Decavanadate Inhibits Mycobacterial Growth More Potently Than Other Oxovanadates

51V 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 Mycobacterium smegmatis (M. smeg) and Mycobacterium tuberculosis (M. tb) was investigated, and speciation was found to be critical for the observed growth inhibition. Specifically, the large orange-colored sodium decavanadate (V10O286-) anion was found to be a stronger inhibitor of growth of two mycobacterial species than the colorless oxovanadate prepared from sodium metavanadate. The vanadium(V) speciation in the growth media and conversion among species under growth conditions was monitored using 51V NMR spectroscopy and speciation calculations. The findings presented in this work is particularly important in considering the many applications of polyoxometalates in biological and medical studies, such as the investigation of the phase-problem in X-ray crystallography for the ribosome. The findings presented in this work investigate the interactions of oxometalates with other biological systems

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

Regulation of Polar Peptidoglycan Biosynthesis by Wag31 Phosphorylation in Mycobacteria

<p>Abstract</p> <p>Background</p> <p>Sensing and responding to environmental changes is a central aspect of cell division regulation. <it>Mycobacterium tuberculosis </it>contains eleven Ser/Thr kinases, two of which, PknA and PknB, are key signaling molecules that regulate cell division/morphology. One substrate of these kinases is Wag31, and we previously showed that partial depletion of Wag31 caused morphological changes indicative of cell wall defects, and that the phosphorylation state of Wag31 affected cell growth in mycobacteria. In the present study, we further characterized the role of the Wag31 phosphorylation in polar peptidoglycan biosynthesis.</p> <p>Results</p> <p>We demonstrate that the differential growth among cells expressing different <it>wag31 </it>alleles (wild-type, phosphoablative, or phosphomimetic) is caused by, at least in part, dissimilar nascent peptidoglycan biosynthesis. The phosphorylation state of Wag31 is found to be important for protein-protein interactions between the Wag31 molecules, and thus, for its polar localization. Consistent with these results, cells expressing a phosphomimetic <it>wag31 </it>allele have a higher enzymatic activity in the peptidoglycan biosynthetic pathway.</p> <p>Conclusions</p> <p>The Wag31_Mtbphosphorylation is a novel molecular mechanism by which Wag31_Mtbregulates peptidoglycan synthesis and thus, optimal growth in mycobacteria.</p

Sansanmycin natural product analogues as potent and selective anti-mycobacterials that inhibit lipid I biosynthesis.

Tuberculosis (TB) is responsible for enormous global morbidity and mortality, and current treatment regimens rely on the use of drugs that have been in use for more than 40 years. Owing to widespread resistance to these therapies, new drugs are desperately needed to control the TB disease burden. Herein, we describe the rapid synthesis of analogues of the sansanmycin uridylpeptide natural products that represent promising new TB drug leads. The compounds exhibit potent and selective inhibition of Mycobacterium tuberculosis, the etiological agent of TB, both in vitro and intracellularly. The natural product analogues were also shown to be nanomolar inhibitors of Mtb phospho-MurNAc-pentapeptide translocase, the enzyme responsible for the synthesis of lipid I in mycobacteria. This work lays the foundation for the development of uridylpeptide natural product analogues as new TB drug candidates that operate through the inhibition of peptidoglycan biosynthesis

Antecedents and consequences of effectuation and causation in the international new venture creation process

The selection of the entry mode in an international market is of key importance for the venture. A process-based perspective on entry mode selection can add to the International Business and International Entrepreneurship literature. Framing the international market entry as an entrepreneurial process, this paper analyzes the antecedents and consequences of causation and effectuation in the entry mode selection. For the analysis, regression-based techniques were used on a sample of 65 gazelles. The results indicate that experienced entrepreneurs tend to apply effectuation rather than causation, while uncertainty does not have a systematic influence. Entrepreneurs using causation-based international new venture creation processes tend to engage in export-type entry modes, while effectuation-based international new venture creation processes do not predetermine the entry mod

Multiple M. tuberculosis Phenotypes in Mouse and Guinea Pig Lung Tissue Revealed by a Dual-Staining Approach

A unique hallmark of tuberculosis is the granulomatous lesions formed in the lung. Granulomas can be heterogeneous in nature and can develop a necrotic, hypoxic core which is surrounded by an acellular, fibrotic rim. Studying bacilli in this in vivo microenvironment is problematic as Mycobacterium tuberculosis can change its phenotype and also become acid-fast negative. Under in vitro models of differing environments, M. tuberculosis alters its metabolism, transcriptional profile and rate of replication. In this study, we investigated whether these phenotypic adaptations of M. tuberculosis are unique for certain environmental conditions and if they could therefore be used as differential markers. Bacilli were studied using fluorescent acid-fast auramine-rhodamine targeting the mycolic acid containing cell wall, and immunofluorescence targeting bacterial proteins using an anti-M. tuberculosis whole cell lysate polyclonal antibody. These techniques were combined and simultaneously applied to M. tuberculosis in vitro culture samples and to lung sections of M. tuberculosis infected mice and guinea pigs. Two phenotypically different subpopulations of M. tuberculosis were found in stationary culture whilst three subpopulations were found in hypoxic culture and in lung sections. Bacilli were either exclusively acid-fast positive, exclusively immunofluorescent positive or acid-fast and immunofluorescent positive. These results suggest that M. tuberculosis exists as multiple populations in most conditions, even within seemingly a single microenvironment. This is relevant information for approaches that study bacillary characteristics in pooled samples (using lipidomics and proteomics) as well as in M. tuberculosis drug development

157 - Jordan Tyler Koehn

Includes bibliographical references.The conformations of long polymers of isoprene units such as those found in the side-chains of lipoquinones involved in bacterial (i.e. Mycobacterium tuberculosis) and eukaryotic electron transport systems (ETS) have not been characterized. First, menaquinone-2 and ubiquinone-2 were synthesized then using 1H-1H 2D NOESY and ROESY NMR spectroscopy, we demonstrated that both molecules adopt a folded, U-shaped conformation in solution and within a model membrane interface. These folded conformations are in stark contrast to the extended conformations seen in literature. Folded conformations likely affect reactivity, function, and recognition within the ETS and provide insight into drug development of potent inhibitors.Great Minds in Research - Honorable Mention

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. For example, Mycobacterium tuberculosis, the causative agent of tuberculosis, uses predominantly MK-9­(II-H2) (saturated at the second isoprene unit) as its electron carrier and depends on the synthesis of MK-9­(II-H2) for survival in host macrophages. Thus, MKs with partially saturated isoprenyl side chains may represent a novel virulence factor. Naturally occurring longer MKs are very hydrophobic, whereas MK analogues that have a truncated (i.e., one to three isoprenes) isoprenyl side chain are less hydrophobic. This improves their solubility in aqueous solutions, allowing rigorous study of their structure and biological activity. We present the synthesis and characterization of two partially saturated MK analogues, MK-2­(II-H2) and MK-3­(II-H2), and two novel fully saturated MK derivatives, MK-2­(I,II-H4) and MK-3­(I,II,III-H6)

Decaprenyl Diphosphate Synthesis in Mycobacterium tuberculosis

Z-prenyl diphosphate synthases catalyze the sequential condensation of isopentenyl diphosphate with allylic diphosphates to synthesize polyprenyl diphosphates. In mycobacteria, these are precursors of decaprenyl phosphate, a molecule which plays a central role in the biosynthesis of essential mycobacterial cell wall components, such as the mycolyl-arabinogalactan-peptidoglycan complex and lipoarabinomannan. Recently, it was demonstrated that open reading frame Rv2361c of the Mycobacterium tuberculosis H(37)Rv genome encodes a unique prenyl diphosphate synthase (M. C. Schulbach, P. J. Brennan, and D. C. Crick, J. Biol. Chem. 275:22876-22881, 2000). We have now purified the enzyme to near homogeneity by using an Escherichia coli expression system and have shown that the product of this enzyme is decaprenyl diphosphate. Rv2361c has an absolute requirement for divalent cations and an optimal pH range of 7.5 to 8.5, and the activity is stimulated by both detergent and dithiothreitol. The enzyme catalyzes the addition of isopentenyl diphosphate to geranyl diphosphate, neryl diphosphate, ω,E,E-farnesyl diphosphate, ω,E,Z-farnesyl diphosphate, or ω,E,E,E-geranylgeranyl diphosphate, with K(m) values for the allylic substrates of 490, 29, 84, 290, and 40 μM, respectively. The K(m) value for isopentenyl diphosphate is 89 μM. The catalytic efficiency is greatest when ω,E,Z-farnesyl diphosphate is used as the allylic acceptor, suggesting that this is the natural substrate in vivo, a conclusion that is supported by previous structural studies of decaprenyl phosphoryl mannose isolated from M. tuberculosis. This is the first report of a bacterial Z-prenyl diphosphate synthase that preferentially utilizes an allylic diphosphate primer having the α-isoprene unit in the Z configuration, indicating that Rv1086 (ω,E,Z-farnesyl diphosphate synthase) and Rv2361c act sequentially in the biosynthetic pathway that leads to the formation of decaprenyl phosphate in M. tuberculosis