Molecular analysis of oriT-independent transfer in the gonococcal R-plasmids

描述(申请人提供)：出现对青霉素和四环素具有显著临床抗药性的淋病奈瑟菌菌株是一个全球性问题。利用共居的41kb的四环素耐药的tet(M)自传质粒，可将7.4kb、5.6kb和5.2kb的3个编码β-内酰胺酶(R)的质粒从致病奈瑟菌动员到共生奈瑟氏菌以及从泌尿生殖道和生殖器外感染部位的菌群。近年来，我们实验室通过鉴定和鉴定转移来源(ORIT)和移动酶(MOB)蛋白，以及启动和转移淋球菌7.4和5.6kb R-质粒所需的遗传因素，在了解淋球菌R-质粒转移的分子机制方面取得了很大进展。这些ORits既可以被同一β-内酰胺酶质粒编码的MOB蛋白识别，也可以被来自自传递质粒的转运体(TrA)的MOB蛋白识别。5.2kb的R-质粒是7.4kb的R-质粒的ORIT/MOB缺失衍生物，其在淋球菌中的动员机制尚不清楚。我们发现，从圣胡安分离到的5.2kb的R-质粒pSJ5.2可以在肠杆菌R64、N3和41kb的tet(M)接合质粒的帮助下通过融合(共整合)被动员到大肠杆菌中。本项目的目的是确定淋球菌中41kb的tet(M)接合质粒对pSJ5.2进行接合转移的分子机制。这项研究的中心假设是pSJ5.2可以在淋球菌中动员，因为它可以利用重组机制导致与41kb的tet(M)连接质粒共整合。将pSJ5.2从交配试验中产生的tet(M)：：pSJ5.2共整合中回收出来，对其两侧的连接点进行测序，整合并阐明重组的中间产物(S)。与转移相关的其他功能ORIT和顺式作用位点将通过体内径流DNA实验、含有pSJ5.2和tet(M)片段的质粒构建体的合成、在大肠杆菌动员实验期间提供的来自反式tet(M)的反式蛋白的互补分析、DNA测序以及使用GenBank数据库的比较分析来确定。将首次在tet(M)质粒中确定TrA装置和其他与转移相关的DNA基因座。这一研究结果将有助于设计创新的方法来控制耐药质粒在细菌中的水平传播。 公共卫生相关性：我们项目的主要目标是了解不同病原菌之间耐药质粒横向转移所涉及的动员的分子机制。 本课题组首次发现淋病奈瑟氏菌的5.2kb的β-内酰胺酶质粒是通过与几个大肠杆菌的接合质粒和淋球菌的tet(M)接合质粒共整合而被动员起来的。这项研究将有助于设计创新的方法来控制耐药质粒在细菌中的水平传播。

DESCRIPTION (provided by applicant): The emergence of strains of Neisseria gonorrhoeae that express clinically significant levels of resistance to penicillin and tetracycline is a global problem. Three beta-lactamase-encoding (R) plasmids of 7.4 kb, 5.6 kb and 5.2 kb can be mobilized from the pathogenic Neisseria to commensal Neisseria and the flora from the urogenital and extragenital sites of infection with the help of the co-resident 41 kb tetracycline- resistance, tet(M) self-transmissible plasmid. In recent years, considerable advances have been made in our laboratory in understanding the molecular machinery of transfer of the gonococcal R-plasmids through the identification and characterization of the origin of transfer (oriT) and mobilase (mob) proteins; the genetic factors required for the initiation and transfer of the gonococcal 7.4 and 5.6 kb R-plasmids. These oriTs can be recognized either by mob proteins coded by the same beta-lactamase plasmid or by mob proteins from the Transfer (Tra) apparatus of the self-transmissible plasmid. The 5.2 kb R- plasmid is an oriT / mob deletion derivative of the 7.4 kb R-plasmid whose mechanism of mobilization in the gonococci remain to be elucidated. We found that the 5.2 kb R- plasmid isolated in San Juan, pSJ5.2, can be mobilized to Escherichia coli by fusion (cointegration) with the help of the Enterobacterial R64, N3 and the 41 kb tet(M) conjugative plasmids. The objective of this project is to determine the molecular mechanism involved in the conjugal transfer of pSJ5.2 by the 41 kb tet(M) conjugative plasmid in the gonococcus. The central hypothesis of the study is that pSJ5.2 can be mobilized in the gonococcus because it can employ mechanisms of recombination leading to cointegration with the 41 kb tet(M) conjugative plasmid. The pSJ5.2 plasmid will be rescued from tet(M)::pSJ5.2 cointegrates generated during mating assays to sequence the junctions flanking the plasmid integrate and elucidate the intermediate(s) for the plasmid recombination. Other functional oriT's and cis-acting sites associated with transfer will be determined by in vivo runoff DNA experiments, synthesis of plasmid constructs containing fragments of pSJ5.2 and tet(M), complementation assays with Tra proteins from tet(M) provided in Trans during mobilization experiments in E. coli, DNA sequencing, and comparative analysis using GenBank databases. For the first time the Tra apparatus and other DNA loci related to transfer will be determined in the tet(M) plasmid. The result of this research will help to design innovative methods to control the horizontal transmission of resistance plasmids among bacteria. PUBLIC HEALTH RELEVANCE: The main goal of our project is to understand the molecular machinery of mobilization involved in the lateral transfer of resistance plasmid among different pathogenic bacteria. For the first time, our group showed that the 5.2 kb beta-lactamase plasmid of Neisseria gonorrhea is mobilized by cointegration with several conjugative plasmids of Escherichia coli and the tet(M) gonococcal conjugative plasmid. This research will help to design innovative methods to control the horizontal transmission of resistance plasmids among bacteria.