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.

病毒宿主相互作用和微生物生态学 该建议包括在细胞和组水平上的微生物学的两个截然不同的方面。 （1） 使用噬菌体MU换位作为我们的工具探测大肠杆菌基因组组织和染色体动力学。亩 换位不仅在高效率和缺乏目标特异性方面是独一无二的 机构是由尼克 - 加入而不是切割途径发生的。在最后一个赠款期，我们 利用这些特性，以测量大肠杆菌中基因组基因局之间相互作用的体内相互作用速率 他们使用新的统计工具的接近度。在大肠杆菌基因组的当前视图的完全逆转中， 我们的分析揭示了一个未分类的，混合良好的基因组，自由地进行翻译 在所有测量的基因座之间。分析还表明，几个基因家族（例如，六个广泛 分布式核糖体RNA Operaons）显示“聚类”，即强3D共定位不管线性如何 基因组距离。 SMC/冷凝蛋白复合物MukBEF和核酸型蛋白质的活性 HU-α负责这些特性。我们建议探索这些现象，以获得高 基因组组织的分辨率，并了解其如何影响细菌中的基因表达。 （2） 在两个特定的生长条件下剖析抗生素耐受性的机制：蜂群（作为一个移动 集体）和C-DI-GMP合成由挖出的循环酶yfin催化。细菌可以 在其浮游物质对应物致命的浓度下承受抗生素的暴露。我们称之为 特异性（非遗传）抗性，Sr。在最后一个赠款期间，我们发现一个子的死亡 抗生素引起的杀戮的人口对促进SR的群是有益的。引入 预杀性细胞确实增强了SR，使我们能够从两个E的杀死细胞中净化SR因子。 大肠杆菌和沙门氏菌。我们确定了SR因子为ACRA，ACRA是三方外排的周长成分 泵;该泵的外膜成分TOLC也是多个药物外排泵的构成。 我们表明，ACRA通过从外部与TOLC相互作用，激活了活细胞中的药物外排 在短期内外排，从长远来看诱导其他类别的外排泵的表达，因此 放大响应并建立SR。我们称这种现象为“死灵信号”， 在革兰氏阳性和革兰氏阴性细菌中发现了规格特异性的坏死。我们也是 发现特定的环化酶yfin生产C-DI-GMP，逮捕了细胞生长以促进 抗生素耐受性持久状状态。我们建议进一步探索这两种反应。鉴于非 - 遗传抗性是已知的孵化器，用于发展遗传抗性，我们的发现与电流有关 遗传性抗生素耐药性的宽度出现，可能与化学疗法抗性有关 癌症，在获取遗传抗性之前，该药物会排出药物。