Mycobacterium tuberculosis eis regulates autophagy, inflammation, and cell death through redox-dependent signaling

The "enhanced intracellular survival" (eis) gene of Mycobacterium tuberculosis (Mtb) is involved in the intracellular survival of M. smegmatis. However, its exact effects on host cell function remain elusive. We herein report that Mtb Eis plays essential roles in modulating macrophage autophagy, inflammatory responses, and cell death via a reactive oxygen species (ROS)-dependent pathway. Macrophages infected with an Mtb eis-deletion mutant H37Rv (Mtb-Δeis) displayed markedly increased accumulation of massive autophagic vacuoles and formation of autophagosomes in vitro and in vivo. Infection of macrophages with Mtb-Δeis increased the production of tumor necrosis factor-α and interleukin-6 over the levels produced by infection with wild-type or complemented strains. Elevated ROS generation in macrophages infected with Mtb-Δeis (for which NADPH oxidase and mitochondria were largely responsible) rendered the cells highly sensitive to autophagy activation and cytokine production. Despite considerable activation of autophagy and proinflammatory responses, macrophages infected with Mtb-Δeis underwent caspase-independent cell death. This cell death was significantly inhibited by blockade of autophagy and c-Jun N-terminal kinase-ROS signaling, suggesting that excessive autophagy and oxidative stress are detrimental to cell survival. Finally, artificial over-expression of Eis or pretreatment with recombinant Eis abrogated production of both ROS and proinflammatory cytokines, which depends on the N-acetyltransferase domain of the Eis protein. Collectively, these data indicate that Mtb Eis suppresses host innate immune defenses by modulating autophagy, inflammation, and cell death in a redox-dependent manner.     

A Random Sequential Mechanism of Aminoglycoside Acetylation by Mycobacterium tuberculosis Eis Protein

An important cause of bacterial resistance to aminoglycoside antibiotics is the enzymatic acetylation of their amino groups by acetyltransferases, which abolishes their binding to and inhibition of the bacterial ribosome. Enhanced intracellular survival (Eis) protein from Mycobacterium tuberculosis (Mt) is one of such acetyltransferases, whose upregulation was recently established as a cause of resistance to aminoglycosides in clinical cases of drug-resistant tuberculosis. The mechanism of aminoglycoside acetylation by MtEis is not completely understood. A systematic analysis of steady-state kinetics of acetylation of kanamycin A and neomycin B by Eis as a function of concentrations of these aminoglycosides and the acetyl donor, acetyl coenzyme A, reveals that MtEis employs a random-sequential bisubstrate mechanism of acetylation and yields the values of the kinetic parameters of this mechanism. The implications of these mechanistic properties for the design of inhibitors of Eis and other aminoglycoside acetyltransferases are discussed.     

Structure of the Mycobacterium tuberculosis proteasome and mechanism of inhibition by a peptidyl boronate

Mycobacterium tuberculosis (Mtb) has the remarkable ability to resist killing by human macrophages. The 750 kDa proteasome, not available in most eubacteria except Actinomycetes, appears to contribute to Mtb's resistance. The crystal structure of the Mtb proteasome at 3.0 A resolution reveals a substrate-binding pocket with composite features of the distinct beta1, beta2 and beta5 substrate binding sites of eukaryotic proteasomes, accounting for the broad specificity of the Mtb proteasome towards oligopeptides described in the companion article [Lin et al. (2006), Mol Microbiol doi:10.1111/j.1365-2958.2005.05035.x]. The substrate entrance at the end of the cylindrical proteasome appears open in the crystal structure due to partial disorder of the alpha-subunit N-terminal residues. However, cryo-electron microscopy of the core particle reveals a closed end, compatible with the density observed in negative-staining electron microscopy that depended on the presence of the N-terminal octapetides of the alpha-subunits in the companion article, suggesting that the Mtb proteasome has a gated structure. We determine for the first time the proteasomal inhibition mechanism of the dipeptidyl boronate N-(4-morpholine)carbonyl-beta-(1-naphthyl)-L-alanine-L-leucine boronic acid (MLN-273), an analogue of the antimyeloma drug bortezomib. The structure improves prospects for designing Mtb-specific proteasomal inhibitors as a novel approach to chemotherapy of tuberculosis.     

Structural analysis of the N‐acetyltransferase Eis1 from Mycobacterium abscessus reveals the molecular determinants of its incapacity to modify aminoglycosides

Enhanced intracellular survival (Eis) proteins belonging to the superfamily of the GCN5‐related N‐acetyltransferases play important functions in mycobacterial pathogenesis. In Mycobacterium tuberculosis, Eis enhances the intracellular survival of the bacilli in macrophages by modulating the host immune response and is capable to chemically modify and inactivate aminoglycosides. In nontuberculous mycobacteria (NTM), Eis shares similar functions. However, Mycobacterium abscessus, a multidrug resistant NTM, possesses two functionally distinct Eis homologues, Eis1_Mab and Eis2_Mab. While Eis2_Mab participates in virulence and aminoglycosides resistance, this is not the case for Eis1_Mab, whose exact biological function remains to be determined. Herein, we show that overexpression of Eis1_Mab in M. abscessus fails to induce resistance to aminoglycosides. To clarify why Eis1_Mab is unable to modify this class of antibiotics, we solved its crystal structure bound to its cofactor, acetyl‐CoA. The structure revealed that Eis1_Mab has a typical homohexameric Eis‐like organization. The structural analysis supported by biochemical approaches demonstrated that while Eis1_Mab can acetylate small substrates, its active site is too narrow to accommodate aminoglycosides. Comparison with other Eis structures showed that an extended loop between strands 9 and 10 is blocking the access of large substrates to the active site and movement of helices 4 and 5 reduces the volume of the substrate‐binding pocket to these compounds in Eis1_Mab. Overall, this study underscores the molecular determinants explaining functional differences between Eis1_Mab and Eis2_Mab, especially those inherent to their capacity to modify aminoglycosides.     

Thermostable hexameric form of Eis (Rv2416c) protein of M. tuberculosis plays an important role for enhanced intracellular survival within macrophages

Eis protein is reported to enhance the intracellular survival of Mycobacterium tuberculosis in human macrophages. Eis protein is not only known to skew away the immunity by disturbing the protective T(H)1 response, but aminoglycoside acetyltransferase activity of Eis is reported to regulate autophagy, inflammation and cell death. Here we have gained insight into the structure-function properties of Eis. Eis protein is a hexameric αβ protein. Although urea and guanidinium hydrochloride (GdmCl) was found to induce one-step unfolding of Eis but size exclusion chromatography showed that GdmCl treated Eis maintained its hexameric form. SDS-PAGE assay confirmed that hexameric form of Eis is partially stable to SDS and converts into trimers and monomers. Out of these three forms, aminoglycoside acetyltransferase activity is found to be associated only with hexamers. The Tm of Eis was found to be ～75°C. Aminoglycoside acetyltransferase Eis demonstrated remarkable heat stability retaining >80% of their activity at 70°C which falls down to ～50% at 75°C and is completely inactive at 80°C. Further, intracellular survival assay with heated samples of M. smegmatis harboring eis gene of M. tuberculosis H37Rv demonstrated a possible role for the thermostability associated with Eis protein in the enhanced intracellular survival within macrophages. In sum, these data reveal that only hexameric form of Eis has a thermostable aminoglycoside acetyltransferase activity. This is the first report showing the thermostability associated with aminoglycoside acetyltransferase activity of Eis protein being one of the essential features for the execution of its biological role.     

The 1.9 A crystal structure of alanine racemase from Mycobacterium tuberculosis contains a conserved entryway into the active site

We report the crystal structure of alanine racemase from Mycobacterium tuberculosis (Alr(Mtb)) at 1.9 A resolution. In our structure, Alr(Mtb) is found to be a dimer formed by two crystallographically different monomers, each comprising 384 residues. The domain makeup of each monomer is similar to that of Bacillus and Pseudomonas alanine racemases and includes both an alpha/beta-barrel at the N-terminus and a C-terminus primarily made of beta-strands. The hinge angle between these two domains is unique for Alr(Mtb), but the active site geometry is conserved. In Alr(Mtb), the PLP cofactor is covalently bound to the protein via an internal aldimine bond with Lys42. No guest substrate is noted in its active site, although some residual electron density is observed in the enzyme's active site pocket. Analysis of the active site pocket, in the context of other known alanine racemases, allows us to propose the inclusion of conserved residues found at the entrance to the binding pocket as additional targets in ongoing structure-aided drug design efforts. Also, as observed in other alanine racemase structures, PLP adopts a conformation that significantly distorts the planarity of the extended conjugated system between the PLP ring and the internal aldimine bond.     

Selective killing of nonreplicating mycobacteria

Antibiotics are typically more effective against replicating rather than nonreplicating bacteria. However, a major need in global health is to eradicate persistent or nonreplicating subpopulations of bacteria such as Mycobacterium tuberculosis (Mtb). Hence, identifying chemical inhibitors that selectively kill bacteria that are not replicating is of practical importance. To address this, we screened for inhibitors of dihydrolipoamide acyltransferase (DlaT), an enzyme required by Mtb to cause tuberculosis in guinea pigs and used by the bacterium to resist nitric oxide-derived reactive nitrogen intermediates, a stress encountered in the host. Chemical screening for inhibitors of Mtb DlaT identified select rhodanines as compounds that almost exclusively kill nonreplicating mycobacteria in synergy with products of host immunity, such as nitric oxide and hypoxia, and are effective on bacteria within macrophages, a cellular reservoir for latent Mtb. Compounds that kill nonreplicating pathogens in cooperation with host immunity could complement the conventional chemotherapy of infectious disease.     

Crystal structures of Mycobacterium tuberculosis S-adenosyl-L-homocysteine hydrolase in ternary complex with substrate and inhibitors

S-adenosylhomocysteine hydrolase (SAHH) is a ubiquitous enzyme that plays a central role in methylation-based processes by maintaining the intracellular balance between S-adenosylhomocysteine (SAH) and S-adenosylmethionine. We report the first prokaryotic crystal structure of SAHH, from Mycobacterium tuberculosis (Mtb), in complex with adenosine (ADO) and nicotinamide adenine dinucleotide. Structures of complexes with three inhibitors are also reported: 3'-keto aristeromycin (ARI), 2-fluoroadenosine, and 3-deazaadenosine. The ARI complex is the first reported structure of SAHH complexed with this inhibitor, and confirms the oxidation of the 3' hydroxyl to a planar keto group, consistent with its prediction as a mechanism-based inhibitor. We demonstrate the in vivo enzyme inhibition activity of the three inhibitors and also show that 2-fluoradenosine has bactericidal activity. While most of the residues lining the ADO-binding pocket are identical between Mtb and human SAHH, less is known about the binding mode of the homocysteine (HCY) appendage of the full substrate. We report the 2.0 A resolution structure of the complex of SAHH cocrystallized with SAH. The most striking change in the structure is that binding of HCY forces a rotation of His363 around the backbone to flip out of contact with the 5' hydroxyl of the ADO and opens access to a nearby channel that leads to the surface. This complex suggests that His363 acts as a switch that opens up to permit binding of substrate, then closes down after release of the cleaved HCY. Differences in the entrance to this access channel between human and Mtb SAHH are identified.     

结核分枝杆菌外排泵研究进展

结核病是由结核分枝杆菌(Mycobacterium tuberculosis,Mtb)引起的一种传染病。随着多药耐药和广泛耐药结核分枝杆菌的出现,结核病的治疗变得更为艰难。近年来研究发现,结核分枝杆菌存在外排泵是其耐药的原因之一,现已发现结核分枝杆菌的主要易化子超家族(major facilitator superfamily,MFS)、三磷酸腺苷(adenosine-triphosphate,ATP)结合盒超家族(ATP-Binding Cassette,ABC)、耐受小节分裂区家族(resistance-nodulation-division,RND)和小耐多药性家族(small multidrug resistance,SMR)外排泵。但是人们对结核分枝杆菌外排泵介导的耐药现象认识不足,仍缺乏从新药发现角度研发外排泵抑制剂的研究。本文拟对结核分枝杆菌的ABC、MFS、RND和SMR外排泵的结构和功能,以及结核分枝杆菌外排泵抑制剂的研究进展进行综述。     

Crystal structures of F420-dependent glucose-6-phosphate dehydrogenase FGD1 involved in the activation of the anti-tuberculosis drug candidate PA-824 reveal the basis of coenzyme and substrate binding

The modified flavin coenzyme F(420) is found in a restricted number of microorganisms. It is widely distributed in mycobacteria, however, where it is important in energy metabolism, and in Mycobacterium tuberculosis (Mtb) is implicated in redox processes related to non-replicating persistence. In Mtb, the F(420)-dependent glucose-6-phosphate dehydrogenase FGD1 provides reduced F(420) for the in vivo activation of the nitroimidazopyran prodrug PA-824, currently being developed for anti-tuberculosis therapy against both replicating and persistent bacteria. The structure of M. tuberculosis FGD1 has been determined by x-ray crystallography both in its apo state and in complex with F(420) and citrate at resolutions of 1.90 and 1.95 A(,) respectively. The structure reveals a highly specific F(420) binding mode, which is shared with several other F(420)-dependent enzymes. Citrate occupies the substrate binding pocket adjacent to F(420) and is shown to be a competitive inhibitor (IC(50) 43 microm). Modeling of the binding of the glucose 6-phosphate (G6P) substrate identifies a positively charged phosphate binding pocket and shows that G6P, like citrate, packs against the isoalloxazine moiety of F(420) and helps promote a butterfly bend conformation that facilitates F(420) reduction and catalysis.     

BCG induces protection against Mycobacterium tuberculosis infection in the Wistar rat model

Our understanding of the correlation of Mycobacterium bovis Bacille Calmette-Guerin (BCG)-mediated immune responses and protection against Mycobacterium tuberculosis (Mtb) infection is still limited. We have recently characterized a Wistar rat model of experimental tuberculosis (TB). In the present study, we evaluated the efficacy of BCG vaccination in this model. Upon Mtb challenge, BCG vaccinated rats controlled growth of the bacilli earlier than unvaccinated rats. Histopathology analysis of infected lungs demonstrated a reduced number of granulomatous lesions and lower parenchymal inflammation in vaccinated animals. Vaccine-mediated protection correlated with the rapid accumulation of antigen specific CD4(+) and CD8(+) T cells in the infected lungs. Immunohistochemistry further revealed higher number of CD8(+) cells in the pulmonary granulomas of vaccinated animals. Evaluation of pulmonary immune responses in vaccinated and Mtb infected rats by real time PCR at day 15 post-challenge showed reduced expression of genes responsible for negative regulation of Th1 immune responses. Thus, early protection observed in BCG vaccinated rats correlated with a similarly timed shift of immunity towards the Th1 type response. Our data support the importance of (i) the Th1-Th2 balance in the control of mycobacterial infection and (ii) the value of the Wistar rats in understanding the biology of TB.     

Allosteric regulation of pyruvate kinase from Mycobacterium tuberculosis by metabolites

Mycobacterium tuberculosis (Mtb) causes both acute tuberculosis and latent, symptom-free infection that affects roughly one-third of the world's population. It is a globally important pathogen that poses multiple dangers. Mtb reprograms its metabolism in response to the host niche, and this adaptation contributes to its pathogenicity. Knowledge of the metabolic regulation mechanisms in Mtb is still limited. Pyruvate kinase, involved in the late stage of glycolysis, helps link various metabolic routes together. Here, we demonstrate that Mtb pyruvate kinase (Mtb PYK) predominantly catalyzes the reaction leading to the production of pyruvate, but its activity is influenced by multiple metabolites from closely interlinked pathways that act as allosteric regulators (activators and inhibitors). We identified allosteric activators and inhibitors of Mtb PYK originating from glycolysis, citrate cycle, nucleotide/nucleoside inter-conversion related pathways that had not been described so far. Enzyme was found to be activated by fructose-1,6-bisphosphate, ribose-5-phosphate, adenine, adenosine, hypoxanthine, inosine, L-2-phosphoglycerate, l-aspartate, glycerol-2-phosphate, glycerol-3-phosphate. On the other hand thiamine pyrophosphate, glyceraldehyde-3-phosphate and L-malate were identified as inhibitors of Mtb PYK. The detailed kinetic analysis indicated a morpheein model of Mtb PYK allosteric control which is strictly dependent on Mg²⁺ and substantially increased by the co-presence of Mg²⁺ and K⁺.     

Crystal structure of Mycobacterium tuberculosis 7,8-dihydropteroate synthase in complex with pterin monophosphate: new insight into the enzymatic mechanism and sulfa-drug action

The enzyme 7,8-dihydropteroate synthase (DHPS) catalyzes the condensation of para-aminobenzoic acid (pABA) with 6-hydroxymethyl-7, 8-dihydropterin-pyrophosphate to form 7,8-dihydropteroate and pyrophosphate. DHPS is essential for the de novo synthesis of folate in prokaryotes, lower eukaryotes, and in plants, but is absent in mammals. Inhibition of this enzyme's activity by sulfonamide and sulfone drugs depletes the folate pool, resulting in growth inhibition and cell death. Here, we report the 1.7 A resolution crystal structure of the binary complex of 6-hydroxymethylpterin monophosphate (PtP) with DHPS from Mycobacterium tuberculosis (Mtb), a pathogen responsible for the death of millions of human beings each year. Comparison to other DHPS structures reveals that the M. tuberculosis DHPS structure is in a unique conformation in which loop 1 closes over the active site. The Mtb DHPS structure hints at a mechanism in which both loops 1 and 2 play important roles in catalysis by shielding the active site from bulk solvent and allowing pyrophosphoryl transfer to occur. A binding mode for pABA, sulfonamides and sulfones is suggested based on: (i) the new conformation of the closed loop 1; (ii) the distribution of dapsone and sulfonamide resistance mutations; (iii) the observed direction of the bond between the 6-methyl carbon atom and the bridging oxygen atom to the alpha-phosphate group in the Mtb DHPS:PtP binary complex; and (iv) the conformation of loop 2 in the Escherichia coli DHPS structure. Finally, the Mtb DHPS structure reveals a highly conserved pterin binding pocket that may be exploited for the design of novel antimycobacterial agents.     

Discovery of novel MDR-Mycobacterium tuberculosis inhibitor by new FRIGATE computational screen

With 1.6 million casualties annually and 2 billion people being infected, tuberculosis is still one of the most pressing healthcare challenges. Here we report on the new computational docking algorithm FRIGATE which unites continuous local optimization techniques (conjugate gradient method) with an inherently discrete computational approach in forcefield computation, resulting in equal or better scoring accuracies than several benchmark docking programs. By utilizing FRIGATE for a virtual screen of the ZINC library against the Mycobacterium tuberculosis (Mtb) enzyme antigen 85C, we identified novel small molecule inhibitors of multiple drug-resistant Mtb, which bind in vitro to the catalytic site of antigen 85C.     

Improved control of tuberculosis and activation of macrophages in mice lacking protein kinase R

Host factors that microbial pathogens exploit for their propagation are potential targets for therapeuic countermeasures. No host enzyme has been identified whose genetic absence benefits the intact mammalian host in vivo during infection with Mycobacterium tuberculosis (Mtb), the leading cause of death from bacterial infection. Here, we report that the dsRNA-dependent protein kinase (PKR) is such an enzyme. PKR-deficient mice contained fewer viable Mtb and showed less pulmonary pathology than wild type mice. We identified two potential mechanisms for the protective effect of PKR deficiency: increased apoptosis of macrophages in response to Mtb and enhanced activation of macrophages in response to IFN-gamma. The restraining effect of PKR on macrophage activation was explained by its mediation of a previously unrecognized ability of IFN-gamma to induce low levels of the macrophage deactivating factor interleukin 10 (IL10). These observations suggest that PKR inhibitors may prove useful as an adjunctive treatment for tuberculosis.     

A conserved dimer and global conformational changes in the structure of apo-PknE Ser/Thr protein kinase from Mycobacterium tuberculosis

The "eukaryotic-like" receptor Ser/Thr protein kinases (STPKs) are candidates for the sensors that mediate environmental adaptations of Mycobacterium tuberculosis (Mtb). To define the mechanisms of regulation and substrate recognition, we determined the crystal structure of the ligand-free, activated kinase domain (KD) of the Mtb STPK, PknE. Remarkably, the PknE KD formed a dimer similar to that first observed in the structure of the ATPgammaS complex of the Mtb paralog, PknB. This structural similarity, which occurs despite little sequence conservation between the PknB and PknE dimer interfaces, supports the idea that dimerization regulates the Mtb receptor STPKs. Insertion of the DFG motif into the ATP-binding site and other conformational differences compared the ATPgammaS:PknB complex suggest that apo-PknE is not pre-organized to bind nucleotides. This structure may represent an inactive conformation stabilized by dimerization or, alternatively, an active conformation that reveals shifts that mediate nucleotide exchange and order substrate binding.     

Sulfolipid-1 biosynthesis restricts Mycobacterium tuberculosis growth in human macrophages

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a highly evolved human pathogen characterized by its formidable cell wall. Many unique lipids and glycolipids from the Mtb cell wall are thought to be virulence factors that mediate host-pathogen interactions. An intriguing example is Sulfolipid-1 (SL-1), a sulfated glycolipid that has been implicated in Mtb pathogenesis, although no direct role for SL-1 in virulence has been established. Previously, we described the biochemical activity of the sulfotransferase Stf0 that initiates SL-1 biosynthesis. Here we show that a stf0-deletion mutant exhibits augmented survival in human but not murine macrophages, suggesting that SL-1 negatively regulates the intracellular growth of Mtb in a species-specific manner. Furthermore, we demonstrate that SL-1 plays a role in mediating the susceptibility of Mtb to a human cationic antimicrobial peptide in vitro, despite being dispensable for maintaining overall cell envelope integrity. Thus, we hypothesize that the species-specific phenotype of the stf0 mutant is reflective of differences in antimycobacterial effector mechanisms of macrophages.     

Lysine acetylation of the Mycobacterium tuberculosis HU protein modulates its DNA binding and genome organization

Nucleoid‐associated protein HU, a conserved protein across eubacteria is necessary for maintaining the nucleoid organization and global regulation of gene expression. Mycobacterium tuberculosis HU (MtHU) is distinct from the other orthologues having 114 amino acid long carboxyl terminal extensions with a high degree of sequence similarity to eukaryotic histones. In this study, we demonstrate that the DNA binding property of MtHU is regulated by posttranslational modifications akin to eukaryotic histones. MtHU purified from M. tuberculosis cells is found to be acetylated on multiple lysine residues unlike the E. coli expressed recombinant protein. Using coimmunoprecipitation assay, we identified Eis as one of the acetyl transferases that interacts with MtHU and modifies it. Although Eis is known to acetylate aminoglycosides, the kinetics of acetylation showed that its protein acetylation activity on MtHU is robust. In vitro Eis modified MtHU at various lysine residues, primarily those located at the carboxyl terminal domain. Acetylation of MtHU caused reduced DNA interaction and alteration in DNA compaction ability of the NAP. Over‐expression of the Eis leads to hyperacetylation of HU and decompaction of genome. These results provide first insights into the modulation of the nucleoid structure by lysine acetylation in bacteria.     

Replication Rates of Mycobacterium tuberculosis in Human Macrophages Do Not Correlate with Mycobacterial Antibiotic Susceptibility

The standard treatment of tuberculosis (TB) takes six to nine months to complete and this lengthy therapy contributes to the emergence of drug-resistant TB. TB is caused by Mycobacterium tuberculosis (Mtb) and the ability of this bacterium to switch to a dormant phenotype has been suggested to be responsible for the slow clearance during treatment. A recent study showed that the replication rate of a non-virulent mycobacterium, Mycobacterium smegmatis, did not correlate with antibiotic susceptibility. However, the question whether this observation also holds true for Mtb remains unanswered. Here, in order to mimic physiological conditions of TB infection, we established a protocol based on long-term infection of primary human macrophages, featuring Mtb replicating at different rates inside the cells. During conditions that restricted Mtb replication, the bacterial phenotype was associated with reduced acid-fastness. However, these phenotypically altered bacteria were as sensitive to isoniazid, pyrazinamide and ethambutol as intracellularly replicating Mtb. In support of the recent findings with M. smegmatis, we conclude that replication rates of Mtb do not correlate with antibiotic tolerance.