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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：
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
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/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

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耐药。此应用程序的目标是验证