Defining the mandate of tuberculosis research in a postgenomic era

The identification of Mycobacterium tuberculosis by Robert Koch in 1882 as the causative agent of tuberculosis, the release of the drug rifampicin in 1970 and the sequencing of the M. tuberculosis genome in 1998 are three major events that have revolutionized tuberculosis research. In spite of these breakthroughs, the continued status of tuberculosis as the largest killer amongst infectious diseases is an issue of major concern. Although directly observed short course chemotherapy exists to treat the disease, the emergence of drug-resistant strains has severely threatened the efficacy of the treatment. The recent sequencing of the M. tuberculosis genome holds promise for the development of new vaccines and the design of new drugs. This is all the more possible when the information from the genome sequence is combined with proteomics and structural and functional genomics. Such an integrated approach has led to the birth of a new field of research christened 'postgenomics' that holds substantial promise for the identification of novel drug targets and the potential to aid the development of new chemotherapeutic compounds to treat tuberculosis. The challenge before the scientific community therefore lies in elucidation of the wealth of information provided by the genome sequence and its translation into the design of novel therapies for the disease. All the major developments in the field of tuberculosis research after the sequencing of the M. tuberculosis genome will be discussed in this review

Genomics of Mycobacterium tuberculosis: old threats & new trends

Tuberculosis (TB) has been declared as a global health emergency by the World Health Organization (WHO). This has been mainly due to the emergence of multiple drug resistant strains and the synergy between tubercle bacilli and the human immunodeficiency virus (HIV). Genomic analysis of strains for outbreak investigations is in vogue for about a decade now. However, information available from whole genome sequencing efforts and comparative genomics of laboratory and field strains is likely to revolutionize efforts towards understanding molecular pathogenesis and dissemination dynamics of this dreaded disease. Genomic information is also going to fuel discovery projects where new targets will be identified and explored towards a new drug for TB. Besides this, efforts of information technologists, chemists, population biologists, freelance workers, media persons, non-governmental organizations and administrators to needed to handle the problem of tuberculosis to prevent it from becoming a pandemic

A 30-kDa host protein binds to two very-late baculovirus promoters

A 30-kDa host factor (polyhedrin-promoter-binding protein; PPBP) specifically binds to sequences critical for transcription from the baculovirus polyhedrin (p29) gene initiator promoter [Burma, S., Mukherjee, B., Jain, A., Habib, S. & Hasnain, S. E. (1994) J. Biol. Chem. 269, 2750-2757; Mukherjee, B., Burma, S. & Hasnain, S. E. (1995) J. Biol. Chem. 270, 4405-4411]. A host factor also binds, in gel shift assays, to the very-late p10 gene promoter through DNA sequence motifs similar to the PPBP p29 interaction. The p10 host factor complex was specifically competed out with oligonucleotides containing p29 cognate sequence motifs AATAAA and TAAGTATT, but this did not occur when these motifs were replaced with random sequences. From ultraviolet cross-linking analysis, the molecular mass of this host factor was estimated to be approximately 30 kDa. Experiments were performed to investigate if this host factor displayed any differences in affinity and turnover with respect to the p29 and p10 untranslated leader sequences known to be important for temporal fine tuning and the late burst of transcription. Half-life determination of the p10-binding protein revealed similar binding affinities for the initiator elements of both the promoters, but higher affinity for the p10 5'-untranslated region (≈30 min versus ≈10 min). The involvement of a similar host factor binding to both the p10 and p29 promoters indicates the possibility of a similar mode of transcription initiation from these two very-late promoters

pheA (Rv3838c) of Mycobacterium tuberculosis encodes an allosterically regulated monofunctional prephenate dehydratase that requires both catalytic and regulatory domains for optimum activity

Prephenate dehydratase (PDT) is a key regulatory enzyme in l-phenylalanine biosynthesis. In Mycobacterium tuberculosis, expression of pheA, the gene encoding PDT, has been earlier reported to be iron-dependent (1, 2). We report that M. tuberculosis pheA is also regulated at the protein level by aromatic amino acids. All of the three aromatic amino acids (phenylalanine, tyrosine, and tryptophan) are potent allosteric activators of M. tuberculosis PDT. We also provide in vitro evidence that M. tuberculosis PDT does not possess any chorismate mutase activity, which suggests that, unlike many other enteric bacteria (where PDT exists as a fusion protein with chorismate mutase), M. tuberculosis PDT is a monofunctional and a non-fusion protein. Finally, the biochemical and biophysical properties of the catalytic and regulatory domains (ACT domain) of M. tuberculosis PDT were studied to observe that, in the absence of the ACT domain, the enzyme not only loses its regulatory activity but also its catalytic activity. These novel results provide evidence for a monofunctional prephenate dehydratase enzyme from a pathogenic bacterium that exhibits extensive allosteric activation by aromatic amino acids and is absolutely dependent upon the presence of catalytic as well as the regulatory domains for optimum enzyme activity

A bifunctional baculovirus homologous region (hr1) sequence: enhancer and orginal of replication functions reside within the same sequence element

Analysis of the synergy between transcription regulation and DNA replication as well as the mechanisms of enhancer action has been of interest to us. Our studies on the Autographa californica multi-nucleocapsid nuclear polyhedrosis virus (AcMNPV) have revealed that the viral homologous region sequence, hr1, can function as an enhancer of polyhedron promoter-driven transcription as well as an origin of DNA replication in transfected host insect cells. Minimal sequence requirements for both these activities of hr1 have been delineated. A host factor that interacts at multiple sites within hr1 has also been implicated in the enhancer function of this sequence. While demonstrating the dual function of hr1, our observations also indicate the importance of host factor(s) in regulating crucial processes in the viral infection cycle

Basic and biomedical product-oriented research - the National Institute of Immunology

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<i>Gut Pathogens</i>: enteric health at the interface of changing microbiology

The International Society for Genomic and Evolutionary Microbiology (ISOGEM) in collaboration with BioMed Central Ltd. has launched Gut Pathogens with the aim of providing a high-quality forum for research on enteric infections of humans and animals. The journal led by three Editors-in-Chief and supported by a highly qualified and organized international Editorial Board publishes open access research articles of repute in areas of biology and the pathogenesis of bacterial, parasitic and viral infections of the gut including their diagnosis, epidemiology and clinical management

The host factor polyhedrin promoter binding protein (PPBP) is involved in transcription from the baculovirus polyhedrin gene promoter

Hypertranscription and temporal expression from the Autographa californica nuclear polyhedrosis (AcNPV) baculovirus polyhedrin promoter involves an &#945;-amanitin-resistant RNA polymerase and requires a trans-acting viral factor(s). We previously reported that a 30-kDa host factor, polyhedrin promoter binding protein (PPBP), binds with unusual affinity, specificity, and stability to the transcriptionally important motif AATAAATAAGTATT within the polyhedrin (polh) initiator promoter and also displays coding strand-specific single-stranded DNA (ssDNA)-binding activity (S. Burma, B. Mukherjee, A. Jain, S. Habib, and S. E. Hasnain, J. Biol. Chem. 269:2750-2757, 1994; B. Mukherjee, S. Burma, and S. E. Hasnain, J. Biol. Chem. 270:4405-4411, 1995). We now present evidence which indicates that an additional factor(s) is involved in stabilizing PPBP-duplex promoter and PPBP-ssDNA interactions. TBP (TATA box binding protein) present in Spodoptera frugiperda (Sf9) cells is characteristically distinct from PPBP and does not interact directly with the polh promoter. Replacement of PPBP cognate sequences within the polh promoter with random nucleotides abolished PPBP binding in vitro and also failed to express the luciferase reporter gene in vivo. Phosphocellulose fractions of total nuclear extract from virus-infected cells which support in vitro transcription from the polh promoter contain PPBP activity. When PPBP was sequestered by the presence of oligonucleotides containing PPBP cognate sequence motifs, in vitro transcription of a C-free reporter cassette was affected but was restored by the exogenous addition of nuclear extract containing PPBP. When PPBP was mopped out in vivo by a plasmid carrying PPBP cognate sequence present in trans, polh promoter-driven expression of the luciferase reporter was abolished, demonstrating that binding of PPBP to the polh promoter is essential for transcription

Specificity of drug transport mediated by CaMDR1: a major facilitator of Candida albicans

CaMDR1 encodes a major facilitator superfamily (MFS) protein inCandida albicans whose expression has been linked to azole resistance and which is frequently encountered in this human pathogenic yeast. In this report we have overexpressed CaMdr1p inS&#402;9 insect cells and demonstrated for the first time that it can mediate methotrexate (MTX) and fluconazole (FLC) transport. MTX appeared to be a better substrate for CaMdr1p among these two tested drugs. Due to severe toxicity of these drugs to insect cells, further characterization of CaMdr1p as a drug transporter could not be done with this system. Therefore, as an alternative, CaMdr1p and Cdr1p, which is an ABC protein (ATP binding cassette) also involved in azole resistance inC. albicans, were independently expressed in a common hypersensitive host JG436 ofSaccharomyces cerevisiae. This allowed a better comparison between the functionality of the two export pumps. We observed that while both FLC and MTX are effluxed by CaMdr1p, MTX appeared to be a poor substrate for Cdr1p. JG436 cells expressing Cdr1p thus conferred resistance to other antifungal drugs but remained hypersensitive to MTX. Since MTX is preferentially transported by CaMdr1p, it can be used for studying the function of this MFS protein

Comparison of Mycobacterium tuberculosis isocitrate dehydrogenases (ICD-1 and ICD-2) reveals differences in coenzyme affinity, oligomeric state, pH tolerance and phylogenetic affiliation

Background: M.tb icd-1 and M.tb icd-2, have been identified in the Mycobacterium tuberculosis genome as probable isocitrate dehydrogenase (ICD) genes. Earlier we demonstrated that the two isoforms can elicit B cell response in TB patients and significantly differentiate TB infected population from healthy, BCG-vaccinated controls. Even though immunoassays suggest that these proteins are closely related in terms of antigenic determinants, we now show that M.tb icd-1 and M.tb icd-2 code for functional energy cycle enzymes and document the differences in their biochemical properties, oligomeric assembly and phylogenetic affiliation. Results: Functionally, both M.tb ICD-1 and ICD-2 proteins are dimers. Zn+2 can act as a cofactor for ICD-1 apart from Mg+2, but not for ICD-2. ICD-1 has higher affinity for metal substrate complex (Km (isocitrate) with Mg++:10 &#956;M &#177; 5) than ICD-2 (Km (isocitrate) with Mg++:20 &#956;M &#177; 1). ICD-1 is active across a wider pH range than ICD-2, retaining 33-35% activity in an acidic pH upto 5.5. Difference in thermal behaviour is also observed with ICD-2 being active across wider temperature range (20&#176;C to 40&#176;C) than ICD-1 (optimum temperature 40&#176;C). The isozymes are NADP+ dependent with distinct phylogenetic affiliations; unlike M.tb ICD-2 that groups with bacterial ICDs, M.tb ICD-1 exhibits a closer lineage to eukaryotic NADP+ dependent ICDs. Conclusion: The data provide experimental evidence to show that the two open reading frames, Rv3339c (ICD-1) and Rv0066c (ICD-2), annotated as probable ICDs are functional TCA cycle enzymes with identical enzymatic function but different physio-chemical and kinetic properties. The differences in biochemical and kinetic properties suggest the possibility of differential expression of the two ICDs during different stages of growth, despite having identical metabolic function