Bioinformatic and functional analysis of antibiotic-responsive small non-coding RNAs in bacterial pathogens

 描述(申请人提供)：抗生素耐药性是我们这个时代最严重的医学挑战之一。迫切需要理解抗生素作用的新范式来指导新的治疗策略。在麻省总医院以及麻省理工学院和哈佛大学的导师的指导下，这位候选人在耐药细菌病原体中发现了数十个对抗生素有反应的小非编码RNA分子(SRNA)，这些分子以前并不知道参与了对抗生素的转录反应。这项建议旨在进一步研究这一有趣的发现，方法是使用生物信息学分析对候选人已经在关键ESKAPE病原体(肠球菌、金黄色葡萄球菌、克雷伯氏菌和大肠杆菌、不动杆菌、假单胞菌、肠杆菌)中产生的转录数据进行生物信息学分析，从而系统地识别抗生素反应sRNA。之所以选择这些病原体进行研究，是因为它们容易导致严重疾病和获得抗药性；它们被美国国立卫生研究院、世卫组织、疾控中心和美国传染病学会视为高度优先的威胁。将分析候选sRNA的序列保守性、与注释基因的表达协变性以及预测的调控目标。最优先的抗生素反应sRNA将被删除或过度表达，这些干扰对抗生素反应的影响将通过抗生素活性的测量、基础和诱导转录图谱以及鉴定结合伙伴和上位基因的分析来评估。初步 工作已经发现了一种sRNA，它的缺失会加速抗生素介导的杀戮。这些研究的结果将是了解这些sRNA在调节转录网络对抗生素暴露的反应中的作用，目的是确定新的治疗靶点和策略。这份K08指导的临床科学家研究职业发展奖提案旨在培训候选人在四年的时间里有效地探索这一有前景的发现，为独立的研究职业生涯做准备。应聘者的临床训练是传染病，之前受过分子生物学和生物化学的博士训练。在这个职业发展奖的过程中，他将完成生物信息学方法的教学和实践培训，通过课程作业和实验来分析基因组规模的数据集，以及来自一个咨询委员会的培训，该委员会由在基因组方法、网络分析和细菌学方面拥有集体专业知识的资深科学家组成。

 DESCRIPTION (provided by applicant): Antibiotic resistance is one of the most critical medical challenges of our time. New paradigms for understanding antibiotic action are desperately needed to direct new therapeutic strategies. Under the guidance of mentors at Massachusetts General Hospital and the Broad Institute of MIT and Harvard, the candidate has found dozens of antibiotic-responsive small non-coding RNA molecules (sRNAs) in drug- resistant bacterial pathogens that were not previously known to participate in the transcriptional response to antibiotics. This proposal seeks to further investigate this intriguing finding by systematically identifying antibiotic-responsive sRNAs using bioinformatic analysis of transcriptomic data that the candidate has already generated via RNA-Seq in the critical ESKAPE pathogens (Enterococcus, Staphylococcus aureus, Klebsiella and E. coli, Acinetobacter, Pseudomonas, Enterobacter). These pathogens were chosen for study because of their propensity to cause serious disease and to acquire drug resistance; they are recognized by the NIH, WHO, CDC, and Infectious Disease Society of America as high-priority threats. Candidate sRNAs will be analyzed for sequence conservation, covariation of expression with annotated genes, and predicted targets of regulation. The highest priority antibiotic-responsive sRNAs will be deleted or overexpressed, and the effects of these perturbations on the antibiotic response will be assessed through measures of antibiotic activity, basal and induced transcriptional profiling, and assays to identify binding partners and epistatic genes. Preliminary work has already identified one sRNA whose deletion accelerates antibiotic-mediated killing. The outcome of these studies will be to understand the role of these sRNAs in regulating transcriptional networks in response to antibiotic exposure, with the goal of identifying new therapeutic targets and strategies. This K08 Mentored Clinical Scientist Research Career Development Award proposal seeks to train the candidate to effectively explore this promising finding over a four-year period in preparation for an independent research career. The candidate's clinical training is in Infectious Disease, with prior doctoral training in molecular biology and biochemistry. During the course of this career development award, he will complete didactic and hands-on training in bioinformatic methodologies for analyzing genomic-scale datasets through coursework and experimentation, as well as training from an advisory committee of established senior scientists with collective expertise in genomic methodologies, network analysis, and bacteriology.