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Updated: Feb 12, 2026
Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins
Published on: July 1, 2011
Leptospiral flagellar sheath protein FcpA interacts with FlaA2 and FlaB1 in Leptospira biflexa
Yuya Sasaki1,2, Akihiro Kawamoto3, Hajime Tahara4
1Graduate School of Bio-Applications & Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan.
Abstract:
Leptospira spp. are spirochete bacteria that possess periplasmic flagella (PFs) underneath the outer membrane; each flagellum is attached to each end of the protoplasmic cylinder. PFs of Leptospira have a coiled shape that bends the end of the cell body. However, the molecular mechanism by which multiple flagellar proteins organize to form the distinctively curled PF of Leptospira remains unclear. Here we obtained a slow-motility mutant of L. biflexa MD4-3 by random insertion mutagenesis using a Himar1 transposon. In MD4-3, the gene encoding the flagellar sheath protein, flagellar-coiling protein A (FcpA), which was recently identified in L. interrogans, was inactivated. As with L. interrogans ΔfcpA strains, the L. biflexa ΔfcpA strain lacked a distinct curvature at both ends of the cell body, and its motility was significantly reduced as compared with that of the wild-type strain. PFs isolated from the ΔfcpA strain were straight and were thinner than those isolated from the wild-type strain. Western blot analysis revealed that flagellar proteins FlaA1, FlaA2, FlaB1, and FlaB2 were expressed in the ΔfcpA strain but the flagellar proteins, except for FlaB2 were not incorporated in its PFs. Immunoprecipitation assay using anti-FcpA antiserum demonstrated that FcpA associates with FlaA2 and FlaB1. The association between FcpA and FlaA2 was also verified using pull-down assay. The regions of FlaA2 and FlaB1 interacting with FcpA were determined using a bacterial two-hybrid assay. These results suggest that FcpA together with FlaA2, produces coiling of PF of the Leptospira, and the interaction between the sheath and core filament may be mediated by FcpA and FlaB1.
Insights
Flagellar-coiling protein A (FcpA) is essential for Leptospira periplasmic flagella coiling. Inactivation of FcpA in L. biflexa disrupts flagellar structure and reduces motility, revealing FcpA
Area of Science:
- Microbiology
- Molecular Biology
- Bacterial Motility
Background:
- Leptospira spp. are spirochete bacteria characterized by periplasmic flagella (PFs) responsible for their distinctive motility.
- The precise molecular mechanisms governing the unique coiled structure of Leptospira PFs remain largely unknown.
- Periplasmic flagella are located beneath the outer membrane and attached to the protoplasmic cylinder at each cell end.
Purpose of the Study:
- To investigate the role of flagellar-coiling protein A (FcpA) in the formation and function of Leptospira periplasmic flagella.
- To elucidate the molecular interactions of FcpA with other flagellar proteins.
- To understand the contribution of FcpA to the characteristic coiled structure and motility of Leptospira.
Main Methods:
- Random insertion mutagenesis using Himar1 transposon in L. biflexa MD4-3 to generate mutants.
- Phenotypic analysis of motility and cell morphology in wild-type and mutant strains.
- Isolation and characterization of periplasmic flagella from wild-type and mutant strains.
- Western blot analysis to detect flagellar protein expression and incorporation.
- Immunoprecipitation and pull-down assays to determine protein-protein interactions.
- Bacterial two-hybrid assays to map interaction regions between proteins.
Main Results:
- A slow-motility mutant (MD4-3) was identified with an inactivated FcpA gene.
- The L. biflexa ΔfcpA strain exhibited a lack of cell body curvature and significantly reduced motility.
- Isolated PFs from the ΔfcpA strain were straight and thinner compared to wild-type PFs.
- FcpA was found to associate with flagellar proteins FlaA2 and FlaB1.
- FcpA and FlaA2 are suggested to be key players in producing PF coiling, with FcpA and FlaB1 mediating sheath-core filament interactions.
Conclusions:
- FcpA is crucial for the coiling of Leptospira periplasmic flagella and proper flagellar assembly.
- FcpA interacts with FlaA2 and FlaB1, suggesting a model where FcpA, FlaA2, and FlaB1 orchestrate the unique coiled structure of PFs.
- Understanding FcpA's function provides insights into the molecular basis of spirochete motility and flagellar organization.
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