Multistep adhesion of Plasmodium sporozoites

The FASEB Journal • Research Communication

Multistep adhesion of Plasmodium sporozoitesStephan Hegge,* Sylvia Munter,* Marion Steinbuchel,* Kirsten Heiss,* Ulrike Engel,†

Kai Matuschewski,*,‡ and Friedrich Frischknecht*,1

*Department of Parasitology, Hygiene Institute, University of Heidelberg Medical School, Heidelberg,Germany; †Nikon Imaging Center, BioQuant, Heidelberg, Germany; and ‡Department of Parasitology,Max Planck Institute for Infection Biology, Berlin, Germany

ABSTRACT Adhesion of eukaryotic cells is a com-plex process during which interactions between extra-cellular ligands and cellular receptors on the plasmamembrane modulate the organization of the cytoskele-ton. Pathogens particularly rely often on adhesion totissues or host cells in order to establish an infection.Here, we examined the adhesion of Plasmodium sporo-zoites, the motile form of the malaria parasite transmit-ted by the mosquito, to flat surfaces. Experimentsusing total internal reflection fluorescence micros-copy and analysis of sporozoites under flow revealeda stepwise and developmentally regulated adhesionprocess. The sporozoite-specific transmembrane pro-teins TRAP and S6 were found to be important forinitial adhesion. The structurally related protein TLPappears to play a specific role in adhesion under staticconditions, as tlp(�) sporozoites move 4 times lessefficiently than wild-type sporozoites. This likely re-flects the decreased intradermal sporozoite movementof sporozoites lacking TLP. Further, these three sporo-zoite surface proteins also act in concert with actinfilaments to organize efficient adhesion of the sporo-zoite prior to initiating motility and host cell invasion.—Hegge, S., Munter, S., Steinbuchel, M., Heiss, K.,Engel, U., Matuschewski, K., Frischknecht, F. Multi-step adhesion of Plasmodium sporozoites. FASEB J.24, 2222–2234 (2010). www.fasebj.org

Key Words: flow chamber � malaria � TRAP � gliding motility

Eukaryotic cells can adhere to each other, to theextracellular matrix, or to artificial substrates, such asglass or plastic. Cell adhesion depends on the physicaland chemical nature of the substrate and is often thefirst step for a cell to initiate motility. Both processesare coordinated in a complex spatial and temporal wayinvolving even in the simplest case many dozens ofproteins (1). Adhesion and motility are important atseveral stages during the life cycle of the malariaparasite and essential for pathogenesis. During theblood stage, which is responsible for the symptoms ofmalaria, Plasmodium merozoites adhere to red bloodcells prior to invasion (2). Triggered by the parasite,infected red blood cells themselves attach to the endo-thelium, using host receptors such as CD36 and ICAM(3). Adhesion of gametes—the sexual stage of Plasmo-

dium parasites—to each other is mediated by the game-tocyte-specific proteins p48 and p45, which are essentialfor fertilization in the mosquito midgut (4). Furtheralong the life cycle of the parasite, adhesion is impor-tant for the ookinete to contact the epithelial cells ofthe midgut, which must be penetrated so the parasitecan transform into an oocyst at the midgut wall (5).Next, sporozoites develop in oocysts, are released intothe hemolymph of the mosquito, and eventually invadethe salivary gland.

Sporozoites isolated from mosquito midguts do nottranslocate, but those isolated from the salivary glandsare highly motile cells that locomote by gliding motility,a substrate-dependent motion during which the highlypolarized parasite does not change its shape (6, 7).Sporozoites move slowly within the salivary ducts andspeed up on transmission into the skin, where theymigrate in the dermis in order to find and enter ablood or lymph vessel (8–10). Following the invasion ofa blood vessel, sporozoites travel with the blood flow tothe liver, where they are arrested and move on theendothelium, before passing through liver-residentmacrophages to invade hepatocytes (11, 12). Sporozo-ite invasion and flow chamber experiments showed adecrease in sporozoite attachment in the presence ofheparin (13), and cellular and biochemical adhesionassays confirmed that binding of sporozoites to differ-ent cell types depends on the sulfation level of surfaceproteoglycans (14). Stellate cells, which reside in thespace of Disse between the endothelium and the hepa-tocytes, express highly sulfated proteoglycans thatreach through the fenestrae of the liver vein endothe-lium and thus can interact with sporozoites. This bind-ing depends partly on the circumsporozoite protein(CSP) and induces processing of CSP, which, in turn,triggers a signal to invade the respective cell layer(14–16). The first identified Plasmodium gene with adirect role in parasite locomotion and cell invasion wasthe sporozoite-specific thrombospondin-related anony-mous protein (TRAP), which encodes a transmem-brane protein containing two adhesion modules, a von

1 Correspondence: Department of Parasitology, HygieneInstitute, University of Heidelberg Medical School, ImNeuenheimer Feld 324, 69120 Heidelberg, Germany. E-mail:[email protected]

doi: 10.1096/fj.09-148700

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Willebrand A-domain and a thrombospondin repeat inits extracellular portion, and a conserved cytoplasmictail that links the protein to the parasite motor machin-ery (17). Sporozoites lacking the membrane proteinTRAP can still attach to but fail to invade salivaryglands. TRAP binds in vitro to Saglin, a receptor onsalivary glands (18), which was found to be essential forinvasion by sporozoites (18, 19).

TRAP knockout parasites [trap(�)] are arrested inthe mosquito hemolymph, due to their incapacity toinvade salivary glands and show no gliding motility(17). In addition, sporozoites posses two surface pro-teins that are structurally related to TRAP, TRAP-likeprotein (TLP) (20, 21) and S6 (TREP) (22, 23). Bothproteins contain the unifying modules of TRAP familyinvasins, i.e., extracellular adhesion domains and thecytoplasmic tail domain. Parasites lacking these pro-teins are no longer able to efficiently invade salivaryglands [s6(�)] and show a partial delay in blood stagedevelopment [tlp(�)], respectively (20–23). Interest-ingly, when injected into the skin, the numbers oftlp(�) sporozoites entering hepatocytes dramaticallydecreased, suggesting that TLP might be involved inmigration within the skin, e.g., by inhibiting transmigra-tion through skin cells (21). The described phenotypesindicate that these proteins might play roles duringadhesion, motility, and/or host cell invasion (24). Inthis study, we performed the first detailed analysis ofsporozoite locomotion under static and flow condi-tions as in vitro models for migration. We employedcomparative real-time imaging of wild-type (WT) andknockout parasites lacking the three sporozoite-specificTRAP adhesins to dissect the roles of these parasitesurface proteins in adhesion and motility under de-fined conditions. To obtain a dynamic view of adhe-sion, we first imaged the settling of sporozoites usingtotal internal reflection fluorescence (TIRF) micros-copy. Subsequently, we filmed WT sporozoites as well astrap(�), tlp(�), and s6(�) sporozoites in flow chambersand under drugs that affect parasite actin dynamics.

MATERIALS AND METHODS

Ethics statement

Experiments with animals were approved by the state author-ities, and all animals were treated according to German law.

Imaging sporozoites

WT, knockout, and GFP-expressing Plasmodium berghei sporo-zoites were produced in Anopheles stephensi mosquitoes, asdescribed previously (10, 20, 22). Briefly, salivary glands frominfected mosquitoes were dissected in RPMI (PAA, Pasching,Austria) tissue culture medium supplemented with 3% BSA.Next, the sporozoites were isolated by gently squeezing with aplastic piston in a 1.5-ml reaction tube. Sporozoites wereobserved with a Zeiss Axiovert 200 (Carl Zeiss, Obernkirchen,Germany) as part of a CellObserver system with variousobjective lenses (see below). Images were recorded on a ZeissAxiocam HRm.

Live-cell imaging to test sporozoite gliding in the absenceand presence of flow was performed according to Hegge et al.(6). In brief, 50 to 250 parasites were filmed at low magnifi-cation (�100), so that sufficient data were available forstatistical classification and quantification of sporozoites, ac-cording to their motility pattern (6). We filmed for 100 s at 1Hz, induced hydrodynamic flow, and continuously filmed thesame field of view for another 100 s.

For TIRF microscopy, parasites were placed on a glass-bottom Petri dish (35-mm No. 1.5; MatTek, Ashland, MA,USA) and imaged on a Nikon TIRF unit on a TE-2000 E(Nikon, Tokyo, Japan) using the 488-nm laser line for exci-tation. An �60 PlanApo TIRF objective NA 1.45 (Nikon) wasused, and images were recorded with a HamamatsuORCA-AG digital camera at 1 Hz. Still images and fluores-cence intensity projection images were processed usingImageJ (U.S. National Institutes of Health, Bethesda, MD,USA). Quantitative analysis of sporozoite movement patternswas performed using ToAST, an ImageJ-based analysis toolimplemented in FIJI (http://pacific.mpi-cbg.de/wiki/index-.php/Main_Page) (6). All figures were created with theAdobe creative suite package(Adobe Systems, San Jose, CA,USA). The original pixel size was maintained during process-ing.

Adhesion experiments

For adhesion analysis, isolated sporozoites were first incu-bated either at 28°C for 2 h when using E-64 (10 �M;Sigma-Aldrich, Steinheim, Germany) or on ice for 30 minwhen using nuclear stains, actin dynamics modulating drugs,or DMSO. For experiments comparing different mutants(without additional drugs), 10 min of incubation time wasapplied. Next, parasites were placed in an uncoated flowchamber (uncoated �-Slide I for Live Cell Analysis; Ibidi,Martinsried, Germany), allowed to adhere for 10 min andimaged using an Axiovert 200 (Carl Zeiss) at room tempera-ture. Movies were saved as zvi (Zeiss Vision Image) andimported to ImageJ for analysis. Images were collected with aZeiss Axiocam HRm using Axiovision 4.6 software, an �10Apoplan objective (NA 0.25) and a GFP filter set. A numberof images containing �500 parasites in total were capturedprior to the flow. Adding 2 ml of the respective mediuminduced hydrodynamic flow forces (Supplemental Fig 1), andthe same number of pictures was recorded after flow once anequilibrium was reached.

Diffusion experiments

For diffusion experiments, sporozoites were placed in anuncoated flow chamber (�-Slide I; Ibidi), and imaging wasperformed as above. For the perfusion of drugs (cytochalasinD and jasplakinolide) 30 �l of a 100 �M stock solution inRPMI with 3% (m/v) BSA were placed 5 mm next to the fieldof view and allowed to diffuse inside the flow chamber duringcontinuous image acquisition.

Flow experiments

The experimental setup was principally identical to the adhesionexperiment mentioned above, except that only one microscopicfield was observed and continuously recorded during the exper-iment. Sporozoites were treated and added to the flow chamberas described above and were allowed to adhere for 10 minbefore image acquisition was started. The flow movies werealways performed at the same position relative to the flow inorder to exclude symmetric variations between experiments.The acquisition rate was 1 or 2 Hz, flow conditions were induced

2223MULTISTEP ADHESION OF PLASMODIUM SPOROZOITES

after 50 or 100 recorded frames by applying 2 ml of medium toone reservoir, and the parasites were filmed for up to 7 min. Theanalysis was performed manually using ImageJ’s Manual Track-ing plugin or semiautomatically using ToAST (6).

Counting experiment

Sporozoites were dissected into one batch. One-third of theparasites were treated and added to a flow chamber asdescribed above; the sporozoites were allowed to adherefor 10 min at room temperature, and manual assignmentwas started using an �63 (NA 1.4, oil; Carl Zeiss) objectiveand fluorescent light. The remaining two-thirds of theparasites were treated with drugs or DMSO (control) 30and 180 min after the initial drug. To even out agingeffects on sporozoite motility or adhesion, all experimentswere performed in triplicate with a different order of drugor DMSO treatment.

All experiments were performed by the same person inorder to avoid subjective differences during the assignment ofmotility and/or adhesion states. The person allocated para-site states for 30 min at 3 (30, 90, 150 min) time points afteraddition of the respective drug/DMSO control. Similar to ref.7, every parasite in the focal plane was observed for �15 sbefore it was allocated to one of 5 states: the sporozoite is1) attaching with one part, 2) attaching with more than onepart, 3) gliding, 4) floating, or 5) waving. Between 41 and 177sporozoites (median�106�30 sporozoites, 3322 in total)were examined per condition.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 5.0bfor Mac OS X (GraphPad, San Diego, CA, USA) and Mi-crosoft Excel for Mac, version 11.5 (Microsoft, Redmond,WA, USA). Normally distributed populations of similar(�10%) cluster sizes were analyzed via Students t test, whilethe significance of populations with different cluster sizes wastested according to Bland and Kerry (25, 26). Significancesfor non-normally distributed values were examined applyingthe Mann Whitney U test.

RESULTS

Stepwise adhesion of sporozoites

To image adhesion of individual sporozoites, we em-ployed TIRF (Fig. 1A) and differential interferencecontrast (DIC) microscopy on isolated Plasmodiumberghei sporozoites expressing cytoplasmic green fluo-rescent protein (27). This allowed the visualization ofwhole sporozoites and simultaneously identified theareas where the parasites approached the glass surface(Fig. 1B). Floating sporozoites adhered initially at asingle random site to the substrate, an event we namedprimary adhesion (Fig. 1B–D). Initial adhesion canoccur at either end, apical or proximal, followed by

Figure 1. Analyzing sporozoite adhesion with TIRF microscopy. A) Schematic of TIRF showing the incoming, totally reflectinglaser and the evanescent field that allows detection of fluorescent signals in the proximity of the coverslip surface. Exampleimages show a sporozoite imaged with differential interference contrast (DIC) and with TIRF. Note that only the sporozoite tipsare positioned within the evanescent field. B) Single frames and maximum intensity projections (0–250 and 1–251 s) showinginverted TIRF (top) and DIC (bottom) images from a sporozoite attached with one end on a glass surface. Green arrowheadindicates TIRF signal at proximal end and corresponding point in DIC images. Red arrowheads indicate regions wheresporozoite approaches glass surface during waving. C) Sequence of inverted TIRF images from a sporozoite attaching to (0–9s) and gliding on (9–12 s) a glass surface in counterclockwise direction. Proximal end attaches first (0–primary adhesion–greenarrow), with apical end attaching subsequently (0–9 s–secondary adhesion). Following apical attachment, TIRF signal growslarger away from the initial attachment site (5–9 s). Sporozoite body approaches glass surface (9 s–tertiary adhesion) beforesporozoite starts gliding (12 s). D) Sporozoite adheres with apical part (0) followed by proximal part (2 s) prior to gliding (4–5s). E) Schematic illustration of multiple adhesion steps seen in D. Red bars indicate adhesion sites; red ellipse indicates nucleusnear the proximal end. Sporozoite establishes a first adhesion side at its apical end (top panel), approaches the coverslip withthe proximal end to form a second adhesion side (middle panel), flips over, and forms a central adhesion and starts gliding(bottom panel). Numbers indicate time (s). Scale bars� 10 �m.

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waving and gliding motility. During transition fromfloating to gliding, the majority of adhering parasiteswent through a stage in which they were seeminglystuck to the substratum with their proximal end. Thisstage was frequently accompanied by active waving (Fig.1B) (7) and could last for several minutes. Followingwaving, a secondary adhesion site was usually formed atthe apical end (Fig. 1C). The apical end then movedslowly forward, increasing the area closely associatedwith the substrate (Fig. 1C, D). This was followed by aslight stretching and flipping over of the sporozoite,such that one side of the entire parasite was in closeproximity to the substrate (tertiary adhesion). Only oncompletion of this series of events could the parasiteinitiate continuous gliding (Fig. 1C–E, SupplementalFig. 2, and Supplemental Movies 1 and 2).

Sporozoite adhesion is an active process requiringparasite actin

Close examination of sporozoite waving revealed threedifferent movement patterns. The free end of sporozo-ites either moved passively with the flow (small changeof frame to frame orientation) (6) or showed suddenmovements, which often changed the orientation ofthe parasite by �90° in �1 s (Fig. 2A, top parasite, andSupplemental Movie 3). We termed this behavior “sud-den waving.” Lastly, the free end of sporozoites cancontinuously move in small-angle increments (Fig. 2B).When this movement occurred with the apical end nearthe surface, we termed this behavior “searching,” as itwas frequently followed by secondary adhesion on theapical end, tertiary adhesion, stretching, and finallygliding (Fig. 2B and Supplemental Movie 2).

Because the pattern of these movements suggestedan active mechanism, we next applied drugs that inter-fere with actin dynamics to adhering sporozoites (Fig.2C). This revealed that inhibitors of actin polymeriza-tion (cytochalasin D and swinholide) stalled parasitewaving and led to an increase in the numbers ofattached sporozoites (Fig. 2C). In contrast, jasplakino-lide, which inhibits actin filament depolymerizationand thereby induces accumulation of actin filaments(28, 29), led to sudden, irregular movements andultimately to detachment of the parasites. To oursurprise, neither latrunculin A nor latrunculin B al-tered parasite behavior compared to control (Fig. 2Cand data not shown). These data show that inhibitingactin dynamics by chemical compounds differentiallyaffect parasite attachment and locomotion.

Primary adhesion is independent of actin dynamics

Most of the sporozoites failed to form a secondaryadhesion site in the presence of high concentrations ofjasplakinolide or cytochalasin D. This finding indicateda role for actin dynamics in cell adhesion prior togliding. Gliding itself was confirmed to be dependenton actin dynamics, since the addition of cytochalasin Dat lower concentrations did not interfere with comple-tion of all adhesion steps, but dramatically reducedgliding speed (ref. 6 and data not shown).

When sporozoites were allowed to settle on thesurface in the presence of actin-depolymerizing drugs,they attached primarily with only one end. We deter-mined whether they preferably attached with the apicalor proximal end by staining the parasite nucleus, whichlocalizes in the proximal half of the parasite (30). In

Figure 2. Sporozoite adhesion is an active, actin-dependent process. A) Inverted and thresholded time-lapse images of asporozoite attached at the proximal end (green arrowhead) and a second gliding sporozoite (red arrowhead indicates apicalend). Note sudden displacement of the attached sporozoite (curved arrow) between 3 and 4 s. Maximum intensity projection(Max) reveals the nearly circular gliding path and illustrates the sudden displacement of the proximal end adhering sporozoite(curved arrow). Adhering sporozoite appears thick due to the collection of out-of-focus light in the wide-field microscope. Seealso Supplemental Movie 3. B) Inverted and thresholded time-lapse images of an attaching sporozoite. During waving, thesporozoite appears thicker due to out-of-focus light (0–2 s). Sporozoite moves in proximity with the apical end over the glasssurface (2–5 s) in a process termed searching. Secondary adhesion is followed (5–7 s) by stretching, during which the sporozoiteseemingly elongates (7–9 s) and starts gliding (10–11 s). Maximum projection includes images from �10 to �15 s, thus showing2 sudden displacements prior to the depicted image sequence. Curved red arrow indicates direction of gliding. Forcomplementary analysis and a further example, see Supplemental Fig. 2. See also Supplemental Movie 2. C) Effects of drugsthat inhibit actin dynamics on sporozoite adhesion and gliding motility. Graphs are normalized to control. Parasites(400 – 800) were manually counted and classified for every condition. Swinholide, cytochalasin D, and jasplakinolide showan inhibiting effect on gliding motility, but only swinholide and cytochalasin D change the parasite adhesion propertiessignificantly. Attached refers to sporozoites that underwent primary, secondary, or tertiary adhesion. Error bars are notshown for better visualization.


the presence of cytochalasin D, most sporozoites at-tached with their apical end. Some also attached at acentral region or with the proximal end (69, 27, and4%, respectively; n�45). In contrast, when cytochalasinD was added to motile sporozoites, the parasitesstopped gliding but remained attached at both the

proximal and apical end (Fig. 3 and SupplementalMovies 4 and 5). In the same experimental assay, theaddition of jasplakinolide also stopped the parasites,but it caused deadhesion of a large fraction of thesporozoites (Fig. 3 and Supplemental Movie 6). When asudden hydrodynamic flow was applied, more sporozo-

Figure 3. Actin filaments play a role in deadhesion of gliding sporozoites. A) Maximum projections showing sporozoites before(i) and during drug diffusion (ii), followed by shear flow (iii–v). Red indicates the first frame contributing to the projections(shown in blue); green indicates the last frame. Asterisks indicate sporozoites that were detached during drug diffusion.Numbers indicate exemplary sporozoites. Maximum projection: before drug, 50 s; diffusion, 100 s. Scale bar � 50 �m. Control(top panel; see Supplemental Movie 4): parasites either keep moving in circles throughout the entire experiment (1), changetheir position during the experiment but stay attached (2), or enter the field of view during the flow and start gliding (notshown). Note that 2 parasites detach during diffusion (ii, asterisks) and another detaches during flow (3). Cytochalasin D(central panel and Supplemental Movie 5): diffusion of cytochalasin D immediately inhibits sporozoite gliding (i, ii; 4–6). Theadhesion of the parasites under high concentrations of cytochalasin D is very stable, since all parasites remain in the sameposition even after application of the flow (iii–v). Jasplakinolide (bottom panel; see Supplemental Movie 6): diffusion ofjasplakinolide inhibits sporozoite gliding and immediately detaches sporozoites even before flow is applied (ii, asterisks).Parasites stop gliding, and most are removed with the flow (7, 9), while some remain attached (8). See also ref. 48.B) Quantitative analysis of diffusion experiments from A. WT sporozoites were filmed before and after drug diffusion. Singlesporozoites were observed and classified as floating, attached, or gliding. Between 59 and 71 transition events were counted from3 independent experiments.


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Figure 4. Sporozoite adhesion is enhanced under flow. A) Two sporozoites reorient within the flow (see Supplemental Movie7). First panel shows 3 sporozoites in the absence of flow. Red arrowhead indicates a waving sporozoite. Blue arrowheadindicates a sporozoite that underwent brief gliding followed by waving. Black arrowhead indicates a floating (out of focus)sporozoite. Flow is applied at 24 s just before the second panel, which results in the immediate reorientation of the adherentsporozoites (red and blue arrowheads) and the floating away of the nonadherent parasite (black arrowhead). Parasites that areadherent at the proximal end undergo further adhesion steps and start gliding at different times, 37 s (blue) and 50 s (red),respectively. The blue parasite eventually detaches (73 s) before gliding 1 complete circle and is dragged away with the flow (77s). Numbers indicate time (s). B) Two sporozoites attach to the substrate after flow is applied (see Supplemental Movie 8). Redarrowheads indicate a gliding sporozoite. White arrowheads indicate parasites in flow. Blue arrowhead indicates a sporozoiteadhering in flow at 11.2 s and starting to glide at 24.2 s. Black arrowhead indicates a sporozoite adhering at 22.6 s and startingto glide at 25.8 s. C) Graphs show transition time for sporozoites from floating to gliding (left) and from floating to gliding for�1 entire circle (right) for flow (�) and no-flow (�) conditions (see Supplemental Movie 9). Whisker plots indicate all observedsporozoites; box indicates 50 percentile; bar indicates the median. Average values are 22.3 s (no flow, 23 sporozoites), and 5.3 s

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ites remained attached under the influence of cytocha-lasin D than in the absence of drugs. In contrast, theyreadily detached under high concentrations of jas-plakinolide. This suggests that more F-actin leads toweaker adhesion (Fig. 3).

Flow enhances adhesion

Taken together, the results obtained with actin inhibi-tors indicated that actin dynamics are important forreinforcing attachment by the second adhesion step. Invivo, this secondary adhesion event could be mediatedor enhanced by the flow of hemolymph within themosquito or blood through the sinusoids of the liver.To mimic this scenario under defined in vitro condi-tions, we applied a sudden flow to sporozoites (Fig. 4).While some parasites were eventually ripped off thesubstrate, most stayed attached and continued gliding.Parasites attached with one end rapidly underwentsecondary adhesion with the free-floating end. A directcomparison of the transition time from floating viaprimary adhesion to gliding under flow and nonflowconditions revealed that the process of multistep adhe-sion of sporozoites was substantially faster under flow(Fig. 4A, B, and Supplemental Movies 7 and 8). Indeed,most sporozoites started to glide within a few secondsafter initial contact under flow (Fig. 4B). However, alarge number of gliding sporozoites only moved for afew seconds before lifting off again at the apical endand reorienting themselves in the flow or being even-tually dragged away with the flow (Fig. 4B and Supple-mental Movie 9). We thus compared the time it tooksporozoites following initial adhesion to start gliding for�1 circle. This quantification showed a 7-fold reductionin transition time under flow conditions (Fig. 4C).

We next probed the influence of actin dynamicsduring the secondary adhesion step by exposing swin-holide-treated sporozoites to flow. Comparing the per-centages of parasites adhering with either one or bothends, we found the majority (80%) of parasites eventu-ally undergoing secondary adhesion (Fig. 4D). Thisfinding demonstrates that external forces applied to aprimary adhering sporozoite can at least partially com-pensate for the loss of dynamic actin required forsecondary adhesion under static conditions. We, there-fore, propose that the vertical vector of the appliedhydrodynamic force results in rapid reinforcement ofsporozoite adhesion (Fig. 4E).

Adhesion of sporozoites is developmentally regulated

During sporozoite maturation, the capacity for glidingincreases (6, 7, 31). To test whether the ability for

adhesion develops similarly, we isolated sporozoitesfrom the salivary glands, hemolymph, and midgut atdifferent time points after mosquito infection andtested for their capacity to adhere under flow (Fig. 5A).Although midgut-associated sporozoites typically donot adhere, we found a substantial age-dependentincrease in adhesion to the substrate (Fig. 5B). Weinterpret these findings as a reflection of �2 parasitesubgroups within each population: an already maturegroup of sporozoites that is ready to exit the oocysts,and a fraction with yet undeveloped adhesion proper-ties. This contrasts with the adhesion capacity of hemo-lymph and salivary gland sporozoites, which show peakadhesions around their time of emergence (Fig. 5B). Asexpected, sporozoites from salivary glands show thebest capacity to adhere, with sporozoites from hemo-lymph falling between the midgut and the salivarygland parasites. Interestingly, at d 13–14 after infec-tion, the capacity to adhere was similar in hemo-lymph and salivary gland sporozoites. This indicates aprominent role of an endogenous developmentalprogram rather than exclusive environmental cuesfrom the target organ for some aspects during sporo-zoite maturation (Fig. 5B).

Because midgut sporozoites largely fail to undergoefficient primary adhesion (Fig. 5B), we suggest that thisevent is likely not dependent on CSP, as this protein isexpressed on the surface of midgut sporozoites (32) (Fig.5B). To probe whether differential CSP processing plays arole in adhesion, as was shown for liver cell invasion (14),we tested adhesion under flow in the presence of E-64, abroad-spectrum cysteine protease inhibitor that alsoblocks CSP processing (33) (Fig. 5C). We did not observea difference in the adhesion characteristics, indicating aminor role, if any, of CSP processing in mediating sporo-zoite attachment to the substratum. This is not surprising,considering that E-64 treatment was shown to have noeffect on gliding motility but is specifically needed forhepatocyte invasion (14, 33).

Since actin plays a dual role in adhesion andmotility, we tested sporozoite adhesion under addi-tional conditions known to reduce their ability toglide (Fig. 5D), such as in the absence of BSA.Although gliding is reduced, adhesion of the para-sites is not affected in the absence of BSA, whichactivates motility in vitro (7) by inducing a signalingcascade with Ca2� and cAMP as second messengers(34). Similarly, in the presence of the calcium iono-phore A23187, which results in micronemal dis-charge and increased sporozoite motility (35), thereis no change in initial adhesion (Fig. 5D). This shows

(flow, 10 sporozoites, left); 43 s (no flow, 20 sporozoites), and 16.5 s (flow, 8 sporozoites, right). D) Single-end-adheringsporozoites undergo secondary adhesion after application of flow in presence of actin depolymerizing drugs. Graph showsnumber of sporozoites that adhered only with one end and those that adhered with both ends before and after flow was appliedin the presence of 2 �M swinholide. E) Cartoon illustrating flow acting on sporozoites that are in the process of adhesion.i) Initial adhesion. ii, iii) Flow-induced pushing of sporozoite toward substrate. iv) Secondary adhesion formation. Horizontalarrows indicate strength of laminar flow, according to distance from substrate; curved arrows indicate resulting movement ofsporozoite; red bars indicate adhesion sites. Scale bars � 10 �m.


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that initial adhesion and gliding are at least partiallybased on different mechanisms.

Distinct adhesion phenotypes of sporozoites lackingTRAP family adhesins

Transmembrane proteins of the TRAP family playdistinct roles in the infectivity of apicomplexan para-sites, including sporozoites (17, 20–23, 36, 37). TRAP-(17), TLP- (20, 21), and S6-knockout parasites (22, 23)show a strong to mild motility impairment, respectively,in sporozoites. Taking advantage of these existingknockout parasites, we thus probed whether the corre-sponding motility phenotypes can be explained by adeficiency in the formation of the adhesion sequenceprior to gliding.

We first examined the adhesion strength of sporozo-ites from these mutant parasite lines under flow condi-tions (Fig. 6). Hemolymph trap(�) or s6(�) sporozo-ites, but not tlp(�) sporozoites, showed significantly

[trap(�) P�0.015; s6(�) P�0.001; tlp(�) P�0.05] weakeradhesion as compared to WT parasites. Intriguingly,when isolated from salivary glands, the proportion ofattached tlp(�) sporozoites was significantly (P�0.014)higher than in WT parasites (Fig. 6). s6(�) sporozo-ites from salivary glands showed weaker adhesion (P�0.002), while trap(�) sporozoites did not invade sali-vary glands (17).

Sporozoite surface protein TLP regulates parasiteadhesion and detachment

Since trap(�) and s6(�) sporozoites are severely im-paired in their capacity to invade salivary glands, whiletlp(�) sporozoites are not (20, 21, 22, 23), we examinedthe latter parasite line in more detail in side-by-sidecomparison to WT parasites.

tlp(�) sporozoites are able to glide normally and toinvade and replicate in the liver, albeit at a slightlyreduced rate, if injected directly into the blood (20,

Figure 5. Adhesion capacity of sporozoites increases during parasite maturation. A) Images of sporozoites isolated from midguts(left), hemolymph (middle), and salivary glands (right) in a flow chamber before (top) and after (bottom) application of flow.Insets: magnified view of boxed areas; arrowheads indicate sporozoites. Scale bars � 50 �m. B) Quantification of sporozoiteadhesion under flow using midgut, hemolymph, and salivary gland sporozoites from different days postinfection. No differencewas found between all populations of sporozoites dissected at d 16/17 and those dissected at d 19/20. Also, no difference wasfound between sporozoites isolated from hemolymph or salivary glands at d 13/14. Note that midgut parasites isolated at d21–26 can adhere as well as hemolymph sporozoites isolated at d 13/14. At least 500 parasites were counted before flow wasapplied. C) E-64 does not influence adhesion. Sporozoites dissected from different compartments were incubated in RPMI �3%BSA at 28°C for 2 h with and without E-64 and tested for adhesion using flow assay. D) Quantification of adhesion under flowusing salivary gland sporozoites (d 17–22) in RPMI in combination with different additives. �, RPMI without additives; BSA,RPMI�3% bovine serum albumin; CytoD, RPMI�2 �M cytochalasin D; CI, RPMI�1 �M calcium ionophore A23187. Errorbars � weighted sd from �3 independent experiments.


21). However, when injected intradermally, the parasiteload in the liver decreased 10-fold (21), indicating thatTLP might be involved in migration out of the inocu-lation site in the skin. To dissect a possible role of TLP

in adhesion, we defined 4 different adhesion-basedpatterns: attached, which describes sporozoites adher-ent with �1 end, while the other shows no or only smalldisplacements; waving, which describes parasites at-tached with one end to the substrate and the other endmoving actively in the medium; gliding, in eithercounterclockwise (CCW) or clockwise (CW) direction;and switching, which describes parasites flipping onceor several times between CCW and CW gliding. Atypical pattern of salivary gland WT sporozoite motilityis shown in Supplemental Movies 4 and 10: the vastmajority of sporozoites are gliding (69�18%), while14% are attached (14�12%) or waving (14�8%) orwere classified as switching (3�2%) (Figs. 3 and 7A).Comparison of the sporozoite numbers and movementpatterns before and during flow revealed an increase ofthe total number of parasites found in the field of view(Fig. 7B). This was due to a larger increase in the rateof sporozoites that adhered compared to the rate ofsporozoites that detached from the surface and disap-peared from the field of view during flow (see Supple-mental Movies 10 and 11). Control parasites showed nosignificant difference in movement patterns, althoughthe number of waving sporozoites decreased duringflow from 14 � 8 to 2 � 3% (P�0.08; Fig. 7B–D).

Figure 6. Distinct adhesion capacities of sporozoites lackingTRAP family adhesins. Quantitative comparison to WT sporo-zoites revealed no significant change in sporozoite adhesionof trap(�), s6(�), or tlp(�) parasites at the midgut stage.When isolated from the hemolymph, trap(�) or s6(�), but nottlp(�) parasites, show a significant decrease in adhesion(P�0.05). When isolated from salivary glands tlp(�), sporo-zoites are retained better than WT (P�0.05), while s6(�)sporozoites show weaker adhesion (P�0.01). Note thattrap(�) sporozoites do not invade salivary glands. Errorbars � weighted sd from �3 independent experiments usingparasites from d 16–19 postinfection.

Figure 7. Adhesion-dependent gliding defects in tlp(�) sporozoites are compensated by flow. A) Movement patterns of WTsporozoites isolated from salivary glands (see Supplemental Movie 10): attached (blue), waving (red), gliding (gray), andswitching (green). Bars are weighted and normalized to percentage. Error bars � weighted sd from 3 independent experimentswith a total of 901 sporozoites. B) The number of adherent WT and tlp(�) sporozoites increases during flow (SupplementalMovie 11). Bars are weighted and normalized to conditions before flow. Error bars � weighted sd from 3 independentexperiments. C) Flow alters motility pattern of tlp(�) but not WT sporozoites significantly, as revealed by clustered t tests. Datafrom B were normalized to total number of sporozoites for each condition. Colors reflect motility patterns as in B.D) Comparative analysis of changes in movement patterns of WT and tlp(�) parasites. Changes in movement patterns for WTparasites occur in the same direction, albeit not at the same scale. Only change in number of switching sporozoites shows asignificant difference, indicating that TLP may play a role in the stability of sporozoite gliding. E) Flow induces a highlysignificant decrease in average number of circles (i.e., sporozoite speed) in WT, but a highly significant increase in tlp(�)sporozoites. Only sporozoites classified as gliding were examined. Whisker plots indicate 10–90% of all observed circles/100 s;box indicates 25–75 percentile; bar indicates median; � indicates arithmetic average. *P�0.05, **P�0.01, ***P�0.001.


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Examination of the normalized pattern for tlp(�) par-asites revealed a number of significant shifts (Fig. 7C):the number of gliding parasites significantly increasedby 25% (P�0.006); the quantity of switching sporozo-ites grew by 7.6-fold (P�0.005); and no more wavingsporozoites were detected (P�0.003).

We next evaluated the stability of gliding sporozoitesand counted the number of circles described by motilesporozoites during a time interval of 100 s before andduring flow (Fig. 7E). The average number of circleswas reduced from 2.9 (n�152 parasites) to 2.4 (n�332parasites) of WT sporozoites under flow conditions(P�0.002). However, tlp(�) sporozoites show a com-pletely different pattern: parasite speed, expressed asthe number of circles per 100 s, was not symmetricallydistributed around the arithmetic mean (median:1circle; mean: 2.2 circles). The majority of circlingsporozoites performed �1 circle/100 s, while otherswere gliding very efficiently. This highly asymmetricdistribution indicates the presence of �2 populationsof motile sporozoites. Interestingly, under flow condi-tions, a symmetric distribution was found around anaverage of 3.0 circles/100 s, which is similar to thespeed of WT sporozoites before flow (P�0.22). Theseresults show that only few tlp(�) sporozoites manage toglide efficiently, while the majority cannot glide for �1parasite length in the absence of flow. The hydrody-namic forces of the flow thus appear to compensatefor this defect and stabilize gliding, although thegeneral instability is still reflected by the increase inthe number of sporozoites that switch movementdirection (Fig. 7C). Taken together, TLP appears toincrease the probability of continued adhesion for-mation during gliding, either by stabilization ofnewly formed adhesion sites or by increasing thespeed of adhesion formation.

DISCUSSION

Multistep and developmentally regulated adhesion ofsporozoites

We showed that the adhesion of Plasmodium sporozoitesis a stepwise process that is partially dependent on thedynamics of parasite actin and membrane proteins ofthe TRAP family. Sporozoites contact a surface witheither their apical or proximal end or with a smallmembrane area between the two ends. Those attachingfirst at a different site than the proximal end usuallymove over the substrate at the newly built contact siteuntil the proximal end is reached. This is followed bytwo outcomes: forward gliding if the apical end has alsoestablished a contact with the substrate, or waving ifnot. During waving, the parasite undergoes actin-de-pendent active movements that lead to the movementof the entire sporozoite body around its attachedproximal end (Fig. 2 and Supplemental Fig 2). In theabsence of flow, waving is a common intermediary stepduring the adhesion sequence. Flow enhances adhe-

sion, likely by pushing the sporozoites toward thesubstrate. Furthermore, the tissue of origin plays adominant role in adhesion properties of sporozoites,with salivary gland sporozoites displaying the largestproportion of adhering parasites. In addition, we no-ticed that sporozoite adhesion increased with age inde-pendent of the origin within the mosquito vector,suggesting that adhesion is also a developmentallyregulated process (Fig. 5B).

Actin-dependent adhesion

The actin cytoskeleton is known to be important forparasite gliding motility (38, 39). Here, we presentevidence that actin filaments and their turnover areimportant for adhesion prior to gliding. In the pres-ence of cytochalasin D, a compound that binds to thegrowing ends of F-actin and thus leads to the depoly-merization of filaments, sporozoites are able to adherewith one end (usually their apical end) to the substrate,suggesting that rapid polymerization of actin is notneeded for this step. This is reminiscent of the adhe-sion of merozoites to red blood cells, as merozoites alsoattach to red blood cells in the presence of cytochalasinB, but do not enter them (40). Notably, actin polymer-ization appears to be required for the second adhesionstep, as sporozoites treated with cytochalasin D can nolonger undergo this step. Curiously, in the presence ofswinholide, which sequesters and inhibits the forma-tion of actin filaments by intercalating between theneighboring protomers in the filaments and by bindingin a 1:2 ratio to G-actin (41, 42), sporozoites can stillundergo a secondary adhesion step, but fail to com-plete adhesion (tertiary adhesion) and are not able toglide. An explanation for this difference between thetwo drugs is currently elusive. Possibly different num-bers of actin filaments remain under the influence ofthe two drugs. Interestingly, in the presence of jas-plakinolide, which leads to the formation of morefilamentous actin in merozoites and the related parasiteToxoplasma gondii (43–45), sporozoites do not adhereeither. Actin filaments appear dispensable for the firstadhesion step (primary adhesion), but too much F-actin leads to detachment, as shown when motilesporozoites were exposed to high concentrations ofjasplakinolide and subsequently to shear stress by ap-plication of flow (Figs. 3 and 8). Thus, the dynamicstate of the actin cytoskeleton or the pool of actinfilaments needs to be tightly regulated for efficientmotility.

Roles of TRAP family transmembrane proteins inparasite adhesion

trap(�) or s6(�) sporozoites were largely deficient inentering the salivary glands (17, 22) and showed areduced capacity to glide (17, 22) and to adhere (Fig.6). This implies that trap(�) and s6(�) sporozoiteseither have a defect in supporting complete adhesionor that they rapidly detach. In any case, these experi-


ments revealed a central role for both proteins instabilizing sporozoite adhesion, which is likely medi-ated by their respective extracellular adhesion do-mains, i.e., the von Willebrand A-domain and thethrombospondin repeat (31). Considering that bothTRAP and S6 are thought to be linked to actin fila-ments via aldolase binding to their cytoplasmic taildomain (46, 47), we tested the possibility that TRAPand S6 also regulate actin filament organization. Whenwe tried to rescue the defects of trap(�) and s6(�)sporozoites in adhesion with varying concentrations ofcytochalasin D and jasplakinolide (data not shown), wedid not see compensation of the adhesion defects.However, we cannot formally rule out an indirect effectby shifting the pool and/or organization of actin fila-ments to a state that does not support adhesion (Fig. 8).We suggest that both TRAP and S6 primarily mediateadhesion through their extracellular interactions withthe surface.

The apparent nonredundant roles for TRAP and S6in adhesion of hemocoel sporozoites is in good agree-ment with the complete life cycle arrest prior to salivarygland invasion of trap(�) sporozoites. This supports thenotion that TRAP function cannot be compensated forby other family members (17). In contrast, s6(�) para-sites retain residual capacity to invade salivary glands(22, 23). In support of these in vivo data, we show

residual, albeit significantly reduced, adhesion in s6(�)salivary gland-associated sporozoites. Apparently, thestrong defects observed in hemocoel sporozoites arepartially compensated for in mature, salivary glandsporozoites. Additional experimental genetics work,such as combinatorial mutants or stage-specific overex-pression of candidate proteins, is required to addressthe question of whether one TRAP family member canpotentially compensate for the partial defects in adhe-sion and deadhesion shown here for the s6(�) andtlp(�) mutants, respectively.

tlp(�) sporozoites were able to enter salivary glandsbut were sparsely found in the hemolymph, showingthat an increase in adhesion strength of the examinedmutants (Fig. 6) correlates directly with the rate ofsalivary gland infection. Not surprisingly, tlp(�) parasitesonly displayed a subtle phenotype by slowing down infec-tion in vivo (20, 21). In our assays, we found that indeedsimilar numbers of tlp(�) and WT sporozoites were ableto glide, but the tlp(�) sporozoites did so with much lesscontinuity than WT sporozoites, as 50% moved only 1circle, while 50% of WT sporozoites moved �3 com-plete circles (Fig. 7 and Supplemental Movies 10 and11). Consequently, gliding tlp(�) sporozoites detached�3-fold more frequently than WT sporozoites from thesubstrate to reorient themselves (Fig. 7 and Supple-mental Movie 11). No difference in the adhesive capac-ities between WT and tlp(�) sporozoites was detectablewhen sporozoites were isolated from midgut or hemo-lymph. However, in contrast to WT parasites, salivarygland tlp(�) sporozoites showed an increased ratio ofadhered parasites under flow (Fig. 6). WT sporozoitesadhered to the substrate and remained adherent (at-tached, searching, gliding) significantly (P�0.0013)longer than tlp(�) sporozoites. tlp(�) parasites may ad-here as often as the WT but lose adhesion more rapidly,resulting in a higher proportion of drifting parasites.Curiously, force-induced adhesion via hydrodynamicflow compensates this phenotype, probably due to thevertical vector component of the flow. Considering thesignificant (P�0.006) increase of gliding tlp(�) para-sites under flow and thereby improved ability for con-tinuous gliding, we suggest the following explanationfor the observed in vivo phenotype of tlp(�) sporozo-ites: the skin represents a tissue in which sporozoitesencounter little flow. In migrating through this tissue,loss of TLP function could result in reduced attach-ment and thus slower progression through the tissue,leading to lower entry rates into blood vessels. However,once tlp(�) sporozoites entered the blood circulation,

TABLE 1. Summary of adhesion outcomes leading to the scheme in Fig. 8

Parameter tlp(�), in flow WT, cyto Da WT tlp(�) s6(�) WT, jasb trap(�)

Initial adhesion � � o o � � �Adhesion rate � deadhesion rate � � o � � � �Circular gliding o / o � � / /

�, increased compared to WT (no-flow conditions); o, no apparent difference to WT (no-flow conditions); �, diminished compared to WT(no-flow conditions); /, feature absent. aUnder �2 �m cytochalasin D or swinholide. bUnder �2 �m jasplakinolide.

Figure 8. Proposed roles of TRAP family adhesins and actinfilaments during sporozoite adhesion and movement. Pool ofF-actin (black bars) contains polymers of heterogeneouslength. Numbers of filaments and their average length areextended in the presence of jasplakinolide (bottom bars) andreduced by cytochalasin D (top bars). TRAP, S6, and TLPmight play a role in modulating actin filament numbers andlength toward the optimal state necessary for continuousgliding (WT, shaded box). See also Table 1.


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the presence of flow would allow them the sameefficiency for motility as the WT. Considering thatmigration through cells is also less efficient in tlp(�)than in WT sporozoites (21), it is possible that adecreased capacity to adhere correlates with a de-creased capacity to invade or migrate through a cell.Parasites likely need to migrate through more cells inthe skin before entering the blood than they need tomigrate through in the liver to establish a liver infec-tion. Therefore, only a small delay in emergence fromthe liver stage could be detected when tlp(�) sporozo-ites were injected into the blood, while a larger delaywas observed when they were injected into the skin.One might be tempted to call for in vivo imaging todirectly visualize this effect. While this might be feasiblein the skin, we think the subtle but important pheno-type of the tlp(�) sporozoite in the mosquito and theliver calls for the development of better in vitro tools todissect similar subtle phenotypes of other parasitemutants. Our quantitative analysis and TIRF micros-copy are the first steps toward a more appropriatetoolkit for analyzing the sporozoite motility during thefirst steps of the malaria parasite in its mammalian host.This study also provides an example illustrating howbiophysical approaches using dynamic imaging of cellmotility can contribute to integrating enigmatic in vivofindings into mechanistic insights.

Probabilistic model of motility

We speculate that the three investigated sporozoitesurface proteins, TRAP, S6, and TLP, act in concert toachieve an optimal regulation of sporozoite attachmentand motility. As a working hypothesis, we propose amodel for sporozoite motility that is based on thelikeliness of adhesion and detachment to occur. Re-moval of different parts of the machinery governingthese steps leads to changes in the respective probabil-ities (Table 1). WT sporozoites coordinate adhesionand detachment in a manner leading to optimal gliding(Fig. 8) (48). Using parasites lacking TRAP, S6, or TLP,we found that TRAP and S6 are required for sporozoiteadhesion, with TRAP playing a more prominent role.TLP appears to be important for the regulation ofadhesion and detachment during movement and there-fore coordinating the continuity of gliding. We furtherpropose that actin filaments play a role in the coordi-nated cycle of adhesion and deadhesion that appearsnecessary during sustained motility (48): the experi-ments with inhibitors suggest that shorter (or fewer)actin filaments enhance the ability to adhere, thusdiminishing the capacity to detach, while longer (ormore) filaments lead to the opposite effect (Fig. 8).During motility, the situation is even more complex, astrap(�) sporozoites that do adhere also show a defect indeadhesion (48).

This study shed light not only on the sequentialadhesion steps that lead to sporozoite motility but alsoidentified distinct roles of actin dynamics and TRAPfamily proteins in this process. While the detailed

molecular mechanisms and the interplay of the pro-teins involved needs to undergo further examination,our study highlighted new roles for the key players anduncovered a molecular link between adhesion andmotility of sporozoites.

The authors thank Kai Uhrig for discussions and com-ments on the manuscript. The work was funded by grantsfrom the Federal German Ministry for Education and Re-search (BMBF-BioFuture), the German Research Foundation(DFG-SFB 544), and the European Network of ExcellenceBioMalPar. The authors gratefully acknowledge support fromthe University of Heidelberg Medical School and the Clusterof Excellence CellNetworks. The authors thank Diana Schep-pan and Iris Arnold for mosquito infection, Jan-Peter Rodererfor help with data analysis, and the Nikon Imaging Center atthe University of Heidelberg for access to their microscopes.The authors declare no conflicts of interest.

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Received for publication October 19, 2009.Accepted for publication January 21, 2010.


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Multistep adhesion of Plasmodium sporozoites

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