Abstract
The Esx and PE/PPE families of proteins are among the most immunodominant mycobacterial antigens and have thus been the focus of research to develop vaccines and immunological tests for diagnosis of bovine and human tuberculosis, mainly caused by Mycobacterium bovis and Mycobacterium tuberculosis, respectively. In non-tuberculous mycobacteria (NTM), multiple copies of genes encoding homologous proteins have mainly been identified in pathogenic Mycobacterium species phylogenically related to Mycobacterium tuberculosis and Mycobacterium bovis. Only ancestral copies of these genes have been identified in nonpathogenic NTM species like Mycobacterium smegmatis, Mycobacterium sp. KMS, Mycobacterium sp. MCS, and Mycobacterium sp. JLS. In this study we elucidated the genomes of four nonpathogenic NTM species, viz Mycobacterium komanii sp. nov., Mycobacterium malmesburii sp. nov., Mycobacterium nonchromogenicum, and Mycobacterium fortuitum ATCC 6841. These genomes were investigated for genes encoding for the Esx and PE/PPE (situated in the esx cluster) family of proteins as well as adjacent genes situated in the ESX-1 to ESX-5 regions. To identify proteins actually expressed, comparative proteomic analyses of purified protein derivatives from three of the NTM as well as Mycobacterium kansasii ATCC 12478 and the commercially available purified protein derivatives from Mycobacterium bovis and Mycobacterium avium was performed. The genomic analysis revealed the occurrence in each of the four NTM, orthologs of the genes encoding for the Esx family, the PE and PPE family proteins in M. bovis and M. tuberculosis. The identification of genes of the ESX-1, ESX-3, and ESX-4 region including esxA, esxB, ppe68, pe5, and pe35 adds to earlier reports of these genes in nonpathogenic NTM like M. smegmatis, Mycobacterium sp. JLS and Mycobacterium KMS. This report is also the first to identify esxN gene situated within the ESX-5 locus in M. nonchromogenicum. Our proteomics analysis identified a total of 609 proteins in the six PPDs and 22 of these were identified as shared between PPD of M.bovis and one or more of the NTM PPDs. Previously characterized M tuberculosis/M. bovis homologous immunogenic proteins detected in one or more of the nonpathogenic NTM in this study included CFP-10 (detected in M. malmesburii sp. nov. PPD), GroES (detected in all NTM PPDs but M. malmesburii sp. nov.), DnaK (detected in all NTM PPDs), and GroEL (detected in all NTM PPDs). This study confirms reports that the ESX-1, ESX-3, and ESX-4 regions are ancestral regions and thus found in the genomes of most mycobacteria. Identification of NTM homologs of immunogenic proteins warrants further investigation of their ability to cause cross-reactive immune responses with MTBC antigens.
Highlights
Purified protein derivatives (PPDs) known as tuberculins have been used for more than 100 years as antigens in the diagnosis of human and bovine tuberculosis (TB), based on the delayed hypersensitivity reaction they induce
PPD prepared from Mycobacterium avium is included as a control antigen representative of the background responses to environmental mycobacterial antigens in the single intradermal comparative cervical tuberculin test (SICCT) as well as the interferon gamma assays used in different countries in bovine tuberculosis (BTB) control strategies (Vordermeier et al, 2006)
For PPD production, liquid cultures were prepared in Middlebrook 7H9 medium (Becton Dickinson, USA) supplemented with 0.1% OADC and glycerol, incubated under continued shaking at 200 g at 37◦C for 4 weeks for the rapid growing mycobacteria (M. fortuitum and M. malmesburii sp. nov.) and 6 weeks for the slow growing non-tuberculous mycobacteria (NTM) (M. kansasii and M. nonchromogenicum) or until turbid growth was observed
Summary
Purified protein derivatives (PPDs) known as tuberculins have been used for more than 100 years as antigens in the diagnosis of human and bovine tuberculosis (TB), based on the delayed hypersensitivity (skin) reaction they induce. While these tests generally show satisfactory sensitivities, the major drawback to the tuberculin based assays is the reduced specificity This is presumably associated with cross-reactive immune responses due to exposure of animals and humans to non-tuberculous mycobacteria (NTM) or environmental mycobacteria and the TB vaccine strain, Bacille Calmette et Guérin (BCG). These crossreactive immune responses are most likely due to the presence of immunogenic proteins conserved across the Mycobacterium genus as major components of PPD derived from M. bovis (Schiller et al, 2010). As an alternative approach to mitigate the frequency of false positive responses to PPD-B induced by environmental mycobacterial antigens a PPD derived from Mycobacterium fortuitum (PPD-F) used in the modified BOVIGAM assay, has shown to increase the test specificity in South Africa (Michel et al, 2011)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
