Exploring the Zoonotic Potential of Mycobacterium avium Subspecies paratuberculosis through Comparative Genomics

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.

本研究采用比较基因组学方法，对比分析了从早发性儿童克罗恩病（Crohn's disease, CD）患者以及约翰氏病（Johne's disease）患病动物体内分离的鸟分枝杆菌副结核亚种（Mycobacterium avium subspecies paratuberculosis, MAP）的基因组。
研究通过Illumina测序技术，对4株CD患者来源、1株溃疡性结肠炎（ulcerative colitis, UC）患者来源以及2株非炎症性肠病（inflammatory bowel disease, IBD）对照个体来源的MAP分离株完成草图基因组测序，并辅以比较基因组杂交（comparative genome hybridisation, CGH）分析。
同时，本研究还对2株牛源与1株羊源的MAP分离株开展了全基因组测序及CGH分析。
系统发育分析结果显示，全部7株人类来源的MAP分离株遗传相似度极高，且与1株牛型分离株聚为同一演化分支；其余3株已测序分离株（包括参考牛源分离株K10）则遗传特征迥异。
人类来源的MAP分离株携带两处大型串联重复序列，经聚合酶链反应（polymerase chain reaction, PCR）验证了其序列组织结构。这两处重复序列分别命名为vGI-17与vGI-18，各自覆盖63个和109个开放阅读框（open reading frames, ORF）。
通过对30余株额外MAP分离株（含3株人类来源、27株动物来源以及1株环境分离株）进行PCR筛查，证实vGI-17与vGI-18在多数MAP分离株中广泛存在。
针对vGI-17的实时定量PCR检测结果显示，携带vGI-17串联重复的细胞比例在不同分离株中介于0.01%至15%之间，且人类来源分离株中vGI-17的携带比例高于多数动物来源分离株。
上述研究结果表明，这两处串联重复属于瞬时基因组重排事件。本研究推测，人类来源MAP菌株中vGI-17的高占比，可能通过提升基因组冗余度，并对生物学重要区域的蛋白质表达实现粗略调控，从而增强其在人类宿主内的感染或定植能力。