The occurrence and molecular characterization of non-tuberculous mycobacteria in cattle, African buffalo (Syncerus caffer) and their environments in South Africa and genomic characterization and proteomic comparison with Mycobacterium bovis

Abstract

The aim of this study was to investigate the diversity and prevalence of non-tuberculous mycobacteria (NTM) in cattle, African buffaloes and their environments in South Africa and the potential of these NTM to elicit cross- reactive immune responses in these animal species which may in turn lead to false diagnosis of bovine tuberculosis. A total of 40 NTM species were identified during a countrywide survey. Mycobacterium terrae, Mycobacterium nonchromogenicum, Mycobacterium vaccae/ Mycobacterium vanbaalenii and a group of isolates closely related to Mycobacterium moriokaense (M. moriokaense-like isolates) were the four most frequently isolated species. Further characterization of M. moriokaense- like isolates revealed two novel NTM species which were named Mycobacterium malmesburii sp.nov. and Mycobacterium komanii sp.nov. respectively. Genomes of M. nonchromogenicum, M. malmesburii sp. nov., M. komanii sp. nov., and M. fortuitum ATCC 6841 were elucidated and investigated for genes encoding homologues of M. bovis predominant immunogenic proteins. These included genes encoding for the Esx family proteins (esx genes), mpb70, mpb63, mpb64, hspX, tpx, Rv1120c, canA and dnaK. The esx gene orthologs encoded in ESX-1 (esxA and esxB), ESX-3 (esxH and esxG), esxR, and ESX-4 (esxT and esxU) loci were identified in the NTM genomes while those encoded in ESX-2 locus were absent in all the four NTM genomes and only esxN (encoded in the ESX-5 locus) and its homologue, esxK were present in M. nonchromogenicum. Gene orthologs encoding for MPB70 (M. malmesburii sp.nov. and M. komanii sp.nov.), DnaK (all four NTM species), CanA (all four NTM species), MPB64 (all four NTM species), Rv1120c (in all four NTM species), TpX, MBP63 and HspX (all in M. nonchromogenicum and M. fortuitum), were found in the NTM genomes. In contrast orthologs of mpb83 and espC were not detected in any of the four NTM. We could not judge just based on the overall protein sequence homologies of the antigens whether the NTM homologues will give rise to cross-reactive immune responses. We consequently checked the existence in NTM of epitopes shown to be immunogenic in M. bovis and M. tuberculosis. Amino acid sequence alignment of the EsxA and EsxB of the NTM sequenced in this study as well as M. smegmatis, M. bovis and M. tuberculosis respectively was done to investigate their similarities at “immunogenic” epitope level. In this analysis, we found that the six bovine T-cell recognized epitopes of M. bovis ESAT-6 described by Vordermeier et al., 2003 and 2007 had similarities to those of M. fortuitum and M. nonchromogenicum (showing sequence similarity of as high as 81.28% and as low as 52.9% ). Likewise a certain degree of sequence similarity between the six M. bovis CFP 10 immunogenic epitopes and those of the NTM species (highest similarity of 75% observed between all NTM and M. bovis and lowest similarity of 50% between M. komanii sp.nov, M. malmesburii sp.nov and M. bovis.) was observed. Still, with sequence homologies of less than 100% between the M. bovis immunogenic epitopes and those of the NTM, it was difficult to unambiguously predict T-cell cross-recognition. Comparison of the EsxR and EsxH amino acid sequences at immunogenic epitope level, revealed higher sequence similarities in the epitopes of NTM and those of M. bovis than the predicted protein sequences of EsxA and EsxB. A sequence similarity of 100% was observed between two of the five M. bovis immunogenic epitopes of EsxR and those of M. fortuitum, M. malmesburii sp. nov. and M. komanii sp.nov. Full cross- recognition of these NTM EsxR epitopes is therefore highly likely, and may lead to misdiagnosis of bovine Tuberculosis (BTB). The other three EsxR/EsxH epitopes shown to be immunogenic in M. bovis also exist in the three NTM showing similarity of as low as 77.7%.