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.     

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.     

Molecular features governing the stability and specificity of functional complex formation by Mycobacterium tuberculosis CFP-10/ESAT-6 family proteins

The Mycobacterium tuberculosis complex CFP-10/ESAT-6 family proteins play essential but poorly defined roles in tuberculosis pathogenesis. In this article we report the results of detailed spectroscopic studies of several members of the CFP-10/ESAT-6 family. This work shows that the CFP-10/ESAT-6 related proteins, Rv0287 and Rv0288, form a tight 1:1 complex, which is predominantly helical in structure and is predicted to closely resemble the complex formed by CFP-10 and ESAT-6. In addition, the Rv0287.Rv0288 complex was found to be significantly more stable to both chemical and temperature induced denaturation than CFP-10.ESAT-6. This approach demonstrated that neither Rv0287.Rv0288 nor the CFP-10.ESAT-6 complexes are destabilized at low pH (4.5), indicating that even in low pH environments, such as the mature phagosome, both Rv0287.Rv0288 and CFP-10.ESAT-6 undoubtedly function as complexes rather than individual proteins. Analysis of the structure of the CFP-10.ESAT-6 complex and optimized amino acid sequence alignments of M. tuberculosis CFP-10/ESAT-6 family proteins revealed that residues involved in the intramolecular contacts between helices are conserved across the CFP-10/ESAT-6 family, but not those involved in primarily intermolecular contacts. This analysis identified the molecular basis for the specificity and stability of complex formation between CFP-10/ESAT-6 family proteins, and indicates that the formation of functional complexes with key roles in pathogenesis will be limited to genome partners, or very closely related family members, such as Rv0287/Rv0288 and Rv3019c/Rv3020c.     

Heterologous expression and biochemical characterization of recombinant alpha phosphoglucomutase from Mycobacterium tuberculosis H37Rv

Phosphoglucomutase (PGM) plays an important role in polysaccharide capsule formation and virulence in a number of bacterial pathogens. However, the enzyme has not yet been characterized from Mycobacterium tuberculosis (Mtb). Here, we report the biochemical properties of recombinant Mtb-PGM as well as the in silico structural analysis from Mtb H37Rv. The purified recombinant enzyme was enzymatically active with a specific activity of 67.5U/mg and experimental k(cat) of 70.31s(-1) for the substrate glucose-1-phosphate. The enzyme was stable in pH range 6.5-7.4 and exhibited temperature optima range between 30 and 40°C. Various kinetic parameters and constants of the rPGM were determined. A structural comparison of Modeller generated 3D Mtb-PGM structure with rabbit muscle PGM revealed that the two enzymes share the same overall heart shape and four-domain architecture, despite having only 17% sequence identity. However, certain interesting differences between the two have been identified, which provide an opportunity for designing new drugs to specifically target the Mtb-PGM. Also, in the absence of the crystal structure of the Mtb-PGM, the modeled structure could be further explored for in silico docking studies with suitable inhibitors.     

Molecular cloning, overexpression and biochemical characterization of hypothetical beta-lactamases of Mycobacterium tuberculosis H37Rv

Aim:  Molecular cloning, overexpression and biochemical characterization of the genes from the Mycobacterium tuberculosis H37Rv genome having hypothetical β-lactamases activity.
Methods and Results:  Analysis of the M. tuberculosis H37Rv genome revealed that Rv 2068c , Rv 0406 c and Rv 3677 c gene products were predicted to exhibit β-lactamases activity. All the three genes were cloned in pET28a vector and overexpressed in C41 (DE3) Escherichia coli cells. The His-tagged recombinant proteins were confirmed by immunoblotting and were shown to have β-lactamase activity by the hydrolysis of nitrocefin and other β-lactams. Catalytic parameters for all the recombinant proteins were derived followed by the enzyme inhibition studies. Antibiotic susceptibility studies using the recombinant strains showed an increased resistance against different classes of β-lactam antibiotics.
Conclusion:  The study revealed the possibility of more than one gene in M. tuberculosis , encoding proteins having β-lactamase or β-lactamase-like activity, giving wide spectrum of resistance against β-lactams.
Significance and Impact of the Study:  Systematic study of hypothetical β-lactamases of M. tuberculosis and related species and their correlation with β-lactam and inhibitor susceptibility profile might be useful in developing new antibiotic regime for the treatment of tuberculosis caused by multiple drug resistant (MDR) strains.     

Deoxyribonucleic acid replication time in Mycobacterium tuberculosis H37 Rv

The DNA increment method, designed for measuring the increment in the amount of DNA after inhibition of initiation of fresh rounds of replication initiation was employed to measure the rate of deoxyribonucleic acid (DNA) chain growth in Mycobacterium tuberculosis H37Rv growing in Youman and Karlson's medium at 37°C with a generation time of 24 h and also in relatively fast growing species like Mycobacterium smegmatis and Escherichia coli. From the results obtained, the time required for a DNA replication fork to traverse the chromosome from origin to terminus (C period) was calculated. The chain elongation rates of DNA of the three organisms was determined from the C period and the known genome sizes assuming that all these genomes have a single replication origin and bidirectional replication fork. The rate for M. tuberculosis was 3,200 nucleotides per min about 11 times slower than that of M. smegmatis and about 13–18 times slower than that of E. coli.Abbreviations DNA deoxyribonucleic acid - td delay in initiation - OD optical density - CAM chloramphenicol - RIF rifampicin     

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 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.     

Biosynthesis of nucleic acid purines in Mycobacterium tuberculosis H37Rv

1. The biosynthesis of nucleic acid purine in Mycobacterium tuberculosis H(37)R(v) has been studied by using (14)C-labelled precursors. 2. The results indicate that C-2 and C-8 of the purine ring are derived most efficiently from serine and glycine and not from formate. 3. [(14)C]Methionine is not incorporated into the ureide carbon atoms of the purine ring.     

Comprehensive analysis of exported proteins from Mycobacterium tuberculosis H37Rv

Proteins secreted by Mycobacterium tuberculosis play an essential role in the pathogenesis of tuberculosis. The culture filtrates of M. tuberculosis H37Rv made by Sadamu Nagai (Japan), are considerably enriched for secreted proteins compared to other culture filtrates. Complementary approaches were used to identify the secreted proteins in these culture filtrates: (i) 2-DE combined with MALDI-TOF MS and (ii) LC coupled MS/MS. Peptides derived from a total of 257 proteins were identified of which 144 were identified by more than one peptide. Several members of the immunologically important early secretory antigenic target-6 (ESAT-6) family of proteins were found to be major components. The majority of the identified proteins, 159 (62%), were predicted to be exported through the general secretory pathway. We experimentally verified that the signal peptides, which mediate translocation through the cell membrane, had been removed in 41 of the identified proteins, and in 35 of those, there was an AXA motif N-terminally to the cleavage site, showing that this motif is important for the recognition and cleavage of signal peptides in mycobacteria. A large fraction of the secreted proteins were unknown, suggesting that we have mapped an unexplored part of the exported proteome of M. tuberculosis. complement.     

Genes of Mycobacterium tuberculosis H37Rv downregulated in the attenuated strain H37Ra are restricted to M. tuberculosis complex species

By comparing gene expression of virulent Mycobacterium tuberculosis H37Rv and attenuated strain H37Ra, we previously detected six genes that appear to be markedly downregulated in the attenuated strain compared with the virulent one. Three of these genes, i.e. Rv1345, Rv2770c, and Rv0288, code for proteins that can be predictively associated to immunological or pathogenetic aspects of M. tuberculosis infection; the other genes, i.e. Rv2336, Rv1320c, and Rv2819c, code for proteins with unknown functions (Rindi et al., 1999). In this paper we searched for the above mentioned genes in Pvu II-digested genomic DNA of a number of mycobacterial species by southern blot analysis employing PCR-generated probes in high-stringency conditions. Hybridization signals were only found in species belonging to the M. tuberculosis complex, i.e., M. tuberculosis, M. bovis, including the BCG strain, and M. microti, but not in other mycobacterial species, including M. avium, M. intracellulare, M. malmoense, M. xenopi, M. kansasii, M. simiae, M. marinum, M. scrofulaceum, M. gordonae, M. fortuitum, and M. smegmantis. These results indicate that genes Rv1345, Rv2770c, Rv0288, Rv2336, Rv1320c, and Rv2819c are associated with the most virulent mycobacteria and further support their potential role in M. tuberculosis virulence.     

Dehalogenation of Haloalkanes by Mycobacterium tuberculosis H37Rv and Other Mycobacteria

Haloalkane dehalogenases convert haloalkanes to their corresponding alcohols by a hydrolytic mechanism. To date, various haloalkane dehalogenases have been isolated from bacteria colonizing environments that are contaminated with halogenated compounds. A search of current databases with the sequences of these known haloalkane dehalogenases revealed the presence of three different genes encoding putative haloalkane dehalogenases in the genome of the human parasite Mycobacterium tuberculosis H37Rv. The ability of M. tuberculosis and several other mycobacterial strains to dehalogenate haloaliphatic compounds was therefore studied. Intact cells of M. tuberculosis H37Rv were found to dehalogenate 1-chlorobutane, 1-chlorodecane, 1-bromobutane, and 1,2-dibromoethane. Nine isolates of mycobacteria from clinical material and four strains from a collection of microorganisms were found to be capable of dehalogenating 1,2-dibromoethane. Crude extracts prepared from two of these strains, Mycobacterium avium MU1 and Mycobacterium smegmatis CCM 4622, showed broad substrate specificity toward a number of halogenated substrates. Dehalogenase activity in the absence of oxygen and the identification of primary alcohols as the products of the reaction suggest a hydrolytic dehalogenation mechanism. The presence of dehalogenases in bacterial isolates from clinical material, including the species colonizing both animal tissues and free environment, indicates a possible role of parasitic microorganisms in the distribution of degradation genes in the environment.     

Purification and properties of DNA polymerase from Mycobacterium tuberculosis H37Rv

DNA polymerase has been purified approximately 2000-fold from Mycobacterium tuberculosis H37Rv. The purified preparation was homogeneous by electrophoretic criteria and has a molecular weight of 135 000. The purified enzyme resembles Escherichia coli polymerase I in its properties, being insensitive to sulfhydryl drugs and possessing 5',3'-exonuclease activity in addition to polymerase and 3',5'-exonuclease activities. However, it differs from the latter in its sensitivity to higher salt concentration and DNA intercalating agents such as 8-aminoquinoline. The polymerase exhibited maximal activity between 37--42 degrees C and pH 8.8--9.5. The polymerase was stable for several months below 0 degree C. However, the 5',3'-exonuclease activity was more labile. The effects of different metal ions, polyamines and drugs on the polymerase activity are presented.     

Cloning and characterization of aspartate-beta-semialdehyde dehydrogenase from Mycobacterium tuberculosis H37 Rv

AIMS: To clone and characterize the aspartate-beta-semialdehyde dehydrogenase of Mycobacterium tuberculosis H37Rv. METHODS AND RESULTS: The asd gene of M. tuberculosis H37Rv was cloned in pGEM-T Easy vector, subcloned in expression vector pQE30 having a T5 promoter, and overexpressed in Escherichia coli. The ASD enzyme was expressed to levels of 40% but was found to be inactive. Functional ASD was obtained by altering induction and growth conditions and the enzyme was purified to near homogeneity using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The K(m) and V(max) values for the three substrates L-ASA, NADP and Pi, the turnover number and specific activity of the enzyme were determined. CONCLUSIONS: Functional ASD enzyme of M. tuberculosis was obtained by gene cloning and protein purification using affinity chromatography. The K(cat) and specific activity of the enzyme were 8.49 s(-1) and 13.4 micromol min(-1) microg(-1) respectively. Significance and Impact of the Study: The ASD enzyme is a validated drug target. We characterized this enzyme from M. tuberculosis and future work would focus on deducing the three-dimensional structure of the enzyme and design of inhibitors, which could be used as drugs against TB.