The role of CshA and CshB in selective mRNA protection in S. aureus

描述(申请人提供)：金黄色葡萄球菌通常对甲氧西林和其他细胞壁活性抗生素具有抗药性。此应用程序的目标是验证 干扰特定信使核糖核酸降解的金黄色葡萄球菌MazF毒素和特定RNA结合蛋白作为抗菌药物发现的新靶点。毒素抗毒素系统(TA)在细菌中很常见。在以蛋白质为基础的不同TA模块中，MazEF系统是迄今为止最具特点的。在我们之前发表的论文中，我们已经证明金黄色葡萄球菌的MazFsa毒素是一种特异的内切核酸酶，它切割在V或V‘可以是A、C或G的VUUV’部位，导致蛋白质合成抑制和随后的生长停滞。 MazFsa介导的生长停滞是独一无二的，因为这些细胞是活的，但不能复制。为了描述这种独特的生长停滞形式，我们已经证明MazFsa切割了大多数细胞mRNAs，但保留了一些家务(例如recA、gyrB)和全球调节因子(SARA)mRNAs，可能允许细菌在不牺牲生存能力的情况下进入代谢停滞状态。我们推测，在MazFsa介导的压力下，特定的RNA结合蛋白(S)可能会保护这些“重要的”mRNAs中的一些。我们假设的一个推论是，这些“特定RNA结合蛋白”的失活，加上MazFsa的激活，将促进细胞死亡，而不是生长停滞，因为这些“必要的”mRNA将不再受到保护。细胞分级分析，结合西北印迹和标记的Sara mRNA探针，使我们能够鉴定CSHA、CshB和SA1641为假定的“RNA结合蛋白”。CSHA和CshB是潜在的死盒RNA解旋酶，而SA1641与RNA甲基转移酶有同源性。此前，已知DEAD-BOX蛋白参与核糖体的生物发生和mRNA的衰变。因此，保护mRNAs不受MazFsa介导的CSHA和CshB的切割将是DEAD-box蛋白的一个新概念。为了验证上述假设，我们制定了以下具体目标：i)确定CSHA和CshB在体外结合和保护选择性mRNAs免受MazFsa介导的降解的能力；ii)评估单和双CSHA和cshB突变体中选择性mRNA的降解，这些突变体在MazFsa诱导下的生存能力以及它们对选择性抗生素的敏感性；iii)鉴定有助于保护选择性mRNA免受MazFsa介导的降解的其他因子(例如SA1641)；IV)在感染小鼠模型中评估单和双CSHA和cshB突变体在MazFsa诱导和不诱导MazFsa诱导下的存活率。这些研究结果将使我们能够验证DEAD-box和其他独特的RNA结合蛋白在金黄色葡萄球菌中的新功能。这些研究还将为我们提供验证，即激活MazFsa，结合CSHA、CshB、SA1641和/或其他因素的失活，是一种杀死MSSA和MRSA的新方法。因此，MazFsa和“特定RNA结合蛋白”代表了抗生素开发的新靶点。由于TA系统、DEAD-box和其他RNA结合蛋白在原核生物中普遍存在，我们的结果可能适用于其他病原体。

DESCRIPTION (provided by applicant): S. aureus bacteria are commonly resistant to methicillin and other cell-wall active antibiotics. The goal of this application is to validate the MazF toxin and specific RNA binding proteins of S. aureus that interfere with specific mRNA degradation as novel targets for antibacterial drug discovery. Toxin antitoxin systems (TA) are common in bacteria. Among the different TA modules that are protein-based, the MazEF system is by far the best characterized. In prior papers that we have published, we have shown that that the MazFsa toxin of S. aureus is a specific endoribonuclease that cuts at the VUUV' site where V or V' can be A, C or G, leading to inhibition of protein synthesis and the ensuing growth arrest. The MazFsa-mediated growth arrest is unique because these cells are viable but cannot replicate. To delineate this unique form of growth arrest, we have shown that MazFsa cleaves most of the cellular mRNAs but spares some housekeeping (e.g. recA, gyrB) and a global regulator (sarA) mRNAs, presumably allowing the bacterium to enter metabolic quiescence without sacrificing viability. We hypothesize that specific RNA-binding protein(s) may protect some of these "important" mRNAs" under MazFsa-mediated stress. A corollary of our hypothesis is that inactivation of these "specific RNA binding proteins", coupled with MazFsa activation, will promote cell death rather than growth arrest since these "essential" mRNA will no longer be protected. Cell fractionation analysis, coupled with Northwestern blots with a labeled sarA mRNA probe, have enabled us to identify CshA, CshB and SA1641 as putative "RNA-binding proteins". CshA and CshB are potential DEAD-box RNA helicases while SA1641 has homology with RNA methyltransferase. Previously, DEAD-box proteins have been known to participate in ribosome biogenesis and mRNA decay. Thus, protection of mRNAs from MazFsa-mediated cleavage by CshA and CshB will be a novel concept for DEAD-box proteins. To validate the above hypotheses, we have developed the following specific aims: I) defining the ability of CshA and CshB to bind and protect selective mRNAs from MazFsa-mediated degradation in vitro; II) assessing the degradation of selective MRNA in single and double cshA and cshB mutants, the viability of these mutants upon MazFsa induction and their sensitivity to selective antibiotics; III) characterization of other factors (e.g. SA1641) that help protect selective mRNA from MazFsa-mediated degradation; IV) evaluating the survival of single and double cshA and cshB mutants with and without MazFsa induction in murine models of infection. The results of these studies will allow us to validate the novel function of DEAD-box and other unique RNA-binding proteins in S. aureus. These studies will also provide us with the validation that activation of MazFsa, coupled with inactivation of CshA, CshB, SA1641 and/or other factors, is a novel approach to kill MSSA and MRSA. Accordingly, MazFsa and "specific RNA binding proteins" represent novel targets for antibiotic development. As TA systems, DEAD-box and other RNA binding proteins are common in prokaryotes, our results may apply to other pathogens.