Raw sequence reads from Mycobacterium tuberculosis OH190 resistant isolates. Mycobacterium tuberculosis H37Rv

OH190 is a synthetic analogue of pyridomycin, whose anti-tuberculosis activity appeared unchanged by episomal overexpression of the pyridomycin target, the enoyl-reductase InhA. In search of alternative/additional protein targets, OH190 and pyridomycin resistant H37Rv isolates were selected on plates containing 10 mg/L and 20 mg/L OH190 and pyridomycin, respectively. For both antibiotics, no colonies were isolated at 40 mg/L. Three OH190 resistant clones randomly selected (from 122 resistant clones) were confirmed to have low level resistance (2-fold) to OH190 and to pyridomycin, while the three selected pyridomycin resistant isolates were >16-fold more resistant to pyridomycin and 8-fold more resistant to OH190. Sanger sequencing of InhA and its promoter confirmed that all three OH190 resistant isolates were wild-type for this DNA region, while all three pyridomycin resistant strains carried a previously undescribed mutation in InhA, namely, a481c (M161L). Whole genome sequencing and variant analysis of the three OH190 resistant isolates and the parental H37Rv wild-type strain, found that 2 of the resistant isolates (named OH-RC-10.4, and OH-RC-20.1) carried a unique non-synonymous SNP in Rv0678 (c212a [A71D] and a208g [N70D] respectively, both located in the helix-turn-helix DNA binding domain). No variants (SNP or small In/Del) were found in the third OH190 resistant isolate (named OH-RC-10.5). Due to the mutations identified in Rv0678, the sequence reads of OH-RC-10.5 mapped to this region were scrutinised. The read depth between bases 28-32 of Rv0678 was found disproportionately higher, and the reads only partially mapped to the reference gene. The unmapped portion of the partially mapped reads was found to align to the extremities of an IS6110, clearly indicating its intragenic insertion in this resistant clone. This finding was confirmed by an increase in the size of a PCR product for Rv0678 and subsequent Sanger sequencing. Rv0678 codes for a transcriptional repressor whose loss of function leads to overexpression of the efflux pump mmpL5/S5, which in turn results in low level resistance to the anti-tuberculosis drugs bedaquiline and clofazimine. The identification of this IS6110 mediated inactivation of Rv0678 and consequential resistance to OH190, led us to question if such IS6110 mediated resistance is also observed clinically.

OH190是吡啶霉素（pyridomycin）的合成类似物，将其靶点烯酰还原酶InhA（enoyl-reductase InhA）进行质粒过表达后，该化合物的抗结核活性未发生改变。为寻找其他或额外的蛋白质靶点，我们分别在含有10 mg/L OH190与20 mg/L吡啶霉素的平板上筛选得到耐OH190与耐吡啶霉素的H37Rv菌株。针对这两种抗生素，在40 mg/L浓度下均未分离得到耐药菌落。从122株耐OH190菌株中随机选取3株，经验证其对OH190与吡啶霉素均表现为2倍的低水平耐药；而选取的3株耐吡啶霉素菌株对吡啶霉素的耐药倍数超过16倍，对OH190的耐药倍数为8倍。对InhA基因及其启动子区域进行桑格测序（Sanger sequencing）后证实，所有3株耐OH190菌株的该DNA区域均为野生型；而3株耐吡啶霉素菌株的InhA基因均携带一种此前未被报道的突变：a481c（M161L）。对3株耐OH190菌株及亲本野生型H37Rv菌株开展全基因组测序与变异分析，结果显示其中2株耐药菌株（命名为OH-RC-10.4与OH-RC-20.1）在Rv0678基因中各携带一个独特的非同义单核苷酸多态性（Single Nucleotide Polymorphism, SNP），分别为c212a[A71D]与a208g[N70D]，二者均位于螺旋-转角-螺旋DNA结合结构域（helix-turn-helix DNA binding domain）内。第三株耐OH190菌株（命名为OH-RC-10.5）未检测到任何变异（单核苷酸多态性或小型插入缺失（Insertion/Deletion, In/Del））。鉴于Rv0678基因中已发现的突变，我们对OH-RC-10.5匹配至该区域的测序读段进行了详细核查，经分析发现，Rv0678基因第28至32位碱基的测序深度异常升高，且测序读段仅部分匹配至参考基因序列。对部分匹配读段的未匹配部分进行序列比对后发现，其与插入序列IS6110（IS6110）的末端序列高度匹配，明确证实该耐药菌株中存在IS6110的基因内插入。该结果通过Rv0678的聚合酶链式反应（Polymerase Chain Reaction, PCR）产物分子量增大及后续桑格测序得到验证。Rv0678编码一种转录阻遏蛋白，该蛋白功能丧失会导致外排泵mmpL5/S5的过表达，进而使菌株对结核治疗药物贝达喹啉（bedaquiline）与氯法齐明（clofazimine）产生低水平耐药。本次发现IS6110介导的Rv0678基因失活及由此产生的OH190耐药性，促使我们探究此类IS6110介导的耐药现象是否也存在于临床分离株中。