Evaluation of Rv0220, Rv2958c,Rv2994 and Rv3347c of Mycobacterium tuberculosis for serodiagnosis of tuberculosis

Tuberculosis (TB), the leading cause of death among infectious diseases worldwide, is caused by Mycobacterium tuberculosis (M. tuberculosis). Early accurate diagnosis means earlier prevention, treatment and control of TB. To confirm efficient diagnostic antigens for M. tuberculosis, the serodiagnosis value of four recombinant proteins including Rv0220, Rv2958c, Rv2994 and Rv3347c was evaluated in this study. The specificities and sensitivities of four recombinant proteins were determined based on enzyme‐linked immunosorbent assay (ELISA) by screening sera from smear‐positive pulmonary TB patients (n = 92), uninfected individuals (n = 60) and patients with Mycoplasma pneumoniae (n = 32) that potentially cross‐react with M. tuberculosis. The ELISAs showed that Rv0220, Rv2958c, Rv2994 and Rv3347c exhibited high specificities and sensitivities in detecting immunoglobulin G (IgG) antibody, with 98.3/91.3%, 91.7/85.9%, 93.3/89.1% and 93.3/80.4% respectively. According to the receiver‐operating characteristic (ROC) analysis, the area under the ROC of the target proteins was 0.988, 0.969, 0.929 and 0.945 respectively. Western blot was established to evaluate the immunoreactivities of target proteins to mice and human sera. Results demonstrated that Rv0220, Rv2958c, Rv2994 and Rv3347c could specifically recognize TB‐positive sera and the sera of mice immunized with the corresponding protein. Thus, Rv0220, Rv2958c, Rv2994 and Rv3347c were valuable potential diagnostic antigens for M. tuberculosis.     

Molecular characterization of secretory proteins Rv3619c and Rv3620c from Mycobacterium tuberculosis H37Rv

Rv3619c and Rv3620c are the secretory, antigenic proteins of the ESAT-6/CFP-10 family of Mycobacterium tuberculosis H37Rv. In this article, we show that Rv3619c interacts with Rv3620c to form a 1 : 1 heterodimeric complex with a dissociation constant (K(d)) of 4.8 × 10(-7) M. The thermal unfolding of the heterodimer was completely reversible, with a T(m) of 48 °C. The comparative thermodynamics and thermal unfolding analysis of the Rv3619c-Rv3620c dimer, the ESAT-6-CFP-10 dimer and another ESAT family heterodimer, Rv0287-Rv0288, revealed that the binding strength and stability of Rv3619c-Rv3620c are relatively lower than those of the other two pairs. Molecular modeling and docking studies predict the structure of Rv3619c-Rv3620c to be similar to that of ESAT-6-CFP-10. Spectroscopic studies revealed that, in an acidic environment, Rv3619c and Rv3620c lose their secondary structure and interact weakly to form a complex with a lower helical content, indicating that Rv3619c-Rv3620c is destabilized at low pH. These results, combined with those of previous studies, suggest that unfolding of the proteins is required for dissociation of the complex and membrane binding. In the presence of membrane mimetics, the α-helical contents of Rv3619c and Rv3620 increased by 42% and 35%, respectively. In mice, the immune response against Rv3619c protein is characterized by increased levels of interferon-γ, interleukin-12 and IgG(2a) , indicating a dominant Th1 response, which is mandatory for protection against mycobacterial infection. This study therefore emphasizes the potential of Rv3619c as a subunit vaccine candidate.     

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.     

Rv0802c acetyltransferase from Mycobacterium tuberculosis H37Rv

Rv0802c acetyltransferase is a mycobacterial RNase E-associated protein. 6His and FLAG-tagged acetyltransferase was cloned from Mycobacterium tuberculosis H37Rv, expressed in Escherichia coli and partially purified. It is a 25 kDa protein showing a modest sequence homology with other acetyltransferases. The R-X-X-G-X-G sequence for acetyl-coenzyme A recognition and binding can be found in the molecule.     

Characterization of culture filtrate proteins Rv1197 and Rv1198 of ESAT-6 family from Mycobacterium tuberculosis H37Rv

BackgroundWe have characterized two immunogenic proteins, Rv1197 and Rv1198, of the Esx-5 system of the ESAT-6 family of Mycobacterium tuberculosis H37Rv.MethodsThe complex formation between Rv1197 and Rv1198 was characterized by biophysical techniques. The reactivity of serum from TB patients towards these proteins was characterized by ELISA. Lymphocyte proliferation and cytokine induction were followed in restimulated splenocytes from immunized mice by using MTT assay and CBA flowcytometry, respectively.ResultsRv1197 and Rv1198 strongly interact to form a heterodimeric complex under reducing conditions, which is characterized by a dissociation constant of 97 × 10^− 9 M and melting temperature, Tm, of 50.5 °C. Strong humoral responses to Rv1197, Rv1198, CFP-10 and MoaC1 (Rv3111) antigens were found in Indian patients with active pulmonary tuberculosis (n = 44), in comparison to non-infected healthy individuals (n = 20). The seroreactivity to Rv1198 was characterized by a sensitivity of 75% and specificity of 90%. In BALB/c mice, immunization with Rv1198-FIA induced a pro-inflammatory response with elevated levels of TNF and IL-6, along with low induction of IFN-γ, IL-2 and IL-10, but no induction of IL-4.ConclusionRv1197 and Rv1198 form a stable complex, which is regulated by the redox state of Rv1198. Rv1198 is immunogenic with highly specific seroreactivity towards TB patients' serum. Rv1198 elicits a pro-inflammatory recall response in immunized mice.General SignificanceThis study characterizes the interaction of Rv1197 and Rv1198, and establishes the immunogenic nature of Rv1198.     

Role of the DinB Homologs Rv1537 and Rv3056 in Mycobacterium tuberculosis

The environment encountered by Mycobacterium tuberculosis during infection is genotoxic. Most bacteria tolerate DNA damage by engaging specialized DNA polymerases that catalyze translesion synthesis (TLS) across sites of damage. M. tuberculosis possesses two putative members of the DinB class of Y-family DNA polymerases, DinB1 (Rv1537) and DinB2 (Rv3056); however, their role in damage tolerance, mutagenesis, and survival is unknown. Here, both dinB1 and dinB2 are shown to be expressed in vitro in a growth phase-dependent manner, with dinB2 levels 12- to 40-fold higher than those of dinB1. Yeast two-hybrid analyses revealed that DinB1, but not DinB2, interacts with the β-clamp, consistent with its canonical C-terminal β-binding motif. However, knockout of dinB1, dinB2, or both had no effect on the susceptibility of M. tuberculosis to compounds that form N²-dG adducts and alkylating agents. Similarly, deletion of these genes individually or in combination did not affect the rate of spontaneous mutation to rifampin resistance or the spectrum of resistance-conferring rpoB mutations and had no impact on growth or survival in human or mouse macrophages or in mice. Moreover, neither gene conferred a mutator phenotype when expressed ectopically in Mycobacterium smegmatis. The lack of the effect of altering the complements or expression levels of dinB1 and/or dinB2 under conditions predicted to be phenotypically revealing suggests that the DinB homologs from M. tuberculosis do not behave like their counterparts from other organisms.The emergence and global spread of multi- and extensively drug-resistant strains of Mycobacterium tuberculosis have further complicated the already daunting challenge of controlling tuberculosis (TB) (15). The mechanisms that underlie the evolution of drug resistance in M. tuberculosis by chromosomal mutagenesis and their association with the conditions that tubercle bacilli encounter during the course of infection are poorly understood (6). It has been postulated that hypoxia, low pH, nutrient deprivation, and nitrosative and oxidative stress impose environmental and host immune-mediated DNA-damaging insults on infecting bacilli (64). In addition, the observed importance of excision repair pathways for the growth and survival of M. tuberculosis in murine models of infection (13, 55) and the upregulation of M. tuberculosis genes involved in DNA repair and modification in pulmonary TB in humans provide compelling evidence that the in vivo environment is DNA damaging (51).Damage tolerance constitutes an integral component of an organism''s response to genotoxic stress, preventing collapse of the replication fork at persisting, replication-blocking lesions through the engagement of specialized DNA polymerases that are able to catalyze translesion synthesis (TLS) across the sites of damage (19, 21, 60). Most TLS polymerases belong to the Y family, which comprises a wide range of structurally related proteins present in bacteria, archaea, and eukaryotes (44). Of these, the DinB subfamily of Y family polymerases, whose founder member is Escherichia coli Pol IV (63), is conserved among all domains of life (44). The association of Y family polymerases with inducible mutagenesis has implicated these enzymes in the adaptation of bacteria to environmental stress (17, 20, 39, 54, 58, 59, 66). Their key properties are exemplified in E. coli Pol IV: the polymerase catalyzes efficient and accurate TLS across certain N²-dG adducts (27, 28, 34, 40, 45, 67) and has been implicated in the tolerance of alkylation damage (4); furthermore, overexpression of Pol IV significantly increases mutation rates in E. coli (reviewed in references 21 and 26), and dinB is the only SOS-regulated gene required at induced levels for stress-induced mutagenesis in this organism (20). Furthermore, overproduction of E. coli Pol IV inhibits replication fork progression through replacement of the replicative polymerase to form an alternate replisome in which Pol IV modulates the rate of unwinding of the DnaB helicase (25) and also reduces colony-forming ability (61).The M. tuberculosis genome encodes two Y family polymerase homologs belonging to the DinB subfamily, designated herein as DinB1 (DinX, encoded by Rv1537) and DinB2 (DinP, encoded by Rv3056), as well as a third, distantly related homolog encoded by Rv3394c (see Fig. S1 in the supplemental material) (9). On the basis of sequence similarity with their counterparts from E. coli (63) and Pseudomonas aeruginosa (54), including the complete conservation of key acidic residues essential for catalysis, DinB1 and DinB2 may be functional DNA polymerases (see Fig. S1). In contrast, Rv3394c lacks these residues and as such is unlikely to have polymerase activity (see Fig. S1). Unlike most Y family polymerase-encoding genes investigated with other bacteria (17, 26, 54, 58), dinB1 and dinB2 expression in M. tuberculosis is not dependent on RecA, the SOS response, or the presence of DNA damage (5, 7, 52). That these genes are regulated by other mechanisms and so may serve distinct roles in DNA metabolism in M. tuberculosis is suggested by the observation that dinB1 is differentially expressed in pulmonary TB (51) and is a member of the SigH regulon (30), whereas expression of dinB2 is induced following exposure to novobiocin (5).In this study, we adopted a genetic approach to investigate the function of dinB1 and dinB2 in M. tuberculosis. Mutants with altered complements or expression levels of dinB1 and/or dinB2 were analyzed in vitro and in vivo under conditions predicted to be phenotypically revealing based on DinB function established with other model organisms. The lack of discernible phenotypes in any of the assays employed suggests that the DinB homologs from M. tuberculosis do not behave like their counterparts from other organisms.     

Characterization of an acid inducible lipase Rv3203 from Mycobacterium tuberculosis H37Rv

The Rv3203 (LipV) of Mycobacterium tuberculosis (Mtb) H37Rv, is annotated as a member of Lip family based on the presence of characteristic consensus esterase motif ‘GXSXG’. In vitro culture studies of Mtb H37Ra indicated that expression of Rv3203 gene was up-regulated during acidic stress as compared to normal whereas no expression was observed under nutrient and oxidative stress conditions. Therefore, detailed characterization of Rv3203 was done by gene cloning and its further expression and purification as his-tagged protein in microbial expression system. The enzyme was purified to homogeneity by affinity chromatography. It demonstrated broad substrate specificity and preferentially hydrolyzed p-nitrophenyl myristate. The purified enzyme demonstrated an optimum activity at pH 8.0 and temperature 50 °C. The specific activity, K _m and V _max of enzyme was determined to be 21.29 U mg^?1 protein, 714.28 μM and 62.5 μmol ml^?1 min^?1, respectively. The pH stability assay and circular dichroism spectroscopic analysis revealed that Rv3203 protein is more stable in acidic condition. Tetrahydrolipstatin, a specific lipase inhibitor and RHC80267, a diacylglycerol lipase inhibitor abolished the activity of this enzyme. The catalytic triad residues were determined to be Ser⁵⁰, Asp¹⁸⁰ and His²⁰³ residues by site-directed mutagenesis.     

Fatty acid regulation of adenylyl cyclase Rv2212 from Mycobacterium tuberculosis H37Rv

Adenylyl cyclase Rv2212 from Mycobacterium tuberculosis has a domain composition identical to the pH-sensing isoform Rv1264, an N-terminal regulatory domain and a C-terminal catalytic domain. The maximal velocity of Rv2212 was the highest of all 10 mycobacterial cyclases investigated to date (3.9 micromol cAMP.mg(-1).min(-1)), whereas ATP substrate affinity was low (SC(50) = 2.1 mm ATP). Guanylyl cyclase side activity was absent. The activities and kinetics of the holoenzyme and of the catalytic domain alone were similar, i.e. in distinct contrast to the Rv1264 adenylyl cyclase, in which the N-terminal domain is autoinhibitory. Unsaturated fatty acids strongly stimulated Rv2212 activity by increasing substrate affinity. In addition, fatty acids greatly enhanced the pH sensitivity of the holoenzyme, thus converting Rv2212 to a pH sensor adenylyl cyclase. Fatty acid binding to Rv2212 was modelled by homology to a recent structure of the N-terminal domain of Rv1264, in which a fatty acid-binding pocket is defined. Rv2212 appears to integrate three cellular parameters: ATP concentration, presence of unsaturated fatty acids, and pH. These regulatory properties open the possibility that novel modes of cAMP-mediated signal transduction exist in the pathogen.     

Characterization of a novel esterase Rv1497 of Mycobacterium tuberculosisH37Rv demonstrating β-lactamase activity

The Rv1497 (LipL) of the Mycobacterium tuberculosis H37Rv was predicted to be similar to hypothetical esterases and penicillin binding proteins ofM. tuberculosis as well as to be involved in lipid metabolism. Sequence alignment revealed that Rv1497 protein contains characteristic consensus β-lactamase motif ‘SXXK’ in addition to a conserve pentapeptide –GXSXG-, characteristic of lipolytic enzymes, at the C-terminus of protein in contrast to its usual N-terminus location. For detailed characterization of protein, the rv1497 gene was cloned, expressed with N-terminal His-tag and purified to homogeneity on Ni-NTA column. Rv1497 demonstrated both esterase and β-lactamase activities. A serine located within consensus β-lactamase motif ‘SXXK’ was identified as catalytic residue in both esterase and β-lactamase enzymatic activities whereas serine residue located within conserved pentapeptide did not show any effect on both enzyme activities. The catalytic residues of Rv1497 for β-lactamase activity were determined to be Ser88, Tyr-175 and His355 residues by site-directed mutagenesis. The enzyme demonstrated preference for short chain esters (pNP-butyrate). The expression of lipL gene was significantly up-regulated during acidic stress as compared to normal conditions in in vitro culture of M. tuberculosis H37Ra. This is perhaps the first report demonstrating an esterase of mycobacterium showing β-lactamase activity.     

Crystal structure of Rv2118c: an AdoMet-dependent methyltransferase from Mycobacterium tuberculosis H37Rv

Rv2118c belongs to the class of conserved hypothetical proteins from Mycobacterium tuberculosis H37Rv. The crystal structure of Rv2118c in complex with S-adenosyl-l-methionine (AdoMet) has been determined at 1.98 A resolution. The crystallographic asymmetric unit consists of a monomer, but symmetry-related subunits interact extensively, leading to a tetrameric structure. The structure of the monomer can be divided functionally into two domains: the larger catalytic C-terminal domain that binds the cofactor AdoMet and is involved in the transfer of methyl group from AdoMet to the substrate and a smaller N-terminal domain. The structure of the catalytic domain is very similar to that of other AdoMet-dependent methyltransferases. The N-terminal domain is primarily a beta-structure with a fold not found in other methyltransferases of known structure. Database searches reveal a conserved family of Rv2118c-like proteins from various organisms. Multiple sequence alignments show several regions of high sequence similarity (motifs) in this family of proteins. Structure analysis and homology to yeast Gcd14p suggest that Rv2118c could be an RNA methyltransferase, but further studies are required to establish its functional role conclusively. Copyright 12001 Academic Press.     

结核分枝杆菌H37Rv新基因Rv2742克隆表达及纯化

Rv2742是本课题组前期基于蛋白质基因组学策略从结核分枝杆菌Mycobacteriumtuberculosis H37Rv中发现、鉴定的遗漏注释基因。文中旨在建立结核分枝杆菌H37Rv漏注释蛋白Rv2742的可溶性诱导表达、纯化体系,为进一步探索Rv2742基因参与的生物学功能奠定基础。前期实验发现构建的pGEX-4T-2-Rv2742、pET-28a-Rv2742、pET-32a-Rv2742及pMAL-c2X-Rv2742原核表达载体均无法实现目的蛋白的诱导表达。但经密码子优化后,仅有pMAL-c2X-Rv2742载体能够实现目的蛋白的可溶性诱导表达。此外,通过比较不同宿主菌、温度及IPTG浓度对目的蛋白表达量的影响,发现目的蛋白在Rosetta (DE3)中,16℃及0.5mmol/LIPTG诱导条件下表达量最高。直链淀粉树脂(Amyloseresin)亲和层析柱纯化获得较纯的产物,经LC-MS/MS验证确认是Rv2742融合蛋白肽段序列。成功获得结核分枝杆菌H37Rv新基因Rv2742的重组蛋白,可进一步开展其潜在相互作用及免疫原性研究工作。     

Rv1268c protein peptide inhibiting Mycobacterium tuberculosis H37Rv entry to target cells

Tuberculosis (TB) remains one of the most worrying infectious diseases affecting public health around the world; 8.7 million new TB cases were reported in 2011. The search for an Mycobacterium tuberculosis H37Rv protein sequence which is functionally important in host-pathogen interaction has been proposed for developing a new vaccine which will allow efficient and safe control of the spread of this disease.The present study thus reports the results obtained for the Rv1268c protein described in the M. tuberculosis H37Rv genome as a hypothetical unknown, probably secreted, protein based on a highly robust, specific, sensitive and functional approach to the search for potential epitopes to be included in an anti-tuberculosis vaccine. Rv1268c presence was determined by immunoblotting after obtaining polyclonal sera against mycobacterial total sonicate or subcellular fractions. Such sera were used in electron immunomicroscopy (EIM) for confirming protein localisation on the M. tuberculosis envelop by recognising colloidal gold-labelled immunoglobulin. Screening assays revealed the presence of two sequences having high binding activity: one binding A549 alveolar epithelial cells (¹⁴¹TGMAALEQYLGSGHAVIVSI¹⁶⁰) and other binding U937 monocyte-derived macrophages (²¹AVALGLASPADAAAGTMYGD⁴⁰). Such sequences’ ability to inhibit mycobacterial entry during in vitro assays was analysed. The structure of synthetic peptides binding to target cells was also determined, bearing in mind the structure–function relationship. These results, together with those obtained for other proteins, have been involved in selecting peptides which might be included in a subunit-based anti-tuberculosis vaccine.     

Matrix-assisted refolding and redox properties of WhiB3/Rv3416 of Mycobacterium tuberculosis H37Rv

Redox stress is one of the major challenges faced by Mycobacterium tuberculosis during early infection and latency. The mechanism of sensing and adaptation to altered redox conditions is poorly understood. whiB family of Mtb is emerging as an important class of stress responsive genes. WhiB3/Rv3416 has been shown to be important for pathogenesis in animal model and was recently shown to co-ordinate a Fe-S cluster. Here, we report a simple, rapid and efficient matrix-assisted refolding method and important redox properties of WhiB3. Similar to other WhiB proteins, WhiB3 also has four conserved cysteine residues, where two of them are present in a CXXC motif. The Fe-S cluster of WhiB3 remained bound in the presence of strong protein denaturant. Upon cluster removal due to oxidation, the four cysteine residues which are ligands of Fe-S cluster, formed two intra-molecular disulfide bridges where one of them is possibly between the cysteines of CXXC motif, an important feature of several thiol-disulfide oxido-reductases. Far-UV CD spectroscopy revealed the presence of both alpha-helices and beta-strands in apo WhiB3. The secondary structural elements of apo WhiB3 were found resistant for thermal denaturation. The results demonstrated that apo WhiB3 functions as a protein disulfide reductase similar to thioredoxins. The importance of WhiB3 in redox sensing and its possible role in mycobacterial physiology has been discussed.     

Characterization of peptidyl-tRNA hydrolase encoded by open reading frame Rv1014c of Mycobacterium tuberculosis H37Rv

The enzyme peptidyl-tRNA hydrolase (Pth, EC 3.1.1.29) is essential for the viability of bacteria. The ORF Rv1014c of Mycobacterium tuberculosis H37Rv, designated as the mtpth gene, was cloned and over-expressed and the product was purified. Generation of polyclonal antibodies against the purified recombinant protein, termed MtPth, facilitated detection of endogenously expressed MtPth in M. tuberculosis H37Rv cell lysate. MtPth could release diacetyl-[(3)H]-lysine from diacetyl-[(3)H]-lysyl-tRNA(Lys) with Michaelis-Menten kinetic parameters of K (m)=0.7+/-0.2 microM and k (cat)=1.22+/-0.2 s(-1). Transformation of a pTrc99c/mtpth vector allowed the growth of E. coli thermosensitive Pth(ts) mutant strain AA7852 at the non-permissive temperature of 42 degrees C, demonstrating the in vivo activity of MtPth. In addition, at 39 degrees C, over-expression of MtPth in AA7852 cells allowed the cells to remain viable in the presence of up to 200 microg/ml erythromycin. A 3D fold based on NMR and a structural model based on the E. coli Pth crystal structure were generated for MtPth. The essential nature of conserved active-site residues N12, H22 and D95 of MtPth for catalysis was demonstrated by mutagenesis and complementation in E. coli mutant strain AA7852. Thermal and urea/guanidinium chloride (GdmCl)-induced unfolding curves for MtPth indicate a simple two-state unfolding process without any intermediates.     

Adenylyl cyclase Rv0386 from Mycobacterium tuberculosis H37Rv uses a novel mode for substrate selection

Class III adenylyl cyclases usually possess six highly conserved catalytic residues. Deviations in these canonical amino acids are observed in several putative adenylyl cyclase genes as apparent in several bacterial genomes. This suggests that a variety of catalytic mechanisms may actually exist. The gene Rv0386 from Mycobacterium tuberculosis codes for an adenylyl cyclase catalytic domain fused to an AAA-ATPase and a helix-turn-helix DNA-binding domain. In Rv0386, the standard substrate, adenine-defining lysine-aspartate couple is replaced by glutamine-asparagine. The recombinant adenylyl cyclase domain was active with a V(max) of 8 nmol cAMP.mg(-1).min(-1). Unusual for adenylyl cyclases, Rv0386 displayed 20% guanylyl cyclase side-activity with GTP as a substrate. Mutation of the glutamine-asparagine pair either to alanine residues or to the canonical lysine-aspartate consensus abolished activity. This argues for a novel mechanism of substrate selection which depends on two non-canonical residues. Data from individual and coordinated point mutations suggest a model for purine definition based on an amide switch related to that previously identified in cyclic nucleotide phosphodiesterases.     

Characterization and site-directed mutagenesis of the putative novel acyl carrier protein Rv0033 and Rv1344 from Mycobacterium tuberculosis

Mycolic acids are generated in Mycobacterium tuberculosis as a result of the interaction of two fatty acid biosynthetic systems: type I fatty acid synthase (FAS) and type II fatty acid synthase. Acyl carrier protein (ACP) is a small, acidic protein in type II FAS systems. It plays a central role in mycolic acid biosynthesis by transferring the acyl groups from one enzyme to another for the completion of the fatty acid synthesis cycle. The nature of the proper recognition between ACPs and its many interactive proteins is not understood. Here, we report the over-expression, purification, and characterization of two putative ACPs: Rv0033 and Rv1344 in M. tuberculosis. In order to study the role of the conserved residues and the conformation of whole protein, some site-directed mutations of recombinant Acp1344 were made and the 3D structure of Acp1344 was modeled.     

A Novel Method of Identifying Mycobacterium tuberculosis Beijing Strains by Detecting SNPs in Rv0444c and Rv2629

A particular genotype of tuberculosis, named Beijing strain, is strongly associated with drug resistance and high virulence. Therefore, rapid prospective identification of Mycobacterium tuberculosis Beijing strains is very important for identifying and controlling tuberculosis of Beijing genotype. In the present study, we found that the co-mutation, A191C in Rv2629 and G243C in Rv0444c, is closely related to Beijing genotype. Gene Rv2629 and Rv0444c of 139 clinical isolates of M. tuberculosis were analyzed by PCR amplification and sequencing. Among 99 Beijing strains, 86 % (n = 85) isolates had the mutation G243C in Rv0444c and 92.93 % (n = 92) isolates had the mutation A191C in Rv2629. Among 40 non-Beijing isolates, only six isolates carried the mutation G243C in Rv0444c and eight isolates carried the mutation A191C in Rv2629. The co-mutation existed in 84.85 % (n = 84) of 99 clinical genome samples of W-Beijing strains and in only 12.5 % (n = 5) of the 40 non-Beijing strains, and the positive predictive value of 94.38 %, obtained in our experiment with a designed ratio of Beijing isolates, is similar to that in China at present. This result suggested that the detection method of the co-mutation, A191C in Rv2629 and G243C in Rv0444c, proposed in this study was a rapid, reliable, and sensitive one for identifying tuberculosis with Beijing genotype.     

Expression and characterization of alpha-(1,4)-glucan branching enzyme Rv1326c of Mycobacterium tuberculosis H37Rv

Glycogen branching enzyme (GlgB, EC 2.4.1.18) catalyzes the third step of glycogen biosynthesis by the cleavage of an alpha-(1,4)-glucosidic linkage and subsequent transfer of cleaved oligosaccharide to form a new alpha-(1,6)-branch. A single glgB gene Rv1326c is present in Mycobacterium tuberculosis. The predicted amino acid sequence of GlgB of M. tuberculosis has all the conserved regions of alpha-amylase family proteins. The overall amino acid identity to other GlgBs ranges from 48.5 to 99%. The glgB gene of M. tuberculosis was cloned and expressed in Escherichia coli. The recombinant protein was purified to homogeneity using metal affinity and ion exchange chromatography. The recombinant protein is a monomer as evidenced by gel filtration chromatography, is active as an enzyme, and uses amylose as the substrate. Enzyme activity was optimal at pH 7.0, 30 degrees C and divalent cations such as Zn2+ and Cu2+ inhibited activity. CD spectroscopy, proteolytic cleavage and mass spectroscopy analyses revealed that cysteine residues of GlgB form structural disulfide bond(s), which allow the protein to exist in two different redox-dependent conformational states. These conformations have different surface hydrophobicities as evidenced by ANS-fluorescence of oxidized and reduced GlgB. Although the conformational change did not affect the branching enzyme activity, the change in surface hydrophobicity could influence the interaction or dissociation of different cellular proteins with GlgB in response to different physiological states.     

结核分枝杆菌Rv2628蛋白的免疫生物学特性

Rv2628蛋白是结核分枝杆菌Mycobacterium tuberculosis(M.tb)DosR调控的潜伏感染相关抗原。本研究对Rv2628蛋白进行了原核表达和纯化,并以巨噬细胞系和小鼠为研究模型,对其免疫生物学特性进行了分析。SDS-PAGE及Western blotting鉴定结果表明,Rv2628-His融合蛋白以包涵体形式表达,能与兔抗H37Rv多抗血清发生特异性反应,具有较好的免疫反应性。与巨噬细胞系RAW264.7的互作实验结果表明,在1–12 h内Rv2628蛋白能诱导前炎性因子IL-6的上调表达。将纯化的Rv2628融合蛋白皮下免疫BALB/c小鼠,夹心ELISA的测定结果表明,Rv2628蛋白免疫组诱导产生的特异性IFN-γ水平显著高于IL-4的水平(P0.000 1),呈现Th1型细胞免疫应答趋势;以Rv262811-30多肽作为包被抗原,通过间接ELISA测定的血清抗体效价能达到11 600,表明Rv2628也能诱导体液免疫应答。总之,Rv2628能促进巨噬细胞炎症反应的发生,激发小鼠产生强烈的Th1型细胞免疫应答和较好的体液免疫应答,具有作为亚单位疫苗的潜力,为M.tb与宿主之间的相互作用奠定了一定的理论基础。