Leicester Tuberculosis Research Group, Department of Respiratory Sciences, University of Leicester, Leicester, LE1 9HN, UK.
FACS Facility Core Biotechnology Services, University of Leicester, Leicester, LE1 9HN, UK.
Commun Biol. 2024 Sep 28;7(1):1206. doi: 10.1038/s42003-024-06912-0.
During infection Mycobacterium tuberculosis (Mtb) forms physiologically distinct subpopulations that are recalcitrant to treatment and undetectable using standard diagnostics. These difficult to culture or differentially culturable (DC) Mtb are revealed in liquid media, their revival is often stimulated by resuscitation-promoting factors (Rpf) and prevented by Rpf inhibitors. Here, we investigated the role of nitric oxide (NO) in promoting the DC phenotype. Rpf-dependent DC Mtb were detected following infection of interferon-γ-induced macrophages capable of producing NO, but not when inducible NO synthase was inactivated. After exposure of Mtb to a new donor for sustained NO release (named NOD), the majority of viable cells were Rpf-dependent and undetectable on solid media. Gene expression analyses revealed a broad transcriptional response to NOD, including down-regulation of all five rpf genes. The DC phenotype was partially reverted by over-expression of Rpfs which promoted peptidoglycan remodelling. Thus, NO plays a central role in the generation of Rpf-dependent Mtb, with implications for improving tuberculosis diagnostics and treatments.
在感染期间，结核分枝杆菌（Mtb）形成生理上不同的亚群，对治疗有抗性，且无法用标准诊断方法检测到。这些难以培养或差异可培养（DC）的 Mtb 在液体培养基中显现出来，它们的复苏通常受到促进复苏因子（Rpf）的刺激，而受到 Rpf 抑制剂的抑制。在这里，我们研究了一氧化氮（NO）在促进 DC 表型中的作用。在能够产生 NO 的干扰素-γ诱导的巨噬细胞感染后，检测到依赖 Rpf 的 DC Mtb，但当诱导型一氧化氮合酶失活时则无法检测到。在将 Mtb 暴露于持续释放新供体 NO 的物质（称为 NOD）后，大多数存活细胞依赖 Rpf，且在固体培养基上无法检测到。基因表达分析显示，NOD 引发了广泛的转录反应，包括五个 rpf 基因的下调。过表达 Rpfs 部分逆转了 DC 表型，促进了肽聚糖重塑。因此，NO 在依赖 Rpf 的 Mtb 的产生中发挥了核心作用，这对改善结核病的诊断和治疗具有重要意义。
