Virus-host interactions and microbial ecology

病毒-宿​​主相互作用和微生物生态学

• 批准号: 10394302
• 负责人: Rasika M Harshey
• 金额: $ 56.08万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-06 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10394302
• 关键词: 3-Dimensional; Animal Model; Antibiotic Resistance; Antibiotics; Bacteria; Bacteriophage mu; Cells; Cessation of life; Chemoresistance; Chromatin Loop; Chromosomes; Complex; Drug Efflux; Ecology; Escherichia coli; Evolution; Exposure to; Gene Expression; Gene Family; Gene Rearrangement; Genome; Genomics; Gram-Negative Bacteria; Grant; Growth; HU Protein; Human; Incubators; Malignant Neoplasms; Measures; Membrane; Microbiology; Motor; Operon; Paste substance; Pathway interactions; Population; Production; Property; Pump; Research; Resolution; Ribosomal RNA; Salmonella; Specificity; Surface; antibiotic tolerance; cell growth; cell killing; condensin; efflux pump; genetic resistance; genomic locus; in vivo; microbial; non-genetic; periplasm; refractory cancer; response; tool; virus host interaction

Virus-host interactions and microbial ecology This proposal encompasses two very different aspects of microbiology, both at cellular and group levels. (1) Probing E. coli genome organization and chromosome dynamics using phage Mu transposition as our tool. Mu transposition is unique not only in its high efficiency and lack of target specificity, but also in its transposition mechanism, which occurs by a nick-join rather than a cut-and-paste pathway. In the last grant period, we exploited these properties to measure in vivo rates of interactions between genomic loci in E. coli, and studied their proximity using new statistical tools. In a complete reversal of the current view of the E. coli genome, our analysis has revealed an uncompartmentalized, well-mixed genome, where transpositions occur freely between all measured loci. The analysis also revealed that several gene families (for example, six widely distributed ribosomal RNA operons) show `clustering' i.e. strong 3D co-localization regardless of linear genomic distance. The activities of the SMC/condensin complex MukBEF and the nucleoid-compacting protein HU-α are responsible for these properties. We propose to explore these phenomena to obtain a high- resolution view of genome organization, and to understand how it influences gene expression in bacteria. (2) Dissecting the mechanism of antibiotic tolerance under two specific growth conditions: swarming (moving as a collective), and c-di-GMP synthesis catalyzed by the diguanulate cyclase YfiN. Swarming bacteria can withstand exposure to antibiotics at concentrations that are lethal to their planktonic counterparts. We call this swarming-specific (non-genetic) resistance, SR. In the last grant period, we discovered that death of a sub- population as a result of antibiotic-induced killing, is beneficial to the swarm in promoting SR. Introduction of pre-killed cells into a swarm indeed enhanced SR, allowing us to purify the SR factor from killed cells of both E. coli and Salmonella. We identified the SR factor to be AcrA, a periplasmic component of a tripartite RND efflux pump; the outer membrane component of this pump, TolC, is also a constituent of multiple drug efflux pumps. We showed that AcrA stimulates drug efflux in live cells by interacting with TolC from the outside, activating efflux in the short term, and inducing the expression of other classes of efflux pumps in the long term, thus amplifying the response and establishing SR. We have called this phenomenon `necrosignaling', and discovered species-specific necrosignaling in both Gram-positive and Gram-negative bacteria. We also discovered that production of c-di-GMP by the specific cyclase YfiN, arrests cell growth to promote an antibiotic-tolerant persister-like state. We propose to explore both these responses further. Given that non- genetic resistance is a known incubator for evolving genetic resistance, our findings are relevant to the current widespread emergence of genetic resistance to antibiotics, and may be relevant to chemotherapy-resistant cancers, which efflux the drugs prior to acquisition of genetic resistance.

病毒-宿主相互作用与微生物生态学 这一建议包括微生物学的两个非常不同的方面，无论是在细胞和群体水平。（一） Probing E.大肠杆菌基因组组织和染色体动力学使用噬菌体Mu转座作为我们的工具。亩 转座的独特之处不仅在于其高效性和缺乏靶特异性，而且在于其转座 机制，这是通过切口连接而不是剪切和粘贴路径发生的。在过去的一段时间里，我们 利用这些特性来测量E.大肠杆菌，并研究 利用新的统计工具来分析它们的接近程度。在一个完全逆转的E。大肠杆菌基因组， 我们的分析揭示了一个非区室化的，混合良好的基因组，在那里转座自由发生 在所有测量的位点之间。分析还显示，几个基因家族（例如，六个广泛存在的 分布的核糖体RNA操纵子）显示“聚类”，即强的3D共定位，而不管线性 基因组距离SMC/凝聚素复合物MukBEF和类核致密蛋白的活性 HU-α负责这些性质。我们将探索这些现象，以获得一个高- 基因组组织的分辨率视图，并了解它如何影响细菌中的基因表达。（二） 在两种特定的生长条件下剖析抗生素耐受性的机制：群集（以 collective），和由二鸟苷酸环化酶YfIN催化的c-di-GMP合成。成群的细菌可以 能够承受暴露于抗生素的浓度，而抗生素的浓度对它们的抗生素对应物来说是致命的。我们称之 在上一个赠款期间，我们发现，一个亚群体的死亡， 种群作为杀虫剂诱导的杀伤结果，有利于蜂群促进SR。 将预先杀死的细胞放入群中确实增强了SR，使我们能够从两种E. 大肠杆菌和沙门氏菌。我们鉴定了SR因子为AcrA，它是RND三重外排的周质组分 该泵的外膜组件TolC也是多药外排泵的组成部分。 我们发现AcrA通过与外部的TolC相互作用，激活细胞，刺激活细胞中的药物流出 在短期内外排，并在长期内诱导其他类型外排泵的表达， 放大反应并建立SR。我们称这种现象为“坏死信号”， 在革兰氏阳性菌和革兰氏阴性菌中都发现了物种特异性坏死信号。我们也 发现通过特异性环化酶YfiN产生c-di-GMP，阻止细胞生长以促进细胞增殖， 耐药性持久性状态。我们建议进一步探讨这两种反应。鉴于非- 遗传抗性是进化遗传抗性的孵化器，我们的发现与当前的研究有关。 广泛出现对抗生素的遗传耐药性，可能与化疗耐药有关。 癌症，在获得遗传抗性之前排出药物。