Gram-positive DsbE proteins function differently from Gram-negative DsbE homologs. A structure to function analysis of DsbE from Mycobacterium tuberculosis

Mycobacterium tuberculosis, a Gram-positive bacterium, encodes a secreted Dsb-like protein annotated as Mtb DsbE (Rv2878c, also known as MPT53). Because Dsb proteins in Escherichia coli and other bacteria seem to catalyze proper folding during protein secretion and because folding of secreted proteins is thought to be coupled to disulfide oxidoreduction, the function of Mtb DsbE may be to ensure that secreted proteins are in their correctly folded states. We have determined the crystal structure of Mtb DsbE to 1.1 A resolution, which reveals a thioredoxin-like domain with a typical CXXC active site. These cysteines are in their reduced state. Biochemical characterization of Mtb DsbE reveals that this disulfide oxidoreductase is an oxidant, unlike Gram-negative bacteria DsbE proteins, which have been shown to be weak reductants. In addition, the pK(a) value of the active site, solvent-exposed cysteine is approximately 2 pH units lower than that of Gram-negative DsbE homologs. Finally, the reduced form of Mtb DsbE is more stable than the oxidized form, and Mtb DsbE is able to oxidatively fold hirudin. Structural and biochemical analysis implies that Mtb DsbE functions differently from Gram-negative DsbE homologs, and we discuss its possible functional role in the bacterium.     

Mycobacterium tuberculosis requires SufT for Fe-S cluster maturation,metabolism, and survival in vivo

Iron-sulfur (Fe-S) cluster proteins carry out essential cellular functions in diverse organisms, including the human pathogen Mycobacterium tuberculosis (Mtb). The mechanisms underlying Fe-S cluster biogenesis are poorly defined in Mtb. Here, we show that Mtb SufT (Rv1466), a DUF59 domain-containing essential protein, is required for the Fe-S cluster maturation. Mtb SufT homodimerizes and interacts with Fe-S cluster biogenesis proteins; SufS and SufU. SufT also interacts with the 4Fe-4S cluster containing proteins; aconitase and SufR. Importantly, a hyperactive cysteine in the DUF59 domain mediates interaction of SufT with SufS, SufU, aconitase, and SufR. We efficiently repressed the expression of SufT to generate a SufT knock-down strain in Mtb (SufT-KD) using CRISPR interference. Depleting SufT reduces aconitase’s enzymatic activity under standard growth conditions and in response to oxidative stress and iron limitation. The SufT-KD strain exhibited defective growth and an altered pool of tricarboxylic acid cycle intermediates, amino acids, and sulfur metabolites. Using Seahorse Extracellular Flux analyzer, we demonstrated that SufT depletion diminishes glycolytic rate and oxidative phosphorylation in Mtb. The SufT-KD strain showed defective survival upon exposure to oxidative stress and nitric oxide. Lastly, SufT depletion reduced the survival of Mtb in macrophages and attenuated the ability of Mtb to persist in mice. Altogether, SufT assists in Fe-S cluster maturation and couples this process to bioenergetics of Mtb for survival under low and high demand for Fe-S clusters.     

The Mycobacterium tuberculosis Rv2745c Plays an Important Role in Responding to Redox Stress

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death from an infectious disease worldwide. Over the course of its life cycle in vivo, Mtb is exposed to a plethora of environmental stress conditions. Temporal regulation of genes involved in sensing and responding to such conditions is therefore crucial for Mtb to establish an infection. The Rv2745c (clgR) gene encodes a Clp protease gene regulator that is induced in response to a variety of stress conditions and potentially plays a role in Mtb pathogenesis. Our isogenic mutant, Mtb:ΔRv2745c, is significantly more sensitive to in vitro redox stress generated by diamide, relative to wild-type Mtb as well as to a complemented strain. Together with the fact that the expression of Rv2745c is strongly induced in response to redox stress, these results strongly implicate a role for ClgR in the management of intraphagosomal redox stress. Additionally, we observed that redox stress led to the dysregulation of the expression of the σ^H/σ^E regulon in the isogenic mutant, Mtb:ΔRv2745c. Furthermore, induction of clgR in Mtb and Mtb:ΔRv2745c (comp) did not lead to Clp protease induction, indicating that clgR has additional functions that need to be elucidated. Our data, when taken together with that obtained by other groups, indicates that ClgR plays diverse roles in multiple regulatory networks in response to different stress conditions. In addition to redox stress, the expression of Rv2745c correlates with the expression of genes involved in sulfate assimilation as well as in response to hypoxia and reaeration. Clearly, the Mtb Rv2745c-encoded ClgR performs different functions during stress response and is important for the pathogenicity of Mtb in-vivo, regardless of its induction of the Clp proteolytic pathway.     

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.     

The Molecular Structure of Ornithine Acetyltransferase from Mycobacterium tuberculosis Bound to Ornithine, a Competitive Inhibitor

Mycobacterium tuberculosis ornithine acetyltransferase (Mtb OAT; E.C. 2.3.1.35) is a key enzyme of the acetyl recycling pathway during arginine biosynthesis. It reversibly catalyzes the transfer of the acetyl group from N-acetylornithine (NAORN) to l-glutamate. Mtb OAT is a member of the N-terminal nucleophile fold family of enzymes. The crystal structures of Mtb OAT in native form and in its complex with ornithine (ORN) have been determined at 1.7 and 2.4 Å resolutions, respectively. ORN is a competitive inhibitor of this enzyme against l-glutamate as substrate. Although the acyl-enzyme complex of Streptomyces clavuligerus ornithine acetyltransferase has been determined, ours is the first crystal structure to be reported of an ornithine acetyltransferase in complex with an inhibitor. ORN binding does not alter the structure of Mtb OAT globally. However, its presence stabilizes the three C-terminal residues that are disordered and not observed in the native structure. Also, stabilization of the C-terminal residues by ORN reduces the size of the active-site pocket volume in the structure of the ORN complex. The interactions of ORN and the protein residues of Mtb OAT unambiguously delineate the active-site residues of this enzyme in Mtb. Moreover, modeling studies carried out with NAORN based on the structure of the ORN-Mtb OAT complex reveal important interactions of the carbonyl oxygen of the acetyl group of NAORN with the main-chain nitrogen atom of Gly128 and with the side-chain oxygen of Thr127. These interactions likely help in the stabilization of oxyanion formation during enzymatic reaction and also will polarize the carbonyl carbon-oxygen bond, thereby enabling the side-chain atom O^γ1 of Thr200 to launch a nucleophilic attack on the carbonyl-carbon atom of the acetyl group of NAORN.     

Effects of mutations in genes for proteins involved in disulphide bond formation in the periplasm on the activities of anaerobically induced electron transfer chains in Escherichia coli K12

The assembly of anaerobically induced electron transfer chains in Escherichia coli strains defective in periplasmic disulphide bond formation was investigated. Strains deficient in DsbA, DsbB or DipZ (DsbD) were unable to catalyse formate-dependent nitrite reduction (Nrf activity) or synthesize any of the known c-type cytochromes. The Nrf⁺ activity and cytochrome c content of mutants defective in DsbC, DsbE or DsbF were similar to those of the parental, wild-type strain. Neither DsbC expressed from a multicopy plasmid nor a second mutation in dipZ (dsbD) was able to compensate for a dsbA mutation by restoring nitrite reductase activity and cytochrome c synthesis. In contrast, only the dsbB and dipZ (dsbD) strains were defective in periplasmic nitrate reductase activity, suggesting that DsbB might fulfil an additional role in anaerobic electron transport. Mutants defective in dipZ (dsbD) were only slightly more sensitive to Cu⁺⁺ ions at concentrations above 5?mM than the parental strain, but strains defective in DsbA, DsbB, DsbC, DsbE or DsbF were unaffected. These results are consistent with our earlier proposals that DsbA, DsbB and DipZ (DsbD) are part of the same pathway for ensuring that haem groups are attached to the correct pairs of cysteine residues of apocytochromes c in the E. coli periplasm. However, neither DsbE nor DsbF are essential for the reduction of DipZ (DsbD).     

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.     

Two endoplasmic reticulum PDI peroxidases increase the efficiency of the use of peroxide during disulfide bond formation

Disulfide bond formation in the endoplasmic reticulum by the sulfhydryl oxidase Ero1 family is thought to be accompanied by the concomitant formation of hydrogen peroxide. Since secretory cells can make substantial amounts of proteins that contain disulfide bonds, the production of this reactive oxygen species could have potentially lethal consequences. Here, we show that two human proteins, GPx7 and GPx8, labeled as secreted glutathione peroxidases, are actually endoplasmic reticulum-resident protein disulfide isomerase peroxidases. In vitro, the addition of GPx7 or GPx8 to a folding protein along with protein disulfide isomerase and peroxide enables the efficient oxidative refolding of a reduced denatured protein. Furthermore, both GPx7 and GPx8 interact with Ero1α in vivo, and GPx7 significantly increases oxygen consumption by Ero1α in vitro. Hence, GPx7 and GPx8 may represent a novel route for the productive use of peroxide produced by Ero1α during disulfide bond formation.     

Rv1915 and Rv1916 from Mycobacterium tuberculosis H37Rv form in vitro protein-protein complex

BackgroundMycobacterium tuberculosis (Mtb) isocitrate lyase (ICL) is an established drug target that facilitates Mtb persistence. Unlike other mycobacterial strains, where ICL2 is a single gene product, H37Rv has a split event, resulting in two tandemly coded icls - rv1915 and rv1916. Our recent report on functionality of individual Rv1915 and Rv1916, led to postulate the cooperative role of these proteins in pathogen's survival under nutrient-limiting conditions. This study investigates the possibility of Rv1915 and Rv1916 interacting and forming a complex.MethodsPull down assay, activity assay, mass spectrometry and site directed mutagenesis was employed to investigate and validate Rv1915-Rv1916 complex formation.ResultsRv1915 and Rv1916 form a stable complex in vitro, with enhanced ICL/MICL activities as opposed to individual proteins. Further, activities monitored in the presence of acetyl-CoA show significant increase for Rv1916 and the complex but not of Rv0467 and Rv1915Δ90CT. Both full length and truncated Rv1915Δ90CT can form complex, implying the absence of its C-terminal disordered region in complex formation. Further, in silico analysis and site-directed mutagenesis studies reveal Y64 and Y65 to be crucial residues for Rv1915-Rv1916 complex formation.ConclusionsThis study uncovers the association between Rv1915 and Rv1916 and supports the role of acetyl-CoA in escalating the ICL/MICL activities of Rv1916 and Rv1915Δ90CT-Rv1916 complex.General significancePartitioning of ICL2 into Rv1915 and Rv1916 that associates to form a complex in Mtb H37Rv, suggests its importance in signaling and regulation of metabolic pathway particularly in carbon assimilation.     

Crystal structure of the arginine repressor protein in complex with the DNA operator from Mycobacterium tuberculosis

The arginine repressor (ArgR) from Mycobacterium tuberculosis (Mtb) is a gene product encoded by the open reading frame Rv1657. It regulates the l-arginine concentration in cells by interacting with ARG boxes in the promoter regions of the arginine biosynthesis and catabolism operons. Here we present a 2.5-Å structure of MtbArgR in complex with a 16-bp DNA operator in the absence of arginine. A biological trimer of the protein-DNA complex is formed via the crystallographic 3-fold symmetry axis. The N-terminal domain of MtbArgR has a winged helix-turn-helix motif that binds to the major groove of the DNA. This structure shows that, in the absence of arginine, the ArgR trimer can bind three ARG box half-sites. It also reveals the structure of the whole MtbArgR molecule itself containing both N-terminal and C-terminal domains.     

Structural and biochemical characterization of Rv2140c,a phosphatidylethanolamine-binding protein from Mycobacterium tuberculosis

Rv2140c is one of many conserved Mycobacterium tuberculosis proteins for which no molecular function has been identified. We have determined a high-resolution crystal structure of the Rv2140c gene product, which reveals a dimeric complex that shares strong structural homology with the phosphatidylethanolamine-binding family of proteins. Rv2140c forms low-millimolar interactions with a selection of soluble phosphatidylethanolamine analogs, indicating that it has a role in lipid metabolism. Furthermore, the small molecule locostatin binds to the Rv2140c ligand-binding site and also inhibits the growth of the model organism Mycobacterium smegmatis.     

Molecular insights into the binding of coenzyme F420 to the conserved protein Rv1155 from Mycobacterium tuberculosis

Coenzyme F₄₂₀ is a deazaflavin hydride carrier with a lower reduction potential than most flavins. In Mycobacterium tuberculosis (Mtb), F₄₂₀ plays an important role in activating PA-824, an antituberculosis drug currently used in clinical trials. Although F₄₂₀ is important to Mtb redox metabolism, little is known about the enzymes that bind F₄₂₀ and the reactions that they catalyze. We have identified a novel F₄₂₀-binding protein, Rv1155, which is annotated in the Mtb genome sequence as a putative flavin mononucleotide (FMN)-binding protein. Using biophysical techniques, we have demonstrated that instead of binding FMN or other flavins, Rv1155 binds coenzyme F₄₂₀. The crystal structure of the complex of Rv1155 and F₄₂₀ reveals one F₄₂₀ molecule bound to each monomer of the Rv1155 dimer. Structural, biophysical, and bioinformatic analyses of the Rv1155–F₄₂₀ complex provide clues about its role in the bacterium.     

Analysis of nucleoside-binding proteins by ligand-specific elution from dye resin: application to Mycobacterium tuberculosis aldehyde dehydrogenases

We show that Cibacron Blue F3GA dye resin chromatography can be used to identify ligands that specifically interact with proteins from Mycobacterium tuberculosis, and that the identification of these ligands can facilitate structure determination by enhancing the quality of crystals. Four native Mtb proteins of the aldehyde dehydrogenase (ALDH) family were previously shown to be specifically eluted from a Cibacron Blue F3GA dye resin with nucleosides. In this study we characterized the nucleoside-binding specificity of one of these ALDH isozymes (recombinant Mtb Rv0223c) and compared these biochemical results with co-crystallization experiments with different Rv0223c-nucleoside pairings. We found that the strongly interacting ligands (NAD and NADH) aided formation of high-quality crystals, permitting solution of the first Mtb ALDH (Rv0223c) structure. Other nucleoside ligands (AMP, FAD, adenosine, GTP and NADP) exhibited weaker binding to Rv0223c, and produced co-crystals diffracting to lower resolution. Difference electron density maps based on crystals of Rv0223c with various nucleoside ligands show most share the binding site where the natural ligand NAD binds. From the high degree of similarity of sequence and structure compared to human mitochondrial ALDH-2 (BLAST Z-score = 53.5 and RMSD = 1.5 Å), Rv0223c appears to belong to the ALDH-2 class. An altered oligomerization domain in the Rv0223c structure seems to keep this protein as monomer whereas native human ALDH-2 is a multimer.     

Mycobacterium tuberculosis Rv1090 and Rv1987 encode functional β-glucan-targeting proteins

Mycobacterium tuberculosis is a facultative intracellular pathogen, and the ability of this bacterium to survive and to grow inside macrophages is central to its virulence. Multiple strategies are employed by M. tuberculosis to ensure survival in macrophages, including secretion of several proteins, which are good candidates to be virulence factors, drug targets for disease intervention, and vaccine antigens. However, some M. tuberculosis secreted proteins do not appear to play any role in the growth or survival of the bacterium in its mammalian host. Among these proteins are three putative cellulose-targeting proteins encoded by the genes Rv0062, Rv1090, and Rv1987. It has been previously shown that Rv0062 encodes an active cellulase. Here we report that Rv1090 and Rv1987 also encode functional proteins. Rv1090 is able to hydrolyze barley β-glucan while Rv1987 displays cellulose-binding activity on filter paper and on microcrystalline cellulose (Avicel). Collectively, these observations point toward a unique unknown relationship between M. tuberculosis and a cellulose-containing host. We hypothesize that amoeba could be such hosts.     

大肠杆菌二硫键形成相关蛋白的结构、功能及其在基因工程表达外源蛋白上的应用

大肠杆菌分泌蛋白二硫键的形成是一系列蛋白协同作用的结果,主要是Dsb家族蛋白,迄今为止共发现了DsbA、DsbB、DsbC、DsbD、DsbE和DsbG。在体内,DsbA负责氧化两个巯基形成二硫键,DsbB则负责DsbA的再氧化。DsbC和DsbG负责校正DsbA导入的异常二硫键,DsbD则负责对DsbC和DsbG进行再还原,DsbE的功能与DsbD类似。除了直接和二硫键的形成相关外,DsbA、DsbC和DsbG都有分子伴侣功能。它们的分子伴侣功能独立于二硫键形成酶的活性并且对二硫键形成酶活性具有明显的促进作用。基于Dsb蛋白的功能特性,利用它们以大肠杆菌为宿主表达外源蛋白,特别是含有二硫键的蛋白,取得了很多成功的例子。本文简要介绍了这方面的进展,显示Dsb蛋白在促进外源蛋白在大肠杆菌中以可溶形式表达方面具有广阔的应用前景。     

Twin-Arginine Translocation of Active Human Tissue Plasminogen Activator in Escherichia coli

When eukaryotic proteins with multiple disulfide bonds are expressed at high levels in Escherichia coli, the efficiency of thiol oxidation and isomerization is typically not sufficient to yield soluble products with native structures. Even when such proteins are secreted into the oxidizing periplasm or expressed in the cytoplasm of cells carrying mutations in the major intracellular disulfide bond reduction systems (e.g., trxB gor mutants), correct folding can be problematic unless a folding modulator is simultaneously coexpressed. In the present study we explored whether the bacterial twin-arginine translocation (Tat) pathway could serve as an alternative expression system for obtaining appreciable levels of recombinant proteins which exhibit complex patterns of disulfide bond formation, such as full-length human tissue plasminogen activator (tPA) (17 disulfides) and a truncated but enzymatically active version of tPA containing nine disulfides (vtPA). Remarkably, targeting of both tPA and vtPA to the Tat pathway resulted in active protein in the periplasmic space. We show here that export by the Tat translocator is dependent upon oxidative protein folding in the cytoplasm of trxB gor cells prior to transport. Whereas previous efforts to produce high levels of active tPA or vtPA in E. coli required coexpression of the disulfide bond isomerase DsbC, we observed that Tat-targeted vtPA and tPA reach a native conformation without thiol-disulfide oxidoreductase coexpression. These results demonstrate that the Tat system may have inherent and unexpected benefits compared with existing expression strategies, making it a viable alternative for biotechnology applications that hinge on protein expression and secretion.     

Homogeneous recombinant Mycobacterium tuberculosis shikimate dehydrogenase production: An essential step towards target-based drug design

Mycobacterium tuberculosis shikimate dehydrogenase (MtbSD) catalyzes the forth reaction in the shikimate pathway. Here we describe production of K69A, K69H, K69I, K69Q, D105A, and D105N mutant proteins. Screening of several conditions was performed to optimize MtbSD production yield, and an improved purification protocol to obtain homogeneous MtbSD is presented. The rational design of new antitubercular drugs hinges on the availability of M. tuberculosis proteins. Our results show that optimization of expression, disruption, and purification protocols resulted in a higher yield of functional MtbSD enzyme.     

NapA (Rv0430), a Novel Nucleoid-Associated Protein that Regulates a Virulence Operon in Mycobacterium tuberculosis in a Supercoiling-Dependent Manner

Comparison of Mycobacterium tuberculosis with Escherichia coli reveals a reduction in the diversity of DNA-managing proteins, such as DNA topoisomerases, although genome sizes are similar for the two species. The same is true for nucleoid-associated proteins (NAPs), important factors in bacterial chromosome compaction, chromosome remodeling, and regulation of gene expression. In a search for still uncharacterized NAPs, we found that M. tuberculosis protein Rv0430 has NAP-like features: it binds to DNA in a length- and supercoil-dependent fashion, prefers A/T-rich DNA sequences, protects DNA from damaging agents, and modulates DNA supercoiling. At a ratio of 1 dimer/40 bps of DNA, Rv0430 bridges distant DNA segments; at 1 dimer/20 bps, it coats DNA, forming inflexible rods. Rv0430 also stimulates the DNA relaxation activity of topoisomerase I. Remarkably, Rv0430 stimulates its own promoter in a supercoil-dependent manner. It is the first gene of an operon harboring two regulators of M. tuberculosis virulence (virR and sodC), and controls the expression of these downstream virulence regulators and therefore itself is a virulence regulator. The sensitivity of rv0430 expression to supercoiling is consistent with supercoiling being important for infection by M. tuberculosis. Thus, Rv0430 is a novel NAP, doubling up as a topology modulator of M. tuberculosis.