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The mechanism of two-phase motility in the spirochete Leptospira: Swimming and crawling

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Science Advances  30 May 2018:
Vol. 4, no. 5, eaar7975
DOI: 10.1126/sciadv.aar7975
  • Fig. 1 Cell structure and swimming motility of Leptospira.

    (A) Schematic diagram of Leptospira cell structure. The thin black arrow indicates the swimming direction. A cross section of the cell body is depicted below the dashed line: outer membrane (OM), PF, peptidoglycan layer (PG), inner membrane (IM), and cytoplasm (CP). The rotational direction is viewed from the Hook-end to the Spiral-end, as indicated by the thick black arrow. Blue and white arrows indicate the rotational directions of PF and the PC, respectively. At the ends of the cell body surrounded by a dashed square, the flagellar motor of each PF is embedded into PG and IM. (B) Kymograph of a cell swimming in motility medium. Yellow lines indicate cell movement. The area surrounded by a red square is enlarged on the right, and dotted lines indicate the apparent movements of the PC helix.

  • Fig. 2 Crawling motility of Leptospira.

    (A) Kymograph of a cell crawling on a glass surface. The PC appears to be fixed (red dotted lines), indicating movement without slip. (B) Effect of an Ab-LPS on crawling motility; 216 and 214 cells were measured on noncoated (−Ab-LPS) or Ab-LPS–coated (+Ab-LPS) glasses, respectively; open bars indicate cells adhering to glass surfaces without translation. The box-and-whisker plot shows the 25th (the bottom line of the box), 50th (middle), and 75th (bottom) percentiles and the minimum and maximum values (whiskers) of crawling speeds obtained from individual cells; statistical analysis was performed by Mann-Whitney U test (*P < 0.01). The schematics represent a hypothetical explanation of Ab-LPS effects. Various external molecules are shown by different symbols; black dots with a bar indicate LPS; red circles with a bar indicate Ab-LPS; and thin and thick arrows indicate slow and fast crawling, respectively (the details are described in the main text). (C) Wild-type (WT) and ΔfcpA cells observed by dark-field microscopy (top) and pairwise plots of crawling speeds and cell rotation rates (bottom). Red lines in the pairwise plots are regression lines fitted to PC data points. Correlation coefficients (R) for PC of WT and ΔfcpA are 0.98 and 0.97, respectively. R between Spiral-end speed and crawling speed in WT is 0.22 (no regression line shown). (D) Effects of CCCP on the cells attached to a glass surface (movie S5). Top: Results of single-cell tracking. Microscopic images captured at 0.1-s intervals were decimated to 5-s intervals and then integrated. Colors indicate time courses in the order of red, green, blue, purple, and yellow. The cell stopped crawling with the addition of 5 μM CCCP (middle), and therefore, all of the colored cell images were superposed. Bottom: Crawling speed of the cell shown in the top panel. Crawling speeds determined at 0.1-s intervals are shown in gray, and data of a 10–data-point moving average are shown in red.

  • Fig. 3 Fluorescent observation of outer membrane dynamics.

    (A) Crawling cell labeled with Cy3-NHS. In a kymograph, the yellow solid line indicates cell migration (top). In an enlarged kymograph (bottom), orange and yellow dashed lines indicate translational movements of fluorescent spots and slip-less crawling of the cell, respectively. (B) A montage shows an example trajectory of a fluorescent spot. (C) The rotation rates of the PC and Cy3 fluorescent spots determined from speed versus time traces for individual cells are shown. The rotations of PC and Cy3 fluorescent spots were simultaneously analyzed at several different time periods for each trace, and data obtained from two different cells are shown by black dots and triangles (total of 11 data points). A gray line with a slope of 1 is shown. (D) Schematic explanation of fluorescent dye movement attached to a cell crawling on the surface with rotation of a helical cell body.

  • Fig. 4 Observation of beads attached to the cell body via an antibody.

    (A) The upper schematic represents the bead assay. A polystyrene bead with a diameter of 200 nm coated with Ab-LPS (red circle) was attached to the cell (black wavy line). Middle: Superposition of sequential video images. The original movie recorded at 4-ms intervals (movie S7) was decimated to 80-ms intervals, and 10 sequential images were then superposed. The red arrow indicates the trajectory and direction of bead movement. The cell ends were arbitrarily designated as End1 and End2. Bottom: Time traces of x and y positions of the bead. (B) Rotation of the bead based on the focal plane determined as shown in (C) and a change in the size of the bead image shown in (D). Bead rotation is depicted by sequential diagrams below the montage, which are observed from the direction indicated by the black arrow. (C) Enlarged image of the part indicated by the yellow square in (B). The focal plane of observation can be deduced from the visualized helix angle of PC (yellow arrows), as schematically explained. (D) Change in the area of the bead image attributed to halation caused by a z axis displacement of the bead. The x axis indicates image numbers shown in (B). (E) Time traces of End1, End2, and PC rotations in the cell shown in (A). Raw data (gray) were smoothed by moving the average (black). End1 displayed the Hook-shape (H) during recording, whereas End2 changed the shape from the Hook- to Spiral-shape (S) at around 0.2 s. PC rotation was not measured from 0.2 to 0.4 s (indicated by N.D.) due to defocusing. (F) Attachment of an aggregated bead to the cell surface. A kymograph (right) shows an apparent PC stillness (red dashed lines), that is, movement without slip.

  • Fig. 5 Model of the motility form transition in Leptospira.

    (A) Swimming is caused by CCW gyration of the Spiral-end and CW rotation of the PC, and adhesive cell-surface molecules (black dots with a bar) rotate with the cell body. Rotations of adhesive molecules are shown by red and purple symbols on the right. (B) When attaching to the surface via mobile adhesins, the cell moves relative to the anchoring points with PC rotation. In the left cartoon, first, the red adhesin attaches to the surface, and then, the purple one participates in the anchoring.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/5/eaar7975/DC1

    fig. S1. Swimming motility of Leptospira.

    fig. S2. Example kymographs of Leptospira cells crawling without slip.

    fig. S3. WT and “unbent” mutant (ΔfcpA) strains of Leptospira.

    fig. S4. Example kymographs of ΔfcpA mutant cells crawling without slip.

    fig. S5. Movement of a microbead attached to the cell surface.

    fig. S6. Movement of a microbead attached to the cell surface.

    fig. S7. Kymographs of Leptospira cells labeled with Ab-LPS–coated beads or noncoated beads.

    fig. S8. Relationship between swimming speed and bead movement.

    fig. S9. Crawling motility of the pathogenic Leptospira.

    movie S1. A L. biflexa cell swimming in a 10% Ficoll solution.

    movie S2. WT L. biflexa cells crawling on a glass surface.

    movie S3. Effect of an anti-LPS antibody on crawling.

    movie S4. ΔfcpA mutant cells crawling on a glass surface.

    movie S5. Effect of CCCP on Leptospira crawling.

    movie S6. Fluorescent observation of the Leptospira outer membrane using Cy3-NHS.

    movie S7. Movement of a small bead aggregate on the Leptospira cell body.

    movie S8. Movement of a single 200-nm bead on the Leptospira cell body.

    movie S9. Movement of a large bead aggregate on the Leptospira cell body.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Swimming motility of Leptospira.
    • fig. S2. Example kymographs of Leptospira cells crawling without slip.
    • fig. S3. WT and “unbent” mutant (ΔfcpA) strains of Leptospira.
    • fig. S4. Example kymographs of ΔfcpA mutant cells crawling without slip.
    • fig. S5. Movement of a microbead attached to the cell surface.
    • fig. S6. Movement of a microbead attached to the cell surface.
    • fig. S7. Kymographs of Leptospira cells labeled with Ab-LPS–coated beads or noncoated beads.
    • fig. S8. Relationship between swimming speed and bead movement.
    • fig. S9. Crawling motility of the pathogenic Leptospira.

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.avi format). A L. biflexa cell swimming in a 10% Ficoll solution.
    • movie S2 (.avi format). WT L. biflexa cells crawling on a glass surface.
    • movie S3 (.avi format). Effect of an anti-LPS antibody on crawling.
    • movie S4 (.avi format). ΔfcpA mutant cells crawling on a glass surface.
    • movie S5 (.avi format). Effect of CCCP on Leptospira crawling.
    • movie S6 (.avi format). Fluorescent observation of the Leptospira outer membrane using Cy3-NHS.
    • movie S7 (.avi format). Movement of a small bead aggregate on the Leptospira cell body.
    • movie S8 (.avi format). Movement of a single 200-nm bead on the Leptospira cell body.
    • movie S9 (.avi format). Movement of a large bead aggregate on the Leptospira cell body.

    Files in this Data Supplement:

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