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抗性。此应用程序的目的是验证金黄色葡萄球菌的MAZF毒素和特异性RNA结合蛋白，这些蛋白会干扰特定的mRNA降解作为抗菌药物发现的新靶标。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系统是迄今为止最佳的特征。在我们发表的先前论文中，我们已经表明，金黄色葡萄球菌的MAZFSA毒素是一种特定的内核酸酶，在Vuuv'Ent上切割V或V'可以是A，C或G，从而导致蛋白质合成及其生长滞留。 MAZFSA介导的生长停滞是独一无二的，因为这些细胞是可行的，但无法复制。为了描绘这种独特的生长停滞形式，我们表明MAZFSA切割了大部分的细胞mRNA，但请助长一些家务（例如Reca，gyrb）和全球调节剂（Sara）mRNA，大概允许细菌允许细菌输入无效的代谢戒烟。我们假设特定的RNA结合蛋白可以在MAZFSA介导的压力下保护其中一些“重要的“ mRNA”。我们的假设的必然学是，这些“特定RNA结合蛋白”的灭活，而不是与MAZF的激活相结合，而不是北部的生长，而不是分析“ MRNA”，因为它会促进“ MRNA”的生长。带有标记的SARA mRNA探针的印迹使我们能够将CSHA，CSHB和SA1641识别为推定的“ RNA结合蛋白”，而SA1641是潜在的Dead-Box RNA Helicase，而sa1641则与RNA甲基转移酶相生。 MAZFSA介导的CSHA和CSHB的裂解将是验证上述假设的新型概念，我们已经开发了以下特定目的：i）定义CSHA和CSHB的能力，定义了MAZFSA介导的MAZFSA介导的降解的能力； ii）评估单个和双CSHA和CSHB突变体中选择性mRNA的降解，这些突变体在MAZFSA诱导时的生存力及其对选择性抗生素的敏感性； iii）表征其他因素（例如SA1641），这些因素有助于保护选择性mRNA免受MAZFSA介导的降解； iv）评估在感染的鼠模型中，有或没有MAZFSA诱导的单个和双CSHA和CSHB突变体的存活率。这些研究的结果将使我们能够验证金黄色葡萄球菌中死盒和其他独特的RNA结合蛋白的新功能。这些研究还将为我们提供验证，即MAZFSA的激活以及CSHA，CSHB，SA1641和/或其他因素的失活是杀死MSSA和MRSA的一种新型方法。因此，MAZFSA和“特定RNA结合蛋白”代表了抗生素发育的新靶标。作为TA系统，死盒和其他RNA结合蛋白在原核生物中很常见，我们的结果可能适用于其他病原体。