Secretome Prediction of Two M. tuberculosis Clinical Isolates Reveals Their High Antigenic Density and Potential Drug Targets

The Excreted/Secreted (ES) proteins play important roles during Mycobacterium tuberculosis invasion, virulence, and survival inside the host and they are a major source of immunogenic proteins. However, the molecular complexity of the bacillus cell wall has made difficult the experimental isolation of the total bacterial ES proteins. Here, we reported the genomes of two Beijing genotype M. tuberculosis clinical isolates obtained from patients from Vietnam (isolate 46) and South Africa (isolate 48). We developed a bioinformatics pipeline to predict their secretomes and observed that ~12% of the genome-encoded proteins are ES, being PE, PE-PGRS, and PPE the most abundant protein domains. Additionally, the Gene Ontology, KEGG pathways and Enzyme Classes annotations supported the expected functions for the secretomes. The ~70% of an experimental secretome compiled from literature was contained in our predicted secretomes, while only the 34–41% of the experimental secretome was contained in the two previously reported secretomes for H37Rv. These results suggest that our bioinformatics pipeline is better to predict a more complete set of ES proteins in M. tuberculosis genomes. The predicted ES proteins showed a significant higher antigenic density measured by Abundance of Antigenic Regions (AAR) value than the non-ES proteins and also compared to random constructed secretomes. Additionally, we predicted the secretomes for H37Rv, H37Ra, and two M. bovis BCG genomes. The antigenic density for BGG and for isolates 46 and 48 was higher than the observed for H37Rv and H37Ra secretomes. In addition, two sets of immunogenic proteins previously reported in patients with tuberculosis also showed a high antigenic density. Interestingly, mice infected with isolate 46 showed a significant lower survival rate than the ones infected with isolate 48 and both survival rates were lower than the one previously reported for the H37Rv in the same murine model. Finally, after a druggability analysis of the secretomes, we found potential drug targets such as cytochrome P450, thiol peroxidase, the Ag85C, and Ribonucleoside Reductase in the secreted proteins that could be used as drug targets for novel treatments against Tuberculosis.

[1]  Oded Shaham,et al.  Paradoxical Hypersusceptibility of Drug-resistant Mycobacteriumtuberculosis to β-lactam Antibiotics , 2016, EBioMedicine.

[2]  R. Hernández-Pando,et al.  Secretome profile analysis of hypervirulent Mycobacterium tuberculosis CPT31 reveals increased production of EsxB and proteins involved in adaptation to intracellular lifestyle. , 2016, Pathogens and disease.

[3]  Wei Wei,et al.  Genome-wide analysis of excretory/secretory proteins in Echinococcus multilocularis: insights into functional characteristics of the tapeworm secretome , 2015, Parasites & Vectors.

[4]  F. Giardina,et al.  M. tuberculosis T Cell Epitope Analysis Reveals Paucity of Antigenic Variation and Identifies Rare Variable TB Antigens. , 2015, Cell host & microbe.

[5]  Sijia Wu,et al.  Protein array identification of protein markers for serodiagnosis of Mycobacterium tuberculosis infection , 2015, Scientific Reports.

[6]  A. Chande,et al.  Selective enrichment of mycobacterial proteins from infected host macrophages , 2015, Scientific Reports.

[7]  R. Warren,et al.  Phylogeny to function: PE/PPE protein evolution and impact on Mycobacterium tuberculosis pathogenicity , 2015, Molecular microbiology.

[8]  A. Ochoa‐Leyva,et al.  Genome analysis of Excretory/Secretory proteins in Taenia solium reveals their Abundance of Antigenic Regions (AAR) , 2015, Scientific Reports.

[9]  H. Hang,et al.  Mycobacterium tuberculosis proteome microarray for global studies of protein function and immunogenicity. , 2014, Cell reports.

[10]  L. Kremer,et al.  β-Lactamase inhibition by avibactam in Mycobacterium abscessus. , 2014, The Journal of antimicrobial chemotherapy.

[11]  Asma Ahmed,et al.  The ESAT-6 Protein of Mycobacterium tuberculosis Interacts with Beta-2-Microglobulin (β2M) Affecting Antigen Presentation Function of Macrophage , 2014, PLoS pathogens.

[12]  D. Ronning,et al.  Mechanism of inhibition of Mycobacterium tuberculosis antigen 85 by ebselen , 2013, Nature Communications.

[13]  A. Roy,et al.  A database for Mycobacterium secretome analysis: 'MycoSec' to accelerate global health research. , 2013, Omics : a journal of integrative biology.

[14]  B. Britigan,et al.  Gallium Nitrate Is Efficacious in Murine Models of Tuberculosis and Inhibits Key Bacterial Fe-Dependent Enzymes , 2013, Antimicrobial Agents and Chemotherapy.

[15]  I. Popescu,et al.  Comparative study of the efficacy and safety of ceftazidime/avibactam plus metronidazole versus meropenem in the treatment of complicated intra-abdominal infections in hospitalized adults: results of a randomized, double-blind, Phase II trial. , 2013, The Journal of antimicrobial chemotherapy.

[16]  Q. Jin,et al.  Analysis of the Secretome and Identification of Novel Constituents from Culture Filtrate of Bacillus Calmette-Guérin Using High-resolution Mass Spectrometry* , 2013, Molecular & Cellular Proteomics.

[17]  Roger G. Linington,et al.  Chemical Inhibitors of the Type Three Secretion System: Disarming Bacterial Pathogens , 2012, Antimicrobial Agents and Chemotherapy.

[18]  S. Fortune,et al.  Rv3615c is a highly immunodominant RD1 (Region of Difference 1)-dependent secreted antigen specific for Mycobacterium tuberculosis infection , 2011, Proceedings of the National Academy of Sciences.

[19]  S. Sampson,et al.  Mycobacterial PE/PPE Proteins at the Host-Pathogen Interface , 2011, Clinical & developmental immunology.

[20]  P. V. van Helden,et al.  Mycobacterium tuberculosis strains with the Beijing genotype demonstrate variability in virulence associated with transmission. , 2010, Tuberculosis.

[21]  Daniel Restrepo-Montoya,et al.  Computational Prediction and Experimental Assessment of Secreted/Surface Proteins from Mycobacterium tuberculosis H37Rv , 2010, PLoS Comput. Biol..

[22]  Daniel Restrepo-Montoya,et al.  Validating subcellular localization prediction tools with mycobacterial proteins , 2009, BMC Bioinformatics.

[23]  K. Onda,et al.  Sofalcone, an anti-ulcer chalcone derivative, suppresses inflammatory crosstalk between macrophages and adipocytes and adipocyte differentiation: implication of heme-oxygenase-1 induction. , 2009, Biochemical and biophysical research communications.

[24]  P. V. van Helden,et al.  Changing Mycobacterium tuberculosis population highlights clade-specific pathogenic characteristics. , 2009, Tuberculosis.

[25]  M S Shaila,et al.  Characterization of T-cell immunogenicity of two PE/PPE proteins of Mycobacterium tuberculosis. , 2008, Journal of medical microbiology.

[26]  Shengyue Wang,et al.  Genetic Basis of Virulence Attenuation Revealed by Comparative Genomic Analysis of Mycobacterium tuberculosis Strain H37Ra versus H37Rv , 2008, PloS one.

[27]  E. Birney,et al.  Velvet: algorithms for de novo short read assembly using de Bruijn graphs. , 2008, Genome research.

[28]  Rick L. Stevens,et al.  The RAST Server: Rapid Annotations using Subsystems Technology , 2008, BMC Genomics.

[29]  N. Villegas-Sepúlveda,et al.  A novel identification scheme for genus Mycobacterium, M. tuberculosis complex, and seven mycobacteria species of human clinical impact , 2008, European Journal of Clinical Microbiology & Infectious Diseases.

[30]  A. Munro,et al.  Structural Biology and Biochemistry of Cytochrome P450 Systems in Mycobacterium tuberculosis , 2008, Drug metabolism reviews.

[31]  A. Clatworthy,et al.  Targeting virulence: a new paradigm for antimicrobial therapy , 2007, Nature Chemical Biology.

[32]  Akiyasu C. Yoshizawa,et al.  KAAS: an automatic genome annotation and pathway reconstruction server , 2007, Environmental health perspectives.

[33]  Erik L. L. Sonnhammer,et al.  Advantages of combined transmembrane topology and signal peptide prediction—the Phobius web server , 2007, Nucleic Acids Res..

[34]  F. Berven,et al.  Comprehensive analysis of exported proteins from Mycobacterium tuberculosis H37Rv , 2007, Proteomics.

[35]  Stefan Götz,et al.  Blast2GO: A Comprehensive Suite for Functional Analysis in Plant Genomics , 2007, International journal of plant genomics.

[36]  S. Leppla,et al.  Cisplatin Inhibition of Anthrax Lethal Toxin , 2006, Antimicrobial Agents and Chemotherapy.

[37]  Morten Nielsen,et al.  Improved method for predicting linear B-cell epitopes , 2006, Immunome research.

[38]  B. Wang,et al.  Antibody response to four secretory proteins from Mycobacterium tuberculosis and their complex antigen in TB patients. , 2005, The international journal of tuberculosis and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease.

[39]  Søren Brunak,et al.  Non-classical protein secretion in bacteria , 2005, BMC Microbiology.

[40]  Søren Brunak,et al.  Prediction of twin-arginine signal peptides , 2005, BMC Bioinformatics.

[41]  J. Flynn.  Mutual attraction: Does it benefit the host or the bug? , 2004, Nature Immunology.

[42]  S. Brunak,et al.  Improved prediction of signal peptides: SignalP 3.0. , 2004, Journal of molecular biology.

[43]  G. Besra,et al.  Phosphonate inhibitors of antigen 85C, a crucial enzyme involved in the biosynthesis of the Mycobacterium tuberculosis cell wall. , 2004, Bioorganic & medicinal chemistry letters.

[44]  Oscar P. Kuipers,et al.  Proteomics of Protein Secretion by Bacillus subtilis: Separating the “Secrets” of the Secretome , 2004, Microbiology and Molecular Biology Reviews.

[45]  A. Krogh,et al.  Prediction of lipoprotein signal peptides in Gram‐negative bacteria , 2003, Protein science : a publication of the Protein Society.

[46]  D. van Soolingen,et al.  A marked difference in pathogenesis and immune response induced by different Mycobacterium tuberculosis genotypes , 2003, Clinical and experimental immunology.

[47]  I. Smith,et al.  Mycobacterium tuberculosis Pathogenesis and Molecular Determinants of Virulence , 2003, Clinical Microbiology Reviews.

[48]  M. Gennaro,et al.  Factors associated with humoral response to ESAT-6, 38 kDa and 14 kDa in patients with a spectrum of tuberculosis. , 2003, The international journal of tuberculosis and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease.

[49]  M. Brennan,et al.  The PE multigene family: a 'molecular mantra' for mycobacteria. , 2002, Trends in microbiology.

[50]  Rolf Apweiler,et al.  InterProScan - an integration platform for the signature-recognition methods in InterPro , 2001, Bioinform..

[51]  A. Krogh,et al.  Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. , 2001, Journal of molecular biology.

[52]  Griffiths,et al.  A secondary structure motif predictive of protein localization to the chlamydial inclusion membrane , 2000, Cellular microbiology.

[53]  J. Wehland,et al.  Listeriolysin and IrpA are major protein targets of the human humoral response against Listeria monocytogenes , 1997, Infection and immunity.

[54]  A. Sampieri,et al.  Correlation between the kinetics of Th1, Th2 cells and pathology in a murine model of experimental pulmonary tuberculosis. , 1996, Immunology.

[55]  S. Kaufmann,et al.  Secreted antigens of Mycobacterium tuberculosis: characterization with T lymphocytes from patients and contacts after two-dimensional separation. , 1992, The Journal of infectious diseases.

[56]  S. Hasnain,et al.  The PE/PPE multigene family codes for virulence factors and is a possible source of mycobacterial antigenic variation: perhaps more? , 2012, Biochimie.

[57]  R. Dorr,et al.  Imexon enhances gemcitabine cytotoxicity by inhibition of ribonucleotide reductase , 2010, Cancer Chemotherapy and Pharmacology.

[58]  A. Munro,et al.  Structural biology and biochemistry of cytochrome P450 systems in Mycobacterium tuberculosis. , 2008, Drug metabolism reviews.