Characterizing the role of antimicrobial peptide resistance in plague transmission

表征抗菌肽耐药性在鼠疫传播中的作用

• 批准号: 9901450
• 负责人: Mark A Fisher
• 金额: $ 38.13万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-12 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9901450
• 关键词: Address; Anabolism; Antibiotics; Antimicrobial Cationic Peptides; Antimicrobial Resistance; Attenuated; Bacteria; Bacterial Infections; Binding; Biological Assay; Biology; Bioterrorism; Cell Membrane Permeability; Communities; Complex; Data; Fleas; Fluorescence Microscopy; Genes; Genetic; Genetic Structures; Genome; Goals; Health; Human; Hypersensitivity; In Vitro; Infection; Investigation; Knowledge; Label; Lead; Libraries; Lipid A; Lipopolysaccharides; Mass Spectrum Analysis; Mediating; Membrane; Methods; Modification; Molecular; Multi-Drug Resistance; Multiple Bacterial Drug Resistance; Mutation; Nature; PAWR protein; Pathogenesis; Phenotype; Plague; Plasmids; Play; Polymyxin Resistance; Polymyxins; Predisposition; Public Health; Publishing; Recording of previous events; Research; Resistance; Role; Southwestern United States; Structure; Testing; Virulent; Yersinia pestis; antimicrobial; antimicrobial peptide; biophysical techniques; colistin resistance; enterobacterial common antigen; gene function; genetic approach; human pathogen; improved; insight; mutant; new therapeutic target; novel; pathogen; periplasm; resistance gene; resistance mechanism; response; screening; transmission process; vector; weapons

PROJECT SUMMARY It is difficult to overstate the impact Yersinia pestis, the bacterial agent of plague, has had on human history. It is of great concern as a potential agent of bioterrorism because of its highly virulent nature, and it consistently infects thousands of people per year in endemic foci around the world, including the southwestern United States. Despite these facts, there is much that is unknown about the ability of Y. pestis to infect both flea vectors and mammalian hosts, and its ability to undergo successful transmission between them. The goal of this project is to determine the genetic and molecular mechanisms of resistance to cationic antimicrobial peptides (CAMPs) in Y. pestis, and the role this resistance plays in allowing it to successfully infect fleas. Data from our lab revealed that numerous Y. pestis transposon mutants that were unable to maintain successful flea infections also showed in vitro hypersensitivity to CAMPs. Two mutants contained insertions in genes predicted to block modification of the outer membrane lipopolysaccharide, and we recently published data that this was in fact the cause of the mutants' CAMP sensitivity. Several mutations however were in novel or hypothetical genes not previously known to be involved in CAMP resistance. Additionally, disruption of genes involved in biosynthesis of the enterobacterial common antigen resulted in unexpected CAMP-susceptibility in Y. pestis by an unknown mechanism. These data, along with the recent discovery of the plasmid-mediated mcr colistin resistance genes, indicate that CAMP resistance is more complex, and less well understood, than previously thought. The first specific aim of this project is to investigate structural and genetic changes present in these CAMP- susceptible mutants to improve our understanding of the mechanisms of CAMP resistance. Since CAMPs, such as polymyxins, are often one of the only classes of antibiotics with activity against multi-drug resistant bacterial infections, identifying novel mechanisms of resistance is of critical importance. The second specific aim involves constructing a comprehensive, ordered transposon mutant library of all non-essential genes in Y. pestis KIM6+. This library will be screened for mutants that are susceptible to structurally diverse CAMPs in order to identify additional and potentially novel mechanisms of resistance. Mutants identified in this screen will be analyzed using the biophysical and genetic approaches from the first aim to elucidate their mechanisms of CAMP resistance. The third specific aim is to determine the potential role of different mechanisms of CAMP resistance in transmission by testing the ability of the mutants to maintain infection, and by determining the distribution of mutants in a natural flea vector. The knowledge obtained from this project on CAMP resistance mechanisms will open up a number of new trajectories in plague and antimicrobial resistance research, and the ordered mutant library could stimulate additional investigation into the biology of this important human pathogen. Improving our understanding of CAMP resistance is crucial given the status of polymyxins as WHO “Critically Important Antimicrobials,” and the recent discovery of transmissible (mcr-1) resistance. Loss of this class of antibiotics could herald the era of untreatable infections. !

项目总结 鼠疫的细菌剂--鼠疫耶尔森氏菌对人类历史的影响怎么说都不为过。它是 由于其高度致命的性质，作为生物恐怖主义的潜在媒介而受到极大关注，并持续感染 每年有数千人在世界各地的地方病疫源地，包括美国西南部。尽管 这些事实，还有许多关于鼠疫杆菌感染跳蚤媒介和哺乳动物的能力是未知的 以及在它们之间成功传输的能力。该项目的目标是确定 鼠疫菌对阳离子抗菌肽(CAMP)耐药的遗传和分子机制及其作用 这种抵抗力使它能够成功地感染跳蚤。我们实验室的数据显示，许多鼠疫杆菌 不能保持跳蚤成功感染的转座子突变体在体外也显示出对 营地。两个突变体含有插入的基因，被预测为阻止外膜的修饰 脂多糖，我们最近发表的数据表明，这实际上是突变体对cAMP敏感的原因。 然而，在新的或假设的基因中有几个突变，这些基因以前并不知道与cAMP有关 抵抗。此外，参与肠杆菌共同抗原生物合成的基因的破坏导致 通过一种未知的机制在鼠疫杆菌中意外地对cAMP敏感。这些数据，以及最近的 质粒介导的mcr粘菌素耐药基因的发现，表明cAMP耐药更为复杂， 而且，人们对此的理解比之前认为的要少。 这个项目的第一个具体目标是调查这些营地中存在的结构和遗传变化- 以提高我们对cAMP抗性机制的理解。由于夏令营，如 多粘菌素，通常是仅有的几类抗生素之一，对多重耐药细菌具有活性。 对于感染而言，确定新的耐药机制至关重要。第二个具体目标涉及 构建鼠疫菌KIM6+中所有非必需基因的完整有序转座子突变文库。这 将对文库进行筛选，以寻找对结构不同的阵营敏感的突变体，以确定其他 以及潜在的新的抗性机制。在此屏幕中识别的突变体将使用 生物物理学和遗传学方法从第一个目的是阐明它们对cAMP耐药的机制。第三 具体目的是通过测试确定cAMP耐药的不同机制在传播中的潜在作用 突变体保持感染的能力，并通过确定突变体在自然跳蚤中的分布 向量。从这个项目中获得的关于cAMP抵抗机制的知识将开辟一些新的 鼠疫和抗菌素耐药性研究的轨迹，以及有序的突变体文库可能会刺激 对这种重要的人类病原体的生物学进行调查。提高我们对cAMP抵抗的认识 鉴于多粘菌素作为世卫组织“至关重要的抗菌剂”的地位，以及最近发现的 可传播(mcr-1)抗性。失去这类抗生素可能预示着无法治愈的感染时代的到来。 好了！