages in similar positions, but nicotinamide and picolinamide tilt at the water-AOT interface to varying degrees. Combined, these studies demonstrate that small structural changes of small N-containing molecules can affect their specific interactions with membrane-like interfaces and specificity toward different membrane components.

A Synthetic Isoprenoid Lipoquinone, Menaquinone-2, Adopts a Folded Conformation in Solution and at a Model Membrane Interface.

Menaquinones (naphthoquinones, MK) are isoprenoids that play key roles in the respiratory electron transport system of some prokaryotes by shuttling electrons between membrane-bound protein complexes acting as electron acceptors and donors. Menaquinone-2 (MK-2), a truncated MK, was synthesized, and the studies presented herein characterize the conformational and chemical properties of the hydrophobic MK-2 molecule. Using 2D NMR spectroscopy, we established for the first time that MK-2 has a folded conformation defined by the isoprenyl side-chain folding back over the napthoquinone in a U-shape, which depends on the specific environmental conditions found in different solvents. We used molecular mechanics to illustrate conformations found by the NMR experiments. The measured redox potentials of MK-2 differed in three organic solvents, where MK-2 was most easily reduced in DMSO, which may suggest a combination of solvent effect (presumably in part because of differences in dielectric constants) and/or conformational differences of MK-2 in different organic solvents. Furthermore, MK-2 was found to associate with the interface of model membranes represented by Langmuir phospholipid monolayers and Aerosol-OT (AOT) reverse micelles. MK-2 adopts a slightly different U-shaped conformation within reverse micelles compared to within solution, which is in sharp contrast to the extended conformations illustrated in literature for MKs.

Antiinfectives targeting enzymes and the proton motive force.

There is a growing need for new antibiotics. Compounds that target the proton motive force (PMF), uncouplers, represent one possible class of compounds that might be developed because they are already used to treat parasitic infections, and there is interest in their use for the treatment of other diseases, such as diabetes. Here, we tested a series of compounds, most with known antiinfective activity, for uncoupler activity. Many cationic amphiphiles tested positive, and some targeted isoprenoid biosynthesis or affected lipid bilayer structure. As an example, we found that clomiphene, a recently discovered undecaprenyl diphosphate synthase inhibitor active against Staphylococcus aureus, is an uncoupler. Using in silico screening, we then found that the anti-glioblastoma multiforme drug lead vacquinol is an inhibitor of Mycobacterium tuberculosis tuberculosinyl adenosine synthase, as well as being an uncoupler. Because vacquinol is also an inhibitor of M. tuberculosis cell growth, we used similarity searches based on the vacquinol structure, finding analogs with potent (∼0.5-2 µg/mL) activity against M. tuberculosis and S. aureus. Our results give a logical explanation of the observation that most new tuberculosis drug leads discovered by phenotypic screens and genome sequencing are highly lipophilic (logP ∼5.7) bases with membrane targets because such species are expected to partition into hydrophobic membranes, inhibiting membrane proteins, in addition to collapsing the PMF. This multiple targeting is expected to be of importance in overcoming the development of drug resistance because targeting membrane physical properties is expected to be less susceptible to the development of resistance.

Differences in Interactions of Benzoic Acid and Benzoate with Interfaces.

The interaction of benzoic acid and benzoate with model membrane systems was characterized to understand the molecular interactions of the two forms of a simple aromatic acid with the components of the membrane. The microemulsion system based on bis(2-ethylhexyl)sulfosuccinate (AOT) allowed determination of the molecular positioning using 1D NMR and 2D NMR spectroscopic methods. Benzoic acid and benzoate were both found to penetrate the membrane/water interfaces; however, the benzoic acid was able to penetrate much deeper and thus is more readily able to traverse a membrane. The Langmuir monolayer model system, using dipalmitoylphosphatidylcholine, was used as a generic membrane lipid for a cell. Compression isotherms of monolayers demonstrated a pH dependent interaction with a lipid monolayer and confirming the pH dependent observations shown in the reverse micellar model system. These studies provide an explanation for the antimicrobial activity of benzoic acid while benzoate is inactive. Furthermore, these studies form the framework upon which we are investigating the mode of bacterial uptake of pyrazinoic acid, the active form of pyrazinamide, a front line drug used to combat tuberculosis.

The Essential Role of Cholesterol Metabolism in the Intracellular Survival of Mycobacterium leprae Is Not Coupled to Central Carbon Metabolism and Energy Production.

UNLABELLED: Mycobacterium leprae induces the formation of lipid droplets, which are recruited to pathogen-containing phagosomes in infected macrophages and Schwann cells. Cholesterol is among the lipids with increased abundance in M. leprae-infected cells, and intracellular survival relies on cholesterol accumulation. The present study investigated the capacity of M. leprae to acquire and metabolize cholesterol. In silico analyses showed that oxidation of cholesterol to cholest-4-en-3-one (cholestenone), the first step of cholesterol degradation catalyzed by the enzyme 3ß-hydroxysteroid dehydrogenase (3ß-HSD), is apparently the only portion of the cholesterol catabolic pathway seen in Mycobacterium tuberculosis preserved by M. leprae. Incubation of bacteria with radiolabeled cholesterol confirmed the in silico predictions. Radiorespirometry and lipid analyses performed after incubating M. leprae with [4-(14)C]cholesterol or [26-(14)C]cholesterol showed the inability of this pathogen to metabolize the sterol rings or the side chain of cholesterol as a source of energy and carbon. However, the bacteria avidly incorporated cholesterol and, as expected, converted it to cholestenone both in vitro and in vivo. Our data indicate that M. leprae has lost the capacity to degrade and utilize cholesterol as a nutritional source but retains the enzyme responsible for its oxidation to cholestenone. Thus, the essential role of cholesterol metabolism in the intracellular survival of M. leprae is uncoupled from central carbon metabolism and energy production. Further elucidation of cholesterol metabolism in the host cell during M. leprae infection will establish the mechanism by which this lipid supports M. leprae intracellular survival and will open new avenues for novel leprosy therapies. IMPORTANCE: Our study focused on the obligate intracellular pathogen Mycobacterium leprae and its capacity to metabolize cholesterol. The data make an important contribution for those interested in understanding the mechanisms of mycobacterial pathogenesis, since they indicate that the essential role of cholesterol for M. leprae intracellular survival does not rely on its utilization as a nutritional source. Our findings reinforce the complexity of cholesterol's role in sustaining M. leprae infection. Further elucidation of cholesterol metabolism in the host cell during M. leprae infection will establish the mechanism by which this lipid supports M. leprae intracellular survival and will open new avenues for novel leprosy therapies.

Inhibition of the first step in synthesis of the mycobacterial cell wall core, catalyzed by the GlcNAc-1-phosphate transferase WecA, by the novel caprazamycin derivative CPZEN-45.

Because tuberculosis is one of the most prevalent and serious infections, countermeasures against it are urgently required. We isolated the antitubercular agents caprazamycins from the culture of an actinomycete strain and created CPZEN-45 as the most promising derivative of the caprazamycins. Herein, we describe the mode of action of CPZEN-45 first against Bacillus subtilis. Unlike the caprazamycins, CPZEN-45 strongly inhibited incorporation of radiolabeled glycerol into growing cultures and showed antibacterial activity against caprazamycin-resistant strains, including a strain overexpressing translocase-I (MraY, involved in the biosynthesis of peptidoglycan), the target of the caprazamycins. By contrast, CPZEN-45 was not effective against a strain overexpressing undecaprenyl-phosphate-GlcNAc-1-phosphate transferase (TagO, involved in the biosynthesis of teichoic acid), and a mutation was found in the tagO gene of the spontaneous CPZEN-45-resistant strain. This suggested that the primary target of CPZEN-45 in B. subtilis is TagO, which is a different target from that of the parent caprazamycins. This suggestion was confirmed by evaluation of the activities of these enzymes. Finally, we showed that CPZEN-45 was effective against WecA (Rv1302, also called Rfe) of Mycobacterium tuberculosis, the ortholog of TagO and involved in the biosynthesis of the mycolylarabinogalactan of the cell wall of M. tuberculosis. The outlook for WecA as a promising target for the development of antituberculous drugs as a countermeasure of drug resistant tuberculosis is discussed.

Structure and inhibition of tuberculosinol synthase and decaprenyl diphosphate synthase from Mycobacterium tuberculosis.

We have obtained the structure of the bacterial diterpene synthase, tuberculosinol/iso-tuberculosinol synthase (Rv3378c) from Mycobacterium tuberculosis , a target for anti-infective therapies that block virulence factor formation. This phosphatase adopts the same fold as found in the Z- or cis-prenyltransferases. We also obtained structures containing the tuberculosinyl diphosphate substrate together with one bisphosphonate inhibitor-bound structure. These structures together with the results of site-directed mutagenesis suggest an unusual mechanism of action involving two Tyr residues. Given the similarity in local and global structure between Rv3378c and the M. tuberculosis cis-decaprenyl diphosphate synthase (DPPS; Rv2361c), the possibility exists for the development of inhibitors that target not only virulence but also cell wall biosynthesis, based in part on the structures reported here.

Novel insights into the mechanism of inhibition of MmpL3, a target of multiple pharmacophores in Mycobacterium tuberculosis.

MmpL3, a resistance-nodulation-division (RND) superfamily transporter, has been implicated in the formation of the outer membrane of Mycobacterium tuberculosis; specifically, MmpL3 is required for the export of mycolic acids in the form of trehalose monomycolates (TMM) to the periplasmic space or outer membrane of M. tuberculosis. Recently, seven series of inhibitors identified by whole-cell screening against M. tuberculosis, including the antituberculosis drug candidate SQ109, were shown to abolish MmpL3-mediated TMM export. However, this mode of action was brought into question by the broad-spectrum activities of some of these inhibitors against a variety of bacterial and fungal pathogens that do not synthesize mycolic acids. This observation, coupled with the ability of three of these classes of inhibitors to kill nonreplicating M. tuberculosis bacilli, led us to investigate alternative mechanisms of action. Our results indicate that the inhibitory effects of adamantyl ureas, indolecarboxamides, tetrahydropyrazolopyrimidines, and the 1,5-diarylpyrrole BM212 on the transport activity of MmpL3 in actively replicating M. tuberculosis bacilli are, like that of SQ109, most likely due to their ability to dissipate the transmembrane electrochemical proton gradient. In addition to providing novel insights into the modes of action of compounds reported to inhibit MmpL3, our results provide the first explanation for the large number of pharmacophores that apparently target this essential inner membrane transporter.

Pyrazinoic acid, the active form of the anti-tuberculosis drug pyrazinamide, and aromatic carboxylic acid analogs are protonophores.

Pyrazinoic acid is the active form of pyrazinamide, a first-line antibiotic used to treat Mycobacterium tuberculosis infections. However, the mechanism of action of pyrazinoic acid remains a subject of debate, and alternatives to pyrazinamide in cases of resistance are not available. The work presented here demonstrates that pyrazinoic acid and known protonophores including salicylic acid, benzoic acid, and carbonyl cyanide m-chlorophenyl hydrazone all exhibit pH-dependent inhibition of mycobacterial growth activity over a physiologically relevant range of pH values. Other anti-tubercular drugs, including rifampin, isoniazid, bedaquiline, and p-aminosalicylic acid, do not exhibit similar pH-dependent growth-inhibitory activities. The growth inhibition curves of pyrazinoic, salicylic, benzoic, and picolinic acids, as well as carbonyl cyanide m-chlorophenyl hydrazone, all fit a quantitative structure-activity relationship (QSAR) derived from acid-base equilibria with R2 values > 0.95. The QSAR model indicates that growth inhibition relies solely on the concentration of the protonated forms of these weak acids (rather than the deprotonated forms). Moreover, pyrazinoic acid, salicylic acid, and carbonyl cyanide m-chlorophenyl hydrazone all caused acidification of the mycobacterial cytoplasm at concentrations that inhibit bacterial growth. Thus, it is concluded that pyrazinoic acid acts as an uncoupler of oxidative phosphorylation and that disruption of proton motive force is the primary mechanism of action of pyrazinoic acid rather than the inhibition of a classic enzyme activity.

Mycobacteriophage Ms6 LysA: a peptidoglycan amidase and a useful analytical tool.

Since the peptidoglycan isolated from Mycobacterium spp. is refractory to commercially available murolytic enzymes, possibly due to the presence of various modifications found on this peptidoglycan, the utility of a mycobacteriophage-derived murolytic enzyme was assessed for an analysis of peptidoglycan from mycobacteria. We cloned, expressed, and purified the lysA gene product, a protein with homology to known peptidoglycan-degrading amidases, from bacteriophage Ms6. The recombinant protein was shown to cleave the bond between l-Ala and d-muramic acid of muramyl pentapeptide and to release up to 70% of the diaminopimelic acid present in the isolated mycobacterial cell wall. In contrast to lysozyme, which, in culture, inhibits the growth of both Mycobacterium smegmatis and Mycobacterium tuberculosis, LysA had no effect on the growth of either species. However, the enzyme is useful for solubilizing the peptide chains of isolated mycobacterial peptidoglycan for analysis. The data indicate that the stem peptides from M. smegmatis are heavily amidated, containing few free carboxylic acids, regardless of the cross-linking status.

SAR study of piperidine derivatives as inhibitors of 1,4-dihydroxy-2-naphthoate isoprenyltransferase (MenA) from Mycobacterium tuberculosis.

The electron transport chain (ETC) in the cell membrane consists of a series of redox complexes that transfer electrons from electron donors to acceptors and couples this electron transfer with the transfer of protons (H+) across a membrane. This process generates proton motive force which is used to produce ATP and a myriad of other functions and is essential for the long-term survival of Mycobacterium tuberculosis (Mtb), the causative organism of tuberculosis (TB), under the hypoxic conditions present within infected granulomas. Menaquinone (MK), an important carrier molecule within the mycobacterial ETC, is synthesized de novo by a cluster of enzymes known as the classic/canonical MK biosynthetic pathway. MenA (1,4-dihydroxy-2-naphthoate prenyltransferase), the antepenultimate enzyme in this pathway, is a verified target for TB therapy. In this study, we explored structure-activity relationships of a previously discovered MenA inhibitor scaffold, seeking to improve potency and drug disposition properties. Focusing our campaign upon three molecular regions, we identified two novel inhibitors with potent activity against MenA and Mtb (IC50 = 13-22 µM, GIC50 = 8-10 µM). These analogs also displayed substantially improved pharmacokinetic parameters and potent synergy with other ETC-targeting agents, achieving nearly complete sterilization of Mtb in combination therapy within two weeks in vivo. These new inhibitors of MK biosynthesis present a promising new strategy to curb the continued spread of TB.

Proline Dehydrogenase and Pyrroline 5 Carboxylate Dehydrogenase from Mycobacterium tuberculosis: Evidence for Substrate Channeling.

In Mycobacterium tuberculosis, proline dehydrogenase (PruB) and ∆1-pyrroline-5-carboxylate (P5C) dehydrogenase (PruA) are monofunctional enzymes that catalyze proline oxidation to glutamate via the intermediates P5C and L-glutamate-γ-semialdehyde. Both enzymes are essential for the replication of pathogenic M. tuberculosis. Highly active enzymes were expressed and purified using a Mycobacterium smegmatis expression system. The purified enzymes were characterized using natural substrates and chemically synthesized analogs. The structural requirements of the quinone electron acceptor were examined. PruB displayed activity with all tested lipoquinone analogs (naphthoquinone or benzoquinone). In PruB assays utilizing analogs of the native naphthoquinone [MK-9 (II-H2)] specificity constants Kcat/Km were an order of magnitude greater for the menaquinone analogs than the benzoquinone analogs. In addition, mycobacterial PruA was enzymatically characterized for the first time using exogenous chemically synthesized P5C. A Km value of 120 ± 0.015 µM was determined for P5C, while the Km value for NAD+ was shown to be 33 ± 4.3 µM. Furthermore, proline competitively inhibited PruA activity and coupled enzyme assays, suggesting that the recombinant purified monofunctional PruB and PruA enzymes of M. tuberculosis channel substrate likely increase metabolic flux and protect the bacterium from methylglyoxal toxicity.

Detailed structural and quantitative analysis reveals the spatial organization of the cell walls of in vivo grown Mycobacterium leprae and in vitro grown Mycobacterium tuberculosis.

The cell wall of mycobacteria consists of an outer membrane, analogous to that of gram-negative bacteria, attached to the peptidoglycan (PG) via a connecting polysaccharide arabinogalactan (AG). Although the primary structure of these components is fairly well deciphered, issues such as the coverage of the PG layer by covalently attached mycolates in the outer membrane and the spatial details of the mycolic acid attachment to the arabinan have remained unknown. It is also not understood how these components work together to lead to the classical acid-fast staining of mycobacteria. Because the majority of Mycobacterium tuberculosis bacteria in established experimental animal infections are acid-fast negative, clearly cell wall changes are occurring. To address both the spatial properties of mycobacterial cell walls and to begin to study the differences between bacteria grown in animals and cultures, the cell walls of Mycobacterium leprae grown in armadillos was characterized and compared with that of M. tuberculosis grown in culture. Most fundamentally, it was determined that the cell wall of M. leprae contained significantly more mycolic acids attached to PG than that of in vitro grown M. tuberculosis (mycolate:PG ratios of 21:10 versus 16:10, respectively). In keeping with this difference, more arabinogalactan (AG) molecules, linking the mycolic acids to PG, were found. Differences in the structures of the AG were also found; the AG of M. leprae is smaller than that of M. tuberculosis, although the same basic structural motifs are retained.

A bioanalytical method to determine the cell wall composition of Mycobacterium tuberculosis grown in vivo.

Mycobacterium tuberculosis bacilli exhibit cell wall alterations during in vivo growth. Development of ultrasensitive analytical techniques with high specificities is required to analyze the cell wall of M. tuberculosis isolated from experimental animals because of the low amounts of bacteria available and contamination by host tissue. Here we present a novel methodology to analyze all three major components (mycolic acids, arabinogalactan, and peptidoglycan) of the mycobacterial cell wall from mycobacteria isolated from animal tissue. In this procedure, the cell wall carbohydrates are analyzed by gas chromatography tandem mass spectrometry (GC/MS/MS) of alditol acetates, the peptidoglycan by GC/MS (mass spectrometry) analysis of the unique amino acid diaminopimelic acid (after derivatization with isopropyl chloroformate), and the mycolic acids by liquid chromatography (LC)/MS (negative ion) without derivatization. The procedure was designed so that all three analyses could be performed starting with a single sample given the difficulty of preparing multiple aliquots in known ratios. Linkage analysis, including an enantiomeric specific procedure, of the arabinogalactan polymer is also presented. These procedures will enable the determination of the cell wall alterations known to occur in the important nongrowing "dormant" M. tuberculosis present during disease. With some adaptations, the methodology is also applicable to the analysis of small amounts of in vivo grown bacteria of other species.

Mycobacterial MenG: Partial Purification, Characterization, and Inhibition.

Menaquinone (MK) is an essential component of the electron transport chain (ETC) in the gram-variable Mycobacterium tuberculosis and many Gram-positive pathogens. Three genes in the M. tuberculosis genome were annotated as methyltransferases involved in lipoquinone synthesis in mycobacteria. Heterologous expression of Rv0558 complemented an ubiE (the quinone C-methyltransferase involved in ubiquinone and menaquinone synthesis) deletion in Escherichia coli, and expression in a wild-type E. coli strain increased quinone C-methyltransferase specific activity by threefold. Rv0558 encodes a canonical C-methyltransferase or, more specifically, a S-adenosylmethionine/demethylmenaquinol methyltransferase. Partially purified recombinant protein catalyzed the formation of MK from demethylmenaquinone (DMK), although the activity of the recombinant protein was low and appeared to require a cofactor or intact membrane structure for activity. Membrane preparations from irradiated M. tuberculosis also showed poor activity; however, membrane preparations from wild-type Mycobacterium smegmatis showed robust, substrate-dependent activity. The apparent Km values for demethylmenaquinone and SAM were 14 ± 5.0 and 17 ± 7.0 µM, respectively. Interestingly, addition of dithiothreitol, dithionite, NADH, or other substrates of primary dehydrogenases to reaction mixtures containing membrane preparations stimulated the activity. Thus, these observations strongly suggest that demethylmenaquinol is the actual substrate of MenG. Ro 48-8071, previously reported to inhibit mycobacterial MK synthesis and growth, inhibited Rv0558 activity with an IC50 value of 5.1 ± 0.5 µM, and DG70 (GSK1733953A), first described as a respiration inhibitor in M. tuberculosis, inhibits MenG activity with an IC50 value of 2.6 ± 0.6 µM.

Do bioactive 8-hydroxyquinolines oxidovanadium(IV) and (V) complexes inhibit the growth of M. smegmatis?

The antiproliferative effects of four series of VIVO- and VVO-based compounds containing 8-hydroxyquinoline ligands on the bacterium Mycolicibacterium smegmatis (M. smeg) were investigated. The effects on M. smeg were compared to the antiproliferative effects on the protozoan parasite Trypanosoma cruzi (T. cruzi), the causative agent for Chagas disease. In this study, we investigate the speciation of these compounds under physiological conditions as well as the antiproliferative effects on the bacterium M. smeg. We find that the complexes are more stable the less H2O is present, and that the stability increases in lipid-like environments. Only one heteroleptic complex and two homoleptic complexes were found to show similar antiproliferative effects on M. smeg as reported for T. cruzi so the responses generally observed by M.smeg. is less than observed by the pathogen. In summary, we find that M. smeg is more sensitive to the detailed structure of the V-complex but overall these complexes are less effective against M. smeg compared to T. cruzi.