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

描述(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成果，但全球结核病负担仍然很重(1、2)，新增900万人 2011年，全球结核病病例和140万人死亡。结核分枝杆菌在宿主中的持久性需要一种持久的状态，这种状态有效地关闭了许多生物过程，这些过程是常见抗生素的靶标(3-5)。这种持久状态可以通过在药物方案(3、6)中加入吡嗪酰胺(PZA)来对抗，这种药物因其对持久状态的独特选择性而将化疗持续时间从9-12个月缩短到6个月。尽管PZA作为一线结核病药物长期使用，但其分子靶点仍不清楚。最近，PZA的一个新靶点被确定为核糖体蛋白S1，或RpsA(7)。RpsA是一种重要的核糖体蛋白，在信使RNA(信使RNA)翻译(8，9)和反译中发挥重要作用，反译是一种独特的过程，使用转移信使RNA(TmRNA)分子来挽救停滞的核糖体(10，11)。在结核分枝杆菌中，RpsA包含四个连续的S1RNA结合域和一个放线杆菌特有的C末端结构域(CTD)。最近的研究结果表明，RpsA的过度表达使结核分枝杆菌对PZA产生耐药性，部分耐PZA的结核分枝杆菌临床分离株存在映射到rpsA基因的突变。此外，属于结核分枝杆菌复合体的致病菌加拿大分枝杆菌(MCA)的临床分离株对PZA具有内在抗药性，并包含rpsA基因突变(12)。先前的生化工作证实，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中的一个临床相关突变取消了POA的结合[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，导致了反式翻译的抑制，这一过程对Mtb的持久状态非常重要。为了验证这一假说并阐明POA活性的机制，我们将测试tmRNA介导的多肽标记以及这一过程的药物敏感性。此外，我们还将对在耐药临床分离株中发现的突变RpsA蛋白进行生化和生物物理表征。此外，我们将从结构上表征RpsA，以深入了解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、更有效的持久性药物以改进结核病和耐药结核病的治疗具有指导意义。

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.