TetR-like regulator BP1026B_II1561 controls aromatic amino acid biosynthesis and intracellular pathogenesis in .

() causes the tropical disease melioidosis that afflicts an estimated 165,000 people each year. is a facultative intracellular pathogen that transits through distinct intracellular stages including attachment to host cells, invasion through the endocytic pathway, escape from the endosome, replication in the cytoplasm, generation of protrusions towards neighboring cells, and host cell fusion allowing infection to spread without exiting the intracellular environment. We have identified a TetR-like transcriptional regulator, BP1026B_II1561, that is up-regulated during the late stages of infection as protrudes toward neighboring cells.

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 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 -chlorophenyl hydrazone all exhibit pH-dependent inhibition of mycobacterial growth activity over a physiologically relevant range of pH values.

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

In , proline dehydrogenase (PruB) and ∆-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 . Highly active enzymes were expressed and purified using a expression system.

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.

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.

Mycobacterial MenG: Partial Purification, Characterization, and Inhibition.

Menaquinone (MK) is an essential component of the electron transport chain (ETC) in the gram-variable and many Gram-positive pathogens. Three genes in the genome were annotated as methyltransferases involved in lipoquinone synthesis in mycobacteria. Heterologous expression of complemented an (the quinone -methyltransferase involved in ubiquinone and menaquinone synthesis) deletion in and expression in a wild-type strain increased quinone -methyltransferase specific activity by threefold.

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

The antiproliferative effects of four series of VO- and VO-based compounds containing 8-hydroxyquinoline ligands on the bacterium Mycolicibacterium smegmatis (M. smeg) were investigated. The effects on M.

Synthetic Sansanmycin Analogues as Potent Translocase I Inhibitors.

Herein, we report the design and synthesis of inhibitors of () phospho-MurNAc-pentapeptide translocase I (MurX), the first membrane-associated step of peptidoglycan synthesis, leveraging the privileged structure of the sansanmycin family of uridylpeptide natural products. A number of analogues bearing hydrophobic amide modifications to the pseudo-peptidic end of the natural product scaffold were generated that exhibited nanomolar inhibitory activity against MurX and potent activity against . We show that a lead analogue bearing an appended neopentylamide moiety possesses rapid antimycobacterial effects with a profile similar to the frontline tuberculosis drug isoniazid.

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.

Pt- or Mo-substituted decavanadates inhibit the growth of Mycobacterium smegmatis.

Inhibitory effects of two monosubstituted decavanadates by Pt in monoplatino(IV)nonavanadate(V) ([HPtVO], VPt), and by Mo in monomolybdo(VI)nonavanadate(V) ([MoVO],VMo) were investigated against the growth of Mycobacterium smegmatis with the EC values of 0.0048 mM and 0.015 mM, respectively.

The Acid-Base Equilibrium of Pyrazinoic Acid Drives the pH Dependence of Pyrazinamide-Induced Growth Inhibition.

Pyrazinamide, a first-line antibiotic used against , has been shown to act in a pH-dependent manner . Why pyrazinamide, an antitubercle prodrug discovered more than 65 years ago, exhibits this pH-dependent activity was unclear. Upon entering mycobacterial cells, pyrazinamide is deamidated to pyrazinoate by an enzymatic process and exists in an acid-base equilibrium with pyrazinoic acid.

Survival in J774A.1 Cells Is Dependent on MenJ Moonlighting Activity, Not Its Enzymatic Activity.

MenJ, a flavoprotein oxidoreductase, is responsible for the saturation of the β-isoprene unit of mycobacterial menaquinone, resulting in the conversion of menaquinone with nine isoprene units (MK-9) to menaquinone with nine isoprene units where the double bond in the second unit is reduced [MK-9(II-H)]. The hydrogenation of MK-9 increases the efficiency of the mycobacterial electron transport system, whereas the deletion of MenJ results in decreased survival of the bacteria inside J774A.1 macrophage-like cells but is not required for growth in culture.

Synthesis and Characterization of Partially and Fully Saturated Menaquinone Derivatives.

Menaquinones (MKs) contain both a redox active quinone moiety and a hydrophobic repeating isoprenyl side chain of varying lengths and degrees of saturation. This characteristic structure allows MKs to play a key role in the respiratory electron transport system of some prokaryotes by shuttling electrons and protons between membrane-bound protein complexes, which act as electron acceptors and donors. Hydrophobic MK molecules with partially and fully saturated isoprenyl side chains are found in a wide range of eubacteria and archaea, and the structural variations of the MK analogues are evolutionarily conserved but poorly understood.

Characterization of MenA (isoprenyl diphosphate:1,4-dihydroxy-2-naphthoate isoprenyltransferase) from Mycobacterium tuberculosis.

The menaquinone biosynthetic pathway presents a promising drug target against Mycobacterium tuberculosis and potentially other Gram-positive pathogens. In the present study, the essentiality, steady state kinetics of MenA from M. tuberculosis and the mechanism of MenA inhibition by Ro 48-8071 were characterized.

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.

Decavanadate Inhibits Mycobacterial Growth More Potently Than Other Oxovanadates.

V NMR spectroscopy is used to document, using speciation analysis, that one oxometalate is a more potent growth inhibitor of two Mycobacterial strains than other oxovanadates, thus demonstrating selectivity in its interaction with cells. Historically, oxometalates have had many applications in biological and medical studies, including study of the phase-problem in X-ray crystallography of the ribosome. The effect of different vanadate salts on the growth of () and () was investigated, and speciation was found to be critical for the observed growth inhibition.

Mechanism of Fluorinated Anthranilate-Induced Growth Inhibition in Mycobacterium tuberculosis.

The biosynthesis of tryptophan in Mycobacterium tuberculosis is initiated by the transformation of chorismate to anthranilate, catalyzed by anthranilate synthase (TrpE/TrpG). Five additional enzymes are required to complete tryptophan biosynthesis. M.

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.

Mycobacterial MenJ: An Oxidoreductase Involved in Menaquinone Biosynthesis.

MenJ, annotated as an oxidoreductase, was recently demonstrated to catalyze the reduction (saturation) of a single double bond in the isoprenyl side-chain of mycobacterial menaquinone. This modification was shown to be essential for bacterial survival in J774A.1 macrophage-like cells, suggesting that MenJ may be a conditional drug target in Mycobacterium tuberculosis and other pathogenic mycobacteria.

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.

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.

Genome-scale analysis of the genes that contribute to Burkholderia pseudomallei biofilm formation identifies a crucial exopolysaccharide biosynthesis gene cluster.

Burkholderia pseudomallei, the causative agent of melioidosis, is an important public health threat due to limited therapeutic options for treatment. Efforts to improve therapeutics for B. pseudomallei infections are dependent on the need to understand the role of B.

Oxidative Phosphorylation as a Target Space for Tuberculosis: Success, Caution, and Future Directions.

The emergence and spread of drug-resistant pathogens, and our inability to develop new antimicrobials to combat resistance, have inspired scientists to seek out new targets for drug development. The complex is a group of obligately aerobic bacteria that have specialized for inhabiting a wide range of intracellular and extracellular environments. Two fundamental features in this adaptation are the flexible utilization of energy sources and continued metabolism in the absence of growth.