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. 2014 Jan 29;9(1):e85763.
doi: 10.1371/journal.pone.0085763. eCollection 2014.

Disruption of TgPHIL1 Alters Specific Parameters of Toxoplasma Gondii Motility Measured in a Quantitative, Three-Dimensional Live Motility Assay

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Free PMC article

Disruption of TgPHIL1 Alters Specific Parameters of Toxoplasma Gondii Motility Measured in a Quantitative, Three-Dimensional Live Motility Assay

Jacqueline M Leung et al. PLoS One. .
Free PMC article

Abstract

T. gondii uses substrate-dependent gliding motility to invade cells of its hosts, egress from these cells at the end of its lytic cycle and disseminate through the host organism during infection. The ability of the parasite to move is therefore critical for its virulence. T. gondii engages in three distinct types of gliding motility on coated two-dimensional surfaces: twirling, circular gliding and helical gliding. We show here that motility in a three-dimensional Matrigel-based environment is strikingly different, in that all parasites move in irregular corkscrew-like trajectories. Methods developed for quantitative analysis of motility parameters along the smoothed trajectories demonstrate a complex but periodic pattern of motility with mean and maximum velocities of 0.58 ± 0.07 µm/s and 2.01 ± 0.17 µm/s, respectively. To test how a change in the parasite's crescent shape might affect trajectory parameters, we compared the motility of Δphil1 parasites, which are shorter and wider than wild type, to the corresponding parental and complemented lines. Although comparable percentages of parasites were moving for all three lines, the Δphil1 mutant exhibited significantly decreased trajectory lengths and mean and maximum velocities compared to the parental parasite line. These effects were either partially or fully restored upon complementation of the Δphil1 mutant. These results show that alterations in morphology may have a significant impact on T. gondii motility in an extracellular matrix-like environment, provide a possible explanation for the decreased fitness of Δphil1 parasites in vivo, and demonstrate the utility of the quantitative three-dimensional assay for studying parasite motility.

Conflict of interest statement

Competing Interests: We have the following interests. Gary E. Ward is a member of the Board of Directors of PLOS. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. Mark A. Rould is employed by Macromolecular Consulting LLC. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. T. gondii moves in a corkscrew-like manner in three dimensions.
(A) RH strain T. gondii tachyzoites expressing a tandem tomato fluorescence cassette (RH-OVA-tdTomato) were injected into a mouse earflap and imaged by two-photon laser scanning microscopy. A maximum intensity projection (MIP) shows parasites that move in a corkscrew-like fashion (e.g., dashed white box). Scale bar = 29 µm. See Video S1 for the corresponding movie. (A') Higher magnification view of dashed white box in (A); scale bar = 12 µm. (B) Assembly and dimensions of the imaging (“Pitta”) chamber. Coverslips were assembled using double-sided tape, and perfused with a 1∶3∶3 mixture of parasites treated with Hoechst 33342, 3D motility media and chilled Matrigel, respectively. (C) Pitta chambers were incubated at 27°C for 7 min, followed by 2 min equilibration in the preheated 35°C microscope enclosure. Fluorescent parasite nuclei were imaged for 60 s in a 402 µm×401 µm×40 µm volume (approximately 67 z-stacks, with 41 z-slices captured every 0.88 s) by time-lapse fluorescence videomicroscopy. Datasets were visualized during acquisition by generating MIPs, tracked using the Imaris software, and then analyzed using the Bugs software as indicated in the workflow. (D) A MIP showing that parasites also move in corkscrew-like trajectories in Matrigel. Scale bar = 50 µm. See Video S3 for the corresponding movie. (D') Higher magnification view of dashed white box in (D); scale bar = 10 µm. The colour scheme for all MIPs was inverted for better visualization of parasite trajectories.
Figure 2
Figure 2. Visualization and analysis of two representative 3D trajectories of parasites in Matrigel.
(A) and (B) Positional coordinates for two representative wild-type (RH) parasite trajectories are visualized as connected, discrete trackpoints (white), and overlaid with the trajectory after smoothing (green). Scale bar = 7 µm. (C) and (D) Plots of velocity (red), curvature (green) and torsion (blue) values along the length of the parasite trajectories shown in panels A and B, respectively. The curvature and torsion values would be constant through time for a regular helix; the variation in these measurements along the parasite trajectories shows that they move in irregular-shaped yet periodically fluctuating corkscrews.
Figure 3
Figure 3. 3D motility of the Δphil1 parasites.
(A) MIPs for wild-type (RH), TgPHIL1 knockout (Δphil1) and complemented (Comp) parasites. Scale bar = 50 µm. The colour scheme for all MIPs was inverted for better visualization of parasite trajectories. The percentage of total parasites moving (B) was comparable for the three parasite lines, but the cumulative frequency distribution (C) and histogram (D) of the smoothed trajectory lengths for RH (black), Δphil1 (red) and Comp parasites (grey) reveal that the Δphil1 parasites do not move as far as the RH or Comp parasites within the same timeframe (Kolmogorov-Smirnov test, D = 0.199, p<0.0001 and D = 0.114, p<0.0001, respectively). The Δphil1 parasites also exhibited significantly decreased mean velocity compared to the RH parasites (E) and significantly reduced maximum velocity compared to both RH and Comp parasites (F) (paired t-test, significance indicated by asterisks). Closed data points are the results from five independent experiments comparing RH and Δphil1 parasites; open data points are the results from four independent experiments comparing Δphil1 and Comp parasites. Each of the independent experiments (assigned a different colour in the scatter plot) was performed in either triplicate or quadruplicate. The total number of parasites analyzed was 6,467 for RH, 9,305 for Δphil1 and 3,743 for Comp. * p<0.05, ** p<0.001, ns = not significant.

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