Genomic diversity and evolution of Mycobacterium ulcerans revealed by next-generation sequencing

Mycobacterium ulcerans is the causative agent of Buruli ulcer, the third most common mycobacterial disease after tuberculosis and leprosy. It is an emerging infectious disease that afflicts mainly children and youths in West Africa. Little is known about the evolution and transmission mode of M. ulcerans, partially due to the lack of known genetic polymorphisms among isolates, limiting the application of genetic epidemiology. To systematically profile single nucleotide polymorphisms (SNPs), we sequenced the genomes of three M. ulcerans strains using 454 and Solexa technologies. Comparison with the reference genome of the Ghanaian classical lineage isolate Agy99 revealed 26,564 SNPs in a Japanese strain representing the ancestral lineage. Only 173 SNPs were found when comparing Agy99 with two other Ghanaian isolates, which belong to the two other types previously distinguished in Ghana by variable number tandem repeat typing. We further analyzed a collection of Ghanaian strains using the SNPs discovered. With 68 SNP loci, we were able to differentiate 54 strains into 13 distinct SNP haplotypes. The average SNP nucleotide diversity was low (average 0.06-0.09 across 68 SNP loci), and 96% of the SNP locus pairs were in complete linkage disequilibrium. We estimated that the divergence of the M. ulcerans Ghanaian clade from the Japanese strain occurred 394 to 529 thousand years ago. The Ghanaian subtypes diverged about 1000 to 3000 years ago, or even much more recently, because we found evidence that they evolved significantly faster than average. Our results offer significant insight into the evolution of M. ulcerans and provide a comprehensive report on genetic diversity within a highly clonal M. ulcerans population from a Buruli ulcer endemic region, which can facilitate further epidemiological studies of this pathogen through the development of high-resolution tools.

溃疡分枝杆菌（Mycobacterium ulcerans）是布鲁里溃疡（Buruli ulcer）的致病原，而布鲁里溃疡是仅次于结核病与麻风病的第三大常见分枝杆菌疾病。该疾病属于新发传染病，主要感染西非地区的儿童与青少年群体。目前学界对溃疡分枝杆菌的演化过程与传播模式所知甚少，部分原因在于分离株间已知的遗传多态性匮乏，这限制了遗传流行病学（genetic epidemiology）的应用。为系统性解析单核苷酸多态性（single nucleotide polymorphisms, SNPs），本研究采用454与Solexa测序技术对3株溃疡分枝杆菌菌株的基因组进行了测序。以代表加纳古典谱系的参考分离株Agy99的基因组作为参考进行比对后，在代表祖先谱系的日本菌株中发现了26564个单核苷酸多态性位点。将Agy99与另外两株加纳分离株进行比对后，仅发现173个单核苷酸多态性位点；这两株分离株属于此前通过可变数目串联重复分型（variable number tandem repeat typing）在加纳区分出的另外两种型别。研究团队进一步利用本次发现的单核苷酸多态性位点对一批加纳菌株开展了分析。通过68个单核苷酸多态性位点，我们可将54株菌株划分为13种不同的单倍型。单核苷酸多态性位点的平均核苷酸多样性较低（68个位点的平均多样性为0.06~0.09），且96%的单核苷酸多态性位点对呈现完全连锁不平衡（linkage disequilibrium）。经估算，溃疡分枝杆菌加纳进化支与日本菌株的分化时间约为39.4万至52.9万年前。加纳亚型的分化时间约为1000至3000年前，甚至更近——研究发现这些亚型的演化速率显著高于平均水平。本研究结果为溃疡分枝杆菌的演化过程提供了重要见解，并全面报道了来自布鲁里溃疡流行区的高度克隆性溃疡分枝杆菌群体的遗传多样性；该研究通过开发高分辨率分析工具，可为该病原菌的后续流行病学研究提供有力支撑。