The POA-RpsA interaction in trans-translation inhibition in M. tuberculosis

描述（由申请人提供）：结核病（TB）是由结核分枝杆菌（Mtb）引起的传染病（1）。当感染者排出细菌时，疾病通过空气传播（1）。尽管最近根除结核病的努力取得了积极的成果，但该疾病的全球负担仍然很高（1，2），每年有900万新的结核病患者。 2011年，全球结核病病例和140万例死亡。Mtb在宿主中的持续存在需要持续状态，其有效地关闭作为常见抗生素靶标的许多生物过程（3-5）。这种持续状态可以通过将吡嗪酰胺（PZA）包括在药物方案中来对抗（3，6），并且由于其对持续状态的独特选择性，这种药物负责将化疗的持续时间从9-12个月缩短至6个月。尽管PZA作为一线抗结核药物长期使用，但其分子靶点仍不清楚。最近，PZA的一个新靶点被鉴定为核糖体蛋白S1或RpsA（7）。RpsA是一种重要的核糖体蛋白，在信使RNA（mRNA）翻译（8，9）和反式翻译（一种使用转移信使RNA（tmRNA）分子拯救停滞核糖体的独特过程）中发挥重要作用（10，11）。在Mtb中，RpsA含有四个连续的S1 RNA结合结构域和放线菌特有的C末端结构域（CTD）。最近的研究结果表明，RpsA过表达赋予结核分枝杆菌PZA抗性，并选择PZA抗性结核分枝杆菌临床分离株携带映射到rpsA基因的突变。此外，临床分离的致病菌属于Mtb复合体，M。canettii（Mca）对PZA具有内在抗性，并且在rpsA基因中含有突变（12）。先前的生物化学工作证实，PZA的活性形式吡嗪酸（POA）与野生型RpsA结合并抑制反式翻译，然而，CTD中的临床相关突变消除了POA结合（7）。目前尚不清楚POA如何与RpsA结合并抑制反式翻译。我们假设POA与RpsA的结合干扰了tmRNA的结合，导致反式翻译的抑制，这是一个对Mtb持续状态非常重要的过程。为了验证这一假设并阐明POA活性的机制，我们将测试tRNA介导的肽标记和这一过程的药物敏感性。此外，我们还将对耐药临床分离株中发现的突变RpsA蛋白进行生化和生物药理学表征。此外，我们将在结构上表征RpsA，以便深入了解POA结合和POA抗性突变的分子决定因素。这些研究将阐明独特的持久性药物PZA的作用机制，并对设计急需的和更强大的持久性药物以改善结核病和耐药结核病的治疗具有影响。

DESCRIPTION (provided by applicant): Tuberculosis (TB) is an infectious disease that is caused by Mycobacterium tuberculosis (Mtb) (1). The disease is spread through the air when infected individuals expel bacteria (1). Despite the fact that recent efforts to eradicate TB have had positive results, the global burden of the disease remains very high (1, 2), with 9 million new cases of tuberculosis and 1.4 million deaths worldwide in 2011. The persistence of Mtb in the host requires a persister state which effectively shuts down many biological processes that are targets for common antibiotics (3-5). This persister state can be combatted by the inclusion of pyrazinamide (PZA) in the drug regimen (3, 6) and this drug is responsible for shortening the duration of the chemotherapy from 9-12 months to 6 months, due to its unique selectivity for the persister state. Despite the long term use of PZA as a first line TB drug, its molecular target remains unclear. Recently, a new target for PZA has been identified as ribosomal protein S1, or RpsA (7). RpsA is a vital ribosomal protein that plays a significant role in messenger RNA (mRNA) translation (8, 9) and in trans-translation, a unique process that uses a transfer-messenger RNA (tmRNA) molecule to rescue stalled ribosomes (10, 11). In Mtb RpsA contains four consecutive S1 RNA- binding domains and a C-terminal domain (CTD) that is unique to actinobacteria. Recent findings have shown that RpsA overexpression confers PZA resistance in Mtb and select PZA-resistant Mtb clinical isolates harbor mutations that map to the rpsA gene. Additionally, clinical isolates of a pathogenic bacterium belonging to Mtb complex, M. canettii (Mca), are intrinsically resistant to PZA and contain mutations in the rpsA gene (12). Previous biochemical work confirmed that the active form of PZA, pyrazinoic acid (POA), binds to wild-type RpsA and inhibits trans-translation, however, a clinically-relevant mutation in the CTD abrogates POA binding (7). It is not clear how POA binds to RpsA and inhibits trans-translation. We hypothesize that POA binding to RpsA interferes with tmRNA binding resulting in an inhibition of trans-translation, a process that is highly important for the persister state o Mtb. In order to test this hypothesis and elucidate the mechanism of POA activity, we will test tmRNA-mediated peptide tagging and the drug susceptibility of this process. Additionally, we will biochemically and biophysically characterize the mutant RpsA proteins that are found in drug resistant clinical isolates. Furthermore, we will structurally characterize RpsA in order to gain insight into the molecular determinants of POA binding and POA resistance mutations. These studies will elucidate a mechanism of action for the unique persister drug PZA and have implications for the design of much needed and more powerful persister drugs for improved treatment of TB and drug-resistant TB.