Genetic Transformation of the Relapsing Fever Spirochete Borrelia hermsii: Stable Integration and Expression of Green Fluorescent Protein from Linear Plasmid 200
Genetic Transformation of the Relapsing Fever Spirochete Borrelia hermsii: Stable Integration and Expression of Green Fluorescent Protein from Linear Plasmid 200
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DOI：
10.1128/jb.05037-11
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发表时间：
2011-07-01
影响因子：
3.2
通讯作者：
Marconi, Richard T.
中科院分区：
文献类型：
--
作者：
Fine, Lindy M.;Earnhart, Christopher G.;Marconi, Richard T.
蜱传回归热（TBRF）是一种由至少15种不同的疏螺旋体引起的螺旋体疾病。在全世界流行地区，这是一个严重的人类健康问题。通过受感染的鸟虱属蜱叮咬传播给人类。在北美，与人类疾病相关的主要疏螺旋体属种是B。hermsii和B. turiclavia。由于缺乏遗传操作系统和完整的基因组序列，直接证明假定的TBRF螺旋体毒力因子在疾病过程中的作用受到阻碍。在这些领域的最新发展的扩展，在这里，我们展示了成功的一代克隆的B。hermsii YOR，其组成型产生绿色荧光蛋白（GFP）（B. hermsii YOR：：kan gfp）。该菌株通过将kan-gfp盒引入200-kb B的非编码区而产生。hermsii线性质粒lp 200。遗传操作不影响生长速率或引发天然质粒的丢失。B。hermsiiYOR：：kanGFP保留感染性并引起宿主血清转化。在体外和体内都证明了GFP的稳定生产。这项研究代表了在开发可用于研究TBRF螺旋体毒力机制的工具方面迈出的重要一步。
Tick-borne relapsing fever (TBRF) is a spirochetal disease caused by at least 15 different Borrelia species. It is a serious human health concern in regions of endemicity throughout the world. Transmission to humans occurs through the bites of infected Ornithodoros ticks. In North America, the primary Borrelia species associated with human disease are B. hermsii and B. turicatae. Direct demonstration of the role of putative TBRF spirochete virulence factors in the disease process has been hindered by the lack of a genetic manipulation system and complete genome sequences. Expanding on recent developments in these areas, here we demonstrate the successful generation of a clone of B. hermsii YOR that constitutively produces green fluorescent protein (GFP) (B. hermsii YOR::kan gfp). This strain was generated through introduction of a kan-gfp cassette into a noncoding region of the 200-kb B. hermsii linear plasmid lp200. Genetic manipulation did not affect the growth rate or trigger the loss of native plasmids. B. hermsii YOR::kan gfp retained infectivity and elicited host seroconversion. Stable production of GFP was demonstrated both in vitro and in vivo. This study represents a significant step forward in the development of tools that can be employed to study the virulence mechanisms of TBRF spirochetes.