Transposon mutagenesis in Mycobacterium abscessus identifies an essential penicillin-binding protein involved in septal peptidoglycan synthesis and antibiotic sensitivity.
Transposon mutagenesis in Mycobacterium abscessus identifies an essential penicillin-binding protein involved in septal peptidoglycan synthesis and antibiotic sensitivity.
复制标题
转座子诱变在结核分枝杆菌鉴定一个重要的青霉素结合蛋白参与中隔肽聚糖合成和抗生素敏感性。
DOI：
10.7554/elife.71947
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发表时间：
2022-06-06
期刊：
影响因子：
7.7
通讯作者：
Kana, Bavesh D.
中科院分区：
文献类型：
--
作者：
Akusobi, Chidiebere;Benghomari, Bouchra S.;Zhu, Junhao;Wolf, Ian D.;Singhvi, Shreya;Dulberger, Charles L.;Ioerger, Thomas R.;Rubin, Eric J.;Kana, Bavesh D.
关键词：
脓肿分枝杆菌（Mab）是一种快速生长的非结核分枝杆菌（NTM），可引起广泛的感染。单克隆抗体感染的治疗是困难的，因为这种细菌对许多种类的抗生素具有内在的耐药性。开发针对Mab的新的有效治疗方法需要更好地了解独特的脆弱性，这些脆弱性可以作为未来药物开发的目标。为了实现这一点，我们通过对参考单抗菌株ATCC 19977进行转座子测序（TnSeq）来鉴定单抗中的必需基因。我们产生了约51,000个独特的转座子突变体，并利用这个高密度文库鉴定出362个体外生长所需的基因。为了研究单抗的物种特异性脆弱性，我们进一步表征了MAB_3167c，这是一种预测的青霉素结合蛋白和假设的脂蛋白（PBP-lipo），在单抗中是必需的，而在结核分枝杆菌（Mtb）中是非必需的。我们发现pbp -脂质主要定位于亚极区，然后在细胞准备分裂时定位于隔膜。单抗pbp -脂质耗竭导致细胞伸长，发展异位分支，形成多个间隔。PBP-lipo与PbpB、DacB1和羧肽酶MAB_0519的敲低导致协同生长阻滞。相比之下，这些遗传相互作用在结核分枝杆菌模式生物耻垢分枝杆菌中不存在，这表明这两个物种中的pbp -脂同源物存在于不同的遗传网络中。最后，抑制pbp -脂质使参考菌株和11株Mab临床分离株对几种抗生素（包括β-内酰胺类、氨苄西林和阿莫西林）增敏超过128倍。总之，本研究表明PBP-lipo是研究细胞壁合成中单克隆抗体特异性过程的关键酶，并将PBP-lipo定位为治疗单克隆抗体感染的有吸引力的药物靶点。
Mycobacterium abscessus (Mab) is a rapidly growing non-tuberculous mycobacterium (NTM) that causes a wide range of infections. Treatment of Mab infections is difficult because the bacterium is intrinsically resistant to many classes of antibiotics. Developing new and effective treatments against Mab requires a better understanding of the unique vulnerabilities that can be targeted for future drug development. To achieve this, we identified essential genes in Mab by conducting transposon sequencing (TnSeq) on the reference Mab strain ATCC 19977. We generated ~51,000 unique transposon mutants and used this high-density library to identify 362 essential genes for in vitro growth. To investigate species-specific vulnerabilities in Mab, we further characterized MAB_3167c, a predicted penicillin-binding protein and hypothetical lipoprotein (PBP-lipo) that is essential in Mab and non-essential in Mycobacterium tuberculosis (Mtb). We found that PBP-lipo primarily localizes to the subpolar region and later to the septum as cells prepare to divide. Depletion of Mab PBP-lipo causes cells to elongate, develop ectopic branches, and form multiple septa. Knockdown of PBP-lipo along with PbpB, DacB1, and a carboxypeptidase, MAB_0519 lead to synergistic growth arrest. In contrast, these genetic interactions were absent in the Mtb model organism, Mycobacterium smegmatis, indicating that the PBP-lipo homologs in the two species exist in distinct genetic networks. Finally, repressing PBP-lipo sensitized the reference strain and 11 Mab clinical isolates to several classes of antibiotics, including the β-lactams, ampicillin, and amoxicillin by greater than 128-fold. Altogether, this study presents PBP-lipo as a key enzyme to study Mab-specific processes in cell wall synthesis and importantly positions PBP-lipo as an attractive drug target to treat Mab infections.