At the bottom of the flagellar structure, there is a basal body composed of MS and C rings [13, 14]. In flagellated bacteria, some proteins in the Fli family form the C ring, which functions as the flagellar rotor and contains the directional switching capability of the flagellar motor

[15–18]. However, a possible role for the leptospiral endoflagella in pathogenicity has never been explored. A complete set of flagella-associated genes BMN 673 mw were found in the genomic sequences of L. interrogans LCZ696 ic50 serovar Lai strain Lai and serovar Copenhageni strain Fiocruz L1-130, including four genes that encode flagellar motor switch proteins (FliG, FliM, FliN and FliY) [19, 20]. In bacteria, the flagellar motor switch proteins play a critical role in control of flagellar motor direction [14, 17, 18]. Thus far FliY has been found in some spirochetes and a few bacteria but does not exist in most bacteria [21, 22]. Particularly, FliY of Bacillus subtilis was shown to be a CheY-P-hydrolyzing protein in the chemotactic signaling cascade [22]. In addition, leptospiral FliY carries a carboxy-terminal domain of 60 amino acid residues that

is homologous to a domain of YscQ in Yersinia pestis [19, 20]. The YscQ protein was identified as a member of the flagellar associated type III secretion system (T3SS), with multiple functions such as controlling the directional this website rotation of flagella and the export of virulence factors including Yop proteins [23, 24].

The C ring of Escherichia coli does not have FliY, but its FliN has a high sequence homology with FliY of L. interrogans strain Lai [19] and FliN is an essential agent for motility and virulence protein export [25]. These data suggest that FliY of pathogenic Leptospira species may have important functions in motility and virulence. In the present study, we constructed a fliY gene Oxalosuccinic acid knock-out (fliY -) mutant of L. interrogans serovar Lai strain Lai based on homologous recombination using a suicide plasmid. To examine the possible role of FliY in pathogenesis, the mutant and wild-type strain were compared in assays of motility in liquid medium and migration on semisolid agar, adhesion to macrophages, stimulation of apoptosis in infected host cells, and lethality to guinea pigs. Results Products of fliY gene amplification and rFliY expression The amplification segments with expected size of the entire fliY gene (1065 bp) from L. interrogans serovar Lai strain Lai were obtained by PCR (Fig 1A). The cloned fliY gene had 100% nucleotide sequence identity with the reported sequences in GenBank (Accession No.: NC_004343, NC_005823) [10, 11]. The recombinant plasmid, E. coli BL21DE3pET32a-fliY , expressed rFliY under inducement of isopropyl-β-D-thiogalactopyranoside (IPTG), and the purified rFliY by Ni-NTA affinity chromatography showed a single band on a polyacrylamide gel after electrophoresis (Fig 1B).