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 这一提议涵盖了微生物学的两个非常不同的方面，无论是在细胞层面还是在群体层面。(1) 以噬菌体Mu转座为工具探索大肠杆菌基因组组织和染色体动力学。穆恩 转座的独特之处不仅在于它的高效率和缺乏靶向性，而且还在于它的转座。 机制，这是通过缺口连接而不是剪切和粘贴路径发生的。在上一次授权期内，我们 利用这些特性在体内测量了大肠杆菌中基因组位点之间的相互作用速度，并研究了 使用新的统计工具，他们的接近程度。与目前对大肠杆菌基因组的看法完全相反， 我们的分析揭示了一个未分割的、混合良好的基因组，其中转位可以自由发生 在所有测量的轨迹之间。分析还揭示了几个基因家族(例如，六个广泛的 分布的核糖体RNA操纵子)表现出聚集，即强3D共定位，而不考虑线性 基因组距离。SMC/凝集素复合体MukBEF和类核致密蛋白的活性 HU-α对这些财产负责。我们建议探索这些现象，以获得更高的- 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。(2) 剖析了两种特定生长条件下的抗生素耐药机制：蜂拥而至(以 和c-di-GMP的合成，由二鸟苷环化酶YfiN催化。成群的细菌可以 耐受暴露于对浮游生物具有致命性的抗生素浓度。我们把这叫做 群集性(非遗传)抗性，SR。在上一次拨款期间，我们发现一名潜水员的死亡- 种群由于抗生素诱导的杀灭，有利于蜂群促进SR。介绍 预灭活的细胞形成一个群体，确实增强了SR，使我们能够从两个E. 大肠埃希菌和沙门氏菌。我们鉴定了SR因子是AcrA，它是一个三体RND外流的周质成分 泵；该泵的外膜组件TolC也是多个药物外排泵的组成部分。 我们发现AcrA通过从外部与TolC相互作用，激活 短期内外排，长期诱导其他类别的外排泵的表达，从而 放大反应并建立SR。我们把这种现象称为‘死亡信号’，而且 在革兰氏阳性和革兰氏阴性细菌中都发现了物种特有的死亡信号。我们也 发现由特定的循环酶YfiN产生c-di-GMP，阻止细胞生长以促进 抗生素耐受类持久病毒症。我们建议进一步探讨这两种反应。鉴于这一点- 遗传抗性是已知的进化遗传抗性的孵化器，我们的发现与目前的 广泛出现的对抗生素的遗传耐药，可能与化疗耐药有关 癌症，在获得遗传耐药性之前将药物排出体外。