Idh Jonna, Andersson Blanka, Lerm Maria, Raffetseder Johanna, Eklund Daniel, Woksepp Hanna, Werngren Jim, Mansjö Mikael, Sundqvist Tommy, Stendahl Olle, Schön Thomas
Division of Microbiology and Molecular Medicine, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
Centre for Infectious Medicine, Karolinska Institute, Stockholm, Sweden.
PLoS One. 2017 Jul 13;12(7):e0181221. doi: 10.1371/journal.pone.0181221. eCollection 2017.
Drugs such as isoniazid (INH) and pretomanid (PRT), used against Mycobacterium tuberculosis are active partly through generation of reactive nitrogen species (RNS). The aim of this study was to explore variability in intracellular susceptibility to nitric oxide (NO) in clinical strains of M. tuberculosis.
Luciferase-expressing clinical M. tuberculosis strains with or without INH resistance were exposed to RNS donors (DETA/NO and SIN-1) in broth cultures and bacterial survival was analysed by luminometry. NO-dependent intracellular killing in a selection of strains was assessed in interferon gamma/lipopolysaccharide-activated murine macrophages using the NO inhibitor L-NMMA.
When M. tuberculosis H37Rv was compared to six clinical isolates and CDC1551, three isolates with inhA mediated INH resistance showed significantly reduced NO-susceptibility in broth culture. All strains showed a variable but dose-dependent susceptibility to RNS donors. Two clinical isolates with increased susceptibility to NO exposure in broth compared to H37Rv were significantly inhibited by activated macrophages whereas there was no effect on growth inhibition when activated macrophages were infected by clinical strains with higher survival to NO exposure in broth. Furthermore, the most NO-tolerant clinical isolate showed increased resistance to PRT both in broth culture and the macrophage model compared to H37Rv in the absence of mutational resistance in genes associated to reduced susceptibility against PRT or NO.
In a limited number of clinical M. tuberculosis isolates we found a significant difference in susceptibility to NO between clinical isolates, both in broth cultures and in macrophages. Our results indicate that mycobacterial susceptibility to cellular host defence mechanisms such as NO need to be taken into consideration when designing new therapeutic strategies.
异烟肼（INH）和普瑞玛尼（PRT）等用于抗结核分枝杆菌的药物部分通过产生活性氮物质（RNS）发挥作用。本研究的目的是探讨结核分枝杆菌临床菌株对一氧化氮（NO）的细胞内敏感性差异。
将表达荧光素酶的有或无异烟肼耐药性的结核分枝杆菌临床菌株在肉汤培养物中暴露于RNS供体（DETA/NO和SIN-1），并通过发光法分析细菌存活率。使用NO抑制剂L-NMMA在干扰素γ/脂多糖激活的小鼠巨噬细胞中评估部分菌株中依赖NO的细胞内杀伤作用。
将结核分枝杆菌H37Rv与六个临床分离株和CDC1551进行比较时，三个具有inhA介导的异烟肼耐药性的分离株在肉汤培养中对NO的敏感性显著降低。所有菌株对RNS供体均表现出可变但剂量依赖性的敏感性。与H37Rv相比，在肉汤中对NO暴露敏感性增加的两个临床分离株被激活的巨噬细胞显著抑制，而当激活的巨噬细胞感染在肉汤中对NO暴露存活率较高的临床菌株时，对生长抑制没有影响。此外，在与对PRT或NO敏感性降低相关基因无突变抗性的情况下，与H37Rv相比，最耐NO的临床分离株在肉汤培养和巨噬细胞模型中对PRT的抗性均增加。
在数量有限的结核分枝杆菌临床分离株中，我们发现临床分离株在肉汤培养和巨噬细胞中对NO的敏感性存在显著差异。我们的结果表明，在设计新的治疗策略时需要考虑分枝杆菌对细胞宿主防御机制如NO的敏感性。
