Genome Of Mycobacterium Tuberculosis H37Rv Research Articles

A Bridging Centrality Plugin for GEPHI and a Case Study for Mycobacterium Tuberculosis H37Rv.

Bridging Centrality (BriCe) is a popular measure that combines the Betweenness centrality and Bridging coefficient metrics to characterize nodes acting as a bridge among clusters. However, there were no implementations of the BriCe plugin that can be readily used in the GEPHI software or any other software dedicated to graph-based studies. In this paper, we present the BriCe plugin for GEPHI. It is available as a third-party functionality from the native GEPHI interface as a handy plugin to add; hence, no additional download and installation process is necessary. The BriCe plugin for GEPHI is open-source, and one can access the code through the GEPHI GitHub repository. As a use case of the BriCe plugin, we analyzed the genome of Mycobacterium tuberculosis H37Rv to identify biological explanations on why some proteins were ranked with top BriCe values? For instance, we were able to formulate a new hypothesis combining the predicted sub cellular localization and high BriCe values concerning lipopolysaccharides (LPS) exportation. Our hypothesis provides a possible link among proteins of a glycosyltransferase group and the type VII Secretion System. The Bridging Centrality plugin for GEPHI is an easy to use tool for analyzing complex graphs and draw novel insights from graphical data.

Domain-wise differentiation of Mycobacterium tuberculosis H37 Rv hypothetical proteins: A roadmap to discover bacterial survival potentials.

Proteomic information revealed approximately 3,923 proteins in Mycobacterium tuberculosis H37 Rv genome of which around ∼25% of proteins are hypothetical proteins (HPs). The present work comprises computational approaches to identify and characterize the HPs of M. tuberculosis that symbolize the putative target for rationale development of a drug or antituberculosis strategy. Proteins were primarily classified based on motif and domain information, which were further analyzed for the presence of virulence factors (VFs), determination of localization, and signal peptide/enzymatic cleavage sites. 863 HPs were found, and 599 HPs were finalized based on motifs, that is, GTP (525), Trx (47), SAM (14), PE-PGRS (5), and CBD (8). 80 HPs contain virulence factor (VF), 24 HPs localized in membrane region, and 4 HPs contain signal peptide/enzymatic cleavage sites. The overall parametric study finalizes four HPs Rv0679c, Rv0906, Rv3627c, and Rv3811 that also comprise GTPase domain. Structure prediction, structure-based function prediction, molecular docking and mutation analysis of selected proteins were done. Docking studies revealed that GTP and GTPase inhibitor (mac0182344) were docked with all four proteins with high affinities. In silico point mutation studies showed that substitution of aspartate with glycine within a GTPase motif showed the largest decrease in stability and pH differentiation also affects protein's stability. This analysis thus fixes a roadmap in the direction of finding potential target of this bacterium for drug development and enlightens the efficacy of GTP as a major regulator of Mycobacterial cellular pathways.

Investigating function roles of hypothetical proteins encoded by the Mycobacterium tuberculosis H37Rv genome

BackgroundMycobacterium tuberculosis (MTB) is a common bacterium causing tuberculosis and remains a major pathogen for mortality. Although the MTB genome has been extensively explored for two decades, the functions of 27% (1051/3906) of encoded proteins have yet to be determined and these proteins are annotated as hypothetical proteins.MethodsWe assigned functions to these hypothetical proteins using SSEalign, a newly designed algorithm utilizing structural information. A set of rigorous criteria was applied to these annotations in order to examine whether they were supported by each parameter. Virulence factors and potential drug targets were also screened among the annotated proteins.ResultsFor 78% (823/1051) of the hypothetical proteins, we could identify homologs in Escherichia coli and Salmonella typhimurium by using SSEalign. Functional classification analysis indicated that 62.2% (512/823) of these annotated proteins were enzymes with catalytic activities and most of these annotations were supported by at least two other independent parameters. A relatively high proportion of transporter was identified in MTB genome, indicating the potential frequent transportation of frequent absorbing essential metabolites and excreting toxic materials in MTB. Twelve virulence factors and ten vaccine candidates were identified within these MTB hypothetical proteins, including two genes (rpoS and pspA) related to stress response to the host immune system. Furthermore, we have identified six novel drug target candidates among our annotated proteins, including Rv0817 and Rv2927c, which could be used for treating MTB infection.ConclusionsOur annotation of the MTB hypothetical proteins will probably serve as a useful dataset for future MTB studies.

Co-Expression of hbha and mtb32C Genes from Mycobacterium tuberculosis H37Rv in a Prokaryotic System

Background: Heparin-binding hemagglutinin (HBHA) protein is a surface adhesin that mediates the attachment of Mycobacterium tuberculosis to host cells by its own unique, carboxyl-terminal region. The methylated HBHA has a specific motif with a lysine-, alanine-, and proline-rich domain. More recently, it has been shown that HBHA protein has potential activity in stimulating immune responses, and is a promising new candidate for diagnostic applications besides a protective antigen against tuberculosis. Objectives: The aim of this study was to isolate a mycobacterial latency gene (hbha), and subsequently produce its protein as a new antigen for the Interferon-Gamma Release Assay test (IGRAs). Methods: In the present work, hbha and mtb32C genes were isolated from the Mycobacterium tuberculosis H37Rv genome using the polymerase chain reaction (PCR) method. The PCR products and pet21+ vector were digested with specific restriction enzymes and then submitted to the ligation procedure. Escherichia coli BL21-CodonPlus (DE3) competent cells were transformed with the recombinant mtb32C-hbha -pet21+ vector. Expression of recombinant protein (Mtb32C-HBHA) was confirmed with Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and western blot methods. Results: Detection of a 500-bp gene and sequencing of recombinant pet-mtb32C-hbha vector, all confirmed the accuracy of the cloning procedure. A 36-KDa band of Mtb32C-HBHA protein was also detected by western blotting. Conclusions: In this study, expression of Mtb32C-HBHA protein was successfully done, in the prokaryotic system. Further studies are needed to evaluate the efficacy of recombinant Mtb32C-HBHA protein in diagnosis of latent tuberculosis.

In silico characterization of hypothetical proteins obtained from Mycobacterium tuberculosis H37Rv

Tuberculosis is one of the oldest diseases with a death rate of 1.5 million per year. Tuberculosis spreads from one person to another through Mycobacterium tuberculosis. This bacteria belongs to the family Mycobacteriaceae, genus Mycobacterium, member of the tuberculosis complex. Mycobacterium tuberculosis is an acid-fast, aerobic, rod-shaped bacteria, ranging from 2 to 4 Â µm in length and 0.2 to 0.5 Â µm in width. Tuberculosis spreads through infected people via sneezing, coughing, etc., with humans acting as the host for the bacteria. The genome of Mycobacterium tuberculosis H37Rv encodes 3906 proteins, of which 1055 are hypothetical proteins (HPs), wherein the functions of the proteins are unknown. The sequences of 1055 HPs of Mycobacterium tuberculosis were analyzed and the functions of 578 HPs were subsequently predicted with a high level of confidence. Several enzymes, transporters and binding proteins of 1055 HPs in M. tuberculosis were analyzed and potential targets were discovered which contribute to the overall survival of the bacteria. The analysis will be of relevance in understanding the mechanism of the bacteria and will prove to be beneficial in the discovery of new drugs.

Structural Characterization and Ligand/Inhibitor Identification Provide Functional Insights into the Mycobacterium tuberculosis Cytochrome P450 CYP126A1

The Mycobacterium tuberculosis H37Rv genome encodes 20 cytochromes P450, including P450s crucial to infection and bacterial viability. Many M. tuberculosis P450s remain uncharacterized, suggesting that their further analysis may provide new insights into M. tuberculosis metabolic processes and new targets for drug discovery. CYP126A1 is representative of a P450 family widely distributed in mycobacteria and other bacteria. Here we explore the biochemical and structural properties of CYP126A1, including its interactions with new chemical ligands. A survey of azole antifungal drugs showed that CYP126A1 is inhibited strongly by azoles containing an imidazole ring but not by those tested containing a triazole ring. To further explore the molecular preferences of CYP126A1 and search for probes of enzyme function, we conducted a high throughput screen. Compounds containing three or more ring structures dominated the screening hits, including nitroaromatic compounds that induce substrate-like shifts in the heme spectrum of CYP126A1. Spectroelectrochemical measurements revealed a 155-mV increase in heme iron potential when bound to one of the newly identified nitroaromatic drugs. CYP126A1 dimers were observed in crystal structures of ligand-free CYP126A1 and for CYP126A1 bound to compounds discovered in the screen. However, ketoconazole binds in an orientation that disrupts the BC-loop regions at the P450 dimer interface and results in a CYP126A1 monomeric crystal form. Structural data also reveal that nitroaromatic ligands “moonlight” as substrates by displacing the CYP126A1 distal water but inhibit enzyme activity. The relatively polar active site of CYP126A1 distinguishes it from its most closely related sterol-binding P450s in M. tuberculosis, suggesting that further investigations will reveal its diverse substrate selectivity.

Crystal structure of VapC21 from Mycobacterium tuberculosis at 1.31 Å resolution

Persisters are individual bacterial cells that exhibit a phenotype characterized by slow growth, low metabolic rate and multidrug tolerance. The processes that drive cells into a persistence state constitute an active but incipient research field, and structural data regarding its components are scarce. The molecular targets of many therapeutic drugs are involved in cell wall synthesis and cell division, and these cellular processes are down-regulated in persister cells, consequently these cells are more likely to survive antibiotic treatment. Toxin-antitoxin systems were shown to have a leading role in the formation of persisters, and several pathogenic bacteria display a wide array of these systems. The Mycobacterium tuberculosis H37Rv genome presents 88 toxin-antitoxin loci, of which 47 code for members of the VapBC protein family. To date, only four crystal structures of Mycobacterium tuberculosis VapBC complexes are available, and all of them present the toxin bound to and inhibited by the antitoxin. We present the 1.31 Å resolution structure of VapC21, the first structure of a Mycobacterium tuberculosis VapC toxin in the absence of its cognate inhibitory antitoxin. Our data show that VapC21 is a dimer in solution, with conserved active site architecture and an extensive antitoxin binding groove. Additionally, the strategy used to mutate a putative catalytic residue allowing the expression and purification of soluble VapC21 will pave the way for the resolution of more toxin structures in the absence of antitoxins. Taken together, our findings represent an important step in unraveling the molecular mechanisms related to persistence, which will contribute for the design of faster and more efficient therapeutic approaches for the treatment of tuberculosis, particularly for infections with multidrug-resistant strains.

Prokaryotic expression, identification and bioinformatics analysis of fbpB–esxA fusing gene from Mycobacterium tuberculosis

Objective To obtain fbpB–esxA fusing gene of Mycobacterium tuberculosis (MTB), express the encoded fusing protein in Escherichia coli ( E. coli), identify protein acquired, and predict the structure and function of the protein utilizing methods of bioinformatics. Methods fbpB and esxA gene were amplified from genome of MTB H37Rv by PCR. The fbpB–esxA fusing gene ligated by (Gly 4Ser) 3 linker was gained by means of Gene Splicing by Overlapping Extension PCR (SOE-PCR), and fusing gene was cloned into expression vector pET-30a. The recombinant plasmid was sequenced and expressed in E. coli BL21(DE3). The protein was identified by Western blot using anti-HIS antibody. Secondary structure and antigenic epitopes of the protein were predicting using tools of bioinformatics. Results The DNA sequences of fbpB–esxA were identical with that published by GenBank. The Ag85B-ESAT-6 fusion protein about 50 kDa comprised 485 amino acids was efficiently produced from expression system in E. coli BL21(DE3) under the induction of IPTG. Bioinformatics analysis showed the protein contained one transmembrane region and fourteen potential antigenic epitopes. Conclusions The Ag85B-ESAT-6 fusion protein is successfully expressed with N-terminal HIS-tag. Gel filtration demonstrated that it exists as insoluble inclusion bodies mainly. The existence of linker doesn't affect immunogenicity of Ag85B and ESAT-6. It will allow for characterization in vitro and establish a foundation of further function research such as vaccine or diagnostic reagent.

Nvestigation of ABC transporter from mycobacterial arabinogalactan biosynthetic cluster

Two genes from the "mycobacterial arabinogalactan biosynthetic cluster" spanning the region from Rv3779 to Rv3809c in the genome of Mycobacterium tuberculosis H37Rv were annotated as possible components of the ATP-binding cassette transporter. Rv3781 encodes a nucleotide-binding domain and Rv3783 determines production of a membrane-spanning domain. We have examined possible roles of these genes in mycobacterial cell wall biosynthesis through inactivation of their respective orthologs in Mycobacterium smegmatis mc(2)155, phenotypic characterization of the mutant strains via metabolic labeling with [U-(14)C]-glucose, cell-free reactions with UDP-[U-(14)C]-galactose monitoring galactan build-up and transcriptional analysis. Several lines of evidence suggest that this ABC transporter is involved in biosynthesis of arabinogalactan, although more investigation is needed to establish its precise role or the transported substrate.

Computational Prediction and Experimental Assessment of Secreted/Surface Proteins from Mycobacterium tuberculosis H37Rv

The mycobacterial cell envelope has been implicated in the pathogenicity of tuberculosis and therefore has been a prime target for the identification and characterization of surface proteins with potential application in drug and vaccine development. In this study, the genome of Mycobacterium tuberculosis H37Rv was screened using Machine Learning tools that included feature-based predictors, general localizers and transmembrane topology predictors to identify proteins that are potentially secreted to the surface of M. tuberculosis, or to the extracellular milieu through different secretory pathways. The subcellular localization of a set of 8 hypothetically secreted/surface candidate proteins was experimentally assessed by cellular fractionation and immunoelectron microscopy (IEM) to determine the reliability of the computational methodology proposed here, using 4 secreted/surface proteins with experimental confirmation as positive controls and 2 cytoplasmic proteins as negative controls. Subcellular fractionation and IEM studies provided evidence that the candidate proteins Rv0403c, Rv3630, Rv1022, Rv0835, Rv0361 and Rv0178 are secreted either to the mycobacterial surface or to the extracellular milieu. Surface localization was also confirmed for the positive controls, whereas negative controls were located on the cytoplasm. Based on statistical learning methods, we obtained computational subcellular localization predictions that were experimentally assessed and allowed us to construct a computational protocol with experimental support that allowed us to identify a new set of secreted/surface proteins as potential vaccine candidates.

Recent Development and Future Perspective of Antitubercular Therapy

Tuberculosis (TB) has remained an enemy of humankind before the beginning of recorded history. Although it is curable and preventable, TB claims the lives of more than 5,000 people every day. Despite of availability of effective chemotherapy and BCG vaccine in 21st century, TB remains one of the most deadly infectious diseases. The concomitant resurgence of TB with the Multi-drug resistant (MDR) or Extremely drug resistant (XDR)-TB and HIV/AIDS pandemic raises the threat posed by untreatable and fatal human TB, exposing the frailties of the current drug armatorium. No new drug is available acting through novel mechanism since last 40 years. The genome of Mycobacterium tuberculosis H37Rv was sequenced in 1998 and reannotated in 2002. This coupled with development of new genetic tools, have greatly contributed to the discovery of potential drug targets for new anti-TB agent. It is increasingly acknowledged that new drugs should not only be active against drug resistant TB, but should also kill persisters and shorten the lengthy TB treatment, which underlies the problem of drug resistance due to poor compliance to the length of therapy. This review article describes the current TB drugs, their merits and demerits as well as the new promising anti-TB agents classified on the basis of molecular framework. It is a comprehensive literature compilation on the present research paradigm of anti-TB drug discovery including advances in the new structural analogs reported in last decade.

Expression and Characterization of Two Functional Methionine Aminopeptidases from Mycobacterium tuberculosis H37Rv

Methionine aminopeptidase (MetAP) carries out an essential posttranslational modification of nascent proteins by removing the N-terminal methionine and is a potential target for discovering antibacterial agents. To characterize and compare the two MetAPs in Mycobacterium tuberculosis, Rv0734 and Rv2861c genes encoding MetAPs from genome of Mycobacterium tuberculosis H37Rv were cloned and expressed in Escherichia coli. Comparative analysis showed that the two recombinant Mycobacterium tuberculosis MetAPs (MtMetAPs, with 6His-tag being cleaved) have different activities in their substrate specificity, divalent ion requirement, and temperature optima. The temperature for MtMetAP1a and MtMetAP1c were 55 and 37 degrees C, respectively, and MtMetAP1a was found to have good temperature stability. The activities of MtMetAPs were increased by Co2+ ions, but were strongly inhibited by Cu2+, Fe2+, and Ni2+. In addition, the MtMetAP1a and MtMetAP1c activities were stimulated by Mg2+ and Zn2+, respectively. Transcriptional comparative analysis of these two genes revealed that, in both H37Ra and H37Rv, there was approximately a 2-fold decrease of Rv0734 transcripts in 60-day-old stationary phase culture comparing to that in 14-day-old log phase bacilli. On the other hand, the transcription level of Rv2861c in tested mycobacteria in log phase culture was nearly 1.5 times lower than that in stationary phase culture. The result suggests that the two MtMetMAPs may perform important function in different growth phases of Mycobacterium tuberculosis.

Molecular cloning, overexpression and biochemical characterization of hypothetical β-lactamases ofMycobacterium tuberculosisH37Rv

Molecular cloning, overexpression and biochemical characterization of the genes from the Mycobacterium tuberculosis H37Rv genome having hypothetical beta-lactamases activity. Analysis of the M. tuberculosis H37Rv genome revealed that Rv2068c, Rv0406c and Rv3677c gene products were predicted to exhibit beta-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 beta-lactamase activity by the hydrolysis of nitrocefin and other beta-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 beta-lactam antibiotics. The study revealed the possibility of more than one gene in M. tuberculosis, encoding proteins having beta-lactamase or beta-lactamase-like activity, giving wide spectrum of resistance against beta-lactams. Systematic study of hypothetical beta-lactamases of M. tuberculosis and related species and their correlation with beta-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.

Expression, essentiality, and a microtiter plate assay for mycobacterial GlmU, the bifunctional glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridyltransferase

UDP- N-acetyl- d-glucosamine (UDP-GlcNAc) is an essential precursor of peptidoglycan and the rhamnose-GlcNAc linker region of mycobacterial cell wall. In Mycobacterium tuberculosis H37Rv genome, Rv1018c shows strong homology to the GlmU protein involved in the formation of UDP-GlcNAc from other bacteria. GlmU is a bifunctional enzyme that catalyzes two sequential steps in UDP-GlcNAc biosynthesis. Glucosamine-1-phosphate acetyl transferase catalyzes the formation of N-acetylglucosamine-1-phosphate, and N-acetylglucosamine-1-phosphate uridylyltransferase catalyzes the formation of UDP-GlcNAc. Since inhibition of peptidoglycan synthesis often results in cell lysis, M. tuberculosis GlmU is a potential anti-tuberculosis (TB) drug target. In this study we cloned M. tuberculosis Rv1018c ( glmU gene) and expressed soluble GlmU protein in E. coli BL21(DE3). Enzymatic assays showed that M. tuberculosis GlmU protein exhibits both glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridylyltransferase activities. We also investigated the effect on Mycobacterium smegmatis when the activity of GlmU is fully removed or reduced via a genetic approach. The results showed that activity of GlmU is required for growth of M. smegmatis as the bacteria did not grow in the absence of active GlmU enzyme. As the amount of functional GlmU enzyme was gradually reduced in a temperature shift experiment, the M. smegmatis cells became non-viable and their morphology changed from a normal rod shape to stubby-rounded morphology and in some cases they lysed. Finally a microtiter plate based assay for GlmU activity with an OD 340 read out was developed. These studies therefore support the further development of M. tuberculosis GlmU enzyme as a target for new anti-tuberculosis drugs.

Evolutionary insights from suffix array-based genome sequence analysis

Gene and protein sequence analyses, central components of studies in modern biology are easily amenable to string matching and pattern recognition algorithms. The growing need of analysing whole genome sequences more efficiently and thoroughly, has led to the emergence of new computational methods. Suffix trees and suffix arrays are data structures, well known in many other areas and are highly suited for sequence analysis too. Here we report an improvement to the design of construction of suffix arrays. Enhancement in versatility and scalability, enabled by this approach, is demonstrated through the use of real-life examples. The scalability of the algorithm to whole genomes renders it suitable to address many biologically interesting problems. One example is the evolutionary insight gained by analysing unigrams, bi-grams and higher n-grams, indicating that the genetic code has a direct influence on the overall composition of the genome. Further, different proteomes have been analysed for the coverage of the possible peptide space, which indicate that as much as a quarter of the total space at the tetra-peptide level is left un-sampled in prokaryotic organisms, although almost all tri-peptides can be seen in one protein or another in a proteome. Besides, distinct patterns begin to emerge for the counts of particular tetra and higher peptides, indicative of a 'meaning' for tetra and higher n-grams. The toolkit has also been used to demonstrate the usefulness of identifying repeats in whole proteomes efficiently. As an example, 16 members of one COG,coded by the genome of Mycobacterium tuberculosis H37Rv have been found to contain a repeating sequence of 300 amino acids.

Scanning the Genome of Mycobacterium tuberculosis to Identify Potential Lectins

Lectins are carbohydrate binding proteins with important roles in many biological processes such as adhesion. Here we have identified 11 potential lectins from Mycobacterium tuberculosis H37Rv genome, using a comprehensive bioinformatics analysis, which will provide helpful clues in molecular mapping of pathogenesis and perhaps also serve as potential drug targets.

Genome-Wide Analysis of Intergenic Regions of Mycobacterium tuberculosis H37Rv Using Affymetrix GeneChips

Sequencing the complete genome of Mycobacterium tuberculosis H37Rv is a major milestone in the genome project and it sheds new light in our fight with tuberculosis. The genome contains around 4000 genes (protein-coding sequences) in the original genome annotation. A subsequent reannotation of the genome has added 80 more genes. However, we have found that the intergenic regions can exhibit expression signals, as evidenced by microarray hybridization. It is then reasonable to suspect that there are unidentified genes in these regions. We conducted a genome-wide analysis using the Affymetrix GeneChip to explore genes contained in the intergenic sequences of the M. tuberculosis H37Rv genome. A working criterion for potential protein-coding genes was based on bioinformatics, consisting of the gene structure, protein coding potential, and presence of ortholog evidence. The bioinformatics criteria in conjunction with transcriptional evidence revealed potential genes with a specific function, such as a DNA-binding protein in the CopG family and a nickel binding GTPase, as well as hypothetical proteins that had not been reported in the H37Rv genome. This study further demonstrated that microarray-based transcriptional evidence would facilitate genome-wide gene finding, and is also the first report concerning intergenic expression in M. tuberculosis genome.

FUNCTIONAL IMPLICATIONS OF MULTIPLE FadD PROTEINS IN THE BIOLOGY OF MYCOBACTERIUM TUBERCULOSIS

Mycobacterium tuberculosis H37Rv genome sequencing revealed an unanticipated variety of genes homologous to proteins involved in β-oxidation machinery. Identification of 34 fadD homologous was quite remarkable and it was tempting to speculate that many of these might not even be expressed in mycobacterial lifecycle. Based on the biochemical function of mycobacterial fadD proteins, we have revealed that there are atleast two distinct families of fadD enzymes. Fatty acyl-AMP ligase (FAAL) activates fatty acids as fatty acyl-adenylates while Fatty acyl-CoA ligase (FACL) activates fatty acids to fatty acyl-CoA thioester analogues. Furthermore, we have demonstrated that FAAL directly transfer fatty acids to cognate polyketide synthase modules to produce complex lipids which are hallmarks of mycobacteria. Recent genome-wide expression studies have emphasized the functional significance of FadD proteins under different conditions. Since these proteins play an important role in mycobacterial biology, we have designed specific inhibitors that may be further developed as antimycobacterial agents. This work was supported by grants from DBT, India to National Institute of Immunology, CSIR to PA and Wellcome Trust International Senior Research Fellowship to RSG.

Modelaje de proteínas de Mycobacterium tuberculosis relacionadas a su defensa frente al estrés oxidativo

En el presente trabajo se describe el uso de procedimientos bioinformáticos e información genómica de acceso libre por Internet, para identificar y caracterizar proteínas que participan en la defensa de Mycobacterium tuberculosis (MT) frente al estrés oxidativo, con el propósito de obtener nuevos blancos farmacológicos en este patógeno. Las secuencias de aminoácidos de las proteínas correspondientes a los genes katG, ahpC, ahpD, sodA, sodC y mgtC fueron comparadas con el genoma de Mycobacteríum tuberculosis H37Rv a través del servidor del National Center for Biotechnology Information, determinando su posible redundancia en el genoma. Paralelamente, se obtuvo información sobre sus residuos más conservados evolutivamente por alineamiento múltiple global usando el programa MAXHOM, así como modelos de sus estructuras tridimensionales usando los métodos de «modelaje molecular comparativo» a través del servidor SWISS-MODEL o modelaje por el método de «reconocimiento de plegamiento» (threading) a través del metaservidor GENESILICO. En base al análisis genómico se puede concluir que las proteínas codificadas por los genes katG, ahpD y mgtC son buenos candidatos como blancos farmacológicos debido a que no tienen similaridad significativa con alguna proteína codificada por genes presentes en el genoma humano y no presentan redundancia en el genoma del Mycobacterium tuberculosis.

Point mutations in the DNA- and cNMP-binding domains of the homologue of the cAMP receptor protein (CRP) in Mycobacterium bovis BCG: implications for the inactivation of a global regulator and strain attenuation

The genome of Mycobacterium tuberculosis H37Rv includes a homologue of the CRP/FNR (cAMP receptor protein/fumarate and nitrate reduction regulator) family of transcription regulators encoded by Rv3676. Sequencing of the orthologous gene from attenuated Mycobacterium bovis Bacille Calmette-Guérin (BCG) strains revealed point mutations that affect the putative DNA-binding and cNMP-binding domains of the encoded protein. These mutations are not present in the published sequences of the Rv3676 orthologues in M. bovis, M. tuberculosis or Mycobacterium leprae. An Escherichia coli lacZ reporter system was used to show that the M. tuberculosis Rv3676 protein binds to DNA sites for CRP, but this DNA binding was decreased or abolished with the Rv3676 protein counterparts from BCG strains. The DNA-binding ability of the M. tuberculosis Rv3676 protein was decreased by the introduction of base changes corresponding to the BCG point mutations. Conversely, the DNA binding of the BCG Rv3676 proteins from BCG strains was restored by removing the mutations. These data show that in this reporter system the point mutations present in the Rv3676 orthologue in BCG strains render its function defective (early strains) or abolished (late strains) and suggest that this protein might be naturally defective in M. bovis BCG strains. This raises the possibility that a contributing factor to the attenuation of BCG strains may be an inability of this global regulator to control the expression of genes required for in vivo survival and persistence.