油酸碳源条件下Rv1372c缺失介导结核分枝杆菌对德拉马尼的非经典耐药机制

Noncanonical mechanism of delamanid resistance mediated by Rv1372c deletion in Mycobacterium tuberculosis under oleate conditions

摘要

目的 针对部分德拉马尼（DLM）耐药结核分枝杆菌临床分离株未检测到经典耐药相关基因突变的现象,基于全基因组测序（WGS）筛选潜在非经典耐药相关基因Rv1372c,并探讨其对结核分枝杆菌（MTB）德拉马尼敏感性的调控作用及可能机制。方法 收集2022-2024年肺结核患者MTB临床分离菌,通过药物敏感性检测（PDST）结合WGS分析,筛选潜在耐药相关候选基因Rv1372c。采用pJV53介导的同源重组系统构建Rv1372c基因敲除株及回补株,在常规碳源及以油酸为唯一碳源条件下比较不同菌株的体外生长特性。通过微量稀释刃天青法测定DLM对Rv1372c突变菌株的最低抑菌浓度,并结合扫描电镜观察菌体形态变化。进一步联合转录组学、脂质组学及蛋白组学分析,系统评估Rv1372c缺失对代谢通路、细胞包膜结构及药物敏感性的影响。结果 本研究纳入341株MTB临床分离菌,其中37株表现为DLM耐药。WGS分析显示,在3株DLM耐药菌株中检测到Rv1372c突变,且均未发现ddn、fgd1及fbiA-C等已知DLM耐药相关基因突变。通过pJV53介导的同源重组法成功构建H37RvΔRv1372c敲除株及回补株。扫描电镜显示敲除株菌体表面更为光滑,分泌物减少。生长动力学分析表明,敲除株在液体培养条件下代时缩短,而在固体培养基上无明显差异。药物敏感性试验显示,在油酸作为唯一碳源条件下,敲除株对DLM的最小抑菌浓度明显升高,而在OADC培养条件下未观察到统计学差异。转录组学、脂质组学及蛋白组学联合分析表明,Rv1372c缺失未导致经典DLM耐药相关基因表达水平改变,但可诱导以ABC转运体家族为主的外排泵相关基因上调,并影响脂质代谢、细胞包膜相关通路及氧化还原过程。结论 Rv1372c缺失可在油酸依赖条件下导致结核分枝杆菌对DLM的耐药表型,其作用机制可能与外排泵激活、脂质代谢重塑及细胞包膜结构改变有关。本研究为阐明缺乏经典耐药突变的DLM耐药临床分离株的耐药机制提供了新线索。

Abstract

Objective To investigate the regulatory role of the non-canonical candidate gene Rv1372c in delamanid (DLM) susceptibility and its underlying mechanisms in Mycobacterium tuberculosis (MTB), based on whole-genome sequencing (WGS) analysis of clinical DLM-resistant isolates lacking known resistance-associated mutations. Methods Clinical MTB isolates were collected from pulmonary tuberculosis patients between 2022 and 2024. Phenotypic drug susceptibility testing (PDST) and WGS were performed to identify mutations associated with Rv1372c. A Rv1372c knockout strain and its complemented strain were constructed using the pJV53-mediated homologous recombination system. Growth characteristics were compared under both standard culture conditions and media containing oleic acid as the sole carbon source. The minimum inhibitory concentrations (MIC) of DLM were determined using the microplate alamar Blue assay. Morphological alterations were examined by scanning electron microscopy (SEM). Furthermore, transcriptomic, lipidomic, and proteomic analyses were integrated to systematically evaluate the effects of Rv1372c deletion on metabolic pathways, cell envelope structure, and drug susceptibility. Results A total of 341 clinical MTB isolates were included, among which 37 exhibited phenotypic resistance to DLM. WGS analysis identified Rv1372c mutations in three DLM-resistant isolates, none of which carried mutations in the known DLM resistance-associated genes ddn, fgd1, fbiA, fbiB, or fbiC. The H37RvΔRv1372c knockout strain and complemented strain were successfully generated using the pJV53 recombineering system. SEM revealed a smoother cell surface and reduced extracellular secretions in the knockout strain. Growth kinetic analysis demonstrated a shortened generation time of the knockout strain in liquid culture, whereas no significant difference was observed on solid medium. Drug susceptibility testing showed that deletion of Rv1372c significantly increased the MIC of DLM under oleic acid-dependent growth conditions, while no obvious change was observed in OADC-supplemented medium. Integrated transcriptomic, lipidomic, and proteomic analyses indicated that Rv1372c deletion did not alter the expression of classical DLM resistance-associated genes. However, it induced the upregulation of efflux pump-related genes, particularly those belonging to the ATP-binding cassette (ABC) transporter family, and affected pathways associated with lipid metabolism, cell envelope remodeling, and redox processes. Conclusion Deletion of Rv1372c confers a non-canonical DLM tolerance phenotype in Mycobacterium tuberculosis under oleic acid-dependent conditions. This phenotype may be associated with efflux pump activation, lipid metabolic remodeling, and alterations in cell envelope structure. These findings provide a potential mechanistic explanation for DLM-resistant clinical isolates lacking known resistance-associated mutations and expand the current understanding of non-canonical mechanisms contributing to reduced DLM susceptibility.

关键词

结核分枝杆菌 / Rv1372c / 德拉马尼 / 非经典耐受 / 外排泵 / 脂质代谢

Key words

Mycobacterium tuberculosis / Rv1372c / delamanid / non-canonical resistance / efflux pumps / lipid metabolism

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基金资助

贵州省科技创新平台项目(黔科合平台CXPTXM(2025)017)

贵州省科技计划项目(黔科合基础-ZK(2023)重点060)

贵州省卫生健康委临床重点专科一攀峰计划建设项目(GZWJWPF2025010)