The heterogeneous motility of the Lyme disease spirochete in gelatin mimics dissemination through tissue

Abstract

The Lyme disease spirochete Borrelia burgdorferi exists in nature in an enzootic cycle that involves the arthropod vector Ixodes scapularis and mammalian reservoirs. To disseminate within and between these hosts, spirochetes must migrate through complex, polymeric environments such as the basement membrane of the tick midgut and the dermis of the mammal. To date, most research on the motility of B. burgdorferi has been done in media that do not resemble the tissue milieus that B. burgdorferi encounter in vivo. Here we show that the motility of Borrelia in gelatin matrices in vitro resembles the pathogen's movements in the chronically infected mouse dermis imaged by intravital microscopy. More specifically, B. burgdorferi motility in mouse dermis and gelatin is heterogeneous, with the bacteria transitioning between at least three different motility states that depend on transient adhesions to the matrix. We also show that B. burgdorferi is able to penetrate matrices with pore sizes much smaller than the diameter of the bacterium. We find a complex relationship between the swimming behavior of B. burgdorferi and the rheological properties of the gelatin, which cannot be accounted for by recent theoretical predictions for microorganism swimming in gels. Our results also emphasize the importance of considering borrelial adhesion as a dynamic rather than a static process.

Fig. 1.

The motility patterns of Lyme disease spirochetes (Bb914) in gelatin resemble those observed in mouse skin. (A) Time course of spirochetal (green) motility in the dermis of a tick-inoculated mouse (Movie S1). Lunging (L) and translocating (T) bacteria are shown. The dermal collagen fibers fluoresce blue due to second-harmonic generation. (B) Time course of Bb motility in 3% gelatin (Movie S2). All four motility states are shown: nonmotile (N), wriggling (W), lunging (L), and translocating (T). (Scale bars: 10 μm.)

Fig. 2.

(A) High-speed image acquisition reveals four distinguishable phases of spirochetal motility in gelatin (Movie S6): nonmotile (N), wriggling bacteria (W) that undulate without locomoting, lunging bacteria (L) that undulate and deform but maintain at least one fixed point along the cell length, and bacteria that translocate through the gelatin matrix (T). (Scale bar, 10 μm.) (B) The fraction of bacteria in each state depends on gelatin concentration. (C) The speed of translocating bacteria decreases with gelatin concentration and is always less than that in liquid medium. (D) The undulation frequency for translocating and wriggling bacteria as a function of gelatin concentration. The wriggling frequency always exceeds the translocating frequency and shows a peak in 3% gelatin solutions. (E) In all concentrations of gelatin and BSK, spirochete wavelengths were measured to be ∼3.3 μm; (F) the amplitude is also independent of gelatin concentration.

Fig. 3.

(A) Time-lapse images of Bb in a 3% gelatin matrix show that adhesions to the matrix are transient (Movie S7). Individual spirochetes are numbered. Most are translocating (spirochetes 1, 3, 4, 6–10). Nonmotile (2) and wriggling (5) spirochetes also are shown. (Scale bar, 10 μm.) (B) Characteristic MSD vs. time plots for the 3–5% gelatin matrices. In all concentrations, the motility over short time intervals is subdiffusive, with a transition to superdiffusive behavior (slope between 1 and 2) that occurs at ∼100 s. (C) Histogram of the slopes of the MSD for the different gelatin concentrations. The 5% matrices show the greatest persistence of motion (i.e., the largest fraction with slopes >1). Black lines show slopes of 1 and 2. (D) The number of bacteria tracked for at least a time Δt decays approximately exponentially. (Inset) Decay time as a function of 1/Φ_tV, which is linear with slope of 114 μm. (B–D) Plot coloring shows 3% (green), 4% (red), and 5% (blue) gelatin.

Fig. 4.

A simple kinetic model determines the effective rate constants for binding and unbinding to the matrix as a function of gelatin concentration. Transitions occur between the nonmotile and wriggling states, between the wriggling and lunging states, and between the lunging and translocating states. If these transitions are in equilibrium, then the equilibrium rate constants are equal to the ratios of the fraction of bacteria in the states; (e.g., the equilibrium constant for the transition between nonmotile and wriggling, K_nw, is equal to the ratio of the fractions of nonmotile bacteria to lunging bacteria). (B) The equilibrium constants as a function of gelatin concentration. The transition from wriggling to nonmotile, K_nw, increases approximately linearly with gelatin concentration. (C) K_wl and K_lt are also proportional to gelatin concentration (φ) and also decrease with the force from the bacterium (which is proportional to the square of the effective viscosity η times the undulation frequency, f). (D) The inverse of the frequency of wrigglers (red) and translocators is proportional to the viscosity. The parameter α = 1 for wrigglers and 7/2 for translocators (SI Text S4). The black line shows the best linear fit to the data.