In Vivo Expression Technology Identifies a Novel Virulence Factor Critical for Borrelia burgdorferi Persistence in Mice

Analysis of the transcriptome of Borrelia burgdorferi, the causative agent of Lyme disease, during infection has proven difficult due to the low spirochete loads in the mammalian tissues. To overcome this challenge, we have developed an In Vivo Expression Technology (IVET) system for identification of B. burgdorferi genes expressed during an active murine infection. Spirochetes lacking linear plasmid (lp) 25 are non-infectious yet highly transformable. Mouse infection can be restored to these spirochetes by expression of the essential lp25-encoded pncA gene alone. Therefore, this IVET-based approach selects for in vivo-expressed promoters that drive expression of pncA resulting in the recovery of infectious spirochetes lacking lp25 following a three week infection in mice. Screening of approximately 15,000 clones in mice identified 289 unique in vivo-expressed DNA fragments from across all 22 replicons of the B. burgdorferi B31 genome. The in vivo-expressed candidate genes putatively encode proteins in various functional categories including antigenicity, metabolism, motility, nutrient transport and unknown functions. Candidate gene bbk46 on essential virulence plasmid lp36 was found to be highly induced in vivo and to be RpoS-independent. Immunocompetent mice inoculated with spirochetes lacking bbk46 seroconverted but no spirochetes were recovered from mouse tissues three weeks post inoculation. However, the bbk46 gene was not required for B. burgdorferi infection of immunodeficient mice. Therefore, through an initial IVET screen in B. burgdorferi we have identified a novel in vivo-induced virulence factor critical for the ability of the spirochete to evade the humoral immune response and persistently infect mice.由于哺乳动物组织内伯氏疏螺旋体（Borrelia burgdorferi）载量极低，对作为莱姆病致病原的该病原菌感染过程中的转录组进行分析极具挑战。为攻克这一技术难题，我们开发了一套体内表达技术（In Vivo Expression Technology, IVET）系统，用于鉴定活性鼠感染过程中伯氏疏螺旋体的表达基因。 缺失线性质粒（linear plasmid, lp）25的疏螺旋体丧失感染能力但转化效率极高，仅需表达lp25编码的必需基因pncA即可恢复这些螺旋体的小鼠感染能力。因此，该基于IVET的筛选策略可选择体内表达的启动子，驱动pncA的表达，进而在小鼠感染三周后回收缺失lp25的感染性疏螺旋体。 我们在小鼠中对约15000个克隆进行筛选，共从伯氏疏螺旋体B31基因组的全部22个复制子中鉴定出289个独特的体内表达DNA片段。这些体内表达的候选基因推定编码多种功能类别的蛋白质，涵盖抗原相关蛋白、代谢相关蛋白、运动相关蛋白、营养转运蛋白以及功能未知蛋白。 研究发现，必需毒力质粒lp36上的候选基因bbk46在体内被高度诱导表达，且该诱导过程不依赖RpoS。免疫健全小鼠接种缺失bbk46的疏螺旋体后可产生血清转换，但接种三周后无法从小鼠组织中分离到疏螺旋体。然而，bbk46基因对于伯氏疏螺旋体感染免疫缺陷小鼠并非必需。 因此，通过本次伯氏疏螺旋体的初始IVET筛选，我们鉴定出一种新型体内诱导毒力因子，其对于疏螺旋体逃逸体液免疫应答并在小鼠体内持续感染至关重要。