Metallochaperones Are Needed for Mycobacterium tuberculosis and Escherichia coli Nicotinamidase-Pyrazinamidase Activity.
Metallochaperones Are Needed for Mycobacterium tuberculosis and Escherichia coli Nicotinamidase-Pyrazinamidase Activity.
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结核分枝杆菌和大肠杆菌烟酰胺酶-吡嗪酰胺酶活性需要金属伴侣。
DOI：
10.1128/jb.00331-19
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发表时间：
2020
影响因子：
3.2
通讯作者：
Zimic,Mirko
中科院分区：
文献类型：
--
作者：
Sheen,Patricia;Monsalve,Anuntxi;Campos,Jhanina;Huerta,Rodolfo;Antiparra,Ricardo;Arteaga,Héctor;Duran,Patricia;Bueno,Carlos;Kirwan,DanielaE;Gilman,RobertH;Zimic,Mirko
结核分枝杆菌烟酰胺酶-吡津酰胺酶(PZAse)是一种催化烟酰胺-吡津酰胺转化为烟酸-吡津酸的金属酶。这项研究调查了PZAse的最佳活性是否需要金属配位体。结核分枝杆菌和大肠杆菌PZAses(分别为PZAse-MT和PZAse-EC)通过金属耗竭(分别给予PZAse-MT-Apo和PZAse-EC-Apo)灭活。用大肠杆菌金属配位酮ZnuA或Rv2059(结核分枝杆菌类似物)重新激活。在对ZnuA和Rv2059进行蛋白质分解和热处理后，重复这一过程。CDC1551结核分枝杆菌参考菌株Rv2059编码基因被敲除，并测定PZA敏感性和PZA外排速率。ZnuA(200 μM)可获得65%的PZAse-EC-Apo复活。Rv2059(1 μM)和ZnuA(1 μM)的PZAse-MT-Apo复活率分别为69%和34.3%。ZnuA和Rv2059的蛋白分解处理以及对ZnuA施加三次(但不是一次)热休克显著降低了PZAse-MT-Apo的重新激活能力。结核分枝杆菌Rv2059基因敲除株是Wayne阳性的，对PZA敏感，POA外排速率与参考菌株没有显著差异，尽管在基因敲除后观察到了较低的外排速率的趋势。金属配位酮Rv2059在体外恢复了去金属的PZAsein的活性。虽然Rv2059在体外是重要的，但它对体内PZA敏感性的影响似乎较小，它可能在结核分枝杆菌的作用机制和对吡津胺的耐药性中具有重要意义。结核病是一种由结核分枝杆菌引起的传染病，仍然是世界范围内导致疾病和死亡的主要原因之一。吡津酰胺是一种治疗结核病的关键药物，但其作用机制尚不完全清楚，利用现有的工具测试结核分枝杆菌对吡津胺的耐药性并不容易。本研究的意义在于，金属配位蛋白样蛋白可能对吡嗪酰胺的作用机制和耐药机制起关键作用。这可能有助于开发更好的检测吡津胺耐药性的工具，这将对结核病患者的临床管理产生重大影响：根据患者个体菌株的耐药性特征适当定制的药物方案可以带来更好的临床结果，减少感染的继续传播，并减少更具挑战性和治疗成本的耐药菌株的发展。
Mycobacterium tuberculosis nicotinamidase-pyrazinamidase (PZAse) is a metalloenzyme that catalyzes conversion of nicotinamide-pyrazinamide to nicotinic acid-pyrazinoic acid. This study investigated whether a metallochaperone is required for optimal PZAse activity. M. tuberculosis and Escherichia coli PZAses (PZAse-MT and PZAse-EC, respectively) were inactivated by metal depletion (giving PZAse-MT–Apo and PZAse-EC–Apo). Reactivation with the E. coli metallochaperone ZnuA or Rv2059 (the M. tuberculosis analog) was measured. This was repeated following proteolytic and thermal treatment of ZnuA and Rv2059. The CDC1551 M. tuberculosis reference strain had the Rv2059 coding gene knocked out, and PZA susceptibility and the pyrazinoic acid (POA) efflux rate were measured. ZnuA (200 μM) achieved 65% PZAse-EC–Apo reactivation. Rv2059 (1 μM) and ZnuA (1 μM) achieved 69% and 34.3% PZAse-MT–Apo reactivation, respectively. Proteolytic treatment of ZnuA and Rv2059 and application of three (but not one) thermal shocks to ZnuA significantly reduced the capacity to reactivate PZAse-MT–Apo. An M. tuberculosis Rv2059 knockout strain was Wayne positive and susceptible to PZA and did not have a significantly different POA efflux rate than the reference strain, although a trend toward a lower efflux rate was observed after knockout. The metallochaperone Rv2059 restored the activity of metal-depleted PZAsein vitro. Although Rv2059 is importantin vitro, it seems to have a smaller effect on PZA susceptibilityin vivo.It may be important to mechanisms of action and resistance to pyrazinamide in M. tuberculosis. Further studies are needed for confirmation.IMPORTANCETuberculosis is an infectious disease caused by the bacterium Mycobacterium tuberculosis and remains one of the major causes of disease and death worldwide. Pyrazinamide is a key drug used in the treatment of tuberculosis, yet its mechanism of action is not fully understood, and testing strains of M. tuberculosis for pyrazinamide resistance is not easy with the tools that are presently available. The significance of the present research is that a metallochaperone-like protein may be crucial to pyrazinamide’s mechanisms of action and of resistance. This may support the development of improved tools to detect pyrazinamide resistance, which would have significant implications for the clinical management of patients with tuberculosis: drug regimens that are appropriately tailored to the resistance profile of a patient’s individual strain lead to better clinical outcomes, reduced onward transmission of infection, and reduction of the development of resistant strains that are more challenging and expensive to treat.