A nonradioactive 96-well plate assay for screening of trans-sialidase activity

Trans-sialidase (E.C. 3.2.1.18) catalyzes the transfer of preferably alpha2,3-linked sialic acid to another glycan or glycoconjugate, forming a new alpha2,3 linkage to galactose or N-acetylgalactosamine. Here, we describe a nonradioactive 96-well plate fluorescence test for monitoring trans-sialidase activity with high sensitivity, specificity, and reproducibility using sialyllactose and 4-methylumbelliferyl-beta-D-galactoside as donor and acceptor substrates, respectively. The assay conditions were optimized using the trans-sialidase from Trypanosoma congolense and its general applicability was confirmed with recombinant trans-sialidase from Trypanosoma cruzi. Using this procedure, a large number of samples can be tested quickly and reliably, for instance in monitoring trans-sialidase during enzyme purification and the production of monoclonal antibodies, for enzyme characterization, and for identifying potential substrates and inhibitors. The trans-sialidase assay reported here was capable of detecting trans-sialidase activity in the low-mU range and may be a valuable tool in the search for further trans-sialidases in various biological systems.     

A duplex real-time PCR assay for detection of mutT4 and mutT2 mutations in Mycobacterium tuberculosis of Beijing genotype

To determine the polymorphism of mutT genes of Mycobacterium tuberculosis of Beijing genotype, we developed a duplex real-time PCR assay based on hybridization probes for the Roche LightCycler instrument. The assay rapidly detects mutations at codons 48 and 58 of genes mutT4 and at mutT2, respectively.     

A high-throughput assay for screening l- or d-amino acid specific aminotransferase mutant libraries

Aminotransferases are pyridoxal phosphate-dependent enzymes whose potential for the biocatalytic production of enantiopure amino acids is increasingly recognized. Because of this, there is a growing interest in engineering them to alter their substrate specificity and to increase their catalytic activity. Here, we report the development of a high-throughput assay for screening α-ketoglutarate-dependent aminotransferase mutant libraries. To achieve this, we exploited the l-glutamate dehydrogenase coupled assay that has previously been shown to allow for aminotransferase activity to be monitored in vitro. We adapted this assay to allow screening of mutant libraries of either l- or d-amino acid specific aminotransferases in a continuous fashion. This assay requiring clarified cell lysates is reproducible, rapid, and sensitive because it allowed for the identification of a catalytically active mutant of Bacillus sp. YM-1 d-amino acid aminotransferase displaying a decrease in k_cat/K_M of more than two orders of magnitude. In addition, this assay allowed us to discover a mutant of Escherichia coli branched-chain amino acid aminotransferase, F36W, which is approximately 60-fold more specific toward the natural substrate l-leucine than l-phenylalanine as compared with wild type. This result demonstrates the potential of our assay for the discovery of mutant aminotransferases displaying altered substrate specificity, an important goal of enzyme engineering.     

Dissection of Rab7 localization on Mycobacterium tuberculosis phagosome

The late endosomal marker Rab7 has been long believed to be absent from the phagosome containing Mycobacterium tuberculosis (M.tb) in macrophage, but the detail kinetics remains elusive. Here, we found that Rab7 is transiently recruited to and subsequently released from M.tb phagosomes. For further understanding of the effect of Rab7 dissociation from the phagosome, we examined the localization of lysosomal markers on the phagosome in the macrophage expressing a dominant-negative Rab7. The localization of lysosomal associated membrane protein-2 (LAMP-2) on the phagosome was Rab7-independent, while that of cathepsin D was Rab7-dependent. These results agree with the localization of each lysosomal marker on M.tb phagosome at 6 h postinfection-i.e., LAMP-2, but not cathepsin D localized on the majority of M.tb phagosomes. These results suggest that the dissociation of Rab7 from M.tb phagosome is the important process in inhibition of phagolysosome biogenesis.     

S-nitrosylation of Mycobacterium tuberculosis tyrosine phosphatase A (PtpA) induces its structural instability

S-nitrosylation is associated with signal transduction and microbicidal activity of nitric oxide (NO). We have recently described the S-nitrosylation of Mycobacterium tuberculosis protein tyrosine phosphatase A, PtpA, an enzyme that plays an important role in mycobacteria survival inside macrophages. This post-translational modification decreases the activity of the enzyme upon modification of a single Cys residue, C53. The aim of the present work was the investigation of the effect of S-nitrosylation in PtpA kinetic parameters, thermal stability and structure. It was observed that the K_M of nitrosylated PtpA was similar to its unmodified form, but the V_max was significantly reduced. In contrast, treatment of PtpA C53A with GSNO, did not alter either K_M or V_max. These results confirmed that PtpA S-nitrosylation occurs specifically in the non-catalytic C53 and that this modification does not affect substrate affinity. Using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy techniques it was shown that PtpA S-nitrosylation decreased protein thermal stability and promoted a local effect in the surroundings of the C53 residue, which interfered in both protein stability and function.     

A rapid microtiter plate assay for measuring the effect of compounds on Staphylococcus aureus membrane potential

We developed a homogenous microtiter based assay using the cationic dye 3, 3′-Diethyloxacarbocyanine iodide, DiOC₂(3), to measure the effect of compounds on membrane potential in Staphylococcus aureus. In a screen of 372 compounds from a synthetic compound collection with anti-Escherichia coli activity due to unknown modes of action at least 17% demonstrated potent membrane activity, enabling rapid discrimination of nuisance compounds.     

Continuous colorimetric screening assay for detection of d-amino acid aminotransferase mutants displaying altered substrate specificity

d-Amino acid aminotransferase (DAAT) catalyzes the synthesis of numerous d-amino acids, making it an attractive biocatalyst for the production of enantiopure d-amino acids. To bolster its biocatalytic applicability, improved variants displaying increased activity toward non-native substrates are desired. Here, we report the development of a high-throughput, colorimetric, continuous coupled enzyme assay for the screening of DAAT mutant libraries that is based on the use of d-amino acid oxidase (DAAO). In this assay, the d-amino acid product of DAAT is oxidized by DAAO with concomitant release of hydrogen peroxide, which is detected colorimetrically by the addition of horseradish peroxidase and o-dianisidine. Using this assay, we measured apparent K_M and k_cat values for DAAT and identified mutants displaying altered substrate specificity via the screening of cell lysates in 96-well plates. The DAAO coupled assay is sensitive in that it allowed the detection of a DAAT mutant displaying an approximately 2000-fold decrease in k_cat/K_M relative to wild type. In addition, the DAAO assay enabled the identification of two DAAT mutants (V33Y and V33G) that are more efficient than wild type at transaminating the non-native acceptor phenylpyruvate. We expect that this assay will be useful for the engineering of additional mutants displaying increased activity toward non-native substrates.     

Inhibition of Mycobacterium tuberculosis topoisomerase I by m-AMSA,a eukaryotic type II topoisomerase poison

m-AMSA, an established inhibitor of eukaryotic type II topoisomerases, exerts its cidal effect by binding to the enzyme–DNA complex thus inhibiting the DNA religation step. The molecule and its analogues have been successfully used as chemotherapeutic agents against different forms of cancer. After virtual screening using a homology model of the Mycobacterium tuberculosis topoisomerase I, we identified m-AMSA as a high scoring hit. We demonstrate that m-AMSA can inhibit the DNA relaxation activity of topoisomerase I from M. tuberculosis and Mycobacterium smegmatis. In a whole cell assay, m-AMSA inhibited the growth of both the mycobacteria.     

Biochemical characterization of recombinant nucleoside hydrolase from Mycobacterium tuberculosis H37Rv

Tuberculosis (TB) is a major global health threat. There is a need for the development of more efficient drugs for the sterilization of the disease’s causative agent, Mycobacterium tuberculosis (MTB). A more comprehensive understanding of the bacilli’s nucleotide metabolic pathways could aid in the development of new anti-mycobacterial drugs. Here we describe expression and purification of recombinant iunH-encoded nucleoside hydrolase from MTB (MtIAGU-NH). Glutaraldehyde cross-linking results indicate that MtIAGU-NH predominates as a monomer, presenting varied oligomeric states depending upon binding of ligands. Steady-state kinetics results show that MtIAGU-NH has broad substrate specificity, accepting inosine, adenosine, guanosine, and uridine as substrates. Inosine and adenosine displayed positive homotropic cooperativity kinetics, whereas guanosine and uridine displayed hyperbolic saturation curves. Measurements of kinetics of ribose binding to MtIAGU-NH by fluorescence spectroscopy suggest two pre-existing forms of enzyme prior to ligand association. The intracellular concentrations of inosine, uridine, hypoxanthine, and uracil were determined and thermodynamic parameters estimated. Thermodynamic activation parameters (E_a, ΔG^#, ΔS^#, ΔH^#) for MtIAGU-NH-catalyzed chemical reaction are presented. Results from mass spectrometry, isothermal titration calorimetry (ITC), pH-rate profile experiment, multiple sequence alignment, and molecular docking experiments are also presented. These data should contribute to our understanding of the biological role played by MtIAGU-NH.     

Competitive inhibitors of type B ribose 5-phosphate isomerases: design, synthesis and kinetic evaluation of new d-allose and d-allulose 6-phosphate derivatives

This study reports syntheses of d-allose 6-phosphate (All6P), d-allulose (or d-psicose) 6-phosphate (Allu6P), and seven d-ribose 5-phosphate isomerase (Rpi) inhibitors. The inhibitors were designed as analogues of the 6-carbon high-energy intermediate postulated for the All6P to Allu6P isomerization reaction (Allpi activity) catalyzed by type B Rpi from Escherichiacoli (EcRpiB). 5-Phospho-d-ribonate, easily obtained through oxidative cleavage of either All6P or Allu6P, led to the original synthon 5-dihydrogenophospho-d-ribono-1,4-lactone from which the other inhibitors could be synthesized through nucleophilic addition in one step. Kinetic evaluation on Allpi activity of EcRpiB shows that two of these compounds, 5-phospho-d-ribonohydroxamic acid and N-(5-phospho-d-ribonoyl)-methylamine, indeed behave as new efficient inhibitors of EcRpiB; further, 5-phospho-d-ribonohydroxamic acid was demonstrated to have competitive inhibition. Kinetic evaluation on Rpi activity of both EcRpiB and RpiB from Mycobacteriumtuberculosis (MtRpiB) shows that several of the designed 6-carbon high-energy intermediate analogues are new competitive inhibitors of both RpiBs. One of them, 5-phospho-d-ribonate, not only appears as the strongest competitive inhibitor of a Rpi ever reported in the literature, with a K_i value of 9 μM for MtRpiB, but also displays specific inhibition of MtRpiB versus EcRpiB.     

Selection of improved Beauveria bassiana (Bals.) Vuill. strains based on 2-deoxy-d-glucose resistance and physiological analysis

A series of 2-deoxy-d-glucose resistant mutants was obtained from wild type Beauveria bassiana 88 (Bb 88) by UV irradiation. Five mutants were characterized on Sabouraud Dextrose Agar and Chitin Agar for both radial extension rate (V_r) and specific growth rate (μ). These values were obtained after adjusting morphometric data to a mathematical model used for filamentous fungi. Additionally, the protease and lipase potency index, conidial size, viability, and production levels were analyzed. The highest values for those physiological measurements were obtained by mutant 882.5 which, relative to Bb 88, showed a 30% reduction in half-life (LT₅₀) on Sphenarium purpurascens, 70% on Acheta domesticus, and 71% on Tenebrio molitor larvae and adults. The half lethal concentration (LC₅₀) on T. molitor larvae was 2.8 × 10⁵ conidia/mL (con/mL) and 1.5 × 10⁶ con/mL, respectively, for mutant 882.5 and Bb 88. This demonstrates that mutant 882.5 is more virulent, with up to an 80% reduction in LC₅₀. This work provides a convenient method for improving strains to be used in biocontrol as a suitable alternative to transgenic constructs.     

Fellutamide B is a potent inhibitor of the Mycobacterium tuberculosis proteasome

Via high-throughput screening of a natural compound library, we have identified a lipopeptide aldehyde, fellutamide B (1), as the most potent inhibitor of the Mycobacterium tuberculosis (Mtb) proteasome tested to date. Kinetic studies reveal that 1 inhibits both Mtb and human proteasomes in a time-dependent manner under steady-state condition. Remarkably, 1 inhibits the Mtb proteasome in a single-step binding mechanism with K_i = 6.8 nM, whereas it inhibits the human proteasome β5 active site following a two-step mechanism with K_i = 11.5 nM and  = 0.93 nM. Co-crystallization of 1 bound to the Mtb proteasome revealed a structural basis for the tight binding of 1 to the active sites of the Mtb proteasome. The hemiacetal group of 1 in the Mtb proteasome takes the (R)-configuration, whereas in the yeast proteasome it takes the (S)-configuration, indicating that the pre-chiral CHO group of 1 binds to the active site Thr1 in a different orientation. Re-examination of the structure of the yeast proteasome in complex with 1 showed significant conformational changes at the substrate-binding cleft along the active site. These structural differences are consistent with the different kinetic mechanisms of 1 against Mtb and human proteasomes.     

Protein kinase and phosphatase signaling in Mycobacterium tuberculosis physiology and pathogenesis

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB), evades the antimicrobial defenses of the host and survives within the infected individual through a complex set of strategies. These include active prevention of host cellular killing processes as well as overwhelming adaptive gene expression. In the past decade, we have gained an increased understanding of how mycobacteria not only have the ability to adapt to a changing host environment but also actively interfere with the signaling machinery within the host cell to counteract or inhibit parts of the killing apparatus employed by the macrophage. Mtb is able to sense its environment via a set of phospho-signaling proteins which mediate its response and interaction with the host in a coordinated manner. In this review, we summarize the current knowledge about selected Mtb serine, threonine, and tyrosine kinase and phosphatase signaling proteins, focusing on the protein kinases, PknG and PtkA, and the protein phosphatase, PtpA.     

Formation of l-quebrachitol from d-bornesitol in leaves of Acer pseudoplatanus

d-Bornesitol and l-quebrachitol have been found in the leaves of Acer pseudoplatanus L. The results of incorporation studies using labeled myo-inositol-¹⁴C, l-inositol-¹⁴C and d-bornesitol-¹⁴C indicate that l-quebrachitol is produced by epimerization of d-bornesitol. In Artemisia vulgaris, however, the precursor of l-quebrachitol is l-inositol.     

The evolutionary landscape of the Mycobacterium tuberculosis genome

Mycobacterium tuberculosis is one of the most deadly human pathogens. The major mechanism for the adaptations of M. tuberculosis is nucleotide substitution. Previous studies have relied on the nonsynonymous-to-synonymous substitution rate (d_N/d_S) ratio as a measurement of selective constraint based on the assumed selective neutrality of synonymous substitutions. However, this assumption has been shown to be untrue in many cases. In this study, we used the substitution rate in intergenic regions (d_i) of the M. tuberculosis genome as the neutral reference, and conducted a genome-wide profiling for d_i, d_S, and the rate of insertions/deletions (indel rate) as compared with the genome of M. canettii using a 50 kb sliding window. We demonstrate significant variations in all of the three evolutionary measurements across the M. tuberculosis genome, even for regions in close vicinity. Furthermore, we identified a total of 233 genes with their d_S deviating significantly from d_i within the same window. Interestingly, d_S also varies significantly in some of the windows, indicating drastic changes in mutation rate and/or selection pressure within relatively short distances in the M. tuberculosis genome. Importantly, our results indicate that selection on synonymous substitutions is common in the M. tuberculosis genome. Therefore, the d_N/d_S ratio test must be applied carefully for measuring selection pressure on M. tuberculosis genes.     

A live-cell high-throughput screening assay for identification of fatty acid uptake inhibitors

We developed a live-cell high-throughput assay system using the baker's yeast Saccharomyces cerevisiae to screen for chemical compounds that will inhibit fatty acid uptake. The target for the inhibitors is a mammalian fatty acid transport protein (mmFATP2), which is involved in the fatty acid transport and activation pathway. The mmFATP2 was expressed in a S. cerevisiae mutant strain deficient in Fat1p-dependent fatty acid uptake and reduced in long-chain fatty acid activation, fat1Deltafaa1Delta. To detect fatty acid import, a fluorescent fatty acid analog, 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (C1-BODIPY-C12), was incubated with cells expressing FATP2 in a 96-well plate. The mmFATP2-dependent C1-BODIPY-C12 uptake was monitored by measuring intracellular C1-BODIPY-C12 fluorescence on a microtiter plate reader, whereas extracellular fluorescence was quenched by a cell viability dye, trypan blue. Using this high-throughput screening method, we demonstrate that the uptake of the fluorescent fatty acid ligand was effectively competed by the natural fatty acid oleate. Inhibition of uptake was also demonstrated to occur when cells were pretreated with sodium azide or Triacsin C. This yeast live-cell-based assay is rapid to execute, inexpensive to implement, and has adequate sensitivity for high-throughput screening. The assay basis and limitations are discussed.     

Temperature dependence of the Langmuir monolayer packing of mycolic acids from Mycobacterium tuberculosis

Phase diagrams of the Langmuir monolayer of dicyclopropyl alpha mycolic acid (α-MA), cyclopropyl methoxy mycolic acid (MeO-MA), and cyclopropyl ketomycolic acids (Keto-MA) from Mycobacterium tuberculosis were obtained by thermodynamic analysis of the surface pressure (π) vs. average molecular area (A) isotherms at temperatures in the range of 10-46 °C. The Langmuir monolayers of MAs were shown to exhibit various phases depending on the temperature (T) and the π values. In the Langmuir monolayer of Keto-MA, the carbonyl group in the meromycolate chain apparently touches the water surface to give the molecule a W-shape in all the temperatures and surface pressures studied. Keto-MA formed a rigid solid condensed film, with four hydrocarbon chains packing together, not observed in the others. In contrast, the monolayer films of α-and MeO-MAs having no such highly hydrophilic intra-chain groups in the meromycolate chain were mostly in liquid condensed phase. This novel insight into the packing of mycolic acids opens up new avenues for the study of the role of mycolic acids in the mycobacterial cell envelopes and pathogenic processes.