The Lyme disease spirochete, Borrelia burgdorferi, exists in a zoonotic cycle involving an arthropod tick and mammalian host. Dissemination of the organism within and between these hosts depends upon the spirochete's ability to traverse through complex tissues. Additionally, the spirochete outruns the host immune cells while migrating through the dermis, suggesting the importance of B. burgdorferi motility in evading host clearance. B. burgdorferi's periplasmic flagellar filaments are composed primarily of a major protein, FlaB, and minor protein, FlaA. By constructing a flaB mutant that is nonmotile, we investigated for the first time the absolute requirement for motility in the mouse-tick life cycle of B. burgdorferi. We found that whereas wildtype cells are motile and have a flat-wave morphology, mutant cells were nonmotile and rod shaped. These mutants were unable to establish infection in C3H/HeN mice via either needle injection or tick bite. In addition, these mutants had decreased viability in fed ticks. Our studies provide substantial evidence that the periplasmic flagella, and consequently motility, are critical not only for optimal survival in ticks but also for infection of the mammalian host by the arthropod tick vector.

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SUMMARY Borrelia burgdorferi, the causative agent of Lyme disease, is a highly motile spirochete, and motility, which is provided by its periplasmic flagella, is critical for every part of the spirochete’s enzootic life cycle. Unlike externally flagellated bacteria, spirochetes possess a unique periplasmic flagellar structure called the collar. This spirochete-specific novel component is linked to the flagellar basal body; however, nothing is known about the proteins encoding the collar or their function in any spirochete. To identify a collar protein and determine its function, we employed a comprehensive strategy that included genetic, biochemical, and microscopic analyses. We found that BB0286 (FlbB) is a novel flagellar motor protein, which is located around the flagellar basal body. Deletion of bb0286 has a profound effect on collar formation, assembly of other flagellar structures, morphology, and motility of the spirochete. Orientation of the flagella toward the cell body is critical for determination of wild-type spirochete’s wave-like morphology and motility. Here, we provide the first evidence that FlbB is a key determinant of normal orientation of the flagella and collar assembly.

c Borrelia burgdorferi must migrate within and between its arthropod and mammalian hosts in order to complete its natural enzootic cycle. During tick feeding, the spirochete transmits from the tick to the host dermis, eventually colonizing and persisting within multiple, distant tissues. This dissemination modality suggests that flagellar motor rotation and, by extension, motility are crucial for infection. We recently reported that a nonmotile flaB mutant that lacks periplasmic flagella is rod shaped and unable to infect mice by needle or tick bite. However, those studies could not differentiate whether motor rotation or merely the possession of the periplasmic flagella was crucial for cellular morphology and host persistence. Here, we constructed and characterized a motB mutant that is nonmotile but retains its periplasmic flagella. Even though ⌬motB bacteria assembled flagella, part of the mutant cell is rod shaped. Cryoelectron tomography revealed that the flagellar ribbons are distorted in the mutant cells, indicating that motor rotation is essential for spirochetal flat-wave morphology. The ⌬motB cells are unable to infect mice, survive in the vector, or migrate out of the tick. Coinfection studies determined that the presence of these nonmotile ⌬motB cells has no effect on the clearance of wild-type spirochetes during murine infection and vice versa. Together, our data demonstrate that while flagellar motor rotation is necessary for spirochetal morphology and motility, the periplasmic flagella display no additional properties related to immune clearance and persistence within relevant hosts. Borrelia burgdorferi, the Lyme disease spirochete, shuttles principally between the Ixodes ticks and a vertebrate host during its natural infection cycle (1-5). In the tick, spirochetes reside primarily in the midgut until the introduction of the host blood meal. During tick feeding on a vertebrate host, spirochetes replicate, and a subset of the motile organisms cross the midgut epithelium into the hemocoel before reaching the salivary glands, where they subsequently are deposited into the host dermis (6-8). Once in the host, B. burgdorferi must migrate through the complex skin tissues, including some hematogenous dissemination, and eventually colonize distant tissues, where they often produce disease symptoms in certain hosts. Subsequently, when a naive tick feeds on an infected reservoir host, spirochetes in the skin migrate into the arthropod to complete the enzootic cycle (3, 4, 9). During the spirochete's migration within and between the hosts, periplasmic flagellar motor rotation and, by extension, motility are thought to be crucial for infection (10, 11). Indeed, nonmotile, reduced-motility, or nonchemotactic mutant spirochetes were found to be attenuated in mouse infection (12)(13)(14). Additionally, our data indicate that B. burgdorferi remains constantly motile in the mouse dermis, even 2 years after needle inoculation (P. Sekar, R. M. Wooten, and M. A. Motaleb, unpublished results). In fact, this Lyme dise...