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

CshA 和 CshB 在金黄色葡萄球菌选择性 mRNA 保护中的作用

基本信息

批准号：
8665389
负责人：
Ambrose Lin Yau Cheung
金额：
$ 40.5万
依托单位：
DARTMOUTH COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-05-24 至 2017-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8665389
关键词：
AIDS/HIV problem Abscess Accident and Emergency department Acute Anti-Bacterial Agents Antibiotics Antitoxins Bacteria Bacterial Physiology Binding Biogenesis Boxing Caring Cell Death Cell Fractionation Cell Wall Cells Cephalosporins Cessation of life Cleaved cell Clinic Complex Coupled DNA Gyrase Daptomycin Data Detection Development Digestion Drops Endoribonucleases Escherichia coli Fractionation Genes Genome Goals Growth Heparin Hospitals Hour Housekeeping In Vitro Individual Infection Infectious Skin Diseases Label Lead Linezolid Link Mediating Messenger RNA Metabolic Methicillin Methyltransferase Mind Modeling Mus Names Normalcy Nutrient Oxacillin Paper Parents Peptide Hydrolases Process Prokaryotic Cells Protein S Protein Synthesis Inhibition Proteins Publishing RNA RNA Helicase RNA-Binding Proteins Radiolabeled Resistance Ribonucleases Ribosomes Role Site Specificity Staining method Stains Staphylococcus aureus Stress System Toxin Translating United States Validation Vancomycin antimicrobial drug bacterial resistance base cellular targeting coping drug discovery empowered endonuclease endoribonuclease helicase in vivo insight interest killings mRNA Decay mRNA Transcript Degradation methicillin resistant Staphylococcus aureus mutant novel novel strategies pathogen pathogenic bacteria public health relevance radiotracer response ribonuclease E

项目摘要

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.

描述（由申请人提供）：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耐药性。该应用程序的目标是验证 MazF 毒素和金黄色葡萄球菌的特异性 RNA 结合蛋白可干扰特定 mRNA 降解，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。在基于蛋白质的不同 TA 模块中，MazEF 系统是迄今为止特征最好的。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 毒素是一种特异性核糖核酸内切酶，可在 VUUV' 位点（其中 V 或 V' 可以是 A、C 或 G）进行切割，从而抑制蛋白质合成并导致生长停滞。 MazFsa 介导的生长停滞是独特的，因为这些细胞是有活力的，但不能复制。为了描述这种独特形式的生长停滞，我们证明 MazFsa 切割大部分细胞 mRNA，但保留一些内务（例如 recA、gyrB）和全局调节器（sarA） mRNA，可能允许细菌进入代谢静止状态而不牺牲活力。我们假设，在 MazFsa 介导的应激下，特定的 RNA 结合蛋白可能会保护其中一些“重要的”mRNA。我们假设的一个推论是，这些“特定的 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“必需的”mRNA 将不再受到保护。细胞分级分离分析，与 Northwestern 相结合使用标记的 sarA mRNA 探针进行印迹，使我们能够将 CshA、CshB 和 SA1641 识别为假定的“RNA 结合蛋白”。 CshA 和 CshB 是潜在的 DEAD-box RNA 解旋酶，而 SA1641 与 RNA 甲基转移酶具有同源性。此前，已知 DEAD-box 蛋白参与核糖体生物发生和 mRNA 衰变。因此，保护 mRNA 免受 MazFsa 介导的 CshA 和 CshB 裂解将是 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) 定义 CshA 和 CshB 在体外结合和保护选择性 mRNA 免受 MazFsa 介导的降解的能力； II) 评估单cshA和双cshB中选择性mRNA的降解突变体，这些突变体在 MazFsa 诱导后的生存能力及其对选择性抗生素的敏感性； III) 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）的表征； IV)评估在小鼠感染模型中在有和没有MazFsa诱导的情况下单和双cshA和cshB突变体的存活率。这些研究的结果将允许我们验证了金黄色葡萄球菌中 DEAD-box 和其他独特 RNA 结合蛋白的新功能。这些研究还将向我们证实，MazFsa 的激活，加上 CshA、CshB、SA1641 和/或其他因子的灭活，是一种杀死 MSSA 和 MRSA 的新方法。因此，MazFsa 和“特异性 RNA 结合蛋白”代表了新的靶点抗生素的开发。由于 TA 系统、DEAD-box 和其他 RNA 结合蛋白在原核生物中很常见，因此我们的结果可能适用于其他病原体。