plasmodium sporozoite biology - cshl...

Plasmodium Sporozoite Biology

Friedrich Frischknecht1 and Kai Matuschewski2

1Integrative Parasitology, Center for Infectious Diseases, University of Heidelberg Medical School,69120 Heidelberg, Germany

2Department of Molecular Parasitology, Institute of Biology, Humboldt University Berlin,10115 Berlin, Germany

Correspondence: [email protected]; [email protected]

Plasmodium sporozoite transmission is a critical population bottleneck in parasite life-cycleprogression and, hence, a target for prophylactic drugs and vaccines. The recent progress of acandidate antisporozoite subunit vaccine formulation to licensure highlights the importanceof sporozoite transmission intervention in the malaria control portfolio. Sporozoites colonizemosquito salivary glands, migrate through the skin, penetrate blood vessels, breach the liversinusoid, and invade hepatocytes. Understanding the molecular and cellular mechanismsthat mediate the remarkable sporozoite journey in the invertebrate vector and the vertebratehost can inform evidence-based next-generation drug development programs and immuneintervention strategies.

Malaria-related pathology is exclusivelycaused by asexual parasite propagation

inside vertebrate erythrocytes, rendering thelife-cycle phases preceding blood infection aprime window of opportunity for targetedprophylactic interventions. The time from aninfectious Anopheles bite to the occurrenceof the first parasites in the peripheral blood,the so-called prepatent period, reflects the obli-gate pre-erythrocytic tissue phase that leadsto a dramatic expansion of the Plasmodiumpopulation from a tiny sporozoite inoculum.This phase is clinically silent and remains di-agnostically inaccessible. Intriguingly, phyloge-netic placement of mammalian Plasmodiumparasites suggests that asexual blood replicationis the exception rather than the rule (Perkins2014). Closely related taxa, including Hepato-

cystis and Nycteria, are nonpathogenic, andfirst-generation tissue merozoites do not rep-licate asexually inside erythrocytes. Instead,these parasites differentiate straight into micro-gametes and macrogametes. In any case, suc-cessful completion of the obligate pre-erythro-cytic development is most critical for hostcolonization, propagation of parasite popula-tions, and onward transmission. Despite the re-cent awareness of the central importance ofsporozoite-based interventions for malaria con-trol many fundamental knowledge gaps remainand a broader molecular, cellular, and immu-nological understanding of sporozoite–hostcross talk is urgently needed. Here, we presentan overview of the foundation, recent discover-ies, and challenges in Plasmodium sporozoitebiology.

Editors: Dyann F. Wirth and Pedro L. Alonso

Additional Perspectives on Malaria: Biology in the Era of Eradication available at www.perspectivesinmedicine.org

Copyright # 2017 Cold Spring Harbor Laboratory Press; all rights reserved

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THE NATURAL HISTORY OF SPOROZOITES

Plasmodium sporogony, an extracellular phaseof asexual replication, occurs in Anophelesmosquitoes, although some species of lizardplasmodia, for instance Plasmodium mexica-num, can also be transmitted by sand flies(Ayala 1971). The onset of sporogony is markedwhen ookinetes settle after their journey fromthe blood meal across the midgut epithelium.They transform and develop into oocysts un-derneath the basal lamina surrounding the di-gestive organ of Anopheles mosquitoes (Beier1998). Curiously, the distribution of oocystscan differ in mosquitoes depending on the in-sect’s ability to agglutinate blood. In mosqui-toes that agglutinate blood, parasites settle atthe bottom of the digestive organ followingthe force of gravity, whereas in those who donot, for example, in Anopheles stephensi, theydistribute randomly (Shortt 1948; Kan et al.2014). Within the oocysts, sporozoites developover the course of 10 days to 4 weeks dependingon the Plasmodium species and ambient tem-perature before they emerge into the circulatoryfluid of the insect, the hemolymph (Figs. 1 and2). They are crescent-shaped, 8- to 14-mm long,and display substrate-dependent locomotionknown as gliding motility (Figs. 3 and 4). With-in the hemolymph, sporozoites are transported

throughout the body cavity but seem to specif-ically interact with and enter into salivaryglands, crossing the basal lamina surroundingthis organ and passing through the saliva-pro-ducing acinar cells (Sterling et al. 1973; Pi-menta et al. 1994; Douglas et al. 2015). Sporo-zoites accumulate in the salivary cavities fromwhere they can also move into the narrow sali-vary ducts that connect to the proboscis. Spo-rozoites are considered mature once they reachthe salivary cavities, but experimental infec-tions with sporozoites liberated from oocystsor collected from the hemolymph can initiatea malaria episode (Shute 1943; Vanderberg1975; Sato et al. 2014). During an infectiousbite, the mosquito ejects saliva and with it asmall fraction of the sporozoites in the gland.Most sporozoites are deposited in the dermis ofthe host during the probing phase when themosquito searches for a blood vessel to punc-ture (Sidjanski and Vanderberg 1997; Mat-suoka et al. 2002; Vanderberg and Frevert2004; Amino et al. 2006). Once the mosquitosucks up blood, sporozoites that continue to beejected with the saliva appear to be largely re-ingested into the mosquito midgut by thestronger flow and, thus, are lost for transmis-sion (Kebaier and Vanderberg 2006). Sporozo-ites in the dermis can actively migrate at high-speed passing through several skin cells and

Figure 1. Sporozoite formation within the oocyst. Transmission electron micrograph showing Plasmodiumberghei sporozoites budding from sporoblasts 10 days after the uptake of parasites by the mosquito. On theright image, some selected structures are highlighted. Blue, plasma membrane; yellow, inner membrane com-plex; green, microtubules; magenta, nascent rhoptries; cyan, nascent micronemes; brown, rootlet fiber; lightblue, nucleus. The image is 4 mm wide. (Image courtesy of Mirko Singer and Stefan Hillmer.)

F. Frischknecht and K. Matuschewski

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enter blood or lymphatic vessels (Amino et al.2006, 2008). Sporozoites of plasmodia that in-fect birds and lizards can enter into and differ-entiate within phagocytic cells of the reticulo-endothelial system, including within the skin.Sporozoites of plasmodia that infect mammalsneed to enter the bloodstream to be transport-ed to the liver, in which they specifically differ-entiate in hepatocytes. To access this niche spo-rozoites arrest in the sinusoids of the liver andpass through endothelial cells or Kupffer cells,liver resident macrophages, to gain access to theunderlying hepatocytes (Baer et al. 2007; Ta-vares et al. 2013). Here again, they usually mi-grate through a few hepatocytes before settlingin one for differentiation into thousands of redblood cell– invading merozoites (Mota et al.2001; Prudencio et al. 2006). Hence, sporozo-ites are the most versatile of Plasmodium stagesbecause they are formed in the invertebrate hostto eventually differentiate in the vertebratehost. On their journey, sporozoites undergomassive changes in their proteomic makeup,migrate actively through different cells in dif-ferent tissues, and use the circulatory fluid ofvector and host for reaching their intermediate(salivary gland) and final (hepatocyte) destina-tion. It is no surprise then that the sporozoite is

as exciting and enigmatic a parasite stage tostudy with still plenty of scope for fundamentaldiscovery.

THE IMMUNE-DOMINANT SPOROZOITEANTIGEN: CSP

The circumsporozoite precipitation test was thefirst antigen assay that permitted a correlationof antisporozoite antibody titers with sterileprotection in a volunteer that was immunizedwith high doses of Plasmodium falciparum–in-fected irradiated mosquitoes (Clyde et al. 1973).In this test, sporozoite surface antigens werebroadly precipitated and the major proteinwas termed circumsporozoite protein (CSP).CSP is thought to be secreted at the anteriorsporozoite tip, translocated backward to the an-terior end, and abundantly shed into the micro-environment (Stewart and Vanderberg 1991).CSP was among the first cloned and sequencedPlasmodium antigens (Dame et al. 1984; Eneaet al. 1984) and is structurally similar in differ-ent Plasmodium species (Sinnis and Nardin2002). CSP is a glycosylphosphatidylinositol(GPI)-anchored membrane protein and con-tains two conserved regions flanking a centralrepeat region of various lengths.

Figure 2. Sporozoites in the Anopheles vector. (A) A mosquito infected with green fluorescent parasites. Note thegreen fluorescence of sporozoites secreted with saliva at the front of the proboscis (arrowhead). (B) Sporozoitesin the veins of a mosquito wing. Arrowheads point to two sporozoites stuck in the veins and two that are streakedout because of the fast movement of the hemolymph. (C) Sporozoites (arrowheads) within the salivary canal ofthe proboscis as they are ejected from the mosquito. (Courtesy of Biology of Parasitism course 2015 at the MarineBiological Laboratory, Woods Hole.)

Plasmodium Sporozoites

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A formulation that contains a fusion proteinof the P. falciparum CSP carboxyl terminus, in-cluding 19 repeats, the hepatitis B surface (S)antigen, and the very strong, investigational ad-juvant AS01, termed RTS,S/AS01, is the firstadvanced candidate antimalaria vaccine (Cohenet al. 2010). The recent completion of the firstmulticenter phase III antimalaria vaccine trialin sub-Saharan Africa (RTS,S Clinical TrialsPartnership 2015) highlighted the reputationof targeting sporozoite transmission on a pop-ulation level. Among the aims of the RTS,S/AS01 vaccine and improved formulations wasto elicit a strong, nonnatural immune responseagainst sporozoites, which is typically not ac-

quired during natural transmission in endemicareas (Offeddu et al. 2012).

As predicted for a stage-specific major sur-face protein, targeted deletion of Plasmodiumberghei CSP did not affect blood infection butaborted sporozoite formation, yielding vacuo-lated oocysts (Menard et al. 1997). This was fur-ther confirmed by down-regulation of CSP,which resulted in aberrant development of oo-cyst membranes and stumpy, noninfectioussporozoites (Thathy et al. 2002). The amino-ter-minal region I apparently mediates sporozoiteinvasion of invertebrate and mammalian hostcells (Sidjanski et al. 1997; Aldrich et al. 2012).Region I and a carboxy-terminal region II bind

Mito

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Figure 3. Shape and subcellular structure of the Plasmodium sporozoite. (A) Schematic showing the cellularorganelles and their position and relative size in the Plasmodium sporozoite. PPR, proximal polar ring; Mito,mitochondrion; PM, plasma membrane (blue); ER, endoplasmic reticulum; IMC, inner membrane complex(yellow); NPC, nuclear pore complex; Ap, apicoplast (yellow); MT, microtubules (green); DG, dense granules(brown); Rho, rhoptries (magenta); Mic, micronemes (cyan); APR, apical polar ring (red). Note the single stackGolgi apparatus and the nucleus being associated to the IMC. (B) Scanning electron micrograph of a Plasmo-dium berghei sporozoite. Sporozoites are an elongated crescent shape and tailored for lasting continuouslocomotion and host cell invasion. When deposited on a substrate sporozoites alternate gliding periods withnongliding phases. Sporozoite material that is shed during gliding locomotion marks the trails. (Image fromMontagna et al. 2012c; adapted, with permission, from the authors.) (C) Computer tomogram of the apical endof a sporozoite. Highlighted are plasma membrane (blue), inner membrane complex (yellow), microtubules(green), rhoptries (magenta), and micronemes (cyan).



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heparin-sulfate proteoglycans on the surface ofliver cells (Cerami et al. 1992; Frevert et al. 1993).On the developmental switch from cell traversalto invasion CSP is proteolytically cleaved there-by exposing region II, indicating that this regionis important for liver colonization and coveredthroughout most of the sporozoite journey toprevent premature cell invasion (Coppi et al.2011). Proteolytic processing of CSP can be tar-geted by protective antibodies (Espinosa et al.2015), indicating that RTS,S/AS01 might beconsiderably improved by replacing the truncat-ed antigen with full length CSP.

An attractive path for in vivo testing ofCSP-based vaccines was the generation of

transgenic P. berghei parasites that harbor aCSP from human-infecting parasites. Thisproved difficult for PfCSP, because comple-mented P. berghei parasites are severely im-paired in salivary gland colonization andyield only very few and impaired sporozoitesfor transmission experiments (Tewari et al.2002). However, P. berghei parasites harboringa chimeric CSP containing the repeat regionfrom P. falciparum or Plasmodium vivax CSP(Persson et al. 2002; Espinosa et al. 2013) and,more recently, complemented Plasmodiumyoelii sporozoites (Zhang et al. 2016) can beused in robust infection models. Naturally ac-quired antisporozoite antibody responses are

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Figure 4. Sporozoite motility in vitro. (A) Three sporozoites move in a circular fashion with their apical endleading (red arrowheads) at speeds above 1 mm/sec. Their near-perfect circular trajectory is revealed by themaximum intensity projection (0–60). Time between images is indicated in seconds. Scale bar, 10 mm. (B)Reflection interference contrast microscopy reveals the contact points between sporozoites and substrate (darkareas). Arrowheads point to the same positions of a migrating sporozoite. Note that at these positions the darkand bright areas appear and disappear highlighting the dynamic adhesion to the substrate. Time between imagesis indicated in seconds. Scale bar, 10 mm. (C) Schematic of the sporozoite pellicle with a focus on the core glidingmotility machinery.



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almost exclusively directed against CSP repeatsbut a potential association with immunityagainst malaria in children and adults remainselusive (Offeddu et al. 2012). This remarkableimmunodominance could well be a mecha-nism of immune evasion (Schofield 1990)and could explain the modest efficacy of theRTS,S/AS01 vaccine (RTS,S Clinical TrialsPartnership 2015). Hence, deciphering thecellular and immunological functions of CSPin parasite–host interactions and discoveringadditional protective sporozoite antigens re-main research priorities (Hafalla et al. 2011,2013).

SPOROZOITE GLIDING MOTILITY, TRAPFAMILY ADHESINS, AND ACTIN FILAMENTS

Sporozoites need to be motile to succeed intheir arduous journey. Evidence suggests thatsporozoites actively exit the oocysts, but the re-quirement of motility has not been formallyshown. Several proteins, including CSP andegress cysteine protease 1 (ECP1) (Aly and Ma-tuschewski 2005; Wang et al. 2005), function insporozoite egress indicating an active partici-pation by the sporozoites, but liberation mightbe further aided by oocyst rupture. Subse-quently, sporozoites need to actively migrateto enter salivary glands. Inside the salivaryglands, most sporozoites stop moving and ag-gregate within the salivary cavities, whereas afew move slowly down the salivary ducts(Frischknecht et al. 2004). Once in the dermis,sporozoites move at average speeds between 1and 2 mm/sec, about 10 times as fast as neutro-phils migrate (Amino et al. 2006). Without thismotility, sporozoites are stuck in the skin andhosts remain largely uninfected after mosquitobites (Montagna et al. 2012a). After enteringblood vessels, sporozoites use their motility toinfect the liver, although they can efficiently ac-complish this with much diminished migrationcapacity (Montagna et al. 2012a).

To move, sporozoites need to attach to asubstrate and generate a force against this sub-strate. Furthermore, they must continuouslygenerate new substrate attachment sites and

turn them over (Fig. 4B). How this is allachieved is not yet understood completely buta complex picture emerges; plasma-membrane-spanning proteins of the thrombosponin-re-lated anonymous protein (TRAP) family, actinfilaments, and actin-binding proteins includ-ing the motor protein myosin and a complexof myosin anchoring proteins form a transientmotor complex that integrates signaling pro-teins and molecules, such as calcium andpossibly cAMP and cGMP (Ono et al. 2008;Montagna et al. 2012b; Carey et al. 2014; Laksh-manan et al. 2015).

The gene coding for TRAP was the first geneto be deleted from the parasite genome thatrevealed an effect in parasite motility (Sultanet al. 1997). TRAP is specifically expressed insporozoites and trap – sporozoites can neithermove continuously nor enter into salivaryglands, resulting in a complete life-cycle arrestjust before sporozoite transmission. Curiously,trap – sporozoites still attach to substrates andmove back and forth over single adhesion sites(Munter et al. 2009). It thus appears that TRAPplays a role in generating directional migration,although precisely how it does this is currentlynot understood. Parasites expressing TRAPwith point mutations in conserved residues ofthe cytoplasmic tail also show aberrant motilitysuggesting that TRAP links extracellular inter-actions with the interior to modulate motility(Kappe et al. 1999). Subtle mutations in theextracellular domains of TRAP that mediate ad-hesion to the substrate lead to no effect on glid-ing motility of sporozoites but abrogate sporo-zoite invasion into salivary glands and liver cells(Matuschewski et al. 2002a). Notably, replace-ment of P. berghei TRAP with the P. falciparumortholog affects gliding motility and partly re-produces the defects observed in trap – sporo-zoites (Wengelnik et al. 1999). This finding is inagreement with species-specific signaling rolesduring motility, as suggested by structural stud-ies of the adhesive domains (Song et al. 2012).

TRAP is the founding member of a largerfamily of TRAP-like adhesins, and sporozoitesexpress two additional members, TRAP-likeprotein (TLP) and TREP/S6/UOS3. Ablationof these genes revealed a major function for



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TREP during sporozoite invasion of salivaryglands (Combe et al. 2009; Steinbuchel andMatuschewski 2009) and a role for TLP duringmigration in the skin (Moreira et al. 2008;Hegge et al. 2010a; Hellmann et al. 2011). Re-placing the cytoplasmic tail of TRAP with thatof TLP showed that sporozoites expressingthis hybrid protein could still move, albeit notas efficiently as wild-type sporozoites (Heisset al. 2008). This result suggests that the cyto-plasmic tails of TRAP and TLP bind to the sameprotein(s) but possibly with different affinities.TRAP family proteins are trafficked to andstored within micronemes and released to thesurface on activation of sporozoites (Ganttet al. 2000; Carey et al. 2014). Sporozoites canbe activated by serum albumin (Vanderberg1974) or small ligands (Perschmann et al.2011), which leads to an increase in intracellu-lar calcium followed by exocytosis of TRAP(Carey et al. 2014). Strikingly, only a small per-centage of the total amount of TRAP familyproteins is on the surface of activated sporozo-ites (Gantt et al. 2000), and it appears that theirnumber is limited for efficient migration(Perschmann et al. 2011). This scarceness ofTRAP family proteins makes investigation oftheir role in motility using classic GFP taggingand microscopy localization approaches a for-midable challenge.

Actin filaments are moved by myosin mo-tors in most examined cells and such is the casealso in Plasmodium. Myosin is anchored to theinner membrane complex (IMC), a flattenedorganelle that subtends the plasma membraneof apicomplexan parasites at a close distance of�30 nm in sporozoites (Fig. 4C). Myosin islikely also connected across the IMC to the sub-pellicular network, which gives the sporozoiteits shape (Khater et al. 2004; Kudryashev et al.2012). The subpellicular network can be con-sidered to be a rigid structure that provides acounterforce for the moving myosin. Thus, my-osin can push actin filaments toward the back ofa sporozoite, and when TRAP family adhesinsare connected to the filaments the adhesins arepushed back simultaneously. This retrogradeflow can be indirectly seen by the motion oftissue debris or small microspheres attached to

activated sporozoites (Munter et al. 2009; Quadtet al. 2016). When sporozoites are attached to arigid substrate they move forward by the samemechanism of force and counterforce.

Yet, actin filaments are elusive structuresin sporozoites. Unlike actin in metazoan cells,which forms long filaments, Plasmodium actinfilaments are short and feature a highly dynamicturnover (Schmitz et al. 2005; Schuler et al.2005). This dynamic turnover is mediated by anumber of actin-binding proteins (Sattler et al.2011) and is also built into the structure of Plas-modium actin itself (Skillman et al. 2011; Vaho-koski et al. 2014). Again, reverse genetic analysishas revealed some insight into which proteinsare important in regulating actin dynamics insporozoites. For example, sporozoites lacking asubunit of the heterodimeric actin filament cap-ping protein (CP) moved in a similar fashion astrap – sporozoites and also failed to enter sali-vary glands (Ganter et al. 2009). The resultinglife-cycle arrest could only be rescued by bothorthologous P. falciparum CP subunits, indicat-ing distinct species-specific adaptations of mi-crofilament regulation in sporozoites (Ganteret al. 2015). One basic open question is howactin filaments are oriented in the sporozoite.For directed forward motility, it is generally en-visaged that actin filaments are aligned alongthe parasite long axis so that myosin motorscan move them backward. The phenotypicback-and-forth movements of trap – and cp –

sporozoites suggest that in these parasites theactin filaments might not be properly orientedthus leading to aberrant movement, but with-out direct visualization of the filaments this hy-pothesis remains conjecture.

Even if we were to understand the interplaybetween TRAP family adhesins and actin, theprocess of movement likely involves moreplayers. TRAP itself is likely cleaved by an intra-membrane protease of the rhomboid family andthis cleavage is important to achieve continuousmigration (Ejigiri et al. 2012). Moreover, an in-hibitor of cysteine proteases is necessary forsporozoite motility, and icp – sporozoites repro-duce the life-cycle arrest observed in trap –-in-fected mosquitoes (Boysen and Matuschewski2013; Lehmann et al. 2014).



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Several Plasmodium-specific membraneproteins that do not belong to the TRAP familyof adhesins, including SIAP-1/ag17/S5, S23/SSP3, PCRMP3, and PCRMP4, play criticalroles in sporozoite transmission (Engelmannet al. 2009; Douradinha et al. 2011; Harupaet al. 2014), indicative of a complex and nonre-dundant protein network that mediates sporo-zoite–host interactions. Deletion of the smallheat shock protein HSP20 also slowed motility,which showed the essential role of sporozoitemigration in the skin, as hsp20 – parasites stillentered the salivary gland but moved at very lowspeed and could not migrate efficiently throughthe dermis (Montagna et al. 2012a,c). Consid-ering the numbers of unexplored proteins pre-sent in sporozoites (Matuschewski et al. 2002b;Kaiser et al. 2004; Lasonder et al. 2008; Lindneret al. 2013a), it is likely that many of these will beimportant for migration, moving a completeunderstanding of this crucial and fascinatingprocess a few decades into the future.

IN AND OUT OF CELLS: FROM GLIDINGTO INVASION

During sporozoite migration the parasites needto pass through cells of the hosts to cross thebarriers constituted by the salivary gland, theskin, and the liver (Fig. 5) (see online Movie 1at perspectivesinmedicine.cshlp.org). To do so,the sporozoite uses CSP, TRAP, and other pro-teins that recognize target cells and allow sporo-zoites to pass them (Ishino et al. 2004, 2005).

The salivary gland surface is likely initially rec-ognized by MAEBL (Kariu et al. 2002; Saenzet al. 2008). Thereafter, TRAP interacts specifi-cally with the mosquito protein saglin to allowentry into the glands (Ghosh et al. 2009), a pro-cess so far only observed by electron microscopy(Sterling et al. 1973; Pimenta et al. 1994). Thesestudies suggest that sporozoites form a tightjunction with the acinar cells of the salivarygland on their way in and curiously “bud” outfrom the cells at the apical end facing the salivarycavities such that they are initially surroundedby a host cell membrane (Pimenta et al. 1994).How they shed this membrane and arrange inlarge nonmotile aggregates is not clear. Once inthe skin, sporozoites pass through dermal cellsby breaching their membranes as revealed by invivo imaging of migrating sporozoites injectedinto mice along with a cell-wounding marker(Formaglio et al. 2014). This wounding phe-nomenon is clearly associated with the capacityof the sporozoite to migrate over long distancesin the dermis because ablation of genes prevent-ing cell wounding inhibits intradermal motility(Bhanot et al. 2005; Amino et al. 2008; Risco-Castillo et al. 2015).

It is currently not clear whether sporozoitesalso need to breach endothelial cells to enter thebloodstream but they clearly can do so whenthey move from the bloodstream toward the he-patocytes (Tavares et al. 2013). They can alsoenter through the liver resident macrophagescalled Kupffer cells (Baer et al. 2007). In bothcases, sporozoites need their capacity to wound

Sporozoites

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Figure 5. Sporozoite migration in vivo. Sporozoite migration after transmission into the dermis. Shown aresporozoites (white) ( far left), the tracks of sporozoites migrating for 50 sec (left), sporozoites (yellow) at the startof the movie overlaid by the tracks (red) (center), and tracks over 50 sec (right) and 500 sec (far right) spatiallycolor-coded such that red, green, and blue colors represent different confocal planes 10 mm apart. (Shots takenfrom movie courtesy of Rogerio Amino, Institut Pasteur, Paris.)



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the cells to progress toward hepatocytes. Evenwhen they reach hepatocytes they continueto wound several cells before settling in a lasthepatocyte (Mota et al. 2001; Frevert et al.2005).

Two competing models aim at explaininghow a sporozoite finally productively infects ahepatocyte. The first model suggests that specif-ic signaling occurs to “prepare” the sporozoitefor entry (Mota et al. 2002), whereas the secondone suggests a switch of the sporozoite from ageneric “wounding and transmigration” modeto an invasion mode (Amino et al. 2008). In thefirst model, the host cells play a role in providinga signal to the sporozoite. The second modelsuggests a stochastic process that stipulatesthat the number of proteins needed for trans-migration is limited and that once they havebeen used up the sporozoite will settle in which-ever cell is the next on its path. Experimentssuggest that both mechanisms might playa role. The lack of one of the major proteinsneeded for transmigration, termed SPECT,causes sporozoites to invade and develop withinfibroblasts in the dermis (Amino et al. 2008;Gueirard et al. 2010). Hence, it is clear that inthe absence of SPECT no further signal is re-quired to a full invasion mode, which apparent-ly is the default activity of sporozoites. However,this does not rule out that in the presence ofSPECT additional regulatory processes alsoplay a role. One such process could be the sens-ing by the sporozoite of the higher potassiumconcentration within cells than in the extracel-lular medium (Kumar et al. 2007; Ono et al.2008). High potassium buffers have been shownto increase the intracellular cAMP concentra-tion, which causes increased intracellular Ca2þ

concentrations followed by exocytosis of micro-nemes. This brings more TRAP onto the surfaceof sporozoites, arrests their motility, and leadsto more invasion (Ono et al. 2008). Also, asdescribed above, the processing of CSP on con-tact to highly sulfated proteoglycans was shownto switch the sporozoite toward an invasivemode (Coppi et al. 2011). Thus, a stepwise pro-cess involving first CSP processing followed bypotassium signaling might, among others, con-tribute to sporozoite invasion.

Intriguingly, we still do not know the recep-tor–ligand pairs involved in the formation of atight junction that appears to be essential forhost cell invasion in Plasmodium and relatedparasites and thus possibly also in sporozoiteinvasion of hepatocytes. TRAP and AMA-1 onthe sporozoite appear to be involved (Matu-schewski et al. 2002a; Silvie et al. 2004). Onthe host cell side, several proteins have beensuggested to be important for invasion includ-ing fetuin (Jethwaney et al. 2005), CD81 (Silvieet al. 2003; Risco-Castillo et al. 2014), and CD68(Cha et al. 2015). Although fetuin might di-rectly interact with TRAP, the roles of CD81and CD68 are likely more complex as no directinteraction with a sporozoite protein could beshown yet.

Once in the host cells, the sporozoite rapidlydedifferentiates, not undetected by the host.During this dedifferentiation, the IMC and thesubpellicular network disassemble leading to around parasite. Curiously, a similar roundingup of sporozoites can be detected in vitro inthe absence of host cells (Kaiser et al. 2003;Hegge et al. 2010b). This transformation pro-gram is initiated prematurely in salivary glandsporozoites that lack UIS1/IK2, a sporozoite-specific protein kinase (Zhang et al. 2010), orPUF2, which encodes a RNA binding protein(Gomes-Santos et al. 2011; Muller et al. 2011;Lindner et al. 2013b). Sporozoites transcribeand store mRNAs that encode proteins requiredfor liver stage development. For instance, trans-lational repression of UIS4, encoding an essen-tial component of the liver stage parasitopho-rous vacuolar membrane, is important forsporozoite infectivity and subsequent stage con-version (Silvie et al. 2014). Deciphering the en-tire signaling network that controls translationalrepression and sporozoite latency is central to abetter understanding of the host switch betweenthe insect and mammalian hosts.

PERSPECTIVES

Considering the potential medical applicationof whole sporozoite vaccination, it would be afundamental progress if initial results of axenicsporozoite cultures (Warburg and Miller 1992;



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Warburg and Schneider 1993; Al-Olayan et al.2002; Porter-Kelley et al. 2006) could be sub-stantially improved to allow mass productionof sporozoites without the need for mosquitocolonies. Especially, if this could be applied tothe different human infective parasite speciesresearch on the sporozoite would be boostedto new heights, as the parasite would becomeworkable without the need of running an insec-tary, which for the case of human parasitesneeds to be in a costly biosafety environment.Furthermore, one of the black boxes of sporo-zoite biology concerns its interaction with thesalivary gland. Also, this would be pushed for-ward if it were possible to culture salivary glandsin vitro for extended periods or generate stablecell lines from the terminally differentiated sali-vary gland cells, which clearly is no minor task.Furthermore, most in vitro assays currentlyused to investigate the sporozoite are not con-sidering the flow of the blood, so incorporatingmicrofluidic devices into sporozoite researchalso promises a molecular and biophysical un-derstanding of the key interactions that arrestthe sporozoite at the liver.

Last, the field would benefit from standard-ized in vitro liver culture systems that wouldallow higher throughput research on the fasci-nating sporozoite liver cell interaction, whichcould lead to the discovery of the molecularmechanism of sporozoite liver cell entry. Ideally,such a culture system would also include otherliver cells with which the sporozoite interactssuch as endothelial and immune cells to retraceand dissect its journey into the liver. Such func-tional assays would then also require a highercomputerized image data throughput, whichshould not be the bottleneck considering theprogress in imaging techniques in other fields.Such a set of new assay systems would be valu-able for investigating innovative approaches tostop the sporozoite, which might ultimatelycontribute to control malaria.

CONCLUDING REMARKS

The Plasmodium sporozoite constitutes the firstform of the malaria parasite entering the hu-man body and, hence, provides the first and

leading targets to control an infection. Onlyfew (�10–100) sporozoites are injected by in-fected mosquitoes, suggesting that they formexcellent intervention targets. Nonetheless, itmight be particularly challenging to eliminateevery one of these few individuals, because asingle sporozoite breakthrough will initiate afulminant blood infection. Sporozoites couldbe targeted by small molecules and/or antibod-ies by either stopping the rapid migration andliver entry of sporozoites or direct killing. Yet,intervention programs focusing on sporozoitesremain scarce and underexplored. Antibodiesagainst the major surface protein CSP havebeen shown to prevent a detectable blood in-fection under experimental conditions. How-ever, the clinical trials results of RTS,S/AS01were unsatisfactory and failed to prevent theoccurrence of natural infections. The searchfor new sporozoite antigens and a better un-derstanding of the molecular mechanisms thatdrive sporozoite motility and liver cell entry areexpected to yield previously unrecognized tar-gets that could turn into urgently needed con-trol measures.

ACKNOWLEDGMENTS

We thank Ross Douglas and Alyssa Ingmund-son for comments on the manuscript.

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published online January 20, 2017Cold Spring Harb Perspect Med Friedrich Frischknecht and Kai Matuschewski

Sporozoite BiologyPlasmodium

Subject Collection Malaria: Biology in the Era of Eradication

Modern Vector ControlNeil F. Lobo, Nicole L. Achee, John Greico, et al.

Malaria PathogenesisDanny A. Milner, Jr.

Malaria ControlVectorial Capacity and Potential Avenues for Anopheline Reproductive Biology: Impacts on

Sara N. Mitchell and Flaminia Catteruccia

Population LevelDeterminants of Malaria Transmission at the

Teun Bousema and Chris Drakeley

Malaria Transmission via the Use of InsecticidesCurrent and Future Prospects for Preventing

Hilary Ranson EliminationMalaria Parasites: Challenges for Malaria Host Cell Tropism and Adaptation of Blood-Stage

Caeul Lim, Selasi Dankwa, Aditya S. Paul, et al.

Malaria Parasites from Host ErythrocytesMolecular Signaling Involved in Entry and Exit of

Shailja Singh and Chetan E. ChitnisEradication: The Role of the EnvironmentMalaria Transmission and Prospects for Malaria

Marcia C. Castro

Eventual EradicationVaccines to Accelerate Malaria Elimination and

al.Julie Healer, Alan F. Cowman, David C. Kaslow, et

Plasmodium vivaxThe Biology of John H. Adams and Ivo Mueller

Immune Responses in MalariaCarole A. Long and Fidel Zavala

Malaria Genomics in the Era of EradicationDaniel E. Neafsey and Sarah K. Volkman

EliminationAntimalarial Drug Resistance: A Threat to Malaria

Didier Menard and Arjen Dondorp

Malaria EpigeneticsAlfred Cortés and Kirk W. Deitsch

Malaria during PregnancyMichal Fried and Patrick E. Duffy Exoerythrocytic Biology

Malaria Parasite Liver Infection and

Ashley M. Vaughan and Stefan H.I. Kappe

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