The Maintenance of Plasmids in Pathogenic Organisms

病原生物中质粒的维持

• 批准号: 7291863
• 负责人: stuart j austin
• 金额: --
• 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/7291863
• 关键词: Maintenance; Plasmids; Pathogenic; Organisms

Low copy number plasmids in bacteria are of interest for two principle reasons. First, they act in many ways like small, dispensible 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 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. This year, 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. By changing the pMT1parS site BoxB sequences to their P1 or P7 equvalents, complete switches of species specificity were acheived. In addition, we were able to show that the specificity of partition mediated plasmid incompatibility, that govens whether two plasmids can be maintained in the same cell is also swiched in these mutants. thus, species psecificity for par protein recognition and partition-mediated incompatibility are co-determined. Thus, the BoxB-ParB recognition is a system for rapid speciation and survival of the plasmid during evolution.

细菌中的低拷贝数质粒由于两个主要原因而令人感兴趣。首先，它们在许多方面像细胞内的小的、可分配的染色体，因此是研究染色体复制和分离的易处理的模型。其次，它们具有相当大的医学意义。它们是在病原菌中传播抗生素耐药性的传播因子，在某些情况下，是人类传染病中细菌感染毒力的决定因素。抗生素耐药性的传播有可能使抗生素治疗在未来几十年内几乎无用。此外，含有毒力质粒的病原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通常因疾病进展或化疗而受损。因此，重要的是要尝试了解这些质粒是如何稳定地维持在细菌种群作为第一步发展战略的感染性疾病的治疗和补救的质粒传播。我们特别感兴趣的机制，质粒使用，以确保其正确的分离到子细胞。我们研究了一个被称为分区基因（P1 par家族）的元素家族，该家族负责分离几种类型的质粒，包括沙门氏菌和志贺氏菌属肠道疾病的毒力质粒，以及鼠疫耶尔森氏菌的毒力质粒;腺鼠疫的病原体。在每种情况下，我们已经表明，隔离是通过识别一个顺式作用位点parS，类似于一个着丝粒，和两个质粒编码的蛋白质，帕拉和ParB。ParB与parS特异性结合，而帕拉是一种ATP酶，可能是分离过程中移动质粒的马达。P1 par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会相互竞争，限制它们在自然界中的传播。我们已经发现，这些物种的特异性驻留在一个新的ParB蛋白和parS网站之间的相互作用。这不是为ParB结合位点提供能量的相互作用。相反，它是ParB N-末端和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，只要改变一个碱基，我们就可以改变系统从一个物种到另一个物种的特异性。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影响。今年，我们进一步探索了这样一个假设，即在顺式作用分区位点的BoxB序列和ParB蛋白之间的接触是负责分区元件的P1 par家族成员的物种特异性。通过将pMT 1 parS位点BoxB序列改变为它们的P1或P7等价物，实现了物种特异性的完全转换。此外，我们能够证明，分配介导的质粒不相容性的特异性，即决定两个质粒是否可以在同一细胞中维持，也在这些突变体中切换。因此共同决定了物种对PAR蛋白识别的特异性和分配介导的不相容性。因此，BoxB-ParB识别是一个在进化过程中质粒快速形成和存活的系统。