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

• 批准号: 8557227
• 负责人: Ambrose Lin Yau Cheung
• 金额: $ 38.05万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 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/8557227
• 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

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）：S。金黄色葡萄球菌通常对甲氧西林和其它细胞壁活性抗生素具有抗性。此应用程序的目标是验证 MazF毒素和S.金黄色葡萄球菌，干扰特定的mRNA降解作为抗菌药物发现的新靶点。毒素抗毒素系统（TA）在细菌中是常见的。在基于蛋白质的不同TA模块中，MazEF系统是迄今为止表征最好的。在我们先前发表的论文中，我们已经表明，S.金黄色葡萄球菌是一种特异性内切核糖核酸酶，其在VUUV'位点切割，其中V或V'可以是A、C或G，导致蛋白质合成的抑制和随后的生长停滞。 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。为了描述这种独特的生长停滞形式，我们已经证明MazFsa切割了大多数细胞mRNA，但保留了一些管家（例如recA，gyrB）和全局调节因子（sarA）mRNA，推测允许细菌进入代谢静止而不牺牲活力。我们假设特定的RNA结合蛋白可能在MazFsa介导的应激下保护这些“重要”mRNA中的一些。我们假设的一个推论是，这些“特异性RNA结合蛋白”的失活，加上MazFsa活化，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，加上西北印迹标记的sarA mRNA探针，使我们能够确定CshA，CshB和SA 1641作为推定的“RNA结合蛋白”。CshA和CshB是潜在的DEAD盒RNA解旋酶，而SA 1641与RNA甲基转移酶具有同源性。以前，已知DEAD盒蛋白参与核糖体生物合成和mRNA衰变。因此，保护mRNA免受CshA和CshB介导的MazFsa切割将是DEAD盒蛋白的新概念。为了验证上述假设，我们开发了以下具体目标：I）确定CshA和CshB结合并保护选择性mRNA免受MazFsa介导的体外降解的能力; II）评估选择性mRNA在单和双cshA和cshB突变体中的降解，这些突变体在MazFsa诱导后的生存力及其对选择性抗生素的敏感性; III）表征有助于保护选择性mRNA免受MazFsa介导的降解的其他因子（例如SA 1641）; IV）在小鼠感染模型中评估有和没有MazFsa诱导的单和双cshA和cshB突变体的存活率。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S.金黄色。这些研究还将为我们提供以下验证：MazFsa的活化与CshA、CshB、SA 1641和/或其他因子的失活结合是杀死MSSA和MRSA的新方法。因此，MazFsa和“特异性RNA结合蛋白”代表了抗生素开发的新靶标。由于TA系统、DEAD-box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。