Virulence Determinants of Borrelia burgdorferi

描述（由申请方提供）：伯氏疏螺旋体和相关螺旋体是侵袭性、非致病性病原体，可引起长期感染和与莱姆病相关的多种表现。直到最近，低传代B。不可能保留感染性的伯氏病。线性质粒Ip 25和Ip 28 -1的存在与感染性相关，最近的研究表明，Ip 25和另一种线性质粒Ip 56大大降低了疏螺旋体的转化率;自发失去Ip 25（从而失去感染性）的罕见克隆优先被穿梭载体转化。本申请中提供的初步数据证实，Ip 25中的大开放阅读框BBE 02编码具有限制性和修饰活性的酶。已经分离出BBE 02的破坏突变体，其易于用穿梭质粒转化并保留感染性。此外，我们小组最近发表的一篇文章表明，引入Ip 25基因BBE 22（pncA）可以恢复缺乏Ip 25的非感染性克隆的感染性。总之，这些发现提供了系统分析B毒力决定因素所需的遗传工具。如本提案所述，Burgdorferi。在特定目标1中，定点诱变将用于破坏被认为参与疏螺旋体生命周期的哺乳动物或蜱阶段的许多基因，包括ospAB、ospC、dbpAB、ospEF和visE。将研究这些突变对C3 H/HeN小鼠和蜱感染的影响，并通过基因互补研究证实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所需的其他基因。将利用体外转座子诱变或可替代地标记标签的诱变来随机破坏感染性的高度可转化的B中的基因。burgdorferiB 31克隆缺乏干扰转化的Ip 25和Ip 56限制修饰系统。这些方法将首次允许对莱姆病疏螺旋体的毒力决定因素进行系统的全球分析，从而深入了解这些高度侵袭性螺旋体的发病机制。

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.