Malaria Parasite Liver Infection and Exoerythrocytic...

Malaria Parasite Liver Infectionand Exoerythrocytic Biology

Ashley M. Vaughan1 and Stefan H.I. Kappe1,2

1Center for Infectious Disease Research, formerly Seattle Biomedical Research Institute, Seattle,Washington 98109

2Department of Global Health, University of Washington, Seattle, Washington 98195

Correspondence: [email protected]

In their infection cycle, malaria parasites undergo replication and population expansionswithin the vertebrate host and the mosquito vector. Host infection initiates with sporozoiteinvasion of hepatocytes, followed by a dramatic parasite amplification event during liverstage parasite growth and replication within hepatocytes. Each liver stage forms up to 90,000exoerythrocytic merozoites, which are in turn capable of initiating a blood stage infection.Liver stages not onlyexploit host hepatocyte resources for nutritional needs but also endeavorto prevent hepatocyte cell death and detection by the host’s immune system. Research overthe past decade has identified numerous parasite factors that play a critical role during liverinfection and has started to delineate a complex web of parasite–host interactions thatsustain successful parasite colonization of the mammalian host. Targeting the parasites’obligatory infection of the liver as a gateway to the blood, with drugs and vaccines, consti-tutes the most effective strategy for malaria eradication, as it would prevent clinical diseaseand onward transmission of the parasite.

The genus Plasmodium consists of many dif-ferent parasite species, each with a narrowhost range and the causative agent of the diseasemalaria in its respective host. Plasmodiumparasites have evolved exceedingly complex lifecycles, using mosquitoes as the vehicle of trans-mission to infect reptiles, birds, and mammals,including humans. Human malaria parasiteinfection inflicts tremendous morbidity andsignificant mortality, mainly caused by two par-asite species, Plasmodium falciparum and Plas-modium vivax. When the parasites’ sporozoitestages are transmitted by a mosquito bite, thesepreerythrocytic forms establish the gateway to

host infection by invading host cells and initi-ating the first round of intracellular replication.Preerythrocytic infection precedes blood stageinfection and, for all Plasmodium species thatinfect mammals, takes place in hepatocyteswithin the liver. Hence, the intrahepatocyticreplication stages are called liver stages or exo-erythrocytic forms (EEFs). Liver infection iscompletely asymptomatic; thus, it cannot bedetected in humans and, in consequence, can-not be directly studied during natural infection.Fritz Schaudinn’s infamous blunder at the turnof the 19th century, the alleged observation thatsporozoites could directly infect red blood cells,

Editors: Dyann F. Wirth and Pedro L. Alonso

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held back research on preerythrocytic infectionfor decades, and it was only in the late 1940s thatthe liver stages of the parasite were discovered.This breakthrough was made possible by thediscovery of rodent malaria parasites such asPlasmodium yoelii and Plasmodium berghei,and their introduction into laboratory research,which provided exceptionally powerful experi-mental tools for the direct analysis of liver in-fection. Indeed, most of the data concerningpreerythrocytic infection have been generatedwith these models. Preerythrocytic stages ofhuman malaria parasites remain exceedinglydifficult to study. However, the developmentof in vitro platforms for the culture of humanhepatocytes and the development of liver-hu-manized mice provide, for the first time, pow-erful preerythrocytic infection models, whichare revolutionizing research on the initial stagesof human malaria parasite infection. Here, wereview the biology of liver infection by sporozo-ites and liver stage development, raise outstand-ing questions regarding the field, highlight op-portunities for future research, and argue forthe liver stages as the essential target for malariaeradication efforts.

HEPATOCYTE INVASION AND TRANSITIONTO LIVER STAGE

After an infectious mosquito bite, sporozoitesmust leave the bite site and find their way tohepatocytes, where liver stage developmentoccurs. The dermis-to-hepatocyte journey (re-viewed in Ejigiri and Sinnis 2009) is complexand perilous for the sporozoites and their num-bers are modest, yet a single infectious mosqui-to bite leads to liver infection, thereby settingthe stage for successful host colonization. Thesporozoite (Fig. 1A) is a unique invasive stage—unlike the parasite’s ookinete stage that onlytraverses across the basal lamina of the mosqui-to midgut (Vinetz 2005) and the merozoitesthat only invade red blood cells (Cowmanet al. 2012)—sporozoites are highly motileand must traverse and invade cells. They enterthe bloodstream by traversing a dermal capil-lary, are transported to the hepatic capillary net-work (the sinusoids), and then traverse across

the endothelial barrier to enter the hepatic pa-renchyma. Here, sporozoites traverse multiplehepatocytes and then finally undergo a func-tional switch that allows them to invade a he-patocyte to take up residence. During and afterinvasion, the sporozoite releases the contents ofa unique set of invasive organelles, the micro-nemes and rhoptries, whose constituent pro-teins mediate molecular interactions with thehost cell (Fig. 1A–C). The molecular mecha-nisms and sequence of events in cell invasionhave been extremely well dissected with regardto merozoite invasion of red blood cells, butmuch less is known about hepatocyte infectionby sporozoites. Merozoite and sporozoite hostcell invasion share commonalities, such as theparasite actin–myosin motor that powers activeentry into the host cell (Fowler et al. 2004) andthe host cell plasma membrane invaginationduring invasion that ensconces the intracellularparasite within a host membrane, known as theparasitophorous vacuole membrane (PVM)(Fig. 1D) (Meis et al. 1983). The PVM is exten-sively modified by the developing liver stageparasite and acts not only as a barrier to thehost cell cytoplasm but also as a conduit forcommunication and nutrient acquisition(Figs. 1E and 2A,B). Research has revealedsome of the key molecular players, both parasiteand host, in the hepatocyte invasion process,many of which are unique to sporozoites.

The major sporozoite surface protein, cir-cumsporozoite protein (CSP), is a key mediatorof the sporozoites’ interaction with the hostduring its journey to the liver and the infectionof hepatocytes. CSP is composed of a centralrepeat region, diverse across Plasmodium spe-cies, flanked by two conserved domains—theamino-terminal region I and a cell-adhesive car-boxy-terminal motif known as the type I throm-bospondin repeat (TSR). Region I masks theadhesive TSR until the sporozoite reaches theliver at which time it is cleaved, allowingthe TSR to mediate hepatocyte adhesion (Coppiet al. 2011). This begs the question, how doesthe sporozoite know it has reached the liver?Localized within the liver sinusoid are highlysulfated heparin sulfate proteoglycans (HSPGs),produced by stellate cells within the space of

A.M. Vaughan and S.H.I. Kappe

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ROP2ROP4RAP2/3

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Figure 1. Sporozoite invasion of host hepatocytes. Graphic representation of the invasion steps that culminate inthe productive infection of the hepatocyte host cell. (A) The surface of the motile sporozoite is coated withcircumsporozoite protein (CSP) (shown in inset), which engages heparin sulfate proteoglycans (HSPGs) on thehepatocyte surface. The apical end of the sporozoite contains organelles whose contents are essential forhepatocyte invasion: rhoptries (Rh, pink), micronemes (Mc, orange), and likely dense granules (DG, blue),although the latter have not been unequivocally identified. (B,C) Contact with an invasion-permissive he-patocyte (brown) triggers CSP processing, which, via an unknown mechanism, triggers the release of invasion-essential proteins from the micronemes (inset). These proteins include thrombospondin-related adhesive pro-tein (TRAP), which binds to HSPGs on the hepatocyte cell surface and to the sporozoite internal glideosomecomplex (not shown) via its cytoplasmic domain to provide traction for the invading sporozoite at the movingjunction (also known as the tight junction, shown as a gray ring). The microneme proteins P52 and P36 are alsoinvolved in the invasion process and may interact with each other as well as interacting with the hepatocyteEphrin A2 receptor (EphA2). The P52/P36/EphA2 axis appears to be critical for the formation of the para-sitophorous vacuole (PV). The hepatocyte receptor CD81 is also important for the invasion process and PVformation, but it is not clear whether the sporozoite directly interacts with it. The inner membrane complex(IMC) (in yellow) anchors the internal glideosome complex, allowing for sporozoite movement into thehepatocyte. Hepatocyte invasion occurs through the moving junction at the point of entry and is accompaniedby the polymerization of host F-actin in association with Arp2/3. Invasion results in the invagination of thehepatocyte plasma membrane and the release of rhoptry proteins, including ROP2, ROP4, and RAP2/3. (D)Successful invasion results in the sporozoite residing within a PV surrounded by a parasitophorous vacuolemembrane (PVM) of hepatocyte origin. The PVM is extensively modified by the parasite and the putative densegranule proteins UIS3 and UIS4 are released and trafficked to the PVM, as is EXP-1 (inset). During sporozoitededifferentiation, the IMC (dashed yellow line) and the apical organelles are broken down. (E) The nascent liverstage trophozoite resides within a PV, has its own parasite plasma membrane still coated by CSP (green), and issurrounded by a PVM (blue).

Liver Stage Malaria

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Disse, and these HSPGs are recognized bythe sporozoite via CSP (Fig. 1B) (Frevert et al.1993). This might lead to a signaling event thatswitches the sporozoite to an invasive pheno-type (Coppi et al. 2007), resulting in the afore-mentioned cleavage of region I, a process that iscatalyzed by a sporozoite protease (Coppi et al.2005).

CSP cleavage appears to be a prerequisitefor productive infection, but what other mole-cules are involved in the process? A number ofsporozoite micronemal proteins are known tobe crucial and they include thrombospondin-

related anonymous protein (TRAP) and the6-cys domain proteins P52 and P36 (Fig. 1B).TRAP is a type 1 transmembrane protein, andlike CSP, it contains a TSR domain but also anintegrin-like domain. TRAP is released onto thesporozoite surface from the micronemes duringinvasion and, during this process, TRAP bindsto the sporozoite actin–myosin motor throughits carboxy-terminal cytoplasmic domain andbinds to HSPGs through its extracellular do-main (Kappe et al. 1999). During invasion,TRAP is translocated from the anterior to theposterior of the sporozoite and is subsequently

TVN

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MSP1ZIPCO

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Figure 2. Liver stage development. Graphic representation of liver stage maturation that leads to the formation ofexoerythrocytic merozoites. (A) The tubulovesicular network (TVN) (blue)—membrane-bound extensionsand whorls that emanate from the parasitophorous vacuole membrane (PVM)—interacts with the hepatocyte’sautophagosomes (which express Atg8/LC3) likely for nutrient uptake. Parasite proteins expressed on the PVM/TVN include IBIS1, EXP1, UIS4, and UIS3, which has been shown to interact with host L-FABP. The parasiteplasma membrane (PPM)-associated protein B9 is known to be important for liver stage development. (B) Asthe liver stage parasite matures, multiple invaginations of the PPM occur (cytomere formation), and this isaccompanied by the expression of MSP1 and ZIPCO. Additionally, LISP1 and LISP2 expression occur on thePVM. (C) Toward the end of liver stage development, individual exoerythrocytic merozoites begin to form, thePVM breaks down (a process that relies on LISP1), and LISP2 is released into the host hepatocyte. (D)Merosomes, merozoites surrounded by hepatocyte plasma membrane, are released into the bloodstreamthrough the liver sinusoid, which is demarcated by epithelial cells (red) and liver resident macrophages, theKupffer cells (yellow).



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shed from the sporozoite by cleavage withinits transmembrane domain, likely by the rhom-boid protease ROM4, which allows for hepato-cyte invasion, because the cleavage releases theTRAP–HSPG bond (Baker et al. 2006; Ejigiriet al. 2012) (Fig. 1B). The micronemal proteinsP52, a predicted GPI-anchored protein, andP36, a predicted secreted protein, are membersof the 6-cys protein family (Templeton and Kas-low 1999), and they are critical for hepatocyteinfection (Fig. 1B,C). Other members of the6-cys family that are expressed in different par-asite stages also have roles in parasite–parasiteor host–parasite interactions (Pradel 2007; vanDijk et al. 2010; Molina-Cruz et al. 2013; Salaet al. 2015). The 6-cys domains are likely in-volved in protein–protein interaction, but evi-dence for this has been scarce. P52 is releasedfrom the micronemes to the sporozoite surfacebefore invasion and disruption of either P52 orP36 in P. berghei (Ishino et al. 2005; van Schaijket al. 2008), and P52 in P. falciparum (vanSchaijk et al. 2008) results in sporozoites thatare defective in hepatocyte invasion. In P. yoelii,the simultaneous deletion of P36 and P52 ren-ders sporozoites unable to form or maintain aPVM, resulting in early abortion of liver stagedevelopment (Labaied et al. 2007). Similarly,P. falciparum sporozoites lacking P36 and P52cannot develop after hepatocyte entry (VanBus-kirk et al. 2009). Recent work revealed the he-patocyte receptor that directly interacts withthese proteins: the transmembrane receptorEphrin A2 (EphA2) (Fig. 1B). EphA2 is essen-tial for sporozoite invasion and concomitantPVM formation in rodent and human malariaparasites and antibodies to EphA2 block spor-ozoite infection (Kaushansky et al. 2015). In-triguingly, its natural ligand EphrinA1 sharesstructural similarity with the 6-cys fold andthere is strong evidence that EphA2 directly in-teracts with P36, thus constituting the first bonafide host receptor–parasite ligand pair with acritical role in hepatocyte infection (Fig. 1B).

In addition to EphA2, other hepatocytesurface proteins are critical for successful spor-ozoite infection. The tetraspanin membraneprotein “cluster of differentiation 81” (CD81)(Fig. 1B) is required on hepatocytes for P. yoelii

and P. falciparum sporozoite invasion withPVM formation but interestingly not forP. berghei. P. yoelii sporozoites are unable to in-fect CD81-deficient mouse hepatocytes, andantibodies against mouse and human CD81 in-hibit the in vitro hepatocyte infection by P. yoeliiand P. falciparum sporozoites (Silvie et al.2003). Yet, a sporozoite ligand for CD81 hasnot been identified. Cholesterol is involved inthe assembly of CD81-rich microdomains onthe cell surface, and this assembly is necessaryfor sporozoite infection (Silvie et al. 2006), in-dicating that it is the organization and compo-sition of microdomains, rather than a directinteraction with CD81, that enable successfulsporozoite infection of hepatocytes. A furtherhepatocyte membrane receptor, scavenger re-ceptor BI (SR-BI), which mediates the selectiveuptake of cholesteryl esters from both high- andlow-density lipoproteins, plays a role in hepato-cyte infection. This role appears rather indirectbecause SR-BI deficiency causes a decreased ex-pression of CD81 on the hepatocyte surface (Ya-laoui et al. 2008). Antibodies to SR-BI do notblock sporozoite invasion (Foquet et al. 2015),adding credence to the idea that SR-BI expres-sion is necessary for CD81 microdomain for-mation but not a receptor for the sporozoite.Recently, an important link between the initialinteraction of the sporozoite with the hepato-cyte surface and the signaling to trigger process-es within sporozoites has been revealed—thepresence of CD81 on hepatocytes appears tobe critical for the subsequent discharge ofthe rhoptries (Risco-Castillo et al. 2014). Re-search thus indicates an orchestrated sequenceof events whereby sporozoite interaction withthe hepatocyte promotes downstream signalingthat triggers the secretion of proteins necessaryfor the formation and modification of the PVM.

Once the sporozoite lies completely en-sconced in the PVM (Fig. 1D), it must meta-morphose into a highly metabolically activeintracellular replication machine, a processthat requires jettisoning or disassembling its in-vasive organelles and inner membrane complex(IMC) (Fig. 1D). IMC breakdown is accompa-nied by spherical plasma membrane expansionin the center of the still elongated parasite and

Liver Stage Malaria


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the contraction of its distal ends (Jayabalasing-ham et al. 2010). This shape conversion is ac-companied by the clearance of the IMC andinvasion-associated organelles, which are dis-charged as large exocytic vesicles or possiblyundergo autophagy. However, the presence ofautophagy in Plasmodium is controversial be-cause the key autophagy marker protein Atg8/LC3 localizes to the parasite apicoplast and doesnot relocalize to autophagosome-like vesiclesor vacuoles (Eickel et al. 2013; Jayabalasinghamet al. 2014). The nascent liver stage preferential-ly develops in the host juxtanuclear region andthe PVM appears to form an association withthe host endoplasmic reticulum (ER) (Banoet al. 2007). Parasite modification of the PVMallows the passage of molecules of up to 855 Dafrom the host cytosol to the PV through openchannels, and it is possible that host-derivedcholesterol that accumulates at the PVM maymodulate channel activity (Bano et al. 2007).These channels ensure that molecules fromthe nutrient-rich hepatocyte can feed the grow-ing liver stage, although a tubulovesicular net-work, described below, likely allows for theuptake of larger molecules. Although the PVMis vital for successful liver stage development,the initial metamorphosis from sporozoite totrophozoite can occur outside a host cell. Thisindicates that the intracellular environmentdoes not provide specific signals to trigger thisprocess but rather that more general changesthat occur with the sporozoites transition intoa mammalian host, such as an elevation in tem-perature and the presence of serum components(Kaiser et al. 2003).

As described above, the initiation of liverstage development is a complex process thatis initiated when the sporozoite detects a suit-able hepatocyte for infection. Once the sporo-zoite has invaded its host cell, it must thendedifferentiate from an invasive extracellularparasite into an intracellular trophozoitecontained within a PVM. During this process,signaling events prevent the parasites’ destruc-tion by the host cell. Finally, the trophozoitetransitions into a highly replicative schizontthat relies on nutrient uptake from its host aswell as its own metabolic processes to ensure its

eventual goal of exoerythrocytic merozoiterelease.

It must be noted that the transition to schi-zogony, which always occurs in P. falciparumliver stage development, does not always occurin P. vivax. The P. vivax sporozoite, once it hasentered a host hepatocyte, dedifferentiatesand can then become a dormant trophozoite,known as the hypnozoite. The hypnozoite canlie dormant for months and even years and thenreactivate and fully develop, leading to P. vivaxmalaria relapses. The processes that lead to hyp-nozoite formation and reactivation are verypoorly understood, and malaria eliminationmust address the radical cure of the dormanthypnozoite.

LIVER STAGE DEVELOPMENT

The liver stage undergoes schizogony—nucleardivision without cell division—during itsmaturation and only in the final phase of thisprocess do invasive exoerythrocytic merozoitesform. Liver stage schizogony constitutes one ofthe most rapid eukaryotic replication events—in a little .2 d, rodent malaria sporozoites cantransform into a mature ellipsoid liver stagecontaining up to 29,000 exoerythrocytic mer-ozoites (Baer et al. 2007). Liver stage develop-ment in P. falciparum takes longer, �6.5 d, withestimates of up to 90,000 exoerythrocytic mer-ozoites packaged in the fully mature parasite(Vaughan et al. 2012). Liver stage schizogonyis characterized by the rapid branching andgrowth of the parasite’s organelles such as themitochondria and the relict plastid organelle,the apicoplast (Stanway et al. 2009), as well asmassive DNA replication. Studies utilizing par-asite and host transgenesis have shed light onthe important pathways that allow for parasitegrowth, and, not surprisingly, a number of par-asite proteins involved in PVM formation andmodification are essential for liver stage forma-tion and development.

The liver stage PVM proteins UIS3 andUIS4 are encoded by genes belonging to theup-regulated in infective sporozoites (UIS)group (Figs. 1D–E, 2A, 3A–C,F). These genesare highly transcribed in sporozoites when they



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A

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Figure 3. Immunofluorescence images of liver stage development. (A–E) Plasmodium yoelii liver stage devel-opment in a mouse liver. (A) The spherical, early liver stage parasite, 12 h after sporozoite invasion, is signifi-cantly smaller than the host hepatocyte nucleus (blue). The liver stage parasitophorous vacuole (PVM) is shownin red (UIS4 expression), the endoplasmic reticulum (ER) is shown in green (BiP expression), and the parasitecontains a single nuclear center (blue). (B) By 24 h, the P. yoelii liver stage parasite (the UIS4-positive PVM isshown in red) has entered schizogony (multiple centers of nuclear replication [blue]). (C) At 30 h of develop-ment, the P. yoelii liver stage (the UIS4 PVM is shown in green) has undergone multiple rounds of nuclearreplication (blue) and contains a highly branched apicoplast (acyl carrier protein expression, shown in red). (D)Late in P. yoelii liver stage development (48 h), the parasite plasma membrane (PPM) undergoes extensiveinvagination resulting in cytomere formation (MSP1, green). LISP2 (red) is expressed on the PVM as well as thehepatocyte membrane (white arrows). The nuclei (blue) of the individual parasites can be seen within the liverstage parasite. (E) At the end of liver stage development (52 h), individual exoerythrocytic merozoites (MSP1expression in green, upper panel, delineates the merozoite membrane) can be seen within the mature liver stageparasite (differential interference contrast, lower panel). (F) Image of a P. vivax hypnozoite in a humanizedmouse liver. The hypnozoite remains latent, is small, and does not replicate its DNA, and its PVM has a uniqueUIS4-positive prominence (green). The hypnozoite mitochondrion (HSP60 expression) is shown in red and isbranched. Scale bars, 10 mm.

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still reside in the mosquito salivary glands butsome are translationally repressed with proteinproduction peaking only after hepatocyte inva-sion. This “just in time” protein expression fromstored mRNAs exemplifies one of the ap-proaches the sporozoite has evolved to readyitself for the rapid transition to liver stage rep-lication and indicates that proteins such as UIS3and UIS4 must be playing an important rolestarting very early in liver stage development.Indeed, deletion of either UIS3 or UIS4 genecauses arrest of early liver stage development(Mueller et al. 2005a,b; Jobe et al. 2007). Thecarboxyl termini of both UIS3 and UIS4 facethe hepatocyte cytoplasm and the amino termi-ni the PV lumen, suggestive of roles in parasite–hepatocyte interactions. Indeed, UIS3 interactswith liver fatty acid protein (L-FABP) (Fig. 2A)(Mikolajczak et al. 2007), suggesting an interac-tion that could serve as a conduit for the trans-port of lipid into the liver stage to supportgrowth. Lipid transport within and betweeneukaryotic cells can occur through membrane-bound ATP-binding cassette (ABC) transport-ers, and the ABCC class of transporters is ex-pressed by both P. berghei and P. falciparum liv-er stages. Interestingly, the deletion of ABCC2(also known as MRP2) in both P. berghei andP. falciparum leads to liver stage arrest, demon-strating the importance of parasite ABC trans-porters in liver stage development and hintingto their role in lipid transport (Rijpma et al.2016). To date, UIS4 function has remainedelusive, as has the function of a second PVM-resident protein exported protein 1 (EXP1, alsoknown as Hep17 in rodent malaria parasites)(Fig. 2A). Later in liver stage development,two further PVM-associated proteins are ex-pressed, LISP1 and LISP2 (Figs. 2B,C and 3D)(Ishino et al. 2009; Orito et al. 2013). LISP1 isinvolved in PVM breakdown and subsequentmerozoite release, whereas LISP2 (which con-tains a modified 6-cys domain) is localized tothe PV and PVM and is also transported to thecytoplasm and plasma membrane of infectedhepatocytes (Figs. 2B,C and 3D). Although thefunctions of these two proteins are not fullyunderstood, gene deletion studies in P. bergheihave indicated their importance in late liver

stage growth because parasites that lack LISP1or LISP2 do not mature effectively. Further crit-ical proteins expressed by liver stage parasitesinclude the 6-cys-like protein, B9, expressedon the parasite plasma membrane (PPM) (Fig.2A) (Annoura et al. 2014) and ZIPCO, an irontransporter expressed on the late liver stage PPM(Fig. 2B) (Sahu et al. 2014). Although an in-creasing number of PV and PVM proteins havebeen identified that play important roles in liverstage development, an important future goal isthe comprehensive identification of all liverstage PVM, PV, and PPM proteins that willsupersede the current identification of impor-tant players using candidate approaches. Impor-tantly, research needs to go beyond the assemblyof “parts lists” and ought to delineate the rela-tionship and interaction among liver stage PVMand PV proteins and their interaction with hostproteins to establish a network of importantliver stage–hepatocyte interactions.

Although the liver stage clearly parasitizesthe extremely metabolically active host hepato-cyte, the massive increase in parasite biomassduring liver stage development likely requiresthe participation of parasite anabolic pathways.Indeed, combined transcriptome and proteomeanalysis of liver stage development showed thatthe parasite de novo type II fatty acid synthesispathway (FAS II) is highly active (Tarun et al.2008). To assess the importance of FAS II, whichis localized to the parasite apicoplast, geneknockouts in rodent malarias have been per-formed and showed that FAS II is critical forlate liver stage development (Yu et al. 2008;Vaughan et al. 2009; Pei et al. 2010; Falkardet al. 2013; Lindner et al. 2014). These reportsshow that the liver stage requires its own fattyacid synthesis, but it is not currently understoodwhy the fatty acids are synthesized—they mightbe incorporated into exoerythrocytic merozoitemembranes, but they could also be incorporat-ed into the growing hepatocyte plasma mem-brane or the PVM. As mentioned above, theUIS3/L-FABP interaction likely allows for lipidacquisition from the hepatocyte. The cholester-ol component of this lipid can be derived fromlow-density lipoprotein internalization by thehepatocyte as well as de novo synthesized hepa-



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tocyte cholesterol (Labaied et al. 2011). Furtherresearch has shown that hepatocyte phosphati-dylcholine, the major membrane phospholipid,is taken up by liver stages, and preventing therate-limiting steps in the synthesis of hepatocytephosphatidylcholine affects liver stage develop-ment (Itoe et al. 2014). All of these studies showthat liver stage parasites critically depend onboth lipid uptake from the host hepatocyteand endogenous lipid synthesis to support theirdramatic growth and differentiation.

During growth, liver stage mass increasesso much that the infected hepatocyte mustsignificantly expand to accommodate the repli-cating parasite. However, the liver stage para-sites remain relatively undifferentiated untilshortly before completion of liver stage matu-ration. Late in development, the PPM under-goes rapid expansion and extensive inva-gination (called cytomere formation) (Figs. 2Band 3D), which dramatically increases parasitesurface area and enables rapid formation of in-dividual exoerythrocytic merozoites (Figs. 2Cand 3E). This process also requires coordinatednuclear and organellar fission to ensure parti-tioning of nuclei and organelles into nascentmerozoites (Stanway et al. 2011). This coordi-nated fission likely relies on both DNA- andRNA-binding proteins, and a liver stage–spe-cific RNA-binding protein, PlasMei2, appearsto be essential for the partitioning of nuclearDNA. The deletion of PlasMei2 in P. yoelii, with-out affecting liver stage growth, prevents thepartitioning of nuclear DNA, and this in turnprevents the formation of exoerythrocytic mer-ozoites (Dankwa et al. 2016). Once merozoiteshave fully formed, the PVM must break down(Fig. 2C) before they can be released, and thisprocess is a result of changes in PVM perme-ability (Sturm et al. 2009) and relies on a para-site phospholipase, which localizes to the PVM(Burda et al. 2015).

Intravital microscopic observations of live-fluorescent P. yoelii and P. berghei mature liverstages in mice have shown that they induce thedetachment of their host hepatocyte, leading tothe budding of merosomes (merozoite-filledvesicles) into the sinusoidal lumen (Fig. 2D)(Sturm et al. 2006; Tarun et al. 2006). The mer-

osome membrane derives from the host hepa-tocyte plasma membrane (Graewe et al. 2011),allowing the merosome to hide from the im-mune system and thereby avoid uptake anddestruction by the numerous liver-residentmacrophages (the Kupffer cells), on its way tothe bloodstream. Merosomes have also beenobserved in mature P. falciparum liver stages(Vaughan et al. 2012), confirming their exis-tence in human malaria parasite liver stages.Most P. yoelii merosomes exit the liver intactand adapt to a relatively uniform size, withinwhich 100 to 200 merozoites reside (Baer et al.2007). Merosomes eventually break up insidepulmonary capillaries, resulting in merozoiteliberation and red blood cell infection.

PARASITE PERTURBATIONS OF HOSTHEPATOCYTES

So far, we have discussed parasite proteins andsome host molecules that are necessary forsporozoite invasion and liver stage develop-ment, but to acquire nutrients and grow, theparasite must not simply reside in the hepato-cyte and exploit it but actively manipulate andsubvert it to avoid its demise. How does thishappen? Already at the point of invasion, theparasite subverts its host. As the sporozoite in-vades, it induces the formation of a ring-shapedstructure in the hepatocyte composed of denovo polymerized host F-actin (Fig. 1C) (Gon-zalez et al. 2009). The hepatocyte Arp2/3 com-plex, an actin-nucleating factor, is recruited tothis ring structure and is necessary for parasiteinvasion (Fig. 1C). The PVM is essential forliver stage development, and the formation ofa productive PVM is a prerequisite for the par-asite’s ability to render its host hepatocyte resis-tant to apoptosis (van de Sand et al. 2005; vanDijk et al. 2005; Kaushansky et al. 2013a). Al-though the sensitivity to apoptosis is dependenton the Bcl-2 family of mitochondrial proteinsthat the parasite subverts (Kaushansky et al.2013a), the parasite molecules that interferewith hepatocyte apoptosis are not known. Aline of defense used by many host cells againstpathogen infection is the synthesis of stressgranules, thereby shutting down protein synthe-

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sis. However, there is no evidence that hepato-cyte infection causes stress granule formation,which implies that the parasite has evolved toprevent this response from occurring in the he-patocyte (Hanson and Mair 2014). In a surpris-ing twist on parasite–host manipulation, liverstage survival actually benefits from the host ERstress response pathway (Inacio et al. 2015),which typically shuts down cellular activity.Proteins and transcripts that act in the unfoldedprotein response leading to ER stress werefound to be elevated in hepatocytes in responseto infection, but why the parasite perturbs itshost in this way is not clear. It is possible that ERstress supports parasite growth by regulatinglipid metabolism, vital for parasite growth(Itoe et al. 2014), or diminishing antigen pre-sentation so the liver stage can remain hiddenfrom the immune system—a trick used by hep-atitis C (Tardif and Siddiqui 2003).

The perturbations of the host hepatocytehave been explored by analyzing changes in he-patocyte transcription and protein expressionearly during liver stage infection (Albuquerqueet al. 2009; Kaushansky et al. 2013b). Transcrip-tome data showed that parasite infection initial-ly causes a global stress response, which thenmorphs into an engagement of host metabolicprocesses and the maintenance of cell viabilityto ensure parasite and host cell survival (Albu-querque et al. 2009). Using cell lysate arrays tosimultaneously monitor many protein pertur-bations, a subset of hepatocyte protein levelsand accompanying posttranslational modifica-tions were measured 24 h postinfection with ro-dent malaria parasites and compared with un-infected hepatocytes (Kaushansky et al. 2013b).This revealed a signaling network aimed atpreventing host cell death but also determinedthat the tumor suppressor P53 is substantiallydampened in infected hepatocytes. This is offunctional significance because increasing P53levels whether chemically or genetically nearlyeliminates liver stage infection and develop-ment and mice lacking P53 are hypersusceptibleto infection (Kaushansky et al. 2013b).

During liver stage development, a tubulo-vesicular network (TVN) forms, which includesmembranous extensions of the PVM as well

as the creation of autonomous vesicular struc-tures within the infected hepatocyte (Fig. 2A)(Mueller et al. 2005a). The TVN likely func-tions in hepatocyte interactions and in access-ing nutrients from the hepatocyte, but thesefunctions need to be experimentally verified.Fluorescent tagging of two P. berghei PVM pro-teins, UIS4 and IBIS1, and the use of correlativelight-electron microscopy have shown that theTVN membranes extend throughout the hostcell and include dynamic vesicles as well aslong tubules that extend and contract from thePVM (Fig. 2A) (Grutzke et al. 2014). Addition-ally, labeling of host hepatocyte compartmentsrevealed an association of late endosomes andlysosomes with the TVN-associated elongatedmembrane clusters. Furthermore, the host au-tophagosome protein Atg8/LC3 colocalizeswith UIS4 at the PVM and TVN (Fig. 2A).These data suggest that the intimate associationbetween the TVN and host endosomes/lyso-somes allows for parasite–hepatocyte signalingas well as nutrient uptake. Further studies haveshown that complete parasite development ac-tually correlates with the gradual loss of autoph-agy marker proteins and associated lysosomesfrom the PVM even though other autophagicevents such as nonselective canonical autoph-agy continue in the host cell (Prado et al. 2015).Thus, it is possible that the parasite continues tobenefit from nonselective canonical autophagyfor its growth.

Supporting the hypothesis that the endo-somal system supports liver stage growth, thegrowth of the parasite is dependent on phos-phoinositide 5-kinase (PIKfyve), which con-verts phosphatidylinositol 3-phosphate intophosphatidylinositol 3,5-bisphosphate (PI(3,5) P2) within the endosomal system—anenzymatic reaction essential for late endocyt-ic membrane fusion (Thieleke-Matos et al.2014). Indeed, the PI (3,5) P2 effector proteinTRPML1, involved in late endocytic membranefusion, was shown to be present in vesicles inclose contact with the PVM and PIKfyve inhibi-tion delayed parasite growth. Additionally, con-tents of late endocytic vesicles have been foundwithin the parasite cytoplasm (Lopes da Silvaet al. 2012). We are only just beginning to un-



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derstand how the parasite manipulates its host,and many questions remain unanswered. Mostnotably, there is a complete lack of understand-ing of how the liver stage, during its own massivegrowth, also enables the growth of the PVM andexpansion of the host hepatocyte to accommo-date its heft—cells do not normally dramaticallyincrease in size, but the mature liver stage–infected hepatocyte can expand to 200 timesits normal volume.

The developing liver stage parasite, as well asinteracting with its host hepatocyte to ensure itssurvival, must also evade the host’s immunesystem to ensure merozoite maturation and re-lease. As noted above, the autophagy markerAtg8/LC3 colocalizes with UIS4 at the PVMand TVN, and aids in liver stage growth. Con-versely, autophagy has been identified as adownstream pathway that is activated in re-sponse to interferon g (IFN-g) in the controlof intracellular infections (Deretic et al. 2013).Indeed, IFN-g, a master regulator of immunefunctions, has been known for many years toinduce the elimination of liver stage parasitesin vivo (Ferreira et al. 1986). Thus, the parasiteneeds to precisely coordinate autophagy for itsbenefit. Research on P. vivax liver stages hasshown that IFN-g-mediated restriction of liverstage P. vivax depends on autophagy-relatedproteins including Beclin-1, PI3K, and Atg5and enhanced the recruitment of LC3 to thePVM. In this case, the LC3 decoration of thePVM led to a noncanonical autophagy pathwayresembling that of LC3-associated phagocyto-sis, promoting the fusion of P. vivax compart-ments with lysosomes and subsequent killing ofthe pathogen (Boonhok et al. 2016). Thus, re-search has uncovered positive and negative as-pects of autophagy on parasite survival, and it isclear that although the parasite can take advan-tage of host cell autophagy, it can also be killedby the pathway and thus must carefully controlthis intracellular process for its own gain.

IFN-g production in response to liver stageparasite detection is part of a programmed in-nate immune response to liver stage infection(Liehl and Mota 2012). This innate immuneresponse to infection is pronounced and canactually lead to the effective killing of a second-

ary liver stage infection (Miller et al. 2014). Thisresponse is in part initiated by liver-residentcells and driven by Plasmodium RNA, whichacts as a pathogen-associated molecular patterncapable of activating the type I IFN response viathe cytosolic pattern recognition receptor Mda5(Liehl et al. 2014). Furthermore, the responseis abrogated in mice deficient in IFN-g, as wellas the type I IFN-a/b receptor (IFNAR), dem-onstrating the important role of IFNAR in driv-ing the innate immune response (Liehl et al.2014; Miller et al. 2014). The cell type linkedto the secretion of IFN-g was shown to beCD49bþCD3þ natural killer T (NKT) cells andan increase in CD1d-restricted NKT cells wascritical in reducing liver stage burden of a sec-ondary infection (Miller et al. 2014). In turn, thelack of IFNAR signaling abrogated the increasein NKT cell numbers in the liver, showing a linkbetween type I IFN signaling, cell recruitment,and parasite elimination (Miller et al. 2014).

The above highlights the extent of our cur-rent knowledge on how the liver stage parasiteinfluences both the host hepatocyte and thehost innate immune system. Far more researchis necessary and will help in the design and im-plementation of drugs to target the liver stageparasite and effective vaccines that engender ap-propriate immune responses to liver stage par-asites. For these advances to take place, we needbetter models for liver stage development andliver stage isolation.

ADVANCES IN MODEL DEVELOPMENTFOR HUMAN MALARIA PARASITE LIVERINFECTION

A natural sporozoite infection typically resultsin the productive invasion of only a small num-ber of hepatocytes. Because of this bottleneck,the liver stage parasite has been extremely diffi-cult to study. However, such studies are essentialto uncover pathways that are necessary for par-asite survival and replication. These pathwayscan then be perturbed to prevent developmentand ultimately to thwart life cycle progression toblood stage disease. To this end, parasite trans-genesis has enabled the creation of fluorescentparasites and this led to the sorting of murine

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hepatocytes infected with P. yoelii fluorescentliver stages, resulting in the first extensive liverstage transcriptomes and proteomes (Tarunet al. 2008; Albuquerque et al. 2009). However,extensive -omic analysis of human malaria liverstages has still not been completed and needs tobe performed. Such analysis is important for thereason that numerous studies have shown sig-nificant biological differences between rodentand human malaria parasites. Nevertheless,platforms for the growth of human malariaparasite liver stages are not easily established,because of the limitation that P. falciparumand P. vivax preerythrocytic stages only infecthuman and some nonhuman primate hepato-cytes and do not grow well in hepatoma cells.Additionally, the P. vivax sporozoite, unlike themodel rodent malaria sporozoite, can developinto either a liver stage schizont or a liver stagehypnozoite (Krotoski et al. 1982)—a dormantliver stage form (Fig. 3F) that can activateand cause malaria relapse infections in affectedindividuals.

In vitro development of P. vivax liver stagesin primary human hepatocytes was first docu-mented in 1984 (Mazier et al. 1984) and forP. falciparum in 1985 (Mazier et al. 1985), andboth parasites were later shown to mature in thehuman hepatocyte cell line HC-04 (Sattabong-kot et al. 2006), albeit poorly. Rapid advancesin cryopreservation methods for primary hu-man hepatocytes make this cell type more easilyavailable, and investigators no longer depend oncells fresh from liver surgery. Recently, an im-proved in vitro system using primary humanhepatocytes in a microscale human liver plat-form was shown to support P. falciparum andP. vivax liver stages development includingP. vivax hypnozoite formation (March et al.2013). An additional promising platform usesinduced pluripotent stem cell-derived hepato-cyte-like cells and liver stage development ofboth P. falciparum and P. vivax appears to mir-ror that observed in primary human hepato-cytes (Ng et al. 2015). Thus, this platform isan attractive alternative because it enables a po-tential unlimited production of hepatocytes.

Although in vitro models for infectionhave greatly improved over the past years, robust

in vivo infection models for preerythrocyticstages of human malaria parasites are highlydesirable. Fortunately, human-liver chimericmouse models that were originally developedfor drug metabolism studies and hepatitis re-search are promising as animal models forhuman malaria liver infection. These modelsare all built around some form of immunocom-promised background plus genetically encodedliver injury that selectively ablates mouse he-patocytes, thereby creating niches in the liverthat can then be repopulated with primaryhuman hepatocytes. An initial study using theSCID Alb-uPA mouse transplanted with hu-man hepatocytes showed that P. falciparum liverstages developed after intravenous sporozoiteinjection (Sacci et al. 2006).

A second liver-chimeric mouse model, theFRG huHep mouse (Azuma et al. 2007), canyield up to 95% human hepatocyte chimerism.After intravenous injection of P. falciparum spo-rozoites into FRG huHep mice, complete liverstage development occurred, resulting in thecreation of schizonts packed with tens of thou-sands of exoerythrocytic merozoites that wereable to transition in vivo to red blood cell infec-tion, which was then continuously maintainedin vitro (Vaughan et al. 2012). This enabled thecreation of P. falciparum genetic crosses, whichwere previously possible only in splenectomizedchimpanzees (Vaughan et al. 2015). The FRGhuHep mouse has, in addition, been used toshow the efficacy of antibodies against sporo-zoite proteins to reduce or prevent P. falciparumliver infection (Sack et al. 2014). This has alsobeen achieved in SCID Alb-uPA mice (Behetet al. 2014; Foquet et al. 2014). Together, thework establishes liver-chimeric mice as an im-portant preclinical tool to assess interventionsagainst human malaria parasite pre-erythrocyt-ic stages and further refinement could ascertaintheir use as a new stepping-stone toward clinicaltesting of interventions in controlled humanmalaria infection trials.

Intriguingly, the FRG huHep mouse wasalso recently used for infections with P. vivax(Mikolajczak et al. 2015). In this study, robustliver infection with P. vivax sporozoites was ob-served that culminated in complete liver stage



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development and exoerythrocytic merozoiterelease 9.5 d after infection and infectious mer-ozoites could be captured by the injection ofhuman reticulocytes. Alongside liver stage de-velopment, the establishment and persistence ofhypnozoites was observed, enabling, for the firsttime, a detailed characterization of this uniquelatent EEF. Although DNA replication did in-deed not occur in hypnozoites as previouslythought, in contrast to previously held assump-tions, latent hypnozoites were not “dormant”but metabolically active, showing limited or-ganelle replication and modest growth overtime. Hypnozoites also exhibited a uniquethickening of the PVM, called a “prominence,”that appears to distinguish them from replicat-ing liver stages (Fig. 3F). The study also showedthat the FRG huHep mouse will be useful tomodel hypnozoite activation, which leadsto the production of second- and third-genera-tion exoerythrocytic merozoites and relapsingblood stage infection, which is a distinguishingclinical feature of P. vivax infection. Thus, theFRG huHep model might prove useful to test theeffectiveness of drugs that can eliminate hypno-zoites and thus prevent relapsing infection withP. vivax.

VACCINATION WITH ATTENUATEDPREERYTHROCYTIC PARASITES

Following earlier research on bird malaria(which do not have a liver stage), a study in1967 showed that mice inoculated with irra-diation-attenuated P. berghei sporozoites in-duced protection from a subsequent infectionwith wild-type sporozoites (Nussenzweig et al.1967) and with this, the concept of an attenu-ated, whole sporozoite malaria vaccine wasborn. Similar studies in humans with irradiatedP. falciparum sporozoites showed equal promise(Clyde et al. 1973; Rieckmann et al. 1974). Sub-sequent work showed that attenuated sporozo-ites have to be infectious and invade hepatocytesto unfold their protective potential likely by vir-tue of expressing new antigens before the cessa-tion of liver stage development. Live sporozoiteimmunization elicits both protective humoralresponses and cellular responses (Doll and

Harty 2014). In the 1970s, irradiation-attenuat-ed sporozoites were not considered a practicalvaccine because of the issues with irradiation,mass production, preservation, and delivery.However, increasing success in producing andstoring P. falciparum sporozoites, irradiatedsporozoites gained a resurgent interest in the2000s and intravenous inoculation of humanswith cryopreserved, irradiated P. falciparumsporozoites recently showed complete pro-tection from infectious sporozoite challenge(Seder et al. 2013).

The discovery of genes that are essential forliver stage development, discussed earlier, gaverise to the idea that attenuation of the parasitecould be accomplished through engineeredgene deletion within the parasite’s complex ge-nome, thus rendering irradiation obsolete. Inthis effort, vaccine development meets parasitebiology and synergizes effectively to create a bi-ologically informed malaria vaccine strategy.Today, numerous genes essential for liver stagedevelopment have been identified, mostly in ro-dent malaria models (Table 1). Gene deletionsarrest the parasite at distinct points during liverstage development, and studies have shown thatlate liver stage–arresting parasites afford supe-rior immunity when compared with both anearly arresting, genetically engineered parasiteand irradiation-attenuated sporozoites. This isdue to the significant increase in antigen loadand breadth, which engenders a broader anddiversified immune response (Butler et al.2011; Sack et al. 2015). Engineering of P. falcip-arum liver stage–attenuated parasites has beenundertaken (VanBuskirk et al. 2009); the para-sites have undergone initial human clinical trialtesting and are now undergoing improvementsin attenuation (Table 1) (Mikolajczak et al.2014; van Schaijk et al. 2014b). Ultimately,this will likely yield an engineered parasite thatcan replace irradiated sporozoites and such avaccine will play a crucial role in malaria elim-ination.

FUTURE PERSPECTIVES

A call for malaria eradication by 2040 isperceived to be an achievable goal (endmalaria

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http://endmalaria2040.org/http://perspectivesinmedicine.cshlp.org/

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2040.org). However, P. falciparum malaria willnot be eradicated globally without an effectivepreerythrocytic vaccine that will prevent theonset of disease-causing blood stage infectionand onward transmission through the mosqui-to vector. In addition, the elusive P. vivax pre-erythrocytic hypnozoite forms must be targetedby radical cure drugs to ensure full malaria erad-ication, and thus drug development ought toplace more emphasis on finding compoundswith activity against liver stages. A robust un-derstanding of hypnozoite biology might wellbe a prerequisite to develop new drugs that af-ford radical cure. A further critical researchquestion in this context is whether preerythro-cytic vaccination can be used to provide an“immunological radical cure” and as suchshould be a major aspect of P. vivax vaccineresearch. Overall, we must be continuouslyguided by the principle that novel and innova-tive intervention strategies will emerge onlyfrom critical research of parasite biology andthe immunology of infection and vaccination.

CONCLUDING REMARKS

The past decade has seen an unprecedentedprogress in research on liver stage malaria.This has brought forth the identification of nu-merous parasite and host factors that are of crit-ical importance to hepatocyte infection andintra-hepatocytic parasite development. With-out doubt, research in the coming years willidentify additional important factors. However,the study of parasite–hepatocyte interactionmust now enter the next phase, by not onlyidentifying critical host–pathogen interactionsbut also assembling these interactions into atemporal and spatial cascading network thatfully delineates parasite infection of and devel-opment within hepatocytes at the molecularlevel, from the point of invasion to the releaseof merozoites. These studies will be likely per-formed with rodent malaria models, but theincreased maturity of in vitro and in vivo hepa-tocyte infection models of human malaria par-asites gives hope that at least some of this workcan be achieved with medically relevant parasitespecies. This is particularly important for the

study of P. vivax hypnozoites in gaining insightsinto the molecular mechanisms of their latency,their activation, and their interaction with he-patocytes during long periods of persistence.

Finally, identification of parasite moleculesthat are critical for liver stage development hastranscended basic biology in that it enabled thedevelopment and use of genetically engineered,attenuated parasite strains for vaccination. It ishoped that the continued biological interroga-tion of liver stage malaria will carry on to bearfruit for parasite immunology and vaccine de-velopment, thereby potentiating its role in help-ing to develop new tools for the eradication ofhuman malaria infection.

ACKNOWLEDGMENTS

Research being performed in the Kappe Labo-ratory is partly funded by the U.S. National In-stitutes of Health and the Bill and MelindaGates Foundation. We thank laboratory mem-bers past and present for their scientific contri-butions as well as our dedicated insectary andvivarium staff.

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