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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 , e sxB, 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.

frontiers in microbiology

Comparative Genomics and Proteomic Analysis of Four Non-tuberculous Mycobacterium Species and Mycobacterium tuberculosis Complex: Occurrence of Shared Immunogenic Proteins