The Maintenance of Plasmids in Pathogenic Organisms

病原生物中质粒的维持

• 批准号: 7592760
• 负责人: stuart j austin
• 金额: $ 36.77万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7592760
• 关键词: ATP phosphohydrolase; Amino Acids; Antibiotic Resistance; Antibiotic Therapy; Bacteria; Bacterial Infections; Binding; Binding Sites; Boxing; CCR; Cancer Patient; Cause of Death; Cells; Centromere; Chromosomes; Collaborations; Communicable Diseases; Complex; DNA; DNA Binding; DNA Fingerprinting; Disease Progression; Elements; Ensure; Enteral; Family; Fluorescence; Genes; Human; Immune system; Individual; Laboratories; Life; Maintenance; Medical; Membrane Proteins; Modeling; Molecular Models; Molecular Structure; Motor; Nature; Numbers; Organism; Plague; Plasmids; Population; Positioning Attribute; Proteins; Publishing; Salmonella; Shigella; Site; Species Specificity; Specific qualifier value; Specificity; Structure; Study models; System; Testing; Virulence; base; chemotherapy; chromosome replication; daughter cell; interest; member; novel; novel strategies; pathogen; pathogenic bacteria; plasmid DNA; remediation; segregation

Low copy number plasmids in bacteria are of interest for two principle reasons. First, they act in many ways like small, dispensable chromosomes within the cell, and are therefore tractable models for the study of chromosome replication and segregation. Second, they are of considerable medical importance. They are the transmissible elements that spread antibiotic resistance among pathogenic bacteria and in some cases, are the determinants of the virulence of bacterial infection in human infectious disease. The spread of antibiotic resistance threatens to make antibiotic therapy virtually useless in the next few decades. In addition, pathogenic bacteria containing virulence plasmids are increasingly the ultimate cause of death of cancer patients whose immune systems are often compromised by disease progression or chemotherapy. It is therefore of importance to try to understand how these plasmids are stably maintained in the bacterial population as a first step toward developing novel strategies for infectious disease therapy and remediation of plasmid spread. We are particularly interested in the mechanisms that plasmids use to ensure their proper segregation to daughter cells. We study a family of elements known as partition genes (the P1par family), that are responsible for the segregation of several types of plasmid including the virulence plasmids of Salmonella and Shigella species responsible for enteric disease, and of Yersinia pestis; the causative organism for bubonic plague. In each case, we have shown that segregation is achieved by recognition of a cis-acting site parS, analogous to a centromere, and two plasmid encoded proteins, ParA and ParB.. ParB binds specifically to parS and ParA is an ATPase that may be a motor for moving the plasmid during segregation. Members of the P1par family show unique species specificities. This is important, because, otherwise, plasmids of different types would compete with each other, limiting their spread in nature. We have discovered that these species specificities reside in a novel interaction between the ParB protein and the parS site. This is not the interaction that provides the energy for ParB binding to the site. Rather, it is a special contact between the ParB N-terminus and a short motif in parS termed the B box. By changing the B box sequence by as little as one base, we can change the specificity of the system from one species to another. This mechanism appears to be a novel type of DNA-protein recognition that may have broad implications for how proteins act at a specific site when other potential binding sites exist. We have further explored the hypothesis that the contact between the BoxB sequences in the cis-acting partition site and the ParB protein are responsible for the species specificity of members of the P1par family of partition elements. Using the recently published crystal structure of the P1 ParB protein as a guide, we have been able to pinpoint the position on the ParB protein surface that specifies recognition of species determinants on the individual plasmid DNA parS sites. By changing a single amino acid in the ParB protein we have been able to switch its recognition specificity completely to that of a different species. This year, we have purified these altered proteins and have been able to show that thier altered specificity does not reside in a change in DNA binding ability. Rather, it appears that the BoxB DNA site acts as a specific activator of the partition complex for plasmid segregation. This has important implications for models of partition. We are currently testing these conclusions by studying the segregation of the plasmid DNA by fluorescence photomicroscopy in living cells. In addition, we have initiated a collaboration with Dr. Xinhua Ji of the Biomolecular Structure Section (Chystallography Laboratory, CCR), to model the molecular structures of the altered interactions

细菌中低拷贝数质粒的研究有两个主要原因。首先，它们在许多方面就像细胞内的小的、可缺性的染色体一样，因此是研究染色体复制和分离的容易处理的模型。其次，它们具有相当大的医学重要性。它们是在致病菌中传播抗生素耐药性的可传播因素，在某些情况下，它们是人类传染病中细菌感染毒性的决定因素。抗生素耐药性的蔓延有可能使抗生素治疗在未来几十年几乎毫无用处。此外，含有毒力质粒的致病菌越来越多地成为癌症患者死亡的最终原因，这些患者的免疫系统往往因疾病进展或化疗而受损。因此，了解这些质粒是如何在细菌种群中稳定维持的，是开发感染性疾病治疗和质粒传播修复新策略的第一步，这一点非常重要。我们特别感兴趣的机制，质粒使用，以确保其适当分离到子细胞。我们研究了一个被称为分割基因的元件家族（P1par家族），它负责分离几种类型的质粒，包括导致肠道疾病的沙门氏菌和志贺氏菌以及鼠疫耶尔森菌的毒力质粒；黑死病腺鼠疫的致病生物在每种情况下，我们已经证明分离是通过识别顺式作用位点parS（类似于着丝粒）和两个质粒编码蛋白ParA和ParB来实现的。ParB与parS特异性结合，而parS是一种atp酶，在分离过程中可能是移动质粒的马达。P1par家族的成员表现出独特的物种特异性。这一点很重要，因为否则，不同类型的质粒就会相互竞争，从而限制它们在自然界的传播。我们发现这些物种特异性存在于ParB蛋白和parS位点之间的一种新的相互作用中。这不是为ParB结合到位点提供能量的相互作用。相反，它是ParB n端与parS中称为B盒的短基序之间的特殊接触。通过改变B盒序列的一个碱基，我们可以改变系统的特异性，从一个物种到另一个物种。这种机制似乎是一种新型的dna -蛋白质识别，可能对蛋白质在其他潜在结合位点存在时如何在特定位点起作用具有广泛的意义。我们进一步探讨了P1par分隔元件家族成员的物种特异性与顺式作用分隔位点BoxB序列与ParB蛋白的接触有关的假设。利用最近发表的P1 ParB蛋白的晶体结构作为指导，我们已经能够精确定位ParB蛋白表面的位置，该位置指定了单个质粒DNA parS位点上物种决定因子的识别。通过改变ParB蛋白中的单个氨基酸，我们已经能够将其识别特异性完全转换为不同物种的识别特异性。今年，我们已经纯化了这些改变的蛋白质，并且已经能够证明它们改变的特异性并不存在于DNA结合能力的变化中。相反，BoxB DNA位点似乎是质粒分离分割复合体的特定激活剂。这对划分模型具有重要的意义。我们目前正通过荧光显微技术在活细胞中研究质粒DNA的分离来验证这些结论。此外，我们还与生物分子结构组（结晶学实验室，CCR）的纪新华博士合作，对改变的相互作用的分子结构进行建模