Exploring the zoonotic potential of Mycobacterium avium subspecies paratuberculosis through comparative genomics

Abstract

A comparative genomics approach was utilised to compare the genomes of Mycobacterium avium subspecies paratuberculosis (MAP) isolated from early onset paediatric Crohn's disease (CD) patients as well as Johne's diseased animals. Draft genome sequences were produced for MAP isolates derived from four CD patients, one ulcerative colitis (UC) patient, and two non-inflammatory bowel disease (IBD) control individuals using Illumina sequencing, complemented by comparative genome hybridisation (CGH). MAP isolates derived from two bovine and one ovine host were also subjected to whole genome sequencing and CGH. All seven human derived MAP isolates were highly genetically similar and clustered together with one bovine type isolate following phylogenetic analysis. Three other sequenced isolates (including the reference bovine derived isolate K10) were genetically distinct. The human isolates contained two large tandem duplications, the organisations of which were confirmed by PCR. Designated vGI-17 and vGI-18 these duplications spanned 63 and 109 open reading frames, respectively. PCR screening of over 30 additional MAP isolates (3 human derived, 27 animal derived and one environmental isolate) confirmed that vGI-17 and vGI-18 are common across many isolates. Quantitative real-time PCR of vGI-17 demonstrated that the proportion of cells containing the vGI-17 duplication varied between 0.01 to 15% amongst isolates with human isolates containing a higher proportion of vGI-17 compared to most animal isolates. These findings suggest these duplications are transient genomic rearrangements. We hypothesise that the over-representation of vGI-17 in human derived MAP strains may enhance their ability to infect or persist within a human host by increasing genome redundancy and conferring crude regulation of protein expression across biologically important regions.

Figure 1. The number of SNPs identified in each isolate compared to the K10 reference genome.

Identical colours represent SNPs shared between isolates. CLIJ = clinical John's disease, Pt = human patient. Note scale change on the y-axis. Origins of isolates are presented in Table 1.

Figure 2. Phylogenetic analysis of human and bovine derived Mycobacterium avium subspecies paratuberculosis.

Neighbour-joining phylogenetic tree based on single nucleotide polymorphisms (SNPs) of Mycobacterium avium subspecies paratuberculosis isolates derived from animal and bovine hosts. The ovine derived isolate CLIJ361 was omitted from this analysis due to its significant genetic divergence from the other isolates.

Figure 3. Schematics and PCR results for vGI-17 and vGI-18.

A: Read depth plots for the seven human derived MAP isolates mapped onto the K10 reference genome. Regions with read depth exceeding the average are shown in red, while regions with below average depth are shown in blue. The inner track is Pt139, then Pt144, Pt145, Pt146, Pt154, Pt155 and the outer track is Pt164. B: MAPAC scatterplot comparing total genomic DNA from human MAP isolate Pt145 with MAP K10 control DNA. Diagonal lines represent −2, 0 and +2 fold differences in signal between test and control spots. vGI-17 and vGI-18 appear as clusters of significantly increased signal over MAP K10. C: PCR results from the human MAP isolate Pt139 and bovine isolate K10 for vGI-17 and vGI-18 duplications. D: IS900 PCR as positive PCR control.

Figure 4. Genome organisation of vGI-17 and vGI-18.

Each vGI consists of a tandem duplication separated by an internal IS4 element. Approximate primer locations are indicated by dashed half arrows. Yellow boxes depict a 244 bp insertion into the duplicated copy of MAPK_1203 (designated MAPK_1203*). This sequence was 100% identical to a region of MAPK_0300 and only was found to be specific to ovine type I MAP strains. Refer to Table S3 and S4 for full lists of genes within vGI-17 and vGI-18.

Figure 5. Proportion of cells containing vGI-17.

Quantification of vGI-17 was determined for 11 isolates using a standard curve method. The copy number ratio of vGI-17 to the reference gene MAPK_3057 was used to determine the proportion of cells containing the vGI-17 duplication. Standard error bars were derived from triplicate PCR reactions.

Figure 6. Schematic representation of genome insertions.

(A) a 165 bp insertion up-stream of MAPK_1669 identified in all isolates compared to the K10 reference genome. The orthologous locus in M. avium subspecies hominissuis (M. av 104) is considered a pseudogene due to frame-shift induced a premature stop codon. (B) a 253 bp insertion up-stream of MAPK_2390 identified in bovine isolate CLIJ644 and ovine isolate CLIJ361.