结核分枝杆菌对吡嗪酰胺耐药的新机制研究

The control of tuberculosis(TB) is becoming one of the most prominent challenges in the world due to the co-infection of TB and HIV, and the emergence of a great number of drug-resistant Mycobacterium tuberculosis(Mtb) strains. Pyrazinamide (PZA) is a unique first-line anti-tuberculosis drug that plays a key role in shortening the TB therapy. Current phenotype based PZA susceptibility testing is not very reliable due to the limitation of the testing techniques. PZA kills Mtb persisters that many other TB drugs are unable to kill, and thus making it an essential drug for inclusion in many drug combinations for treating both drug-susceptible and drug-resistant TB. Resistance to PZA is mostly caused by mutations in the pncA gene encoding pyrazinamidase involved in conversion of the prodrug PZA to the active form POA. However, mechanism of action of PZA has not been well understood. We have found and verified 2 clinical isolated PZA-resistant strains that do not have mutations in reported PZA resistance related genes, such as pncA, rpsA, panD and even hadC. They still had strong pyrazinamidase activity. We found the mutations in Rv2783c gene could possibly be responsible for their PZA-resistance. We have proved that Rv2783c protein was probably the new target of PZA(POA): 1) The over-expression of the mutated Rv2783c gene could cause Mtb H37Rv, the common laboratory strain, resistant to PZA. 2) The purified Rv2783c could bind to POA but not PZA. In addition, we found the Rv2783c is an essential gene. The study aims to uncover the mechanism of POA further at both the gene and the protein levels and to explore the role of Rv2783c gene in the PZA susceptibilities of clinical Mtb isolates. This study will be helpful to uncover the mechanism of persistence of Mtb and can be the theoretical foundation of developing a new type of drugs and new regimens for improved treatment of not only TB but also other persistent bacterial infections. At the same time, it may provide new molecular diagnostic marker for PZA resistance and can give some very good clues for developing better PZA susceptibility testing methods.

当今世界TB控制面临严峻挑战。吡嗪酰胺(PZA)是作用机制最独特的一线抗TB药物，与新、老药组合均表现出极强的协同杀灭作用，因此，对Mtb，包括耐药Mtb的治疗起着重要作用。然而，其作用机制研究进展缓慢，至今成迷。我们发现并验证了2株临床对PZA耐药但无已知相关基因突变的Mtb，它们仍具有较强的将PZA转化为活性形式POA的酶活性。经研究，我们已锁定Rv2783c基因突变可能导致其耐药并证明：突变的Rv2783c基因导入标准Mtb可导致其耐PZA，Rv2783c蛋白可与POA结合但不结合PZA，初步显示该蛋白很可能是PZA的新靶标。本研究将进一步从基因和蛋白质水平深入揭示PZA作用机理，并进一步验证其临床意义。本研究有助于揭示持留菌形成机制，为研发新型抗TB药物及新疗法提供理论基础。同时，可能为诊断PZA的敏感性提供新分子标记，也可能为研制更好的PZA敏感性检测技术提供有益启示。

吡嗪酰胺(PZA)体外几乎无活性，体内单独使用效果也很差，但与多种新、老药组合均可极大缩短疗程，是作用机制最独特、最神秘的抗结核药。前药PZA需要通过PZase活化成POA才有活性，但是POA的作用靶标一直未有定论。.发现新机理：1）过表达突变的Rv2783c基因可导致Mtb对PZA耐药。2）POA可与Rv2783结合，但不与突变的Rv2783结合。3）首次证实了Rv2783具有聚核苷酸磷酸化酶(PNPase)功能和鸟苷五磷酸合成酶(GPS I) 活性。在研究PNPase功能时，我们开创性地探索使用荧光分析方法并取得成功，结果显示：Rv2783蛋白具有不依赖模板而合成ssDNA和ssRNA的功能，同时可降解ssDNA和ssRNA。GPS I主要负责水解而非合成(p)ppGpp。POA可干扰这些功能而PZA本身并不干扰这些Mtb(特别是在逆境)存活的重要功能。4）Rv2783可能是PZA新靶标，可能成为药物研发的新靶标，可解释PZA独特作用机制：POA干扰DNA修复，或者干扰各种RNA的稳定性，甚至是合成错误的RNA进而合成突变的蛋白可能很好解释“持留”现象，即群体中只有小部细菌因为产生了突变的蛋白/RNA从而对多种药物表型耐药/耐受，但是它们的基因组并未发生改变。另外，Rv2783也可能通过抑制Mtb水解 (p)ppGpp从而阻止Mtb从逆境中恢复过来而杀死处于持留状态Mtb。.对448例Mtb临床分离株进行表型和基因型耐药检测发现：1）59.8%耐药Mtb对PZA耐药，故新疗法中若含PZA，使用该疗法时做PZA耐药诊断非常必要。2）发现57个在PZA耐药相关基因/或上游区域的新突变可能成为潜在的PZA耐药分子诊断标记，包含Rv2783突变，印证了我们实验室的发现。3）PZase活性与pncA基因测序结果不一致，突出了PZase活性测试对快速可靠诊断PZA耐药的重要性。4）获得了无任何已知PZA耐药相关基因突变且无PZase活性的9个PZA耐药株或者有活性的17株，说明存在未知PZA耐药机制且pncA基因表达调控机制这可能在PZA耐药中发挥重要作用。.本研究有助于揭示持留Mtb形成机制，为研发新型抗结核药物及新疗法提供理论基础。同时，可能为诊断PZA的敏感性提供新分子标记，也可能为研制更好的PZA敏感性检测技术提供有益启示。