Project description:Mycobacterial tuberculosis (Mtb) is able to preserve its intrabacterial pH (pHIB) near neutrality in the acidic phagosomes of immunologically activated macrophages and to cause lethal pathology in immunocompetent mice. In contrast, when its ability to maintain pHIB homeostasis is genetically compromised, Mtb dies in acidic phagosomes and is attenuated in the mouse. Compounds that phenocopy the genetic disruption of Mtb's pHIB homeostasis could serve as starting points for drug development in their own right or through identification of their targets. A previously reported screen of a natural product library identified a phloroglucinol, agrimophol, that lowered Mtb's pHIB and killed Mtb at an acidic extrabacterial pH. Inability to identify agrimophol-resistant mutants of Mtb suggested that the compound may have more than one target. Given that polyphenolic compounds may undergo covalent reactions, we attempted an affinity-based method for target identification. The structure-activity relationship of synthetically tractable polyhydroxy diphenylmethane analogs with equivalent bioactivity informed the design of a bioactive agrimophol alkyne. After click-chemistry reaction with azido-biotin and capture on streptavidin, the biotinylated agrimophol analog pulled down the Mtb protein Rv3852, a predicted membrane protein that binds DNA in vitro. A ligand-protein interaction between agrimophol and recombinant Rv3852 was confirmed by isothermal calorimetry (ITC) and led to disruption of Rv3852's DNA binding function. However, genetic deletion of rv3852 in Mtb did not phenocopy the effect of agrimophol on Mtb, perhaps because of redundancy of its function.
Project description:Computational prediction of protein function is frequently error-prone and incomplete. In Mycobacterium tuberculosis (Mtb), ~25% of all genes have no predicted function and are annotated as hypothetical proteins, severely limiting our understanding of Mtb pathogenicity. Here, we utilize a high-throughput quantitative activity-based protein profiling (ABPP) platform to probe, annotate, and validate ATP-binding proteins in Mtb. We experimentally validate prior in silico predictions of >240 proteins and identify 72 hypothetical proteins as ATP binders. ATP interacts with proteins with diverse and unrelated sequences, providing an expanded view of adenosine nucleotide binding in Mtb. Several hypothetical ATP binders are essential or taxonomically limited, suggesting specialized functions in mycobacterial physiology and pathogenicity.
Project description:F(420) is a unique cofactor present in a restricted range of microorganisms, including mycobacteria. It has been proposed that F(420) has an important role in the oxidoreductive reactions of Mycobacterium tuberculosis, possibly associated with anaerobic survival and persistence. The protein encoded by Rv0132c has a predicted N-terminal signal sequence and is annotated as an F(420)-dependent glucose-6-phosphate dehydrogenase. Here we show that Rv0132c protein does not have the annotated activity. It does, however, co-purify with F(420) during expression experiments in M. smegmatis. We also show that the Rv0132c-F(420) complex is a substrate for the Tat pathway, which mediates translocation of the complex across the cytoplasmic membrane, where Rv0132c is anchored to the cell envelope. This is the first report of any F(420)-binding protein being a substrate for the Tat pathway and of the presence of F(420) outside of the cytosol in any F(420)-producing microorganism. The Rv0132c protein and its Tat export sequence are essentially invariant in the Mycobacterium tuberculosis complex. Taken together, these results show that current understanding of F(420) biology in mycobacteria should be expanded to include activities occurring in the extra-cytoplasmic cell envelope.
Project description:The lack of effective and accurate diagnostic tools contributes to the high prevalence of tuberculosis (TB) worldwide. The current serodiagnostics for TB are inadequate mainly due to lack of TB-specific antigens with highly accurate diagnosis. In the current study, we aimed to identify novel diagnostic antigens using glutathione S-transferase (GST)-fusion protein technique. We determined the reactivity of these recombinant proteins arrayed in solution and on GSH-immobilized microplates with TB patient sera. Of 409 TB proteins produced, ninety-two yielded seropositive reactions, fourteen including eight novel proteins showed strong immunoreactivity. Further, six were selected and constructed as a multiple-antigen combination set through analysis of various combinations. A comparative study of the multiple-antigen combination set and a commercially available kit revealed that the combination set showed 66.3% (95% CI 60.5-71.8) sensitivity, which was significantly higher than that of the commercial kit [31.6% (95% CI 26.3-37.3)]. The specificity of both methods was similar at 89.6% (95% CI 83.3-95.4) and 90.6% (95% CI 83.0-95.6), respectively. This study provides a set of novel diagnostic protein markers with great potential for the development of novel diagnostic tools for active TB.
Project description:Mycobacterium tuberculosis adenosine-5'-phosphosulfate (APS) reductase is an iron-sulfur protein and a validated target to develop new antitubercular agents, particularly for the treatment of latent infection. To facilitate the development of potent and specific inhibitors of APS reductase, we have probed the molecular determinants that underlie binding and specificity through a series of substrate and product analogues. Our study highlights the importance of specific substitutent groups for substrate binding and provides functional evidence for ligand-specific conformational states. An active site model has been developed for M. tuberculosis APS reductase that is in accord with the results presented here as well as prior structural data reported for Pseudomonas aeruginosa APS reductase and related enzymes. This model illustrates the functional features required for the interaction of APS reductase with a ligand and provides a pharmacological roadmap for the rational design of small molecules as potential inhibitors of APS reductase present in human pathogens, including M. tuberculosis.
Project description:The gene encoding the Mycobacterium tuberculosis Rv2536 protein is present in the Mycobacterium tuberculosis complex (as assayed by PCR) and transcribed (as determined by RT-PCR) in M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. bovis BCG, and M. africanum strains. Rabbits immunized with synthetic polymer peptides from this protein produced antibodies specifically recognizing a 25-kDa band in mycobacterial sonicate. U937 and A549 cells were used in binding assays involving 20-amino-acid-long synthetic peptides covering the whole Rv2536 protein sequence. Peptide 11207 (161DVFSAVRADDSPTGEMQVAQY180) presented high specific binding to both types of cells; the binding was saturable and presented nanomolar affinity constants. Cross-linking assays revealed that this peptide specifically binds to 50 kDa U937 cell membrane and 45 kDa A549 cell membrane proteins.
Project description:Little is known about cyclic AMP (cAMP) function in Mycobacterium tuberculosis, despite its ability to encode 15 adenylate cyclases and 10 cNMP-binding proteins. M. tuberculosis Rv3676, which we have designated CRP(Mt), is predicted to be a cAMP-dependent transcription factor. In this study, we characterized CRP(Mt)'s interactions with DNA and cAMP, using experimental and computational approaches. We used Gibbs sampling to define a CRP(Mt) DNA motif that resembles the cAMP receptor protein (CRP) binding motif model for Escherichia coli. CRP(Mt) binding sites were identified in a total of 73 promoter regions regulating 114 genes in the M. tuberculosis genome, which are being explored as a regulon. Specific CRP(Mt) binding caused DNA bending, and the substitution of highly conserved nucleotides in the binding site resulted in a complete loss of binding to CRP(Mt). cAMP enhanced CRP(Mt)'s ability to bind DNA and caused allosteric alterations in CRP(Mt) conformation. These results provide the first direct evidence for cAMP binding to a transcription factor in M. tuberculosis, suggesting a role for cAMP signal transduction in M. tuberculosis and implicating CRP(Mt) as a cAMP-responsive global regulator.
Project description:BACKGROUND: Obg is a highly conserved GTP-binding protein that has homologues in bacteria, archaea and eukaryotes. In bacteria, Obg proteins are essential for growth, and they participate in spore formation, stress adaptation, ribosome assembly and chromosomal partitioning. This study was undertaken to investigate the biochemical and physiological characteristics of Obg in Mycobacterium tuberculosis, which causes tuberculosis in humans. RESULTS: We overexpressed M. tuberculosis Obg in Escherichia coli and then purified the protein. This protein binds to, hydrolyzes and is phosphorylated with GTP. An anti-Obg antiserum, raised against the purified Obg, detects a 55 kDa protein in immunoblots of M. tuberculosis extracts. Immunoblotting also discloses that cultured M. tuberculosis cells contain increased amounts of Obg in the late log phase and in the stationary phase. Obg is also associated with ribosomes in M. tuberculosis, and it is distributed to all three ribosomal fractions (30 S, 50 S and 70 S). Finally, yeast two-hybrid analysis reveals that Obg interacts with the stress protein UsfX, indicating that M. tuberculosis Obg, like other bacterial Obgs, is a stress related protein. CONCLUSIONS: Although its GTP-hydrolyzing and phosphorylating activities resemble those of other bacterial Obg homologues, M. tuberculosis Obg differs from them in these respects: (a) preferential association with the bacterial membrane; (b) association with all three ribosomal subunits, and (c) binding to the stress protein UsfX, rather than to RelA. Generation of mutant alleles of Obg of M. tuberculosis, and their characterization in vivo, may provide additional insights regarding its role in this important human pathogen.
Project description:Mycobacterium tuberculosis (MTB) infects 30% of all humans and kills someone every 20-30 s. Here we report genome-wide binding for ~80% of all predicted MTB transcription factors (TFs), and assayed global expression following induction of each TF. The MTB DNA-binding network consists of ~16,000 binding events from 154 TFs. We identify >50 TF-DNA consensus motifs and >1,150 promoter-binding events directly associated with proximal gene regulation. An additional ~4,200 binding events are in promoter windows and represent strong candidates for direct transcriptional regulation under appropriate environmental conditions. However, we also identify >10,000 'dormant' DNA-binding events that cannot be linked directly with proximal transcriptional control, suggesting that widespread DNA binding may be a common feature that should be considered when developing global models of coordinated gene expression.