Conformational behavior of oxygenated mycobacterial mycolic acids from Mycobacterium bovis BCG

Phase diagrams of Langmuir monolayers of oxygenated mycolic acids, i.e. methoxy mycolic acid (MeO-MA), ketomycolic acid (Keto-MA), and artificially obtained deoxo-mycolic acid (deoxo-MA) from Mycobacterium bovis BCG were obtained by thermodynamic analysis of the surface pressure (π) vs. average molecular area (A) isotherms. At lower temperatures and lower surface pressures, both Keto- and MeO-MAs formed rigid condensed monolayers where each MA molecule was considered to be in a 4-chain form, in which the three carbon chain segments due to bending of the 3-hydroxy aliphatic carboxylate chain and the 2-side chain were in compact parallel arrangement. At higher temperatures and surface pressures, MeO-MA and deoxo-MA tended to take stretched-out conformations in which the 3-hydroxy aliphatic carboxylate chain was more or less in an extended form, but Keto-MA retained the original 4-chain structure. The thickness measurement of the monolayers in situ by ellipsometry at different π values and temperatures supported the above conclusions derived from the phase diagrams. The enthalpy changes associated with the phase transitions of MeO-MA and deoxo-MA implied that the MeO-MA needed larger energy to change from a compact conformation to an extended one, possibly and partly due to the dehydration of the methoxy group from water surface involved. Molecular dynamics studies of MA models derived from Monte Carlo calculations were also performed, which confirmed the conformational behavior of MAs suggested by the thermodynamic studies on the Langmuir monolayers.     

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.     

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.     

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

Phase diagrams of the Langmuir monolayer of dicyclopropyl alpha mycolic acid (alpha-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 (pi) vs. average molecular area (A) isotherms at temperatures in the range of 10-46 degrees C. The Langmuir monolayers of MAs were shown to exhibit various phases depending on the temperature (T) and the pi 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 alpha-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.     

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.     

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.     

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.     

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.     

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.     

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.     

Unusual Diheme Conformation of the Heme-Degrading Protein from Mycobacterium tuberculosis

Heme degradation plays a pivotal role in the availability of the essential nutrient, iron, in pathogenic bacteria. A previously unannotated protein from Mycobacterium tuberculosis, Rv3592, which shares homology to heme-degrading enzymes, has been identified. Biochemical analyses confirm that Rv3592, which we have termed MhuD (mycobacterial heme utilization, degrader), is able to bind and degrade heme. Interestingly, contrary to previously reported stoichiometry for the Staphylococcus aureus heme degraders, iron-regulated surface determinant (Isd)G and IsdI, MhuD has the ability to bind heme in a 1:2 protein-to-heme ratio, although the MhuD-diheme complex is inactive. Furthermore, the 1.75-Å crystal structure of the MhuD-diheme complex reveals two stacked hemes forming extensive contacts with residues in the active site. In particular, the solvent-exposed heme is axially liganded by His75 and is stacked planar upon the solvent-protected heme. The solvent-protected heme is coordinated by a chloride ion, which is, in turn, stabilized by Asn7. Structural comparison between MhuD-diheme and inactive IsdG and IsdI bound to only one highly distorted metalloporphyrin ring reveals that several residues located in α-helix 2 and the subsequent loop appear to be responsible for heme stoichiometric differences and suggest open and closed conformations for substrate entry and product exit.     

A novel firefly luciferase biosensor enhances the detection of apoptosis induced by ESAT-6 family proteins of Mycobacterium tuberculosis

The activation of caspase-3 is a key surrogate marker for detecting apoptosis. To quantitate caspase-3 activity, we constructed a biosensor comprising a recombinant firefly luciferase containing a caspase-3 cleavage site. When apoptosis was induced, caspase-3 cleavage of the biosensor activated firefly luciferase by a factor greater than 25. The assay conveniently detected apoptosis in real time, indicating that it will facilitate drug discovery. We screened ESAT-6 family proteins of Mycobacterium tuberculosis and found that esxA, esxT and esxL induced apoptosis. Further, activation of nuclear factor-κB (NF-κB) and the NF-κB-regulated genes encoding tumor necrosis factor-α (TNF-α) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) participated in esxT-induced apoptosis. We conclude that this assay is useful for high-throughput screening to identify and characterize proteins and drugs that regulate apoptosis.     

Interaction analysis of TcrX/Y two component system from Mycobacterium tuberculosis

TcrX/Y is one of the twelve two component system (TCS) present in Mycobacterium tuberculosis. We have investigated the TcrX/Y interaction by in silico studies, pull down assay, radioactive phosphotransfer, surface plasmon resonance as well as crosstalk analysis of TcrY with TcrA – a non-cognate response regulator. Sequence alignment of TcrY with other histidine kinases revealed His256 as the residue responsible for autophosphorylation. The modeled structure of TcrX/Y was docked with each other by GRAMM-X revealing the interaction of TcrY/His256 with TcrX/Asp54. TcrY dimerization via the formation of four helix bundle was also observed by protein–protein docking. Autophosphorylation of TcrY has been observed followed by the phosphate transfer from TcrY to TcrX. The phosphorylation process required divalent metal ions like Mg²⁺ or Ca²⁺ ions as evident from the radioactive phosphorylation studies. Interaction was not observed between TcrY and TcrA suggesting the signal transduction process is specific in TcrX/Y system. TcrY hydrolyzes ATP and the K_m value has been found to be 10 mM which is comparable to that of Hsp104. TcrX/Y interaction has been determined by surface plasmon resonance and dissociation constant (K_D) was evaluated to be 3.6 μM. We conclude from our results that TcrX and TcrY are part of the same signal transduction pathway without their involvement in crosstalk with non-cognate counterpart.     

Characterization of a chromosomal toxin-antitoxin, Rv1102c-Rv1103c system in Mycobacterium tuberculosis

Toxin-antitoxin systems, ubiquitous in prokaryotic genomes, have been proposed to play an important role in several stress responses. While Mycobacterium tuberculosis contains more than 80 putative TA loci, the roles they play in this pathogen are yet to be studied. Here, we characterize a chromosomal Rv1102c-Rv1103c TA system in M. tuberculosis. We found that the Rv1102c toxin interacts with the Rv1103c antitoxin in a pull-down assay and the yeast two-hybrid system. Rv1102c cleaved the era mRNA in Escherichia coli, and cleavage was inhibited by co-expression of Rv1103c. Heterologous expression of Rv1102c led to growth arrest in E. coli, which was fully recovered only when Rv1103c was co-expressed in cis with Rv1102c, suggesting that the production and assembly of Rv1102c and Rv1103c are tightly linked. Our additional results indicate that translational coupling of the Rv1102c and Rv1103c genes is important for Rv1102c-Rv1103c binding. Finally, we discovered that the expression of Rv1102c induced growth arrest and increased the level of persister cells in Mycobacterium smegmatis. These results suggest that the Rv1102c-Rv1103c TA system could play a role in M. tuberculosis pathogenesis via generating bacilli that survive in the face of multidrug therapy.     

TLR2 and its co-receptors determine responses of macrophages and dendritic cells to lipoproteins of Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) signals through Toll-like receptor 2 (TLR2) to regulate antigen presenting cells (APCs). Mtb lipoproteins, including LpqH, LprA, LprG and PhoS1, are TLR2 agonists, but their co-receptor requirements are unknown. We studied Mtb lipoprotein-induced responses in TLR2^−/−, TLR1^−/−, TLR6^−/−, CD14^−/− and CD36^−/− macrophages. Responses to LprA, LprG, LpqH and PhoS1 were completely dependent on TLR2. LprG, LpqH, and PhoS1 were dependent on TLR1, but LprA did not require TLR1. None of the lipoproteins required TLR6, although a redundant contribution by TLR6 cannot be excluded. CD14 contributed to detection of LprA, LprG and LpqH, whereas CD36 contributed only to detection of LprA. Studies of lung APC subsets revealed lower TLR2 expression by CD11b^high/CD11c^low lung macrophages than CD11b^low/CD11c^high alveolar macrophages, which correlated with hyporesponsiveness of lung macrophages to LpqH. Thus, lung APC subsets differ in TLR expression, which may determine differences in responses to Mtb.     

Structure of translation initiation factor 1 from Mycobacterium tuberculosis and inferred binding to the 30S ribosomal subunit

The crystal structure of the free form of IF1 from Mycobacterium tuberculosis has been determined at 1.47 Å resolution. The structure adopts the expected OB fold and matches the high structural conservation among IF1 orthologues. In order to further explore the function of Mtb-IF1, we built a model of its interaction with the 30S ribosomal subunit based on the crystal structure of the complex from Thermus thermophilus. The model suggests that several functionally important side chain residues undergo large movements while the rest of the protein in complex shows only very limited conformational change as compared to its form in solution.