PMC

The figure is a schematic representation of the fcpA loci in the WT Fiocruz L1-130 strain and Fiocruz L1-130 fcpA^- strain. The expanded circle shows the site of the frameshift mutation, which occurred in the fcpA gene of the motility-deficient Fiocruz LV2756 strain.

PF from wt Fiocruz L1-130 [bar=200nm] and Fiocruz L1-130 fcpA^- [bar=500nm] strains were purified and labeled with antibodies against FcpA (α-FcpA) and FlaB1 (α-FlaB1). Anti-rabbit IgG anti-sera conjugated with 5nm gold nanoparticles were used to detect bound antibodies. PF were visualized using 2% PTA negative staining.

(A) Coomassie-stained SDS-PAGE of purified flagella from motile Fiocruz LV2756 [lane 1], motility-deficient Fiocruz LV2756 [lane 2], Fiocruz LV2756 fcpA^-/+ [lane 3], WT Fiocruz L1-130 [lane 4], Fiocruz L1-130 fcpA^- [lane 5], and Fiocruz L1-130 fcpA^-/+ [lane 6] strains. In Fig. 3A, arrows indicate the position of FcpA and FlaB1 proteins, which were identified by mass spectroscopy. (B) Immunoblotting analysis of purified flagella incubated with a mixture of polyclonal antibodies against FcpA and control antibodies against flagella-associated proteins, FlaA1 and FlaA2. Arrows indicate the positions of these three proteins in Fig. 3B.

(A) Motility assay for which 10⁵ bacteria were inoculated on 0.5% agarose plates [each square, 1 cm²] and incubated for 10 days at 29°C; (B) Dark-field microscopy [bar=10μm]; (C) Scanning electron microscopy [bar=2μm]; and (D) Transmission electron microscopy of negatively stained, purified periplasmic flagella [bar=100nm]. Motility-deficient Fiocruz LV2756 strain was isolated from a clinical isolate. Fiocruz L1-130 fcpA^- strain was generated by allelic exchange and complemented strains Fiocruz LV2756 fcpA^-/+ and Fiocruz L1-130 fcpA^-/+ were obtained by reintroducing the fcpA gene. See also .

Hamsters were inoculated with 10⁸ bacteria of the motile (white columns) and motility-deficient (gray columns) strains by intraperitoneal (A and B) or conjunctival (C) routes. Quantitative PCR analysis was performed on blood and tissues harvested one (A) and four (B) days after intraperitoneal inoculation and 7 days (C) after conjunctival inoculation. Geometric mean values and standard deviations are shown for genome equivalents of leptospiral DNA per ml of blood and gram of tissue, which were obtained in two independent experiments. Leptospiral DNA load for the motile strain was significantly (p<0.0001) higher than the motility-deficient strain for all tissues and time points. ND, not detected. See also .

Cryo-electron tomography was performed for Fiocruz L1-130 WT (A), Fiocruz L1-130 fcpA^- (B) and Fiocruz L1-130 fcpA^-/+ (C) strains. Panels D, E and F show one slice of a tomographic reconstruction for the regions (boxes in panels A, B and C) of WT Fiocruz L1-130, Fiocruz L1-130 fcpA^- and Fiocruz L1-130 fcpA^-/+ strains, respectively. Arrows indicate the location of PF. Inserts in panel D, E and F depict averaged maps of PF segments for each of the strains. The diameter of the flagellar filament in Fiocruz L1-130 fcpA^- mutant was 15.7 nm, whereas the diameter of filaments in the WT and complemented strains was 20.5 nm. Surface renderings of the corresponding 3-D reconstructions of Fiocruz L1-130 fcpA^- (G) and Fiocruz L1-130 fcpA^-/+ (H) strains are shown, with prominent structural features including the outer membrane (OM), cytoplasmic membrane (IM) and flagellar filament. See also .