Flagellar export apparatus protein, FliH, is involved in post-transcriptional regulation of FlaB, motility and virulence of the relapsing fever spirochete Borrelia hermsii. Borrelia hermsii

Spirochetes are long, thin, motile, helical or flat wave bacteria that are unique among the prokaryotes by having flagella or axial filaments confined to an internal periplasmic space. These flagella are complex organelles that can play major roles in bacterial pathogenicity and are used as propellers allowing bacteria to move through liquids, viscous environments or along surfaces. While most bacteria species use transcriptional regulatory cascades to regulate the synthesis and assembly of their flagella, spirochetes employ an unusual post-transcriptional mechanism. Using next generation sequencing, we characterized a natural mutant of the relapsing fever spirochete Borrelia hermsii lacking the flagellar export apparatus protein FliH. The mutant was non-motile, uncoiled and straight compared to the wild-type spirochetes. The B. hermsii fliH mutant produced only a residual amount of the major flagellin protein FlaB, which was correlated with a reduced number of periplasmic flagella. The amounts of flaB transcript were comparable in the fliH mutant and the wild-type strain. The synthesis of FlaB, the motility and the infectivity of the fliH mutant were rescued by trans-complementation. This report reveals a new function for FliH, and we propose that this regulator of the flagellar export apparatus influences the post-transcriptional processing of the flagella, motility and virulence of the relapsing fever spirochete Borrelia hermsii. Spirochetes are bacteria characterized in part by rotating periplasmic flagella that impart their flat-wave morphology and motility. While other bacteria rely on a transcriptional cascade to regulate the expression of motility genes, spirochetes employ posttranscriptional mechanism(s) that are only partially known. In the present study, we characterize a non-motile mutant of the relapsing fever spirochete Borrelia hermsii that was straight, non-motile and deficient in flagella. We used next generation DNA sequencing of the mutant’s genome, which when compared to the wild-type genome identified a 142 bp deletion in the chromosomal gene encoding the flagellar export apparatus protein FliH. Immunoblot and transcriptome analyses showed that the mutant phenotype was linked to the posttranscriptional deficiency in the synthesis of the major flagellar filament core protein FlaB. The turnover of the residual pool of FlaB produced by the fliH mutant was comparable to the wild-type spirochete while the amount of FlaA was similar to the wild-type level. The non-motile mutant was not infectious in mice and its inoculation did not induce an antibody response. Trans-complementation of the mutant with an intact fliH gene restored the synthesis of FlaB, a normal morphology, motility and infectivity in mice. Therefore, we propose that the flagellar export apparatus protein regulates motility of B. hermsii at the posttranscriptional level by influencing the synthesis of FlaB. Overall design: Borrelia hermsii wild type vs. motility mutant

螺旋体（Spirochetes）是一类细长、具运动性、呈螺旋状或平波状的细菌，区别于其他原核生物（prokaryotes）的独特之处在于其鞭毛或轴丝被限制在内部周质空间（periplasmic space）内。这些鞭毛是复杂的细胞器，在细菌致病性中发挥关键作用，可作为推进器帮助细菌在液体、粘性环境或沿表面移动。多数细菌利用转录调控级联反应调控鞭毛的合成与组装，而螺旋体则采用一种非常规的转录后调控机制。本研究借助下一代测序技术，对缺失鞭毛输出装置蛋白FliH的回归热致病菌赫氏疏螺旋体（Borrelia hermsii）自然突变株进行了表征。与野生型螺旋体相比，该突变株丧失运动能力，菌体呈直杆状且失去螺旋结构。赫氏疏螺旋体fliH突变株仅能产生微量主要鞭毛丝核心蛋白FlaB，这与周质鞭毛数量减少相关。fliH突变株与野生型菌株的flaB转录本水平相当。通过反式互补（trans-complementation）技术，可恢复fliH突变株的FlaB合成、运动能力与感染性。本研究揭示了FliH的全新功能，我们推测该鞭毛输出装置调控蛋白可通过影响转录后加工过程，调控赫氏疏螺旋体的鞭毛合成、运动能力与毒力。螺旋体的特征之一是拥有旋转的周质鞭毛，这赋予其平波状形态与运动能力。其他细菌依赖转录级联反应调控运动基因的表达，而螺旋体所采用的转录后调控机制目前仍未完全阐明。本研究对一株直杆状、丧失运动能力且鞭毛缺陷的回归热赫氏疏螺旋体非运动突变株进行了表征。我们对该突变株的基因组进行了下一代DNA测序，并与野生型基因组比对，发现其编码鞭毛输出装置蛋白FliH的染色体基因存在一段142 bp的缺失。免疫印迹与转录组分析表明，该突变株的表型与主要鞭毛丝核心蛋白FlaB合成的转录后缺陷相关。fliH突变株产生的微量FlaB的周转速率与野生型螺旋体相当，而FlaA的含量与野生型水平一致。该非运动突变株无法在小鼠体内造成感染，且接种小鼠不会诱导抗体应答。通过向突变株导入完整的fliH基因进行反式互补，可恢复其FlaB合成能力、正常形态、运动能力与小鼠感染性。因此，我们认为鞭毛输出装置蛋白FliH可通过影响FlaB的合成，在转录后水平调控赫氏疏螺旋体的运动能力。实验整体设计：赫氏疏螺旋体野生型菌株 vs. 运动缺陷突变株