Proteins Of Mycobacterium Tuberculosis H37Rv Research Articles

Immunotherapeutic potential of an N-formylated peptide of Listeria monocytogenes in experimental tuberculosis

Objective: The current therapeutic regimens for tuberculosis (TB) are complex and involve the prolonged use of multiple antibiotics with diverse side effects that lead to therapeutic failure and bacterial resistance. The standard appliance of immunotherapy may aid as a powerful tool to combat the ensuing threat of TB. We have earlier reported the immunotherapeutic potential of N-formylated peptides of two secretory proteins of Mycobacterium tuberculosis H37Rv. Here, we investigated the immunotherapeutic effect of an N-formylated peptide from Listeria monocytogenes in experimental TB.Methods: The N-terminally formylated listerial peptide with amino acid sequence ‘f-MIGWII’ was tested for its adjunctive therapeutic efficacy in combination with anti-tuberculosis drugs (ATDs) in the mouse model of TB. In addition, its potential to generate reactive oxygen species (ROS) in murine neutrophils was also evaluated.Results: The LemA peptide (f-MIGWII) induced a significant increase in the intracellular ROS levels of mouse neutrophils (p ≤ .05). The ATD treatment reduced the colony forming units (CFU) in lungs and spleen of infected mice by 2.39 and 1.67 log10 units, respectively (p < .001). Treatment of the infected mice with combination of ATDs and LemA peptide elicited higher therapeutic efficacy over ATDs alone. The histopathological changes in the lungs of infected mice also correlated well with the CFU data.Conclusions: Our results clearly indicate that LemA peptide conferred an additional therapeutic effect when given in combination with the ATDss (p < .01) and hence can be used as adjunct to the conventional chemotherapy against TB.

Biochemical characterization of PE_PGRS61 family protein of Mycobacterium tuberculosis H37Rv reveals the binding ability to fibronectin.

The periodic binding of protein expressed by Mycobacterium tuberculosis H37Rv with the host cell receptor molecules i.e. fibronectin (Fn) is gaining significance because of its adhesive properties. The genome sequencing of M. tuberculosis H37Rv revealed that the proline-glutamic (PE) proteins contain polymorphic GC-rich repetitive sequences (PGRS) which have clinical importance in pathogenesis events when the host encounters M. tuberculosis H37Rv. The functional parts of PE_PGRS family proteins, have not been extensively studied in tuberculosis biology. Fibronectin (10 ng and 20 ng) were used for FnBP assay and its enzymatic activities were observed by using various protein concentrations. Therefore, in the present work, we cloned, expressed, purified and identified a novel PE_PGRS61 (Rv3653) family protein in M. tuberculosis H37Rv. Our experiment, observation suggested that at particular concentrations of 10 ng and 20 ng of Fn exhibits optimum binding to the purified Fibronectin Binding Protein (FnBP), a PE_PGRS61 family protein at 0.20 μg and 0.25 μg concentrations, respectively. Moreover, for better understanding the computational analysis, the B-cell and T-cell epitopes prediction prospect some amino acid propensity scales with hydrophilicity and antigenic variation index at their respective locations. Thus, the current findings provide an opportunity to illuminate the functions of PE_PGRS61 family protein. So, in this point of view, it could be useful to develop a novel therapeutic approach or diagnostic pipeline through targeting these fibronectin binding protein (FnBP) expressing genes.

Functional, structural and epitopic prediction of hypothetical proteins of Mycobacterium tuberculosis H37Rv: An in silico approach for prioritizing the targets

The global control of tuberculosis (TB) remains a great challenge from the standpoint of diagnosis, detection of drug resistance, and treatment. Major serodiagnostic limitations include low sensitivity and high cost in detecting TB. On the other hand, treatment measures are often hindered by low efficacies of commonly used drugs and resistance developed by the bacteria. Hence, there is a need to look into newer diagnostic and therapeutic targets. The proteome information available suggests that among the 3906 proteins in Mycobacterium tuberculosis H37Rv, about quarter remain classified as hypothetical uncharacterized set. This study involves a combination of a number of bioinformatics tools to analyze those hypothetical proteins (HPs). An entire set of 999 proteins was primarily screened for protein sequences having conserved domains with high confidence using a combination of the latest versions of protein family databases. Subsequently, 98 of such potential target proteins were extensively analyzed by means of physicochemical characteristics, protein-protein interaction, sub-cellular localization, structural similarity and functional classification. Next, we predicted antigenic proteins from the entire set and identified B and T cell epitopes of these proteins in M. tuberculosis H37Rv. We predicted the function of these HPs belong to various classes of proteins such as enzymes, transporters, receptors, structural proteins, transcription regulators and other proteins. However, the structural similarity prediction of the annotated proteins substantiated the functional classification of those proteins. Consequently, based on higher antigenicity score and sub-cellular localization, we choose two (NP_216420.1, NP_216903.1) of the antigenic proteins to exemplify B and T cell epitope prediction approach. Finally we found 15 epitopes those located partially or fully in the linear epitope region. We found 21 conformational epitopes by using Ellipro server as well. In silico methodology used in this study and the data thus generated for HPs of M. tuberculosis H37Rv may facilitate swift experimental identification of potential serodiagnostic and therapeutic targets for treatment and control.

Potential non homologous protein targets of mycobacterium tuberculosis H37Rv identified from protein–protein interaction network

Bacillus mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis and H37Rv is the most studied strain. Identification of new drug targets for Mtb is among one of the priorities since it is still a major global health problem by being a cause of morbidity and mortality for millions of people each year. We used centrality measures to identify the most central proteins from protein–protein interaction network of mycobacterium tuberculosis H37Rv which was retrieved from STRING database by hypothesizing these proteins would be important to alter the function of the network. We then refined the result by using a dataset obtained from Drug Target Protein Database to identify non-human homologous proteins since in host–parasite diseases like tuberculosis; non-homologous proteins (enzymes) as drug target are the primary choices. We also tried to compare our proposed potential non-human homologous protein target lists against previously reported targets. Moreover, the structural coverage of the proposed target list has been identified. The analysis shows that 807 proteins in mycobacterium tuberculosis H37Rv were found at the center of gravity of the functional network of which 390 were non-human homologous, which are thought to be potential drug targets. 119 (30.51%) of the 390 proteins were reported as drug targets and only 33 (8.46%) of the non-human homologous proposed target lists have solved structure.

WhiB2/Rv3260c, a cell division‐associated protein of Mycobacterium tuberculosis H37Rv, has properties of a chaperone

whiB-like genes have been found in all actinomycetes sequenced so far. The amino-acid sequences of WhiB proteins of Mycobacterium tuberculosis H37Rv are highly conserved and participate in several cellular functions. Unlike other WhiB proteins of M. tuberculosis that have properties of protein disulfide reductases, WhiB2 showed properties like a chaperone as it suppressed the aggregation of several model substrates (e.g. citrate synthase, rhodanese and luciferase). Suppression of aggregation of the model substrates did not require ATP. Four cysteine residues of WhiB2 form two intramolecular disulfide bonds; however, chaperone function was unaffected by the redox state of the cysteines. WhiB2 also restored the activity of chemically denatured citrate synthase and did not require either ATP or a co-chaperone for refolding. The results indicate that WhiB2, which has been shown to be associated with cell division in mycobacteria and streptomyces, has evolved independently of other WhiBs, although it retains basic properties of this group of proteins. This is the first report to show that a WhiB protein has chaperone-like function; therefore, this report will have major implications in attempts to understand the role of WhiB proteins in mycobacteria, particularly in cell division.

In Silico Prediction of Epitopes in Virulence Proteins of Mycobacterium Tuberculosis H37Rv for Diagnostic and Subunit Vaccine Design

The role of secretory and cell surface proteins of Mycobacterium tuberculosis for stimulation of specific host immune response, in different cell types like T –cells, B- cells, macrophages in the pathogenesis of the disease. Therefore, the identification of T-cells epitopes from the set of proteins may serve to define candidate antigens with vaccine potential. In this study by examine the complete genome of M. tuberculosis H37Rv strain, 16 significant virulent proteins total 69 promiscuous nanomer candidate epitope for HTL were recognized against MHC Class II and 47 promiscuous epitope are recognized against CTL for MHC class I for secretory and cell surface proteins. Attempts have been made to provide the implication on the basis of predicted epitopes in the virulence proteins from M. tuberculosis may be useful in the formulation of immunodiagnostic kit and the peptides based safe subunit vaccine design against the tuberculosis (TB).

Induction of cytotoxic T lymphocyte response against Mycobacterial antigen using domain I of anthrax edema factor as antigen delivery system

We have investigated the efficiency of N-terminal 1–260 residues of Edema factor (EFn) as a delivery system for ESAT-6, an antigenic protein of Mycobacterium tuberculosis H37Rv, into the cytosol of mammalian cells. The EFn.ESAT-6 recombinant protein was obtained by genetic fusion of EFn and ESAT-6 DNA. Our data shows that in the presence of PA, EFn.ESAT-6 fusion protein is internalized into the cytosol of antigen presenting cells, and the splenocytes produced both Th1 and Th2 cytokines in vitro. Further, EFn.ESAT-6 elicited effective cytotoxicT lymphocyte (CTL) response in an in vitro CTL assay. This study for the first time demonstrates that EFn can be used as a vehicle to deliver heterologous proteins of therapeutic importance.

Domain Analysis of Fatty Acid Synthase Protein (NP_217040) from Mycobacterium tuberculosis H37Rv—A Bioinformatics Study

Different domains of fatty acid synthase (FAS) protein of Mycobacterium tuberculosis H37Rv, involved in mycolic acid synthesis were analyzed using various bioinformatics tools. Based on different database searches (CDD and Pfam), FAS protein of Mycobacterium tuberculosis was grouped into eight domains, five of which showed close similarity with pdb templates (1MLA, 1IQ6A, 2BMOA, and 1J3NA). Based on the PSI blast analysis, 3D structures of only five domains were predicted using MODELLER software, and loop modeling was done for only those regions that were predicted as loops by predict protein server. Compared to the original structure, the loop modeled structure showed a lower DOPE score value for FAS protein. The X-ray determined templates that were used for predicting the 3D structure suggest that, FAS protein has “Malo- nyl-coenzyme A-Hydratase-Nitrobenzene dioxygenase-3-oxoacyl-(acp) synthase” activity. Accuracy of the prediction of 3D structure of different domains of FAS protein was further validated by Ramachandran plot and PROCHECK (G-value).

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

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

Identification ofMycobacteriumtuberculosisH37Rv Integral Membrane Proteins by One-Dimensional Gel Electrophoresis and Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry

Because many membrane-associated proteins represent potential drug targets, diagnostic probes, and components of vaccines, we have chosen to study the membrane proteins of Mycobacterium tuberculosis H37Rv. To remove cytosolic proteins and facilitate access to the integral membrane proteins, membrane fractions of M. tuberculosis H37Rv were intensely washed with 5 M urea and high pH carbonate solution. One-dimensional SDS-PAGE, followed by enzymatic hydrolysis and nanoLC electrospray ionization MS/MS, proved to be the most efficient way to identify the proteins contained within the membrane fraction. Here we report 349 protein identifications in total, validated by at least two tryptic peptide matches and MOWSE scores greater than 75. Of those 349 proteins, 100 are integral membrane proteins with at least one predicted transmembrane alpha helix (excluding the possible signal sequence). 84 M. tuberculosis H37Rv proteins, including 42 integral membrane proteins, are described for the first time.

Protein identification and tracking in two-dimensional electrophoretic gels by minimal protein identifiers.

Protein identification by matrix-assisted laser desorption/ionization mass-spectrometry peptide mass fingerprinting (MALDI-MS PMF) represents a cornerstone of proteomics. However, it often fails to identify low-molecular-mass proteins, protein fragments, and protein mixtures reliably. To overcome these limitations, PMF can be complemented by tandem mass spectrometry and other search strategies for unambiguous protein identification. The present study explores the advantages of using a MALDI-MS-based approach, designated minimal protein identifier (MPI) approach, for protein identification. This is illustrated for culture supernatant (CSN) proteins of Mycobacterium tuberculosis H37Rv after separation by two-dimensional gel electrophoresis (2-DE). The MPI approach takes into consideration that proteins yield characteristic peptides upon proteolytic cleavage. In this study, peptide mixtures derived from tryptic protein cleavage were analyzed by MALDI-MS and the resulting spectra were compared with template spectra of previously identified counterparts. The MPI approach allowed protein identification by few protein-specific signature peptide masses and revealed truncated variants of mycobacterial elongation factor EF-Tu, previously not identified by PMF. Furthermore, the MPI approach can be employed to track proteins in 2-DE gels, as demonstrated for the 14 kDa antigen, the 10 kDa chaperone, and the conserved hypothetical protein Rv0569 of M. tuberculosis H37Rv. Furthermore, it is shown that the power of the MPI approach strongly depends on distinct factors, most notably on the complexity of the proteome analyzed and accuracy of the mass spectrometer used for peptide mass determination.

Functional annotation of putative aminoglycoside antibiotic modifying proteins in Mycobacterium tuberculosis H37Rv.

The growing availability of sequences of bacterial genomes has revealed a number of open reading frames predicted by sequence alignment to encode antibiotic resistance proteins. The presence of these putative resistance genes within bacterial genomes raises important questions regarding potential reservoirs of resistance elements and their evolution. Here we examine four gene products encoding predicted aminoglycoside-aminocyclitol antibiotic modifying enzymes, two phosphotransferases and two acetyltransferases, derived from analysis of the genome sequence of Mycobacterium tuberculosis strain H37Rv with the goal of assigning biochemical function by purification of each protein and characterization of their ability to modify aminoglycoside antibiotics. Only one of these enzymes, the previously characterized aminoglycoside acetyltransferase AAC(2')-Ic, displayed compelling aminoglycoside modifying activity. While the putative phosphotransferase encoded by the Rv3225c gene did display low levels of aminoglycoside kinase activity, the predicted kinase encoded by the Rv3817 gene lacked any such activity. A potential aminoglycoside 6'-acetyltransferase, encoded by the Rv1347c gene, did not show antibiotic acylation activity but did demonstrate selective thioesterase activity with numerous acyl-CoAs. This activity, together with the genomic environment of the Rv1347c gene in a likely polyketide synthesis cluster, suggests a role for this protein in secondary metabolism and not in antibiotic modification. It was thus shown that only one of four putative aminoglycosides modifying enzymes derived from the whole genome sequencing of M. tuberculosis H37Rv showed sufficient predicted enzyme activity to be annotated as an aminoglycoside resistance element. This study demonstrates the necessity of biochemical annotation methods as a follow up to in silico sequence alignment-based methods of assigning gene product function.

Isoniazid induces expression of the antigen 85 complex in Mycobacterium tuberculosis.

Exposure to isoniazid induced the expression of several secreted proteins in Mycobacterium tuberculosis H37Rv. Two-dimensional gel electrophoresis and immunoblot analyses indicated that two of the prominent isonicotinic acid hydrazide-inducible polypeptides were members of the antigen 85 complex, recently demonstrated to have mycolyltransferase activity. We postulate the existence of an intermediate, whose production is inhibited by isonicotinic acid hydrazide, which plays a negative feedback regulatory role in the metabolism of mycolic acids are revealed by the overexpression of the antigen 85 complex. The approach described here relies on analyses of differential gene expression following exposure to inhibitors and may become a more general tool in dissecting the effects of antimicrobial agents.

Purification and partial characterisation of a novel 66-kDa seroreactive protein of Mycobacterium tuberculosis H37Rv.

A seroreactive protein (TB66) was purified from culture filtrate (CF) and cell sonicate (CS) of Mycobacterium tuberculosis H37Rv by immobilised metal affinity chromatography (IMAC) on a Ni-nitrilotriacetic acid (NTA) column. The TB66 preparations obtained by IMAC contained predominantly a 66-kDa protein with a pI of c. 5.5 as determined by two-dimensional electrophoresis. TB66 was detected in the CF as early as 1 week of growth of H37Rv. The NH2-terminal amino-acid sequence showed 85% homology with the N-terminal sequence of bovine serum albumin (BSA) and 80% homology with human serum albumin. Amino-acid analysis indicated a difference in the amino-acid content of TB66 when compared to BSA, with an abundance of acidic amino acids. A monoclonal antibody (MAb) OD4AG3, raised in this laboratory against an M. avium complex (MAC 101) sonicate cross-reacting with H37Rv sonicate, recognised a heat-stable and trypsin-sensitive epitope of this protein. TB66 was also recognised by MAbs IT1 and IT20 which also react with the 14-kDa antigen of the M. tuberculosis complex. Antibodies against TB66 were present in the sera of 62 of 64 patients with tuberculosis; sera from normal healthy individuals showed no significant reactivity. TB66 appears to be a predominant secretory protein of M. tuberculosis and could play an important role in the pathogenesis of this organism.

Immunological evaluation of a 12-kilodalton protein of Mycobacterium tuberculosis by enzyme-linked immunosorbent assay

Objective: To purify and study the seroreactivity of native and recombinant 12-kilodalton protein of Mycobacterium tuberculosis H37Rv. Design: M. tuberculosis H37Rv cells and Escherichia coli XL-1 containing the plasmid PRL4 encoding the M. tuberculosis heat shock protein GroES homolog were used as sources for the purification of native and recombinant 12 kD of M. tuberculosis respectively. The seroreactivity of the 12 kDs was studied by ELISA using sera from 35 leprosy and 25 active pulmonary tuberculosis (TB) patients, and from 10 normal healthy controls. Results: The 12 kD protein was purified from H37Rv extract (s12 kD) and from recombinant E. coli (r12 kD) by ultrafiltration and MonoQ fast pressure liquid chromatography (FPLC). Analysis of s12 kD and r12 kD by SDS-PAGE revealed a single protein band in both cases with an approximate molecular weight of 12 000 which was recognized by monoclonal antibody SA-12 in immunoblotting. Both the proteins exhibited a pI of ~4.6 by isoelectric focusing. Both the 12 kD proteins exhibited 96% positivity with TB sera as compared to normal control sera ( P < 0.01). Only one serum sample from the 35 leprosy sera tested exhibited binding to both the s12 kD and r12 kD proteins. Delayed type hypersensitivity reaction to the 12 kD proteins was elicited in guinea pigs that had been immunized with H37Rv sonicate. Conclusion: The 12 kD protein could be easily purified and could serve as a valuable serodiagnostic tool in the screening of TB cases from a large population in an endemic area.

Serodiagnostic efficiency of phospholipid associated protein of Mycobacterium tuberculosis H37Rv.

The phospholipid-associated protein (55-67 kDa) fraction of Mycobacterium tuberculosis H37Rv was purified as the DE-V protein fraction. This DE-V fraction was used for diagnosis of tuberculosis by enzyme-linked immunosorbent assay (ELISA), detecting IgG antibody in sera collected from different categories of tuberculosis patients, i.e. with acid fast bacilli (AFB) culture-positive pulmonary tuberculosis, with AFB culture-negative, but radiologically suspected, pulmonary tuberculosis, extrapulmonary tuberculosis, and control groups of patients suffering from diseases other than tuberculosis (asthma and/or rhinitis, lepromatous leprosy) as well as from healthy volunteers. Encouraging operational ELISA validity could be achieved with 93% sensitivity, 100% specificity, 97% efficiency, 100% positive predictivity and 95% negative predictability even among the extrapulmonary and suspected pulmonary tuberculosis patients. The above assay was insensitive but with 100% specificity among control group of patients suffering from diseases other than tuberculosis. The DE-V protein fraction was associated with phosphatidyl inositol and phosphatidyl inositol mannosides. The dissociation of phospholipid-protein complex decreased ELISA specificity. ELISA reactivity of the DE-V fraction appeared to be thermostable; thus, it may have serodiagnostic utility in developing countries.

Immunological activity of a 14-kilodalton recombinant protein of Mycobacterium tuberculosis H37Rv.

A 14-kilodalton peptide antigen from Mycobacterium tuberculosis was isolated from an Escherichia coli lambda gt 11 recombinant DNA clone and was identified by Western blotting (immunoblotting) with monoclonal antibody TB68. Immunization of mice and guinea pigs with the recombinant peptide (rTB68) induced in vitro lymphoproliferative responses in draining lymph node lymphocyte cultures as well as in vivo delayed-type hypersensitivity reactions. Moreover, rTB68 was found both to induce and to cross-react with Mycobacterium leprae immune lymphocytes, but did not generate protective effects against live M. leprae challenge in mice. These findings showed that a 14-kilodalton peptide which has been characterized as specific for M. tuberculosis on the basis of B-cell recognition was capable of generating cell-mediated immune responses and moreover contained T-cell epitopes which were cross-reactive with M. leprae antigens.