Peptide Pheromones in Enterococcal Plasmid Maintenance

描述(申请人提供)：粪肠球菌在当前的抗生素耐药性危机中扮演着主要角色。它是抗药性医院感染的主要原因之一，作为肠道正常菌群的一部分，也是抗药性基因的储存库。多肽信息素诱导的接合质粒转移有助于这些抗生素耐药基因和其他肠球菌毒力因子的传播。本研究的重点是多肽信号在质粒维持中的作用，特别是在粪肠球菌中四环素耐药质粒pCF10的作用。在接合质粒生物学中信息素信号的最初观察发现，无质粒受体细胞产生的肽信号cCF10被用来诱导接合的pCF10转移。CCF10是在细胞表面产生的，并通过cCF10受体与寡肽通透酶的相互作用被含有pCF10的供体细胞内化。一旦进入细胞，cCF10就会与细胞内的效应分子相互作用，诱导结合。最近，我们发现了供体细胞产生的cCF10在粪肠球菌细胞群中维持pCF10的第二个作用。在供体细胞群体中，也会产生cCF10，在明显没有接合的情况下，需要cCF10的内化来维持pCF10。维持功能发生在最小的pCF10复制子结构中，该复制子不能被接合转移，这表明cCF10在pCF10维持中的作用不同于它在接合转移中的作用。 本申请中提出的研究的主要目标是基本了解cCF10是如何影响pCF10维护的。(1)cCF10是否作为维持pCF10的信息素？(2)cCF10的有效转运(细胞外产生)是否需要寡肽渗透酶和cCF10受体PrgZ？(3)pCF10复制子的基本特征是什么？(4)依赖cCF10维持pCF10的细胞内效应分子是什么？这将为未来的实验奠定基础，以确定如何在不诱导可检测到的接合水平的情况下将相同的多肽用于质粒维护。

DESCRIPTION (provided by applicant): Enterococcus faecalis plays a major role in the current antibiotic resistance crisis. It is one of the leading causes of antibiotic-resistant nosocomial infections and, as part of the normal flora of the intestinal tract, also serves as a reservoir of antibiotic resistance genes. Peptide pheromone-induced conjugative plasmid transfer contributes to the spread of these antibiotic resistance genes and of other enterococcal virulence factors. The focus of this research is on the role of peptide signaling in plasmid maintenance, specifically of the tetracycline-resistance plasmid pCF10 in Enterococcus faecalis. The original observations of pheromone signaling in conjugative plasmid biology found that the peptide signal cCF10 produced by plasmid-free recipient cells is used for the induction of conjugative pCF10 transfer. cCF10 is produced at the cell surface and internalized by pCF10-containing donor cells by interaction of the cCF10 receptor with the oligopeptide permease. Once inside the cell cCF10 interacts with intracellular effector molecules to induce conjugation. Recently, we have found a second role, for donor-cell produced cCF10, in maintaining pCF10 in the Enterococcus faecalis cell population. In a donor cell population, cCF10 is also produced and its internalization is required to maintain pCF10 in the apparent absence of conjugation. The maintenance functions occur with a minimum pCF10 replicon construct that is unable to be conjugatively transferred, suggesting the role of cCF10 in pCF10 maintenance is distinct from its role in conjugative transfer. The main goal of the research proposed in this application is to gain a basic understanding of how cCF10 is affecting pCF10 maintenance. (1) Does cCF10 act as a pheromone in maintenance of pCF10? (2) Are the oligopeptide permease and the cCF10 receptor PrgZ required for efficient transport of cCF10 (produced extracellularly)? (3) What are the basic features of the pCF1 0 replicon? (4) What are the intracellular effector molecules responsible for cCF10-dependent pCF10 maintenance? This will lay the groundwork for future experiments to determine how the same peptide can be used for plasmid maintenance without inducing detectable levels of conjugation.