Improved cellular immune response elicited by a ubiquitin-fused ESAT-6 DNA vaccine against Mycobacterium tuberculosis

The present study evaluated the immune response elicited by a ubiquitin-fused ESAT-6 DNA vaccine against Mycobacterium tuberculosis. BALB/c mice were vaccinated with plasmid DNA encoding ESAT-6 protein, ubiquitin-fused ESAT-6 DNA vaccine (UbGR-ESAT-6), pcDNA3-ubiquitin and blank vector, respectively. ESAT-6 DNA vaccine immunization induced a Thl-polarized immune response. The production of Thl-type cytokine (IFN-γ) and proliferative T-cell responses was enhanced significantly in mice immunized with UbGR-ESAT-6 fusion DNA vaccine, compared to non-fusion DNA vaccine. This fusion DNA vaccine also resulted in an increased relative ratio of IgG_2a to IgG_l and the cytotoxicity of T cells. Thus, the present study demonstrated that the UbGR-ESAT-6 fusion DNA vaccine inoculation improved antigen-specific cellular immune responses, which is helpful for protection against tuberculosis infection.     

The ongoing challenge of latent tuberculosis

The global health community has set itself the task of eliminating tuberculosis (TB) as a public health problem by 2050. Although progress has been made in global TB control, the current decline in incidence of 2% yr⁻¹ is far from the rate needed to achieve this. If we are to succeed in this endeavour, new strategies to reduce the reservoir of latently infected persons (from which new cases arise) would be advantageous. However, ascertainment of the extent and risk posed by this group is poor. The current diagnostics tests (tuberculin skin test and interferon-gamma release assays) poorly predict who will develop active disease and the therapeutic options available are not optimal for the scale of the intervention that may be required. In this article, we outline a basis for our current understanding of latent TB and highlight areas where innovation leading to development of novel diagnostic tests, drug regimens and vaccines may assist progress. We argue that the pool of individuals at high risk of progression may be significantly smaller than the 2.33 billion thought to be immune sensitized by Mycobacterium tuberculosis and that identifying and targeting this group will be an important strategy in the road to elimination.     

An approach for studying the mediators of pathogenesis inMycobacterium tuberculosis

Mycobacterium tuberculosis is an example of an intracellular pathogen that mediates the disease state through complex interactions with the host’s immune system. Not only does this organism replicate in the hostile environment prevailing within the infected macrophage, but it has also developed intricate mechanisms to inhibit several defence mechanisms of the host’s immune system. It is postulated here that the mediators of these interactions with the host are products of differentially expressed genes in the pathogen. B and T cell responses of the host are hence to be used as tools to identify such gene products from an expression library of theMycobacterium tuberculosis genome. The various pathways of generating a productive immune response that may be targeted by the pathogen are discussed     

Naturally produced opsonizing antibodies restrict the survival of Mycobacterium tuberculosis in human macrophages by augmenting phagosome maturation

This study investigated the hypothesis that serum antibodies against Mycobacterium tuberculosis present in naturally infected healthy subjects of a tuberculosis (TB) endemic area could create and/or sustain the latent form of infection. All five apparently healthy Indian donors showed high titres of serum antibodies against M. tuberculosis cell membrane antigens, including lipoarabinomannan and alpha crystallin. Uptake and killing of bacilli by the donor macrophages was significantly enhanced following their opsonization with antibody-rich, heat-inactivated autologous sera. However, the capability to opsonize was apparent for antibodies against some and not other antigens. High-content cell imaging of infected macrophages revealed significantly enhanced colocalization of the phagosome maturation marker LAMP-1, though not of calmodulin, with antibody-opsonized compared with unopsonized M. tuberculosis. Key enablers of macrophage microbicidal action—proinflammatory cytokines (IFN-γ and IL-6), phagosome acidification, inducible NO synthase and nitric oxide—were also significantly enhanced following antibody opsonization. Interestingly, heat-killed M. tuberculosis also elevated these mediators to the levels comparable to, if not higher than, opsonized M. tuberculosis. Results of the study support the emerging view that an efficacious vaccine against TB should, apart from targeting cell-mediated immunity, also generate ‘protective’ antibodies.     

Sex differences in the protection of host immune systems by a polyembryonic parasitoid

Endoparasitoids have the ability to evade the cellular immune responses of a host and to create an environment suitable for survival of their progeny within a host. Generally, the host immune system is suppressed by endoparasitoids. However, polyembryonic endoparasitoids appear to invade their hosts using molecular mimicry rather than immune system suppression. It is not known how the host immune system is modified by polyembryonic endoparasitoids. Using haemocyte counts and measurement of cellular immune responses, we evaluated modification of the host immune system after separate infestations by a polyembryonic parasitoid (Copidosoma floridanum) and another parasitoid (Glyptapanteles pallipes) and by both together (multi-parasitism). We found that the polyembryonic parasitoid maintains and enhances the host immune system, whereas the other parasitoid strongly suppresses the immune system. Multi-parasitization analysis revealed that C. floridanum cancelled the immune suppression by G. pallipes and strengthened the host immunity. This enhancement was much stronger with male than with female C. floridanum.     

Mycobacterium tuberculosis Rv2536 protein implicated in specific binding to human cell lines

The gene encoding the Mycobacterium tuberculosis Rv2536 protein is present in the Mycobacterium tuberculosis complex (as assayed by PCR) and transcribed (as determined by RT-PCR) in M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. bovis BCG, and M. africanum strains. Rabbits immunized with synthetic polymer peptides from this protein produced antibodies specifically recognizing a 25-kDa band in mycobacterial sonicate. U937 and A549 cells were used in binding assays involving 20-amino-acid-long synthetic peptides covering the whole Rv2536 protein sequence. Peptide 11207 (161DVFSAVRADDSPTGEMQVAQY180) presented high specific binding to both types of cells; the binding was saturable and presented nanomolar affinity constants. Cross-linking assays revealed that this peptide specifically binds to 50 kDa U937 cell membrane and 45 kDa A549 cell membrane proteins.     

Sublineages of Beijing Strain of Mycobacterium tuberculosis in Sri Lanka

Strains of the Beijing/W genotype of Mycobacterium tuberculosis have been responsible for large outbreaks of tuberculosis around the world, sometimes involving multi-drug resistance. It has been shown that more recently evolved Beijing sublineages are prone to cause outbreaks. Furthermore Beijing is the single predominant cluster in Sri Lanka. The present study identifies that recently evolved sublineages of Beijing strains are present in the study population. The majority of Beijing isolates (92.85%) were pan-susceptible. However, these findings may have important implications for the control and prevention of tuberculosis in Sri Lanka.     

Hidden face of tuberculosis

Occurrence of cell wall deficiency (L-form conversion) in hosts suggests one of the possible pathways by which tubercle bacilli can survive, replicate and persist within the body for a long period harboring latent tuberculosis. Non-acid fast and morphologically modified L-forms of Mycobacterium tuberculosis are difficult to identify and remain often unrecognized or are mistaken for contaminants.     

Analysis of DevR regulated genes in Mycobacterium tuberculosis

The DevRS two component system of Mycobacterium tuberculosis is responsible for its dormancy in host and becomes operative under hypoxic condition. It is experimentally known that phosphorylated DevR controls the expression of several downstream genes in a complex manner. In the present work we propose a theoretical model to show role of binding sites in DevR mediated gene expression. Individual and collective role of binding sites in regulating DevR mediated gene expression has been shown via modeling. Objective of the present work is twofold. First, to describe qualitatively the temporal dynamics of wild type genes and their known mutants. Based on these results we propose that DevR controlled gene expression follows a specific pattern which is efficient in describing other DevR mediated gene expression. Second, to analyze behavior of the system from information theoretical point of view. Using the tools of information theory we have calculated molecular efficiency of the system and have shown that it is close to the maximum limit of isothermal efficiency.     

X-ray structure of putative acyl-ACP desaturase DesA2 from Mycobacterium tuberculosis H37Rv

Genome sequencing showed that two proteins in Mycobacterium tuberculosis H37Rv contain the metal binding motif (D/E)X(2)HX(approximately 100)(D/E)X(2)H characteristic of the soluble diiron enzyme superfamily. These putative acyl-ACP desaturase genes desA1 and desA2 were cloned from genomic DNA and expressed in Escherichia coli BL21(DE3). DesA1 was found to be insoluble, but in contrast, DesA2 was a soluble protein amenable to biophysical characterization. Here, we report the 2.0 A resolution X-ray structure of DesA2 determined by multiple anomalous dispersion (MAD) phasing from a Se-met derivative and refinement against diffraction data obtained on the native protein. The X-ray structure shows that DesA2 is a homodimeric protein with a four-helix bundle core flanked by five additional helices that overlay with 192 structurally equivalent amino acids in the structure of stearoyl-ACP Delta9 desaturase from castor plant with an rms difference 1.42 A. In the DesA2 crystals, one metal (likely Mn from the crystallization buffer) was bound in high occupancy at the B-site of the conserved metal binding motif, while the A-site was not occupied by a metal ion. Instead, the amino group of Lys-76 occupied this position. The relationships between DesA2 and known diiron enzymes are discussed.     

Origin of asymmetry in adenylyl cyclases: structures of Mycobacterium tuberculosis Rv1900c

Rv1900c, a Mycobacterium tuberculosis adenylyl cyclase, is composed of an N-terminal alpha/beta-hydrolase domain and a C-terminal cyclase homology domain. It has an unusual 7% guanylyl cyclase side-activity. A canonical substrate-defining lysine and a catalytic asparagine indispensable for mammalian adenylyl cyclase activity correspond to N342 and H402 in Rv1900c. Mutagenic analysis indicates that these residues are dispensable for activity of Rv1900c. Structures of the cyclase homology domain, solved to 2.4 A both with and without an ATP analog, form isologous, but asymmetric homodimers. The noncanonical N342 and H402 do not interact with the substrate. Subunits of the unliganded open dimer move substantially upon binding substrate, forming a closed dimer similar to the mammalian cyclase heterodimers, in which one interfacial active site is occupied and the quasi-dyad-related active site is occluded. This asymmetry indicates that both active sites cannot simultaneously be catalytically active. Such a mechanism of half-of-sites-reactivity suggests that mammalian heterodimeric adenylyl cyclases may have evolved from gene duplication of a primitive prokaryote-type cyclase, followed by loss of function in one active site.     

Mycobacterium tuberculosis Rv2179c Protein Establishes a New Exoribonuclease Family with Broad Phylogenetic Distribution

Ribonucleases (RNases) maintain the cellular RNA pool by RNA processing and degradation. In many bacteria, including the human pathogen Mycobacterium tuberculosis (Mtb), the enzymes mediating several central RNA processing functions are still unknown. Here, we identify the hypothetical Mtb protein Rv2179c as a highly divergent exoribonuclease. Although the primary sequence of Rv2179c has no detectable similarity to any known RNase, the Rv2179c crystal structure reveals an RNase fold. Active site residues are equivalent to those in the DEDD family of RNases, and Rv2179c has close structural homology to Escherichia coli RNase T. Consistent with the DEDD fold, Rv2179c has exoribonuclease activity, cleaving the 3′ single-strand overhangs of duplex RNA. Functional orthologs of Rv2179c are prevalent in actinobacteria and found in bacteria as phylogenetically distant as proteobacteria. Thus, Rv2179c is the founding member of a new, large RNase family with hundreds of members across the bacterial kingdom.     

Modulation of cooperativity in Mycobacterium tuberculosis NADPH-ferredoxin reductase: cation-and pH-induced alterations in native conformation and destabilization of the NADP+-binding domain

FprA, a Mycobacterium tuberculosis NADPH-ferredoxin reductase, consists of two structural domains, a FAD-binding and a NADP-binding domain, respectively. For the first time, we demonstrated that native FprA, on thermal treatment underwent partial denaturation with unfolding of only the FAD-binding domain and release of the protein-bound flavin. The NADP-binding domain of this protein is highly resistant to denaturation under these conditions. However, the presence of either 150 mM NaCl or KCl or 10 muM MgCl(2) or CaCl(2) or slightly acidic pH of 6.0 resulted in a highly cooperative and complete thermal unfolding of the protein. Physicochemical investigations showed that the monovalent cations or low concentrations of divalent cations induced compaction of the protein conformation. However, divalent cations at higher concentrations resulted in FAD release leading to stabilization of an enzymatically inactive apoenzyme. Detailed thermal denaturation studies on the native protein and the isolated NADP-binding domain showed that cations and pH 6.0 destabilized only the heat-stable NADP-binding domain. The experimental studies demonstrate that modulation of intramolecular ionic interactions induce significant conformational changes in the NADP-binding domain of FprA, resulting in a substantial increase in the structural cooperativity of the whole molecule. The results presented in this paper are of importance as they demonstrate alterations in the native three-dimensional structure of FprA and cooperativity in protein molecule on slight alteration of pH or modification of ionic interactions in protein.     

Resistome analysis of Mycobacterium tuberculosis: Identification of aminoglycoside 2'-Nacetyltransferase (AAC) as co-target for drug desigining

The emergence of multidrug resistant tuberculosis (MDRTB) highlights the urgent need to understand the mechanisms of resistance to the drugs and to develop a new arena of therapeutics to treat the disease. Ethambutol, isonazid, pyrazinamide, rifampicin are first line of drugs against TB, whereas aminoglycoside, polypeptides, fluoroquinolone, ethionamide are important second line of bactericidal drugs used to treat MDRTB, and resistance to one or both of these drugs are defining characteristic of extensively drug resistant TB. We retrieved 1,221 resistant genes from Antibiotic Resistance Gene Database (ARDB), which are responsible for resistance against first and second line antibiotics used in treatment of Mycobacterium tuberculosis infection. From network analysis of these resistance genes, 53 genes were found to be common. Phylogenetic analysis shows that more than 60% of these genes code for acetyltransferase. Acetyltransferases detoxify antibiotics by acetylation, this mechanism plays central role in antibiotic resistance. Seven acetyltransferase (AT-1 to AT-7) were selected from phylogenetic analysis. Structural alignment shows that these acetyltransferases share common ancestral core, which can be used as a template for structure based drug designing. From STRING analysis it is found that acetyltransferase interact with 10 different proteins and it shows that, all these interaction were specific to M. tuberculosis. These results have important implications in designing new therapeutic strategies with acetyltransferase as lead co-target to combat against MDR as well as Extreme drug resistant (XDR) tuberculosis.

Abbreviations

AA - amino acid, AT - Acetyltransferase, AAC - Aminoglycoside 2''-N-acetyltransferase, XDR - Extreme drug-resistant, MDR - Multidrug-resistant, Mtb - Mycobacterium tuberculosis, TB - Tuberculosis.     

结核分枝杆菌海藻糖磷酸磷酸酶诱导小鼠体液和细胞免疫应答

目的 研究结核分枝杆菌(M．tuberculosis)海藻糖磷酸磷酸酶(TPP)诱导小鼠体液和细胞免疫。方法 差速离心分离结核分枝杆菌H37Rv和卡介苗(BCG)的各细胞组分,通过Western杂交检测抗原TPP在结核分枝杆菌H37Rv和BCG中的亚细胞定位情况。分别用5×10~6CFU的BCG和50μg的TPP蛋白免疫C57BL/6小鼠,检测小鼠血清中抗TPP的IgG1和IgG2a抗体效价。取免疫小鼠的脾细胞,体外抗原刺激,用酶联免疫斑点试验(ELISPOT)检测γ干扰素(IFN-γ)分泌细胞。结果 TPP亚细胞定位于结核分枝杆菌H37Rv和BCG的胞壁和细胞膜组分。TPP蛋白免疫后小鼠产生的TPP特异性IgG1和IgG2a抗体效价明显高于BCG免疫小鼠,并且IgG2a的抗体效价高于IgG1。体外抗原刺激TPP蛋白和BCG免疫小鼠的脾细胞,都能诱导较高的IFN-γ分泌。结论 结核分枝杆菌细胞壁蛋白TPP能诱导小鼠Ⅰ型辅助性T细胞介导的免疫反应,可作为抗结核疫苗的候选抗原。     

The molecular structure of epoxide hydrolase B from Mycobacterium tuberculosis and its complex with a urea-based inhibitor

Mycobacterium tuberculosis (Mtb), the intracellular pathogen that infects macrophages primarily, is the causative agent of the infectious disease tuberculosis in humans. The Mtb genome encodes at least six epoxide hydrolases (EHs A to F). EHs convert epoxides to trans-dihydrodiols and have roles in drug metabolism as well as in the processing of signaling molecules. Herein, we report the crystal structures of unbound Mtb EHB and Mtb EHB bound to a potent, low-nanomolar (IC₅₀ ≈ 19 nM) urea-based inhibitor at 2.1 and 2.4 Å resolution, respectively. The enzyme is a homodimer; each monomer adopts the classical α/β hydrolase fold that composes the catalytic domain; there is a cap domain that regulates access to the active site. The catalytic triad, comprising Asp104, His333 and Asp302, protrudes from the catalytic domain into the substrate binding cavity between the two domains. The urea portion of the inhibitor is bound in the catalytic cavity, mimicking, in part, the substrate binding; the two urea nitrogen atoms donate hydrogen bonds to the nucleophilic carboxylate of Asp104, and the carbonyl oxygen of the urea moiety receives hydrogen bonds from the phenolic oxygen atoms of Tyr164 and Tyr272. The phenolic oxygen groups of these two residues provide electrophilic assistance during the epoxide hydrolytic cleavage. Upon inhibitor binding, the binding-site residues undergo subtle structural rearrangement. In particular, the side chain of Ile137 exhibits a rotation of around 120° about its C^α-C^β bond in order to accommodate the inhibitor. These findings have not only shed light on the enzyme mechanism but also have opened a path for the development of potent inhibitors with good pharmacokinetic profiles against all Mtb EHs of the α/β type.     

Potential of Mycobacterium tuberculosis resuscitation-promoting factors as antigens in novel tuberculosis sub-unit vaccines

Novel vaccines are needed to control tuberculosis (TB), the bacterial infectious disease that together with malaria and HIV is worldwide responsible for high levels of morbidity and mortality. TB can result from the reactivation of an initially controlled latent infection by Mycobacterium tuberculosis (Mtb). Mtb proteins for which a possible role in this reactivation process has been hypothesized are the five homologs of the resuscitation-promoting factor of Micrococcus luteus, namely Mtb Rv0867c (rpfA), Rv1009 (rpfB), Rv1884c (rpfC), Rv2389c (rpfD) and Rv2450c (rpfE). Analysis of the immune recognition of these 5 proteins following Mtb infection or Mycobacterium bovis BCG vaccination of mice showed that Rv1009 (rpfB) and Rv2389c (rpfD) are the most antigenic in the tested models. We therefore selected rpfB and rpfD for testing their vaccine potential as plasmid DNA vaccines. Elevated cellular immune responses and modest but significant protection against intra-tracheal Mtb challenge were induced by immunization with the rpfB encoding DNA vaccine. The results indicate that rpfB is the most promising candidate of the five rpf-like proteins of Mtb in terms of its immunogenicity and protective efficacy and warrants further analysis for inclusion as an antigen in novel TB vaccines.     

Probing mechanisms of resistance to the tuberculosis drug isoniazid: Conformational changes caused by inhibition of InhA, the enoyl reductase from Mycobacterium tuberculosis

The frontline tuberculosis drug isoniazid (INH) inhibits InhA, the NADH-dependent fatty acid biosynthesis (FAS-II) enoyl reductase from Mycobacterium tuberculosis (MTB), via formation of a covalent adduct with NAD(+) (the INH-NAD adduct). Resistance to INH can be correlated with many mutations in MTB, some of which are localized in the InhA cofactor binding site. While the InhA mutations cause a substantial decrease in the affinity of InhA for NADH, surprisingly the same mutations result in only a small impact on binding of the INH-NAD adduct. Based on the knowledge that InhA interacts in vivo with other components of the FAS-II pathway, we have initiated experiments to determine whether enzyme inhibition results in structural changes that could affect protein-protein interactions involving InhA and how these ligand-induced conformational changes are modulated in the InhA mutants. Significantly, while NADH binding to wild-type InhA is hyperbolic, the InhA mutants bind the cofactor with positive cooperativity, suggesting that the mutations permit access to a second conformational state of the protein. While cross-linking studies indicate that enzyme inhibition causes dissociation of the InhA tetramer into dimers, analytical ultracentrifugation and size exclusion chromatography reveal that ligand binding causes a conformational change in the protein that prevents cross-linking across one of the dimer-dimer interfaces in the InhA tetramer. Interestingly, a similar ligand-induced conformational change is also observed for the InhA mutants, indicating that the mutations modulate communication between the subunits without affecting the two conformational states of the protein that are present.     

Synthesis of various 3-nitropropionamides as Mycobacterium tuberculosis isocitrate lyase inhibitor

Various 3-nitropropionamides were synthesized and evaluated for in vitro activities against log and starved phase culture of two mycobacterial species and Mycobacterium tuberculosis (MTB) isocitrate lyase (ICL) enzyme inhibition studies. Among 22 compounds, 1-cyclopropyl-7-(3,5-dimethyl-4-(3-nitropropanoyl)piperazin-1-yl)-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (22) was found to be the most active compound in vitro with MICs of 0.16 and 0.04 μM against log- and starved-phase culture of MTB. Compound 22 also showed good enzyme inhibition of MTB ICL with IC(50) of 0.10 ± 0.01 μM. The docking studies also confirmed the binding potential of the compounds at the ICL active site.