Adhesion forces and mechanics in mannose-mediated acanthamoeba interactions

The human pathogenic amoeba Acanthamoeba castellanii (A. castellanii) causes severe diseases, including acanthamoeba keratitis and encephalitis. Pathogenicity arises from the killing of target-cells by an extracellular killing mechanism, where the crucial first step is the formation of a close contact between A. castellanii and the target-cell. This process is medi- ated by the glycocalix of the target-cell and mannose has been identified as key mediator. The aim of the present study was to carry out a detailed biophysical investigation of man- nose-mediated adhesion of A. castellanii using force spectroscopy on single trophozoites. In detail, we studied the interaction of a mannose-coated cantilever with an A. castellanii tro- phozoite, as mannose is the decisive part of the cellular glycocalix in mediating pathogenic- ity. We observed a clear increase of the force to initiate cantilever detachment from the trophozoite with increasing contact time. This increase is also associated with an increase in the work of detachment. Furthermore, we also analyzed single rupture events during the detachment process and found that single rupture processes are associated with mem- brane tether formation, suggesting that the cytoskeleton is not involved in mannose binding events during the first few seconds of contact. Our study provides an experimental and conceptual basis for measuring interactions between pathogens and target-cells at different levels of complexity and as a function of interaction time, thus leading to new insights into the biophysical mechanisms of parasite pathogenicity

Data from: Adhesion forces and mechanics in mannose-mediated acanthamoeba interactions

The human pathogenic amoeba Acanthamoeba castellanii (A. castellanii) causes severe diseases, including acanthamoeba keratitis and encephalitis. Pathogenicity arises from the killing of target-cells by an extracellular killing mechanism, where the crucial first step is the formation of a close contact between A. castellanii and the target-cell. This process is mediated by the glycocalix of the target-cell and mannose has been identified as key mediator. The aim of the present study was to carry out a detailed biophysical investigation of mannose-mediated adhesion of A. castellanii using force spectroscopy on single trophozoites. In detail, we studied the interaction of a mannose-coated cantilever with an A. castellanii trophozoite, as mannose is the decisive part of the cellular glycocalix in mediating pathogenicity. We observed a clear increase of the force to initiate cantilever detachment from the trophozoite with increasing contact time. This increase is also associated with an increase in the work of detachment. Furthermore, we also analyzed single rupture events during the detachment process and found that single rupture processes are associated with membrane tether formation, suggesting that the cytoskeleton is not involved in mannose binding events during the first few seconds of contact. Our study provides an experimental and conceptual basis for measuring interactions between pathogens and target-cells at different levels of complexity and as a function of interaction time, thus leading to new insights into the biophysical mechanisms of parasite pathogenicity

Single rupture analysis of the last rupture event between a mannose-coated AFM cantilever and an <i>A. castellanii</i> cell after 0.5 s, 5 s and 10 s contact time.

<p>Histograms (left) and boxplots (right) of rupture forces (top), slopes (center) and tether lenghts (bottom) of the last rupture event. Significant changes in rupture forces were observed between 0 s and 5 s (*, p≤0.05), and 0 s and 10 s (***, p≤0.001) contact time. The slopes were not significantly changed by the duration of cantilever-amoeba contact. All measured slopes were above -100 pN/s. Significant changes of tether lengths were only recorded between 0 s and 10 s contact time (***, p≤0.001).</p

2D probability density plots of slopes versus position of the last rupture event when breaking the contact between a mannose coated AFM cantilever and a <i>A. castellanii</i> cell after 0.5 s (top-left), 5 s (top-right) and 10 s (bottom) contact time.

<p>Higher contact times result in higher probability densities at higher rupture positions and narrower slope distributions. Sariisik et al. stated that slopes above -30 pN/<i>μ</i>m indicate tethering prior to the rupture event [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176207#pone.0176207.ref031" target="_blank">31</a>]. Since our cantilever retraction speed was 5 <i>μ</i>m, tethers occur if slopes are above -150 pN/s, which is the case for all our measurements. The figure on the bottom also includes the corresponding histograms for slope and rupture position distributions. The axes of ordinates of these histograms represent counts.</p

Pathogenic <i>A. castellanii</i> adhere to mannose-agarose coated beads.

<p><i>Left</i>: <i>A. castellanii</i> trophozoites adhering to a mannose-agarose coated bead. White arrows indicate trophozoites at the edge of the sphere. The amount of acanthamoebae adhering to the bead and the total amount of acanthamoebae were counted directly, 2 h and 4 h after cell seeding. <i>Right</i>: Comparison of <i>A. castellanii</i> and <i>A. comandoni</i> adhesion to mannose-agarose and sepharose beads. Only a few <i>A. comandoni</i> adhered to the sepharose or mannose-agarose coated beads. A large amount of <i>A. castellanii</i> adhered already at short times and the number of adhering acanthamoeba strongly increased with time. Only a few <i>A. castellanii</i> adhered to sepharose beads. Scalebar: 100<i>μ</i>m.</p

Superdiffusion dominates intracellular particle motion in the supercrowded cytoplasm of pathogenic Acanthamoeba castellanii

Acanthamoebae are free-living protists and human pathogens, whose cellular functions and pathogenicity strongly depend on the transport of intracellular vesicles and granules through the cytosol. Using high-speed live cell imaging in combination with single-particle tracking analysis, we show here that the motion of endogenous intracellular particles in the size range from a few hundred nanometers to several micrometers in Acanthamoeba castellanii is strongly superdiffusive and influenced by cell locomotion, cytoskeletal elements, and myosin II. We demonstrate that cell locomotion significantly contributes to intracellular particle motion, but is clearly not the only origin of superdiffusivity. By analyzing the contribution of microtubules, actin, and myosin II motors we show that myosin II is a major driving force of intracellular motion in A. castellanii. The cytoplasm of A. castellanii is supercrowded with intracellular vesicles and granules, such that significant intracellular motion can only be achieved by actively driven motion, while purely thermally driven diffusion is negligible.115243sciescopu

Histogram of total rupture forces (left) and work of detachment (right) between a mannose coated AFM cantilever and an <i>A. castellanii</i> trophozoite after 0.5 s, 5 s and 10 s contact time, respectively.

<p>Adhesion forces and work of detachment increase with time. Comparably high forces between 0.5 nN and 30 nN were measured.</p