In silico analysis of cholesterol catabolic genes/proteins in the genus mycobacterium

Published ThesisIt is well known that Mycobacterium tuberculosis, the causative agent of one of the deadliest human diseases, tuberculosis, uses human cholesterol as a carbon source both in the latent and active phases of its lifestyle. The discovery of the ability of M. tuberculosis to degrade and use cholesterol as a sole source of carbon and energy has opened up the possibility of using genes/proteins involved in cholesterol degradation as novel drug targets. If one can find the highly conserved genes/proteins across the mycobacteria that are capable of degrading cholesterol, then in future these genes can possibly be used as universal drug targets against mycobacterial infections. However, to date, data on how many mycobacterial species utilise cholesterol has not been reported. Furthermore, performing laboratory experiments is laborious and time- and money-consuming, considering each of the mycobacterial species has different lifestyle and culture conditions. The study is aimed at using the available genomic data to perform comparative genomic studies to unravel the nature of cholesterol catabolic genes/proteins in the genus Mycobacterium to determine which mycobacterial species are capable of degrading cholesterol. This study is a first of its kind comprehensive analysis of the genes/proteins involved in cholesterol degradation across 93 mycobacterial species, using bioinformatic tools. Ninety-three mycobacterial species whose genomes are available for public use at the KEGG database were used in this study. Literature on cholesterol degradation by bacteria was collected and the cholesterol degradation pathway was deduced. The intermediate metabolites and enzymes involved in each of the steps were identified and mapped using ChemDraw software. A software program that extracts homolog data across 93 mycobacterial species was developed. The hit proteins’ domains/functions were identified using software programs: NCBI Batch Web CD-search tool and the KEGG functional database. Based on the sequence identity, functional motifs and functional data, if available, the hit proteins were sorted into specific enzymatic reactions of cholesterol degradation. After thorough literature analysis, 152 genes/proteins were identified as cholesterol catabolic genes/proteins and grouped into four different categories. The four categories are: (i) genes predicted to be specifically required for growth on cholesterol, (ii) cholesterol catabolic genes proven to be or predicted to be essential for the survival of M. tuberculosis in macrophage cells and in murine infection, (iii) genes/proteins that are up-regulated during growth on cholesterol, and (iv) genes involved in cholesterol degradation by M. tuberculosis H37Rv, but not confirmed or predicted to be essential. In silico analysis of 152 genes across 93 mycobacterial species revealed that 51 mycobacterial species are unable to degrade cholesterol

PPE Antigen Rv2430c of Mycobacterium tuberculosis induces a strong B-cell response

The variation in sequence and length in the C-terminal region among members of the unique PE (Pro-Glu) and PPE (Pro-Pro-Glu) protein families of Mycobacterium tuberculosis is a likely source of antigenic variation, giving rise to the speculation that these protein families could be immunologically important. Based on in silico analysis, we selected a hypothetical open reading frame (ORF) encoding a protein belonging to the PPE family and having epitopes with predictably higher antigenic indexes. Reverse transcriptase PCR using total RNA extracted from in vitro-cultured M. tuberculosis H37Rv generated an mRNA product corresponding to this gene, indicating the expression of this ORF (Rv2430c) at the mRNA level. Recombinant protein expressed in Escherichia coli was used to screen the sera of M. tuberculosis-infected patients, as well as those of clinically healthy controls (n = 10), by enzyme-linked immunosorbent assay. The panel of patient sera comprised sera from fresh infection cases (category 1; n = 32), patients with relapsed tuberculosis (category 2; n = 30), and extrapulmonary cases (category 3; n = 30). Category 2 and 3 sera had strong antibody responses to the PPE antigen, equal to or higher than those to other well-known antigens, such as Hsp10 or purified protein derivative (PPD). However, a higher percentage of patients belonging to category 1, as opposed to clinically healthy controls, showed stronger antibody response against the PPE protein when probed with anti-immunoglobulin M (IgM) (71 versus 37.5%) or anti-IgG (62.5 versus 28.12%). Our results reveal that this PPE ORF induces a strong B-cell response compared to that generated by M. tuberculosis Hsp10 or PPD, pointing to the immunodominant nature of the protein

The PE-PPE Domain in Mycobacterium Reveals a Serine α/β Hydrolase Fold and Function: An In-Silico Analysis

The PE and PPE proteins first reported in the genome sequence of Mycobacterium tuberculosis strain H37Rv are now identified in all mycobacterial species. The PE-PPE domain (Pfam ID: PF08237) is a 225 amino acid residue conserved region located towards the C-terminus of some PE and PPE proteins and hypothetical proteins. Our in-silico sequence analysis revealed that this domain is present in all Mycobacteria, some Rhodococcus and Nocardia farcinica genomes. This domain comprises a pentapeptide sequence motif GxSxG/S at the N-terminus and conserved amino acid residues Ser, Asp and His that constitute a catalytic triad characteristic of lipase, esterase and cutinase activity. The fold prediction and comparative modeling of the 3-D structure of the PE-PPE domain revealed a “serine α/β hydrolase” structure with a central β-sheet flanked by α-helices on either side. The structure comprises a lid insertion with a closed structure conformation and has a solvent inaccessible active site. The oxyanion hole that stabilizes the negative charge on the tetrahedral intermediate has been identified. Our findings add to the growing list of serine hydrolases in mycobacterium, which are essential for the maintenance of their impermeable cell wall and virulence. These results provide the directions for the design of experiments to establish the function of PE and PPE proteins

Whole genome identification of Mycobacterium tuberculosis vaccine candidates by comprehensive data mining and bioinformatic analyses

<p>Abstract</p> <p>Background</p> <p><it>Mycobacterium tuberculosis</it>, the causative agent of tuberculosis (TB), infects ~8 million annually culminating in ~2 million deaths. Moreover, about one third of the population is latently infected, 10% of which develop disease during lifetime. Current approved prophylactic TB vaccines (BCG and derivatives thereof) are of variable efficiency in adult protection against pulmonary TB (0%–80%), and directed essentially against early phase infection.</p> <p>Methods</p> <p>A genome-scale dataset was constructed by analyzing published data of: (1) global gene expression studies under conditions which simulate intra-macrophage stress, dormancy, persistence and/or reactivation; (2) cellular and humoral immunity, and vaccine potential. This information was compiled along with revised annotation/bioinformatic characterization of selected gene products and <it>in silico </it>mapping of T-cell epitopes. Protocols for scoring, ranking and prioritization of the antigens were developed and applied.</p> <p>Results</p> <p>Cross-matching of literature and <it>in silico</it>-derived data, in conjunction with the prioritization scheme and biological rationale, allowed for selection of 189 putative vaccine candidates from the entire genome. Within the 189 set, the relative distribution of antigens in 3 functional categories differs significantly from their distribution in the whole genome, with reduction in the Conserved hypothetical category (due to improved annotation) and enrichment in Lipid and in Virulence categories. Other prominent representatives in the 189 set are the PE/PPE proteins; iron sequestration, nitroreductases and proteases, all within the Intermediary metabolism and respiration category; ESX secretion systems, resuscitation promoting factors and lipoproteins, all within the Cell wall category. Application of a ranking scheme based on qualitative and quantitative scores, resulted in a list of 45 best-scoring antigens, of which: 74% belong to the dormancy/reactivation/resuscitation classes; 30% belong to the Cell wall category; 13% are classical vaccine candidates; 9% are categorized Conserved hypotheticals, all potentially very potent T-cell antigens.</p> <p>Conclusion</p> <p>The comprehensive literature and <it>in silico</it>-based analyses allowed for the selection of a repertoire of 189 vaccine candidates, out of the whole-genome 3989 ORF products. This repertoire, which was ranked to generate a list of 45 top-hits antigens, is a platform for selection of genes covering all stages of <it>M. tuberculosis </it>infection, to be incorporated in rBCG or subunit-based vaccines.</p

Genetic Basis of Virulence Attenuation Revealed by Comparative Genomic Analysis of Mycobacterium tuberculosis Strain H37Ra versus H37Rv

Tuberculosis, caused by Mycobacterium tuberculosis, remains a leading infectious disease despite the availability of chemotherapy and BCG vaccine. The commonly used avirulent M. tuberculosis strain H37Ra was derived from virulent strain H37 in 1935 but the basis of virulence attenuation has remained obscure despite numerous studies. We determined the complete genomic sequence of H37Ra ATCC25177 and compared that with its virulent counterpart H37Rv and a clinical isolate CDC1551. The H37Ra genome is highly similar to that of H37Rv with respect to gene content and order but is 8,445 bp larger as a result of 53 insertions and 21 deletions in H37Ra relative to H37Rv. Variations in repetitive sequences such as IS6110 and PE/PPE/PE-PGRS family genes are responsible for most of the gross genetic changes. A total of 198 single nucleotide variations (SNVs) that are different between H37Ra and H37Rv were identified, yet 119 of them are identical between H37Ra and CDC1551 and 3 are due to H37Rv strain variation, leaving only 76 H37Ra-specific SNVs that affect only 32 genes. The biological impact of missense mutations in protein coding sequences was analyzed in silico while nucleotide variations in potential promoter regions of several important genes were verified by quantitative RT-PCR. Mutations affecting transcription factors and/or global metabolic regulations related to in vitro survival under aging stress, and mutations affecting cell envelope, primary metabolism, in vivo growth as well as variations in the PE/PPE/PE-PGRS family genes, may underlie the basis of virulence attenuation. These findings have implications not only for improved understanding of pathogenesis of M. tuberculosis but also for development of new vaccines and new therapeutic agents

Proteomic analysis of streptomycin resistant and sensitive clinical isolates of Mycobacterium tuberculosis

<p>Abstract</p> <p>Background</p> <p>Streptomycin (SM) is a broad spectrum antibiotic and is an important component of any anti-tuberculosis therapy regimen. Several mechanisms have been proposed to explain the emergence of resistance but still our knowledge is inadequate. Proteins form a very complex network and drugs are countered by their modification/efflux or over expression/modification of targets. As proteins manifest most of the biological processes, these are attractive targets for developing drugs, immunodiagnostics or therapeutics. The aim of present study was to analyze and compare the protein profile of whole cell extracts from <it>Mycobacterium tuberculosis </it>clinical isolates susceptible and resistant to SM.</p> <p>Results</p> <p>Two-dimensional gel electrophoresis (2DE) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was employed for analyzing the protein profiles. Homology and <it>in silico </it>characterization for identified proteins was assessed using BLAST, InterProScan and KEGG database searches. Computational studies on the possible interactions between SM and identified proteins were carried out by a battery of online servers and softwares, namely, CLUSTALW (KEGG), I-TASSER, VMD, PatchDock and FireDock. On comparing 2DE patterns, nine proteins were found consistently overexpressed in SM resistant isolates and were identified as Rv0350, Rv0440, Rv1240, Rv3075c, Rv2971, Rv3028c, Rv2145c, Rv2031c and Rv0569. <it>In silico </it>docking analysis showed significant interactions of SM with essential (Rv0350, Rv0440 and Rv2971) and non essential (Rv1240, Rv3075c and Rv2031c) genes.</p> <p>Conclusions</p> <p>The computational results suggest high protein binding affinity of SM and suggested many possible interactions between identified proteins and the drug. Bioinformatic analysis proves attributive for analysis of diversity of proteins identified by whole proteome analysis. In-depth study of the these proteins will give an insight into probable sites of drug action other than established primary sites and hence may help in search of novel chemotherapeutic agents at these new sites as inhibitors.</p

Flux Balance Analysis of Mycolic Acid Pathway: Targets for Anti-Tubercular Drugs

Mycobacterium tuberculosis is the focus of several investigations for design of newer drugs, as tuberculosis remains a major epidemic despite the availability of several drugs and a vaccine. Mycobacteria owe many of their unique qualities to mycolic acids, which are known to be important for their growth, survival, and pathogenicity. Mycolic acid biosynthesis has therefore been the focus of a number of biochemical and genetic studies. It also turns out to be the pathway inhibited by front-line anti-tubercular drugs such as isoniazid and ethionamide. Recent years have seen the emergence of systems-based methodologies that can be used to study microbial metabolism. Here, we seek to apply insights from flux balance analyses of the mycolic acid pathway (MAP) for the identification of anti-tubercular drug targets. We present a comprehensive model of mycolic acid synthesis in the pathogen M. tuberculosis involving 197 metabolites participating in 219 reactions catalysed by 28 proteins. Flux balance analysis (FBA) has been performed on the MAP model, which has provided insights into the metabolic capabilities of the pathway. In silico systematic gene deletions and inhibition of InhA by isoniazid, studied here, provide clues about proteins essential for the pathway and hence lead to a rational identification of possible drug targets. Feasibility studies using sequence analysis of the M. tuberculosis H37Rv and human proteomes indicate that, apart from the known InhA, potential targets for anti-tubercular drug design are AccD3, Fas, FabH, Pks13, DesA1/2, and DesA3. Proteins identified as essential by FBA correlate well with those previously identified experimentally through transposon site hybridisation mutagenesis. This study demonstrates the application of FBA for rational identification of potential anti-tubercular drug targets, which can indeed be a general strategy in drug design. The targets, chosen based on the critical points in the pathway, form a ready shortlist for experimental testing

Detecting the molecular scars of evolution in the Mycobacterium tuberculosis complex by analyzing interrupted coding sequences

<p>Abstract</p> <p>Background</p> <p>Computer-assisted analyses have shown that all bacterial genomes contain a small percentage of open reading frames with a frameshift or in-frame stop codon We report here a comparative analysis of these interrupted coding sequences (ICDSs) in six isolates of <it>M. tuberculosis</it>, two of <it>M. bovis </it>and one of <it>M. africanum </it>and question their phenotypic impact and evolutionary significance.</p> <p>Results</p> <p>ICDSs were classified as "common to all strains" or "strain-specific". Common ICDSs are believed to result from mutations acquired before the divergence of the species, whereas strain-specific ICDSs were acquired after this divergence. Comparative analyses of these ICDSs therefore define the molecular signature of a particular strain, phylogenetic lineage or species, which may be useful for inferring phenotypic traits such as virulence and molecular relationships. For instance, <it>in silico </it>analysis of the W-Beijing lineage of <it>M. tuberculosis</it>, an emergent family involved in several outbreaks, is readily distinguishable from other phyla by its smaller number of common ICDSs, including at least one known to be associated with virulence. Our observation was confirmed through the sequencing analysis of ICDSs in a panel of 21 clinical <it>M. tuberculosis </it>strains. This analysis further illustrates the divergence of the W-Beijing lineage from other phyla in terms of the number of full-length ORFs not containing a frameshift. We further show that ICDS formation is not associated with the presence of a mutated promoter, and suggest that promoter extinction is not the main cause of pseudogene formation.</p> <p>Conclusion</p> <p>The correlation between ICDSs, function and phenotypes could have important evolutionary implications. This study provides population geneticists with a list of targets, which could undergo selective pressure and thus alters relationships between the various lineages of <it>M. tuberculosis </it>strains and their host. This approach could be applied to any closely related bacterial strains or species for which several genome sequences are available.</p

Genomic analysis of a household tuberculosis transmission cluster over a ten-year period

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, 2020, Universidade de Lisboa, Faculdade de Farmácia.A tuberculose continua a ser um grave problema de saúde pública, apesar de ser uma doença evitável e tratável. A Organização Mundial de Saúde estima que 1,5 milhões de pessoas morrem de tuberculose ano após ano e acredita-se que cerca de um quarto da população mundial esteja infectada. A Letónia é um dos países europeus de alta prioridade no controlo da tuberculose e tem uma das taxas mais altas de tuberculose multirresistente do mundo, apesar de ter um programa de controlo bem estabelecido. Prevenção, detecção precoce e resposta rápida e eficaz aos surtos são elementos essenciais para controlar a propagação da tuberculose. Durante um período de dez anos, foram colhidas sete amostras de uma família de cinco pessoas da Letónia. Realizámos análises genómicas dos sete isolados com o intuito de desvendar a cadeia de transmissão, investigar a origem de dois casos recorrentes e revelar a possível existência de resistência aos medicamentos. Preparámos bibliotecas genómicas e sequenciámos os isolados com o Ion Proton. Para analisar as sequências genómicas, efectuámos uma análise bioinformática para a detecção de variantes em todo o genoma, que incluiu o alinhamento das reads contra o genoma de referência H37Rv, o local indel realignment, variant calling e a detecção de variantes estruturais. No total, foram encontrados 6 variantes estruturais, e detectámos 1029 SNPs de alta qualidade, dos quais 9 eram filogeneticamente informativos e 17 diferenciavam os isolados. Com base Spoligotyping in silico, os isolados pertenciam à sub-família T1. Ao comparar os nossos dados com os da lista de SNPs filogeneticamente específicos, as estirpes estudadas faziam parte da sub-linhagem Haarlem. Não foram encontrados polimorfismos robustos nos genes associados à resistência aos medicamentos, pelo que os isolados foram classificados como susceptíveis a todas os medicamentos anti-tuberculose. Dois doentes tiveram casos recorrentes que definimos como reinfecções. Gerámos hipóteses para estabelecer a cadeia de transmissão, apoiadas pelos limites definidos no número de SNPs e pelos dados das árvores filogenéticas de máxima verossimilhança. Embora tenhamos utilizado um método de alta resolução, os dados do WGS não foram suficientes para determinar sem ambiguidade a direcção da transmissão do surto. Os dados da epidemiologia molecular precisavam da epidemiologia clássica e da informação clínica para investigar eficazmente este surto.Tuberculosis remains a serious public health problem even though it is a preventable and treatable disease. World Health Organization estimates that 1.5 million people die from tuberculosis year after year and roughly one quarter of the world’s population is believed to be infected. Latvia is one of Europe’s high-priority countries for tuberculosis control and has one of the highest rates of multi-drug resistant tuberculosis in the world, despite having a well-established control programme. Prevention, early detection and quick and effective response to outbreaks are essential elements to control the spread of tuberculosis. Over a period of ten years, seven samples were collected from a family of five people from Latvia. We performed genomic analysis of the seven isolates in order to unravel the chain of transmission, investigate the origin of two recurrent cases and reveal the possible existence of drug resistance. We prepared genomic libraries and we sequenced the isolates using the Ion Proton platform. To analyze the genomic sequences, we caried out bioinformatic analysis using a pipeline for genome-wide variant detection, that included alignment of the reads against the reference H37Rv genome, local indel realignment, variant calling and structural variant detection. Overall, 6 structural variants were found, and we detected 1029 high-quality SNPs, from which 9 were phylogenetically informative and 17 differentiated the isolates. Based on in silico Spoligotyping the isolates belonged to the T1 sub-family and when using phylogenetic specific SNPs, the studied strains were determined to be part of the Haarlem sub-lineage. No robust polymorphisms in genes associated with drug resistance were found, therefore the isolates were classified susceptible to all anti-tuberculosis drugs. Two patients had recurrent cases that we defined as re-infections. We generated hypotheses in order to establish the routes of transmission, supported by the defined cut-offs in the number of SNPs and the data from the maximum likelihood phylogenetic trees. Although we used a high-resolution method, the WGS data was not enough to determine the direction of transmission within the cluster unambiguously. The molecular epidemiology data needed to be combined with classical epidemiology and clinical information to effectively investigate this household transmission cluster.Com o patrocínio da Latvian Biomedical Research And Study Centre

High accuracy mass spectrometry analysis as a tool to verify and improve gene annotation using Mycobacterium tuberculosis as an example

Background: While the genomic annotations of diverse lineages of the Mycobacterium tuberculosis complex are available, divergences between gene prediction methods are still a challenge for unbiased protein dataset generation. M. tuberculosis gene annotation is an example, where the most used datasets from two independent institutions (Sanger Institute and Institute of Genomic Research-TIGR) differ up to 12% in the number of annotated open reading frames, and 46% of the genes contained in both annotations have different start codons. Such differences emphasize the importance of the identification of the sequence of protein products to validate each gene annotation including its sequence coding area. Results: With this objective, we submitted a culture filtrate sample from M. tuberculosis to a highaccuracy LTQ-Orbitrap mass spectrometer analysis and applied refined N-terminal prediction to perform comparison of two gene annotations. From a total of 449 proteins identified from the MS data, we validated 35 tryptic peptides that were specific to one of the two datasets, representing 24 different proteins. From those, 5 proteins were only annotated in the Sanger database. In the remaining proteins, the observed differences were due to differences in annotation of transcriptional start sites. Conclusion: Our results indicate that, even in a less complex sample likely to represent only 10% of the bacterial proteome, we were still able to detect major differences between different gene annotation approaches. This gives hope that high-throughput proteomics techniques can be used to improve and validate gene annotations, and in particular for verification of high-throughput, automatic gene annotations.publishedVersio