Control of Flagellar Filament Length by FlaG in Polarly-Flagellated Bacterial Pathogens

项目摘要 细菌产生各种大分子表面细胞器，这些细胞器在不同的过程中发挥作用。对许多人来说 细胞器，还不清楚细菌是如何调节它们的尺寸的，甚至不知道是否存在特定的机制来 控制细胞器的大小或长度。鞭毛是促进游泳运动的表面细胞器。 以及病原体迁移到宿主的理想生态位以启动感染和疾病的发病机制。二 在鞭毛的生物发生中发现了调节周质长度的特定机制 吊杆和表面钩。然而，目前的一种假说表明，细菌没有特定的和活跃的 调节细胞外鞭毛丝长度的机制，它起着推进器的作用 鞭毛运动，在沙门氏菌等富含模式细菌的表面延伸约10微米 和E.Coli.挑战这一假说的是观察到许多极鞭毛的细菌病原体， 如空肠弯曲杆菌、霍乱弧菌和铜绿假单胞菌等产生鞭毛细丝 ~70%，比富营养化细菌短。此外，删除保存在这些文件中的标志 病原体和许多其他极生鞭毛虫，但在周围生物体中不存在，导致鞭毛细丝 延伸到WT细胞长度的两倍。此外，空肠弯曲菌DflG产生细长的鞭毛 对于天然禽类宿主的盲肠定植，细丝显示出28倍的缺陷，这表明适当的 调节鞭毛长度对于寄主的最佳定殖是必要的。总而言之，这些发现 表明极地鞭毛虫中标志参与了一种特定和活跃的机制来控制细胞外 鞭毛细丝的长度，对一些病原体定植寄主很重要，但分子 FLAG如何控制灯丝长度的机制尚不清楚。在这项提案中，我们将使用空肠弯曲菌作为模型 极地鞭毛虫系统，以了解细菌如何使用FLAG来控制鞭毛的长度 停止细丝聚合，这是一种在很远的距离(~2-3细胞体长度)发生的事件 从细菌表面。我们将测试两个不相互排斥的假设，即FLAG控制鞭毛 花丝长度受以下机制影响：1)旗帜干扰花丝形成之间的相互作用 鞭毛蛋白亚基和Flis伴侣减少鞭毛蛋白的分泌；和/或2)作为鞭毛蛋白的FLAG 模仿以阻止鞭毛蛋白聚合成鞭毛顶端的细丝。我们还将调查 为什么空肠弯曲杆菌DflG产生细长的鞭毛纤维会降低宿主的定殖力 从不同的生理角度分析长丝运动对运动效率和速度的影响 寄主相互作用所需的相关黏度和通过鞭毛分泌的蛋白质。结果 这项研究将为细菌如何控制大分子的维度提供新的知识 表面细胞器，以维持宿主相互作用的适当功能，并在疾病发病机制中采取步骤。

Project Summary Bacteria produce various macromolecular surface organelles that function in different processes. For many organelles, it is unclear how bacteria regulate their dimensions or even if a specific mechanism exists to govern the size or length of the organelle. The flagellum is a surface organelle that facilitates swimming motility and migration of pathogens to ideal niches in hosts to initiate infection and pathogenesis of disease. Two specific mechanisms have been discovered in flagellar biogenesis that regulate the length of the periplasmic rod and surface hook. However, a current hypothesis states that bacteria do not have a specific and active mechanism to regulate the length of the extracellular flagellar filament, which functions as the propeller of the flagellar motor and extends ~10 µm on the surface of model peritrichous bacteria such as Salmonella species and E. coli. Challenging this hypothesis are observations that many polarly-flagellated bacterial pathogens, such as Campylobacter jejuni, Vibrio cholerae, and Pseudomonas aeruginosa, produce flagellar filaments ~70% shorter than those of peritrichous bacteria. Furthermore, deletion of flaG that is conserved in these pathogens and many other polar flagellates but absent in peritrichous organisms, caused flagellar filaments to extend up to twice the length as those of WT cells. In addition, C. jejuni DflaG producing elongated flagellar filaments displayed a 28-fold defect for cecal colonization of the natural avian host, suggesting that properly regulating flagellar filament length is necessary for optimal colonization of a host. Together, these findings indicate that FlaG in polar flagellates is involved in a specific and active mechanism to control the extracellular length of the flagellar filament and is important for some pathogens to colonize hosts, but a molecular mechanism for how FlaG controls filament length is unknown. In this proposal, we will use C. jejuni as a model system for polar flagellates to understand how a bacterium employs FlaG to control the length of the flagellar filament by halting filament polymerization, an event that occurs at a large distance (~2-3 cell body lengths) from the bacterial surface. We will test two non-mutually exclusive hypotheses that FlaG controls flagellar filament length by the following mechanisms: 1) FlaG interfering with interactions between the filament-building flagellin subunits and the FliS chaperone to reduce flagellin secretion; and/or 2) FlaG functioning as a flagellin mimic to block flagellins from polymerizing into the filament at the tip of the flagellum. We will also investigate why production of elongated flagellar filaments by C. jejuni DflaG reduces the ability to colonize a host by analyzing the effects of elongated filaments on motility efficiency and velocity through different physiologically- relevant viscosities and secretion of proteins through the flagellum required for host interactions. Outcomes from this study will provide new knowledge regarding how bacteria control dimensions of macromolecular surface organelles to maintain proper function for host interactions and steps in pathogenesis of disease.