Virulence Determinants of Borrelia burgdorferi

描述(申请人提供)：伯氏疏螺旋体和相关螺旋体是侵袭性的、非产毒的病原体，会导致莱姆疏螺旋体病的长期感染和多种表现。直到最近，保持传染性的低传代伯氏杆菌的遗传操作还是不可能的。IP25和IP28-1线性质粒的存在与传染性相关，最近的研究表明，IP25和另一种线性质粒IP56极大地降低了疏螺旋体的转化率；自然失去IP25(从而具有感染性)的稀有克隆优先通过穿梭载体转化。本申请中提供的初步数据证实，IP25中的大开放阅读框BBE02编码一种具有限制和修饰活性的酶。BBE02的一个中断突变体已经被分离出来，它可以很容易地与穿梭质粒一起转化，并保持感染性。此外，我们团队最近发表的一篇文章表明，导入IP25基因BBE22(PncA)可以恢复缺乏IP25的非传染性克隆的感染性。综上所述，这些发现提供了系统分析伯氏杆菌毒力决定因素所需的遗传工具，如本提案所述。在具体目标1中，定点突变将被用来干扰一些被认为与疏螺旋体生命周期的哺乳动物或壁虱阶段有关的基因，包括SpAB、OspC、DBpAB、OspEF和vise。将研究这些突变对C3H/HEN小鼠和扁虱感染的影响，并通过基因互补研究证实其与基因功能的相关性。具体目标2的目标是确定感染周期中需要的其他基因。在体外，转座子突变或另一种签名标记突变将被用来随机破坏具有感染性的、高度转化的伯氏杆菌B31克隆中的基因，该克隆缺乏干扰转化的IP25和Ip56限制修饰系统。这些方法将首次对莱姆病螺旋体的毒力决定因素进行系统的、全球的分析，从而为深入了解这些高度侵袭性螺旋体的致病机制提供依据。

DESCRIPTION (provided by applicant): Borrelia burgdorferi and related spirochetes are invasive, nontoxigenic pathogens that cause the long-term infection and multiple manifestations associated with Lyme borreliosis. Until recently, genetic manipulations of low passage B. burgdorferi with retention of infectivity have not been possible. Presence of the linear plasmids Ip25 and Ip28-1 correlates with infectivity, and recent studies have shown that Ip25 and another linear plasmid, Ip56, greatly reduce the transformation rates of Borrelia; rare clones that have spontaneously lost Ip25 (and thus infectivity) are preferentially transformed by shuttle vectors. Preliminary data presented in this application confirms that the large open reading frame BBE02 in Ip25 encodes an enzyme with both restriction and modification activities. A disruption mutant of BBE02 has been isolated that is readily transformable with a shuttle plasmid and retains infectivity. In addition, a recent publication by our group demonstrates that introduction of the Ip25 gene BBE22 (pncA) restores infectivity to noninfectious clones lacking Ip25. Taken together, these findings provide the genetic tools required for a systematic analysis of the virulence determinants of B. burgdorferi, as described in this proposal. In Specific Aim 1, site-directed mutagenesis will be used to disrupt a number of genes thought to be involved in the mammalian or tick phases of the Borrelia life cycle, including ospAB, ospC, dbpAB, ospEF, and visE. The effects of these mutations on infection of C3H/HeN mice and ticks will be investigated, and the correlation with gene function confirmed by gene complementation studies. The goal of Specific Aim 2 is to identify additional genes that are required in the infectious cycle. In vitro transposon mutagenesis or alternatively signature-tagged mutagenesis will be utilized to randomly disrupt genes in an infectious, highly transformable B. burgdorferi B31 clone lacking the Ip25 and Ip56 restriction-modification systems that interfere with transformation. These approaches will, for the first time, permit a systematic, global analysis of virulence determinants of Lyme disease Borrelia and will thus provide insight into the mechanisms of pathogenesis of these highly invasive spirochetes.