Pathania Ranjana, Navani Naveen K, Gardner Anne M, Gardner Paul R, Dikshit Kanak L
Institute of Microbial Technology, Sector 39A, Chandigarh, 160-036 India.
Mol Microbiol. 2002 Sep;45(5):1303-14. doi: 10.1046/j.1365-2958.2002.03095.x.
Nitric oxide (NO), generated in large amounts within the macrophages, controls and restricts the growth of internalized human pathogen, Mycobacterium tuberculosis H37Rv. The molecular mechanism by which tubercle bacilli survive within macrophages is currently of intense interest. In this work, we have demonstrated that dimeric haemoglobin, HbN, from M. tuberculosis exhibits distinct nitric oxide dioxygenase (NOD) activity and protects growth and cellular respiration of heterologous hosts, Escherichia coli and Mycobacterium smegmatis, from the toxic effect of exogenous NO and the NO-releasing compounds. A flavohaemoglobin (HMP)-deficient mutant of E. coli, unable to metabolize NO, acquired an oxygen-dependent NO consumption activity in the presence of HbN. On the basis of cellular haem content, the specific NOD activity of HbN was nearly 35-fold higher than the single-domain Vitreoscilla haemoglobin (VHb) but was sevenfold lower than the two-domain flavohaemoglobin. HbN-dependent NO consumption was sustained with repeated addition of NO, demonstrating that HbN is catalytically reduced within E. coli. Aerobic growth and respiration of a flavohaemoglobin (HMP) mutant of E. coli was inhibited in the presence of exogenous NO but remained insensitive to NO inhibition when these cells produced HbN, VHb or flavohaemoglobin. M. smegmatis, carrying a native HbN very similar to M. tuberculosis HbN, exhibited a 7.5-fold increase in NO uptake when exposed to gaseous NO, suggesting NO-induced NOD activity in these cells. In addition, expression of plasmid-encoded HbN of M. tuberculosis in M. smegmatis resulted in 100-fold higher NO consumption activity than the isogenic control cells. These results provide strong experimental evidence in support of NO scavenging and detoxification function for the M. tuberculosis HbN. The catalytic NO scavenging by HbN may be highly advantageous for the survival of tubercle bacilli during infection and pathogenesis.
巨噬细胞内大量生成的一氧化氮（NO）可控制并限制内化的人类病原体结核分枝杆菌H37Rv的生长。目前，结核杆菌在巨噬细胞内存活的分子机制备受关注。在本研究中，我们证明了来自结核分枝杆菌的二聚体血红蛋白HbN具有独特的一氧化氮双加氧酶（NOD）活性，可保护异源宿主大肠杆菌和耻垢分枝杆菌的生长及细胞呼吸免受外源性NO和NO释放化合物的毒性影响。无法代谢NO的大肠杆菌黄素血红蛋白（HMP）缺陷型突变体在有HbN存在时获得了氧依赖性的NO消耗活性。基于细胞血红素含量，HbN的特异性NOD活性比单结构域透明颤菌血红蛋白（VHb）高近35倍，但比双结构域黄素血红蛋白低7倍。随着NO的反复添加，HbN依赖的NO消耗得以持续，这表明HbN在大肠杆菌内被催化还原。在存在外源性NO的情况下，大肠杆菌黄素血红蛋白（HMP）突变体的有氧生长和呼吸受到抑制，但当这些细胞产生HbN、VHb或黄素血红蛋白时，对NO抑制仍不敏感。携带与结核分枝杆菌HbN非常相似的天然HbN的耻垢分枝杆菌在暴露于气态NO时，NO摄取增加了7.5倍，表明这些细胞中存在NO诱导的NOD活性。此外，结核分枝杆菌质粒编码的HbN在耻垢分枝杆菌中的表达导致NO消耗活性比同基因对照细胞高100倍。这些结果为结核分枝杆菌HbN的NO清除和解毒功能提供了有力的实验证据。HbN对NO的催化清除可能对结核杆菌在感染和发病过程中的存活极为有利。
