Life and times:synthesis, trafficking, and evolution of VSG

Evasion of the acquired immune response in African trypanosomes is principally mediated by antigenic variation, the sequential expression of distinct variant surface glycoproteins (VSGs) at extremely high density on the cell surface. Sequence diversity between VSGs facilitates escape of a subpopulation of trypanosomes from antibody-mediated killing. Significant advances have increased understanding of the mechanisms underpinning synthesis and maintenance of the VSG coat. In this review, we discuss the biosynthesis, trafficking, and turnover of VSG, emphasising those unusual mechanisms that act to maintain coat integrity and to protect against immunological attack. We also highlight new findings that suggest the presence of unique or highly divergent proteins that may offer therapeutic opportunities, as well as considering aspects of VSG biology that remain to be fully explored

Exploiting the Achilles' heel of membrane trafficking in trypanosomes

Pathogenic protozoa are evolutionarily highly divergent from their metazoan hosts, reflected in many aspects of their biology. One particularly important parasite taxon is the trypanosomatids. Multiple transmission modes, distinct life cycles and exploitation of many host species attests to great prowess as parasites, and adaptability for efficient, chronic infection. Genome sequencing has begun uncovering how trypanosomatids are well suited to parasitism, and recent genetic screening and cell biology are revealing new aspects of how to control these organisms and prevent disease. Importantly, several lines of evidence suggest that membrane transport processes are central for the sensitivity towards several frontline drugs

Conservation and divergence within the clathrin interactome of <i>Trypanosoma cruzi</i>

Trypanosomatids are parasitic protozoa with a significant burden on human health. African and American trypanosomes are causative agents of Nagana and Chagas disease respectively, and speciated about 300 million years ago. These parasites have highly distinct life cycles, pathologies, transmission strategies and surface proteomes, being dominated by the variant surface glycoprotein (African) or mucins (American) respectively. In African trypanosomes clathrin-mediated trafficking is responsible for endocytosis and post-Golgi transport, with several mechanistic aspects distinct from higher organisms. Using clathrin light chain (TcCLC) and EpsinR (TcEpsinR) as affinity handles, we identified candidate clathrin-associated proteins (CAPs) in Trypanosoma cruzi; the cohort includes orthologs of many proteins known to mediate vesicle trafficking, but significantly not the AP-2 adaptor complex. Several trypanosome-specific proteins common with African trypanosomes, were also identified. Fluorescence microscopy revealed localisations for TcEpsinR, TcCLC and TcCHC at the posterior region of trypomastigote cells, coincident with the flagellar pocket and Golgi apparatus. These data provide the first systematic analysis of clathrin-mediated trafficking in T. cruzi, allowing comparison between protein cohorts and other trypanosomes and also suggest that clathrin trafficking in at least some life stages of T. cruzi may be AP-2-independent

Trypanosomes show species-specific changes in motile behaviour in response to viscosity changes.

<p>A) The effect of viscosity on the proportion of swimming trypanosomes. The parasites were isolated from infected mice and resuspended in fresh trypanosome dilution buffer (TDB) in the absence or presence of methylcellulose (0.2, 0.4, 0.6 and 0.8%), generating medium viscosities between 1 mPa·s (TDB) and 35 mPa·s (0.8% methylcellulose). For each condition the trajectories of 300 parasites were analysed. B) The viscosity of the micro-environment influences the average persistent swimming speed of trypanosomes in a species-dependent manner (n ≥ 100; data are means ± SD). The trypanosomes were incubated in TDB-buffer supplemented with or without methylcellulose. While <i>T</i>. <i>vivax</i> motility was negatively affected by conditions above blood viscosity (i.e. 4 mPa·s), <i>T</i>. <i>brucei</i> and <i>T</i>. <i>evansi</i> parasites swam faster even in very viscous medium. <i>T</i>. <i>congolense</i> motion was not consistently influenced by viscosity. C) The influence of increasing viscosity on trypanosome swimming speed in mouse blood (n ≥ 100; data are means ± SD). Increased viscosity in the presence of blood cells led to reduction of speeds in <i>T</i>. <i>vivax</i>. In contrast, average speeds of <i>T</i>. <i>brucei</i> and <i>T</i>. <i>evansi</i> increased with increasing viscosity. Average speeds of <i>T</i>. <i>congolense</i> parasites did not vary significantly with increased media viscosity.</p

Three-dimensional modelling of trypanosome cells reveals characteristic morphologies correlating to the species-specific motility behaviour.

<p>The parasites were harvested from infected mice and the cell surface was fluorescently labelled with sulfo-NHS dyes. On the left of each panel representative surface-rendered models of fluorescently surface-labelled trypanosome cells are shown. The fluorescence labelling allows the simultaneous visualisation of the cell body and the flagellar membrane. The complete trace of the flagellum (yellow) attached along the cell body (grey) is shown in the three-dimensional representation. The model was orientated to show the view onto the posterior tip, allowing to evaluate the course of the flagellar attachment along the cell body, starting at the exit from the flagellar pocket at the top of the cell and tracing it towards the anterior end. A selection of 3D-models is presented to illustrate the variety of shapes the trypanosomes adopt, based on their cellular waveform. The numbers present the average length of the trypanosomes L_av (n = 100) and the average maximum width of the cells W_av (n = 100) as measured in video stills (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005448#ppat.1005448.g001" target="_blank">Fig 1</a>). The actual length L, volume V and cells surface area A of the surface-rendered models are given below in grey letters. The first cell in each row was chosen for surface-modelling. (A) Examples of normal waveform <i>T</i>. <i>vivax</i> IL 1392 cells. The flagellum is attached in a shallow 180° right hand turn. (B) <i>T</i>. <i>vivax</i> IL 2136 normal waveforms. The flagellum exhibits a similar course to IL 1392 running along a slightly stiffer cell body. (C) The <i>T</i>. <i>brucei</i> ILTat 1.4 strain typically shows a more prominent 180° right handed turn around the posterior third of the cell body. (D) The pleomorphic <i>T</i>. <i>brucei</i> AnTat 1.1 strain slender and stumpy forms. The three cells on the right are short stumpy trypanosomes that are characterised by the absence of a free part of the flagellum. The slender forms show a similar 180° right hand turn when compared to ILTat 1.4 in C). Morphometric data are for the slender stage (E) <i>T</i>. <i>evansi</i> reveals a curlier waveform than <i>T</i>. <i>brucei</i> while the flagellum turns completely (360°) around the cell body.(F) <i>T</i>. <i>congolense</i> IL 1180 cells are small with a stiff cell body and a flagellum running along it in a relatively straight course. (G) <i>T</i>. <i>congolense</i> KETRI 3827 is larger than the IL 1180 strain, but reveals the same characteristic waveform.</p

Trypanosome velocities in mouse blood.

<p>Each bar represents the analysis of 100 high-speed videos (500 fps, 16 s recording time). The bars present the mean swimming speed +/- SD. Black lines depict the individual maximum and minimum speeds recorded. The maximum speeds for persistent swimmers are annotated. The average population swimming speed of a species was calculated from all 300 trajectories and is marked by the blue dot.</p

Representative examples of trypanosomes swimming in blood from different host animals.

<p>The images are stills of the corresponding <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005448#ppat.1005448.s007" target="_blank">S6 Video</a>. Videos were captured with a frame rate of 500 fps. The swimming paths were traced using the Fiji plugin MTrackJ [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005448#ppat.1005448.ref026" target="_blank">26</a>]. The trajectories of one example each of tumbling (red), intermediate (yellow) and persistent (green) swimmers are shown. The numbers are maximum speeds (n = 20).</p

Defined motility patterns characterise the behaviour of trypanosome species in the blood of different hosts.

<p>A) Representative examples of three motility types visualised by single cell trajectories. Videos were captured with a frame rate of 500 fps. The swimming paths were traced (white lines), (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005448#ppat.1005448.s002" target="_blank">S1</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005448#ppat.1005448.s006" target="_blank">S5</a> Videos). Persistent swimmers were defined as cells that swam without a tumbling phase for the duration of a single on-chip recording period (16 s) of the high speed camera. Within this time, intermediate swimmers exhibited at least one tumbling phase. This is a period of two seconds or longer, in which the cells do not leave a circular area with a diameter of 25 μm (white circle). Tumbling cells stay within this area for the complete observation period of 16 s. B) Comparative motility analysis of <i>T</i>. <i>vivax</i> and <i>T</i>. <i>congolense</i> in blood freshly harvested from infected mouse (m), rat (r) and sheep (s). The host animals revealed a comparable parasitaemia in the range of 10⁷ trypanosomes/ml. For comparison, parasites grown in mice were incubated in sheep blood (sb). C) Comparison of the locomotion behaviour of four trypanosome species in mouse blood. All parasites were isolated from infected mice and analysed in fresh neat mouse blood. At least 300 cells were analysed per infection and the motility types were scored as shown in (A).</p

Extended single flagellar beat analysis of trypanosome motility in wet mouse blood.

<p>The graphs plot the velocities produced by single, consecutive flagellar beats (red dots), together with the beat frequency (black dots). The velocities, which were derived from measurement of the translocation of the posterior tip after each flagellar beat (white marks in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005448#ppat.1005448.s009" target="_blank">S8</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005448#ppat.1005448.s017" target="_blank">S16</a> Videos), are averaged over 5 beats to generate the average directional speeds (blue dots). The red number is the overall observation period (ms). A) Fast type of a <i>T</i>. <i>vivax</i> IL 1392 persistent swimmer with a slim waveform (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005448#ppat.1005448.s009" target="_blank">S8 Video</a>), showing a 30 ms—stop upon mechanical hindrance. The flagellar tip directly hit an erythrocyte, whereupon the trypanosome stopped without flagellar beating being interrupted. Translocation resumed with the next flagellar beat. Note that neither beat frequency nor average velocity (blue line) were markedly changed by this stop. B) Example of a <i>T</i>. <i>vivax</i> IL 1392 normal waveform swimmer revealing a short (< 1 second) period characterised by several base-to-tip beats, resulting in backward movement, i.e. negative speed (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005448#ppat.1005448.s010" target="_blank">S9 Video</a>). C) <i>T</i>. <i>vivax</i> IL 2136 exhibited lower beat frequencies than the IL 1392 strain (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005448#ppat.1005448.s011" target="_blank">S10 Video</a>). D) An intermediate <i>T</i>. <i>brucei</i> ILTat 1.4 swimmer exhibited two persistent swimming stretches interrupted by a short tumbling phase. This was followed by a period of beat reversal and backward swimming (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005448#ppat.1005448.s012" target="_blank">S11 Video</a>). E) A persistently swimming <i>T</i>. <i>evansi</i> parasite (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005448#ppat.1005448.s013" target="_blank">S12 Video</a>) reducing velocity by half, while the flagellar beat frequency remained in a constant range. (F) <i>T</i>. <i>congolense</i> IL 1180 showed a short period of rather fast backward motion (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005448#ppat.1005448.s014" target="_blank">S13 Video</a>). (G) <i>T</i>. <i>congolense</i> KETRI 3827 switching between fast forward and slow backward movement (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005448#ppat.1005448.s015" target="_blank">S14 Video</a>). H) <i>T</i>. <i>congolense</i> KETRI 3827 isolated from infected sheep revealing persistent slow forward motion (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005448#ppat.1005448.s016" target="_blank">S15 Video</a>).</p

High-resolution single cell analysis of persistently swimming trypanosomes.

<p>The images on the left are single frames of the corresponding <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005448#ppat.1005448.s009" target="_blank">S8</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005448#ppat.1005448.s017" target="_blank">S16</a> Videos, showing the cells at the beginning of an analysed flagellar beat, while two later positions of the parasites are shown as white outlines to depict the trajectory of the cell. The speed (v) is the highest average velocity reached during the video sequence analysed. The panel to the right presents the trypanosome outlines from each recorded frame for one complete flagellar beat. The successive image frames were stacked along the time axis in a three-dimensional surface representation. This allows the visualisation of the cellular waveform produced by the beating flagellum and the attached cell body. The anterior tip of the flagellum is marked in blue. The duration of the complete flagellar beat is annotated on the t-axis (ms). The right panel reveals the same 3D-surface representation, however, turned to view on to the anterior tip of the flagellum (along the x-axis of movement). This illustrates the sinusoidal oscillation of the flagellar tip, starting the travelling wave running along the body to the posterior end. The average frequency (Hz) of the flagellar beats of the analysed video is given on the right (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005448#sec011" target="_blank">Materials and Methods</a>). A) A representative slim waveform of <i>T</i>. <i>vivax</i> IL 1392 swimming forward with continuous tip-to-base beats in a wet blood film from the mouse (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005448#ppat.1005448.s009" target="_blank">S8 Video</a>). This waveform represents a small subset (1%) within the parasite population and swims significantly faster than cells exhibiting normal waveforms. B) Normal waveform of <i>T</i>. <i>vivax</i> IL 1392 in mouse blood from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005448#ppat.1005448.s010" target="_blank">S9 Video</a>, swimming with a frequency similar to that of the fast waveform in A) but reaching less than half the speed. C) <i>T</i>. <i>vivax</i> IL 2136 in mouse blood reveals slower motion when compared to the IL 1392 strain (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005448#ppat.1005448.s011" target="_blank">S10 Video</a>). D) <i>T</i>. <i>brucei</i> ILTat 1.4 swims with a lower beat frequency than normal waveform <i>T</i>. <i>vivax</i> cells in B) but reaches similar speeds (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005448#ppat.1005448.s012" target="_blank">S11 Video</a>). E) <i>T</i>. <i>evansi</i> KETRI 2479 swimming in mouse blood (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005448#ppat.1005448.s013" target="_blank">S12 Video</a>), with an intermediate beat frequency. The <i>T</i>. <i>evansi</i> waveform typically appears more elastic and curly when compared to the other trypanosomes, propelling the cells to relatively high velocities along curvy trajectories. F) <i>T</i>. <i>congolense</i> IL 1180 swimming with characteristic low frequency beats (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005448#ppat.1005448.s014" target="_blank">S13 Video</a>). The anterior tip shows far shallower oscillations due to the missing free anterior part of the flagellum and the stiff cell body causes a waveform clearly less effective for propulsion than that of the other species. Nevertheless the cell shows persistent forward swimming periods with speeds of around 20 μm/s. G) <i>T</i>. <i>congolense</i> KETRI 3827 swimming in mouse blood (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005448#ppat.1005448.s015" target="_blank">S14 Video</a>), with a lower frequency than strain IL 1180, but with an apparently more effective waveform. H) <i>T</i>. <i>congolense</i> KETRI 3827 from infected sheep (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005448#ppat.1005448.s016" target="_blank">S15 Video</a>) beat with the same frequency as in mouse blood, but with significantly reduced forward swimming speed, showing the influence of the specific host environment. I) <i>T</i>. <i>vivax</i> IL 1392 from infected sheep (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005448#ppat.1005448.s017" target="_blank">S16 Video</a>) swim slower and with decreased flagellar beat frequency when compared to cells in mouse blood (B).</p