Cyclic Amp-Dependent Resuscitation of Dormant Mycobacteria by Exogenous Free Fatty Acids
Cyclic Amp-Dependent Resuscitation of Dormant Mycobacteria by Exogenous Free Fatty Acids
复制标题
DOI：
10.1371/journal.pone.0082914
复制
发表时间：
2013-12-23
期刊：
影响因子：
3.7
通讯作者：
Kaprelyants, Arseny
中科院分区：
文献类型：
--
作者：
Shleeva, Margarita;Goncharenko, Anna;Kaprelyants, Arseny
世界上三分之一的人口携带潜伏性结核病感染，这种感染可能重新激活，导致活动性疾病。尽管人们多年前就知道结核潜伏期，但对它的了解仍然很少。特别是，宿主来源的物质，可能诱导休眠分枝杆菌的复苏，尚未被描述。采用耻垢分枝杆菌（mc(2) 155）和结核分枝杆菌H37Rv休眠（“不可培养”）细胞的体外模型。我们发现，在液体培养基中添加游离不饱和脂肪酸（FA），包括花生四烯酸，浓度为1.6-10 μ m，可以刺激休眠的耻垢分枝杆菌和结核分枝杆菌细胞的复苏。添加游离不饱和脂肪酸（FA）可以提高活化的耻垢分枝杆菌细胞中的cAMP水平，外源添加cAMP （3-10 mM）或二丁基cAMP （0.5-1 mM）替代FA，可以使耻垢分枝杆菌和结核分枝杆菌休眠细胞复苏。缺乏编码FA激活的腺苷酸环化酶（AC）的MSMEG_4279的耻毛分枝杆菌零突变体不能被FA复苏，但可以被cAMP复苏。高表达AC的耻毛分枝杆菌和结核分枝杆菌细胞不能形成不可培养的细胞，AC的特异性抑制剂（8-溴- camp）阻止了fa依赖性复苏。RT-PCR分析显示，随着fa诱导的细胞活化滞后期cAMP的增加，在指数生长开始时，裸鼠的rpfA（编码复苏促进因子A）上调。一种特异性Rpf抑制剂（4-苯甲酰-2-硝基苯基硫氰酸酯）抑制fa诱导的复苏。我们提出了一种休眠分枝杆菌复苏的新途径，包括FAs激活腺苷酸环化酶MSMEG_4279，导致细胞代谢激活，随后RpfA活性增加，刺激细胞在指数期增殖。该研究揭示了宿主来源的脂质在休眠分枝杆菌复苏中的可能作用，它可能在潜伏结核的再激活过程中起作用。
One third of the world population carries a latent tuberculosis (TB) infection, which may reactivate leading to active disease. Although TB latency has been known for many years it remains poorly understood. In particular, substances of host origin, which may induce the resuscitation of dormant mycobacteria, have not yet been described. In vitro models of dormant ("non-culturable") cells of Mycobacterium smegmatis (mc(2) 155) and Mycobacterium tuberculosis H37Rv were used. We found that the resuscitation of dormant M. smegmatis and M. tuberculosis cells in liquid medium was stimulated by adding free unsaturated fatty acids (FA), including arachidonic acid, at concentrations of 1.6-10 mu M. FA addition enhanced cAMP levels in reactivating M. smegmatis cells and exogenously added cAMP (3-10 mM) or dibutyryl-cAMP (0.5-1 mM) substituted for FA, causing resuscitation of M. smegmatis and M. tuberculosis dormant cells. A M. smegmatis null-mutant lacking MSMEG_4279, which encodes a FA-activated adenylyl cyclase (AC), could not be resuscitated by FA but it was resuscitated by cAMP. M. smegmatis and M. tuberculosis cells hyper-expressing AC were unable to form non-culturable cells and a specific inhibitor of AC (8-bromo-cAMP) prevented FA-dependent resuscitation. RT-PCR analysis revealed that rpfA (coding for resuscitation promoting factor A) is up-regulated in M. smegmatis in the beginning of exponential growth following the cAMP increase in lag phase caused by FA-induced cell activation. A specific Rpf inhibitor(4-benzoyl-2-nitrophenylthiocyanate) suppressed FA-induced resuscitation. We propose a novel pathway for the resuscitation of dormant mycobacteria involving the activation of adenylyl cyclase MSMEG_4279 by FAs resulted in activation of cellular metabolism followed later by increase of RpfA activity which stimulates cell multiplication in exponential phase. The study reveals a probable role for lipids of host origin in the resuscitation of dormant mycobacteria, which may function during the reactivation of latent TB.