Comparative genomics between human and animal associated subspecies of the Mycobacterium avium complex: a basis for pathogenicity

Timms et al. BMC Genomics (2015) 16:695 DOI 10.1186/s12864-015-1889-2 RESEARCH ARTICLE Open Access Comparative genomics between human and animal associated subspecies of the Mycobacterium avium complex: a basis for pathogenicity Verlaine J. Timms1,3, Karl A. Hassan2, Hazel M. Mitchell1 and Brett A. Neilan1* Abstract Background: A human isolate of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis 43525) was sequenced and compared genomically to other mycobacterial pathogens. M. paratuberculosis 43525 was recently isolated from a patient with ulcerative colitis and belongs to the M. avium complex, a group known to infect both humans and animals. While M. paratuberculosis is a known pathogen of livestock, there are only 20 human isolates from the last 20 years, therefore we took the opportunity to perform a whole genome comparison between human and animal mycobacterial pathogens. We also compared virulence determinants such as the mycobactin cluster, PE/ PPE genes and mammalian cell entry (mce) operons between MAC subspecies that infect animals and those that infect humans. M. tuberculosis was also included in these analyses given its predominant role as a human pathogen. Results: This genome comparison showed the PE/PPE profile of M. paratuberculosis 43525 to be largely the same as other M. paratuberculosis isolates, except that it had one PPE and one PE_PGRS protein that are only present in human MAC strains and M. tuberculosis. PE/PPE proteins that were unique to M. paratuberculosis 43525, M. avium subsp. hominissuis and a caprine M. paratuberculosis isolate, were also identified. In addition, the mycobactin cluster differed between human and animal isolates and a unique mce operon flanked by two mycobactin genes, mbtA and mbtJ, was identified in all available M. paratuberculosis genomes. Conclusions: Despite the whole genome comparison placing M. paratuberculosis 43525 as closely related to bovine M. paratuberculosis, key virulence factors were similar to human mycobacterial pathogens. This study highlights key factors of mycobacterial pathogenesis in humans and forms the basis for future functional studies. Keywords: Mycobacterium avium, Mycobacterium paratuberculosis, Genome analysis, Pathogenicity, PE/PPE family, Mycobactin, Mammalian cell entry, Inflammatory bowel disease, Johne’s disease, Host-pathogen interactions Background M. avium subsp. paratuberculosis (M. paratuberculosis), of the M. avium complex (MAC), is one of the slowest growing mycobacteria and like other pathogenic mycobacteria, is difficult to detect and treat. It is widely recognised as the cause of Johne’s disease, a gastrointestinal disease of livestock, and is also implicated in human Crohn’s disease [1–3]. The MAC contain * Correspondence: 1 School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney 2052, Australia Full list of author information is available at the end of the article subspecies that infect animals and subspecies pathogenic to humans [4]. The closely related MAC display slight genomic differences depending on their host and comparison of these differences has the potential to identify host specific pathogenicity factors, leading to improved diagnosis and treatment. In the current study we compared the genome of a newly isolated strain of M. paratuberculosis (M. paratuberculosis 43525) from a female patient with ulcerative colitis [5], to other pathogenic mycobacteria using Single Nucleotide Polymorphism (SNP) analysis, BLASTp (homology based) and phmmer (non-homology based) © 2015 Timms et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Timms et al. BMC Genomics (2015) 16:695 algorithms. While M. paratuberculosis normally infects animals, its isolation from humans is rare, with less than 20 isolates reported in the last 20 years [6–9]. Like other M. paratuberculosis from humans, M. paratuberculosis 43525 is cattle type (C-Type) [5]. Therefore, further analysis of this strain provides a unique opportunity to explore other possible variations in host pathogenicity factors. While genomic studies have compared a number of these isolates to other M. paratuberculosis strains and M. hominissuis [10–15], all sequences of M. paratuberculosis to date have been obtained from laboratory strains of unknown subculture number, and often have undergone many years of laboratory passage. Current evidence would suggest that multiple subculture of M. tuberculosis may affect virulence properties with, for example, marked changes in cell wall lipids observed after extensive laboratory passage [16]. Important to note is that M. bovis BCG, widely used in vaccines due to its attenuation in immunocompetent hosts, was produced by multiple subculture in vitro [17]. In contrast, the genome of M. paratuberculosis 43525 was sequenced after only four subcultures and therefore provides a more accurate representation of the wild-type in vivo mycobacterial genome. The virulence factors explored in this study, the PE/ PPE (proline-glutamate/proline-proline-glutamate motif ) genes, mammalian cell entry (mce) operons and the mycobactin cluster, were chosen based on studies into M. tuberculosis and M. avium pathogenicity. The analysis of these genomic loci afford the representation of pathogenicity elements present in M. tuberculosis isolated from human infections and M. paratuberculosis isolated from livestock infections [4, 18–22]. The PE/PPE families are unique to mycobacteria and were first identified for their ability to stimulate IFN-γ Page 2 of 11 [19]. They are GC rich and thought to be the main source of strain variability within the MAC [4]. PE and PPE refer to the residues, Proline-Glutamate and Proline-ProlineGlutamate, respectively, located at the N termini of their encoded proteins. The M. tuberculosis genome devotes 10 % of its protein coding potential to this protein family with various functions attributed to them [23]. Similar to M. tuberculosis, some PPE of M. paratuberculosis are expressed on the cell surface, while others are cell wall associated and interact with the immune system via TLR-2 [19], however, this gene family only represents 2.5 % of the M. paratuberculosis genome [24]. The mammalian cell entry (mce) operons of M. tuberculosis were first discovered in studies to elucidate how M. tuberculosis enters non-phagocytic cells [25]. The genes exist in many bacterial species, however, only in the my (...truncated)