Leptospiral flagellar sheath protein FcpA interacts with FlaA2 and FlaB1 in <i>Leptospira biflexa</i>

<div><p><i>Leptospira</i> 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 <i>Leptospira</i> 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 <i>Leptospira</i> remains unclear. Here we obtained a slow-motility mutant of <i>L</i>. <i>biflexa</i> MD4-3 by random insertion mutagenesis using a <i>Himar1</i> transposon. In MD4-3, the gene encoding the flagellar sheath protein, flagellar-coiling protein A (FcpA), which was recently identified in <i>L</i>. <i>interrogans</i>, was inactivated. As with <i>L</i>. <i>interrogans</i> Δ<i>fcpA</i> strains, the <i>L</i>. <i>biflexa</i> Δ<i>fcpA</i> 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 Δ<i>fcpA</i> 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 Δ<i>fcpA</i> 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 <i>Leptospira</i>, and the interaction between the sheath and core filament may be mediated by FcpA and FlaB1.</p>