Leptospiral flagellar sheath protein FcpA interacts with FlaA2 and FlaB1 in Leptospira biflexa

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.

Spirochete Flagella and Motility

“…Deletion of flaA1 and flaA2 does not affect the synthesis of the sheath [20], whereas fcpA knockout mutants lack a sheath [19,26]. Immunoprecipitations showed the interaction of FcpA with FlaB1 and FlaA2 [19]. These results suggest that FcpA is a major sheath component and plays a central role in coiling via its interaction with the core filament.…”

“…The remaining four proteins are involved in synthesizing the sheath or in coiling the PF through core-sheath interactions; however, their roles are not fully elucidated. Deletion of flaA1 and flaA2 does not affect the synthesis of the sheath [20], whereas fcpA knockout mutants lack a sheath [19,26]. Immunoprecipitations showed the interaction of FcpA with FlaB1 and FlaA2 [19].…”

“…Another important role of the PF is to establish a wavy morphology, similar to a cytoskeleton, and the PF dependence of spirochete morphology has been observed in the periodontal disease-associated spirochetes Treponema denticola [25], B. burgdorferi [26,27], and Leptospira spp. [15,[19][20][21][22]. For example, the loss of the PF in B. burgdorferi straightens the entire cell body [26].…”

“…In Leptospira spp., PF also consists of the core and the sheath, and six proteins have been identified as PF components: FlaA1, FlaA2, FlaB1, FlaB2, FcpA, and FcpB. PFs isolated from leptospiral cells exhibit a coiled shape [15], but the core filament is straight in the absence of a sheath, indicating that the sheath is indispensable for bending the leptospiral PF [19,21]. The PF core filament of the non-pathogenic species Leptospira biflexa is formed by FlaB1 and FlaB2 [19].…”

“…PFs isolated from leptospiral cells exhibit a coiled shape [15], but the core filament is straight in the absence of a sheath, indicating that the sheath is indispensable for bending the leptospiral PF [19,21]. The PF core filament of the non-pathogenic species Leptospira biflexa is formed by FlaB1 and FlaB2 [19]. The remaining four proteins are involved in synthesizing the sheath or in coiling the PF through core-sheath interactions; however, their roles are not fully elucidated.…”