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Trypanosome Motion Represents an Adaptation to the Crowded Environment of the Vertebrate Bloodstream
(2012)
Blood is a remarkable habitat: it is highly viscous, contains a dense packaging of cells and perpetually flows at velocities varying over three orders of magnitude. Only few pathogens endure the harsh physical conditions within the vertebrate bloodstream and prosper despite being constantly attacked by host antibodies. African trypanosomes are strictly extracellular blood parasites, which evade the immune response through a system of antigenic variation and incessant motility. How the flagellates actually swim in blood remains to be elucidated. Here, we show that the mode and dynamics of trypanosome locomotion are a trait of life within a crowded environment. Using high-speed fluorescence microscopy and ordered micro-pillar arrays we show that the parasites mode of motility is adapted to the density of cells in blood. Trypanosomes are pulled forward by the planar beat of the single flagellum. Hydrodynamic flow across the asymmetrically shaped cell body translates into its rotational movement. Importantly, the presence of particles with the shape, size and spacing of blood cells is required and sufficient for trypanosomes to reach maximum forward velocity. If the density of obstacles, however, is further increased to resemble collagen networks or tissue spaces, the parasites reverse their flagellar beat and consequently swim backwards, in this way avoiding getting trapped. In the absence of obstacles, this flagellar beat reversal occurs randomly resulting in irregular waveforms and apparent cell tumbling. Thus, the swimming behavior of trypanosomes is a surprising example of micro-adaptation to life at low Reynolds numbers. For a precise physical interpretation, we compare our high-resolution microscopic data to results from a simulation technique that combines the method of multi-particle collision dynamics with a triangulated surface model. The simulation produces a rotating cell body and a helical swimming path, providing a functioning simulation method for a microorganism with a complex swimming strategy.
Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304\(\pm\)0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of \(\pm\)0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.
In the Lyme disease spirochete Borrelia burgdorferi, the outer membrane protein P66 is capable of pore formation with an atypical high single-channel conductance of 11 nS in 1 M KCl, which suggested that it could have a larger diameter than ‘normal’ Gram-negative bacterial porins. We studied the diameter of the P66 channel by analyzing its single-channel conductance in black lipid bilayers in the presence of different nonelectrolytes with known hydrodynamic radii. We calculated the filling of the channel with these nonelectrolytes and the results suggested that nonelectrolytes (NEs) with hydrodynamic radii of 0.34 nm or smaller pass through the pore, whereas neutral molecules with greater radii only partially filled the channel or were not able to enter it at all. The diameter of the entrance of the P66 channel was determined to be \(\leq\)1.9 nm and the channel has a central constriction of about 0.8 nm. The size of the channel appeared to be symmetrical as judged from one-sidedness of addition of NEs. Furthermore, the P66-induced membrane conductance could be blocked by 80–90% by the addition of the nonelectrolytes PEG 400, PEG 600 and maltohexaose to the aqueous phase in the low millimolar range. The analysis of the power density spectra of ion current through P66 after blockage with these NEs revealed no chemical reaction responsible for channel block. Interestingly, the blockage of the single-channel conductance of P66 by these NEs occurred in about eight subconductance states, indicating that the P66 channel could be an oligomer of about eight individual channels. The organization of P66 as a possible octamer was confirmed by Blue Native PAGE and immunoblot analysis, which both demonstrated that P66 forms a complex with a mass of approximately 460 kDa. Two dimension SDS PAGE revealed that P66 is the only polypeptide in the complex.