Plasmodium Nesting: Remaking the Erythrocyte from the Inside Out

MI67CH12-Cowman ARI 5 August 2013 16:34

Plasmodium Nesting: Remakingthe Erythrocyte from theInside OutJustin A. Boddey and Alan F. CowmanDivision of Infection and Immunity, The Walter and Eliza Hall Institute of Medical Research,Parkville, Victoria 3052, Australia; email: [email protected], [email protected]

Department of Medical Biology, University of Melbourne, Parkville, Victoria 3010, Australia

Annu. Rev. Microbiol. 2013. 67:243–69

First published online as a Review in Advance onJune 26, 2013

The Annual Review of Microbiology is online atmicro.annualreviews.org

This article’s doi:10.1146/annurev-micro-092412-155730

Copyright c© 2013 by Annual Reviews.All rights reserved

Keywords

malaria, trafficking, export, PEXEL, translocon

Abstract

One of the most fascinating and remarkable features of Plasmodium parasites,which cause malaria, is their choice of erythrocytes as the principal host cellsin which to reside during infection of a vertebrate host. Parasites completelyrenovate the terminally differentiated cells, which lack most of the normalorganelles and functions of other cells, such as a nucleus and the machin-ery to express and transport proteins to subcellular locations. Erythrocyteremodeling begins immediately after invasion by the Plasmodium parasite,by expression and export of many hundreds of proteins that assemble intomolecular machinery in the host cell that permit protein trafficking, har-vesting of nutrients, and mechanisms to evade host immune responses. Inthis review, we discuss recent studies on erythrocyte remodeling, includingmechanisms of protein export as well as the identity, functions, and subcel-lular locations of key exported proteins.

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Protein export:transport of malarialproteins from withinthe parasite into theinfected erythrocyte

Contents

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244MALARIA THE DISEASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244ERYTHROCYTE REMODELING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246MECHANISMS OF PROTEIN EXPORT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248

Role of the PEXEL/VTS in Export. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248Plasmepsin V and the Matured N Terminus of PEXEL Proteins . . . . . . . . . . . . . . . . . . 249How Are PNEPs Exported? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249How Is PfEMP1 Exported? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251Role of Phosphatidylinositol-3-Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257

PROTEIN EXPORT MACHINERY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258EXPORTED PROTEINS AND THEIR FUNCTIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . 259

The Exportome of Plasmodium Spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259Cytoadherence Complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260PfEMP1 Trafficking and Display on the Erythrocyte Surface . . . . . . . . . . . . . . . . . . . . . 261Enhancing Erythrocyte Rigidity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261New Permeability Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261

TIMING OF EVENTS IN EXPORT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262CONCLUSIONS AND OUTLOOK. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262

INTRODUCTION

Malaria is a mosquito-borne disease caused by infection with Plasmodium parasites. The parasitesare transmitted by an arthropod vector (Anopheles mosquitoes) to vertebrate hosts, which includebirds, reptiles, rodents, and primates (including humans). Five Plasmodium species infect humans,namely P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi; however, infection withP. falciparum causes the most severe disease. Establishment of infection is dependent on the par-asite’s ability to profoundly remodel erythrocytes. Cellular remodeling engineers an intracellularniche that fosters evasion of host immune responses and scavenging of host nutrients, permittingthe parasite to replicate and thrive. This remarkable remodeling process occurs through the ex-pression of a large repertoire of specialized proteins that are trafficked through a sophisticatedexport pathway into the host cell (38, 56, 78, 94). The renovation of host erythrocytes by exportedparasite proteins ensures the parasite’s survival but also contributes to virulence and pathogenesisof malaria (reviewed in 55).

In this chapter, we discuss the current state of knowledge of protein export and host cell remod-eling by Plasmodium, with an emphasis on P. falciparum, for which most information is known. Wediscuss the key exported proteins deployed by parasites, their functions in virulence and remod-eling, and the mechanisms that deliver them to their subcellular locations within erythrocytes.

MALARIA THE DISEASE

In 2010, malaria caused 216 million infections and 655,000 deaths; 91% of cases occurred inAfrica and 86% of all deaths occurred in children less than five years old (103). Almost half ofthe world’s population is at risk of malaria infection and over 100 countries are endemic for thedisease, primarily in sub-Saharan Africa, Southeast Asia, the Indian subcontinent, South America,

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Sporozoite invasion

Macrogametocyte

Exflagellatedmicrogametocyte

Zygote

Ookinete

Oocyst

Sporozites

Salivaryglands

Mosquito

Liver stage

Ring

Sporozites

Trophozoite

Schizont

Egress

Bloodstage

Sexualstage

Merozoite invasion

Gametocytes

Mosquitoblood meal

Mosquitobloodmeal

Oocyst rupture

Temperatureshift

Midgut

Basallamina

Hepatocyteegress

Hepatocyte

Human host

Figure 1The Plasmodium life cycle. A bite from a Plasmodium falciparum–infected mosquito during a blood meal deposits sporozoites into thehost. Sporozoites migrate to and invade hepatocytes, initiating the liver stage of infection. Sporozoites replicate inside hepatocytes,developing into thousands of merozoites, which are released into the bloodstream during hepatocyte egress. Merozoites invadeerythrocytes and initiate the blood stage of infection, with parasites developing into a ring, followed by the trophozoite stage, and thennuclear division occurs, producing a schizont containing 16–32 merozoites. Erythrocyte egress releases merozoites into thebloodstream, where the blood-stage cycle recommences. A small proportion of blood-stage parasites develop into sexual-stagegametocytes. Male gametocytes ingested during a mosquito blood meal exflagellate in the mosquito midgut and fuse with a femalemicrogametocyte to produce zygotes that develop into ookinetes. Ookinetes penetrate the midgut epithelium and develop into oocystson the basal lamina. Oocysts fill with developing sporozoites and rupture releases thousands that migrate to salivary glands. Here theyawait the next mosquito blood meal and transmission into another human host.

and Oceania. Residents in endemic regions are at risk of repeated infection, which, over time,can induce immunity to clinical symptoms; however, immunity to malaria is variable, complex,and unstable (reviewed in 82). Although the use of bednets, insecticide spraying, and antimalarialshas reduced the global incidence of malaria since 2000, there is still a large burden of disease inAfrica and other locations, and eradication will be difficult to achieve with current approaches.Currently, there is no vaccine deployed, and drug resistance to the best-known antimalarials,including artemisinin, is emerging (13, 91).

The complex life cycle of malaria parasites involves repeated transmission between mosquitoesand vertebrate hosts (Figure 1). Sporozoite forms injected into the host by a mosquito infecthepatocytes in the liver and develop into exoerythrocytic merozoites, which are released into the

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J-dot: vesicle in theerythrocyte cytoplasmcontaining exportedparasite chaperones

Maurer’s cleft:flattened membranousstructures produced byparasites in theinfected erythrocytefor protein transport

PVM:parasitophorousvacuole membrane

KAHRP:knob-associatedhistidine-rich protein

PfEMP1: Plasmodiumfalciparum erythrocytemembrane protein 1

NPP: newpermeability pathway

bloodstream, where they invade circulating erythrocytes. The asexual bloodstage of P. falciparumis cyclic and involves differentiation from invading merozoite to ring (0–5 h), to trophozoite (10–40 h), and finally schizont (40–48 h) stages (Figures 1 and 2). During intracellular development,deformability of P. falciparum–infected erythrocytes is dramatically reduced and the cell is endowedwith the ability to adhere to host endothelial cells and other erythrocytes (Figure 3) (reviewedin 2). Cytoadherence confers on P. falciparum–infected erythrocytes the ability to sequester inmajor organs of the host, preventing circulation through the spleen and destruction by splenicmacrophages. Sequestration of P. falciparum–infected erythrocytes can lead to occlusion of bloodflow and infiltration of immune effector cells, which is linked to severe pathogenesis and death(reviewed in 2, 60).

ERYTHROCYTE REMODELING

The extraordinary process of erythrocyte remodeling is achieved by large-scale export of pro-teins beyond the confines of the parasite and the encasing parasitophorous vacuole membrane(PVM) (Figure 3) (reviewed in 52). Remodeling is dependent on construction of an extraparasitictrafficking network in the host cell that distributes and sorts exported proteins to their subcel-lular locations. A key feature of the extraparasite protein trafficking network is exomembranousstructures of various sizes (93), including Maurer’s clefts (57), electron-dense vesicles (∼80 nmdiameter) (33, 35, 36), and J-dots (49) (Figures 2 and 3). Maurer’s clefts and J-dots are mobilestructures in the erythrocyte cytoplasm during early stages of parasite development (31). Mau-rer’s clefts are large, flattened membranous structures that bud from the PVM in early ring-stagedevelopment and they migrate toward the erythrocyte membrane, where they become physicallytethered as the parasite differentiates into a trophozoite (34, 86, 98). Maurer’s cleft tethering isimportant for trafficking of protein complexes from within them to the underside and surfaceof the host cell membrane (34, 98, 105). The function of electron-dense vesicles and J-dots isunknown, although they may facilitate trafficking of adherence proteins from Maurer’s clefts ontothe erythrocyte membrane (48, 49).

Cytoadherence of P. falciparum–infected erythrocytes is conferred by expression of a high-molecular-weight protein, P. falciparum erythrocyte membrane protein 1 (PfEMP1) (3, 81, 89).The PfEMP1 protein family displays enormous sequence diversity and host-cell-binding selec-tivity, and these features contribute to the spectrum of disease severity and pathology (reviewedin 2). Sequestration in the capillaries of the brain and placenta during pregnancy contributes sig-nificantly to the morbidity and mortality rates from malaria. PfEMP1 is assembled into electron-dense knobs, consisting primarily of the knob-associated histidine-rich protein (KAHRP), andthese structures are inserted under the erythrocyte membrane (67, 95, 105). The PfEMP1 familyof proteins are encoded by approximately 60 var (variable) genes per haploid genome (25) and theyshow monoallelic expression (79). The ability to switch var gene expression provides a mechanismof antigenic variation important for evasion of host immune responses. The PfEMP1 family pro-teins are targets of acquired immunity during infection and may be candidates for development ofinterventions against malaria. The significant diversity of this protein family in the P. falciparumpopulation is a possible impediment to this goal.

Another key feature of erythrocyte remodeling is the establishment of a new permeability path-way (NPP) that confers a profound increase in permeability of the infected erythrocyte membrane(1, 27, 88). The NPP acts as a conduit for the import of nutrients and exchange of waste products,allowing parasites to condition the intracellular ionic environment. The NPP has also been namedthe plasmodial surface anion channel (65). The proteins involved in each of these processes arefurther described below.


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PV

PVM

N

Red

blood cell

FV

N

N

FV

RB

PM

K

Merozoites

MC

N

RB

CMer

N

MC

N

FV

0–5 hRing

5–10 hTrophozoite

10–20 hTrophozoite

>40 hSchizont

MC

FV

K

N

C

200 nm

200 nm

200 nm

200 nm

Ring parasiteRing parasiteRing parasite

Figure 2The blood-stage cycle of malaria parasite development. (Top) Parasites produce a parasitophorous vacuole membrane (PVM) duringinvasion that encases the parasite as it develops. Within 5 h the parasite has developed into a ring and has already begun exportingproteins and generating Maurer’s clefts (MCs). (Right) From 5 to 10 h, parasites generate more MCs from the PVM. As parasitesconsume hemoglobin, hemozoin crystals can be observed in the food vacuole (FV). (Bottom) Parasite development increases between 10and 20 h, and hemoglobin uptake via cytostomes (C; arrow) provides nutrients. The parasite and FV enlarge and erythrocyteremodeling continues as MCs become tethered beneath the erythrocyte membrane, and the formation of knobs occurs on theerythrocyte surface (K; arrowheads). (Left) To complete intraerythrocytic development and produce merozoites (Mer), parasitesundergo nuclear division by cytokinesis, leaving a remnant FV called a residual body (RB). Between 16 and 32 merozoites are producedand their egress allows invasion of new erythrocytes. Abbreviations: N, nucleus; PM, parasite membrane; PV, parasitophorous vacuole.


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Immediatelypostinvasion

Late ring/earlytrophozoite

Densegranulefusion

Early ring

RESA

PTEX

Vesicles

KAHRP

Maurer’s cleft

PfEMP1

Wasteexchange

Nutrientuptake

NPP/PSAC

Nucleus

Knobs

PM

PVM

EM

PV

PTEX

Nutrientuptake

Wasteexchange

Figure 3Erythrocyte remodeling during parasite development. (Top) Immediately postinvasion, parasite-dense granules fuse with the PM,releasing the PTEX and exported proteins such as RESA into the PV. PTEX assembles in the PVM, enabling export. Within minutesafter invasion, RESA can be observed inside the erythrocyte and beneath the EM. (Right) Early ring-stage parasites export manyproteins, including KAHRP and PfEMP1. PfEMP1 associates with Maurer’s clefts, and other vesicular structures can be observedwithin the erythrocyte. Following export, KAHRP and other proteins localize to the cytosolic face of Maurer’s clefts. (Left) Proteinexport continues throughout late rings/early trophozoites via discrete regions in the PV that contain the PTEX translocon (see inset).KAHRP reaches its final destination beneath the EM, binding spectrin and forming knobs. Maurer’s clefts begin to tether beneath theEM and translocation of PfEMP1 from Maurer’s clefts onto the erythrocyte surface begins. PfEMP1 is presented on the cell surfaceand NPPs are established, mediated by the PSAC. Abbreviations: EM, erythrocyte membrane; KAHRP, knob-associated histidine-richprotein; NPP, new permeability pathway; PM, parasite membrane; PSAC, plasmodial surface anion channel; PV, parasitophorousvacuole; PVM, parasitophorous vacuole membrane; PfEMP1, Plasmodium falciparum erythrocyte membrane protein 1; PTEX,Plasmodium translocon of exported proteins; RESA, ring-infected erythrocyte surface antigen.

Parasitophorousvacuole (PV):a membrane-boundcompartment ininfected cellscontaining the malariaparasite

MECHANISMS OF PROTEIN EXPORT

Role of the PEXEL/VTS in Export

Exported proteins must pass through the parasite membrane, the parasitophorous vacuole (PV),and the PVM to reach the host cell cytosol (Figure 3). They are then sorted to their final subcellularlocation, which can include insertion under the erythrocyte membrane in complexes or on thecell surface. An important advance in understanding protein export was the identification of a


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Plasmodium exportelement (PEXEL):an amino acidsignature in the Nterminus of proteinsthat is necessary forexport

Endoplasmicreticulum (ER):a secretory organelle;the first step in thesecretory and exportpathways in malariaparasites

Plasmepsin V:a parasite protease;cleaves the PEXELmotif in proteinsdestined for export

PEXEL-negativeexported protein(PNEP): an exportedmalarial proteinlacking a PEXELmotif

pentameric amino acid sequence (RxLxE/D/Q) required for export, named the Plasmodium exportelement (PEXEL) (56) or vacuolar targeting sequence (VTS) (38), located 20–30 amino acids C-terminal from the signal sequence in proteins destined for export. A similar export motif (RxLR)was identified in the N terminus of effector proteins from oomycete pathogens (102), and despitesuggestions that the mechanism of effector trafficking for Plasmodium spp. and oomycetes wassimilar, subsequent work has shown substantial differences, and it is unlikely they are functionallyequivalent (23, 42, 100, 107).

Plasmepsin V and the Matured N Terminus of PEXEL Proteins

Although it was clear that the PEXEL is required for the export of proteins, its role was notunderstood (38, 56). It has been demonstrated that exported proteins are proteolytically cleavedon the C-terminal side of the conserved leucine (i.e., RxL↓ xE/D/Q) and N-acetylated (i.e.,Ac-xE/Q/D), which showed that the PEXEL motif is a recognition site for a protease that actsin the endoplasmic reticulum (ER), as cleavage was sensitive to brefeldin A (12). Subsequently,the ER resident protease, plasmepsin V, was shown to be responsible for PEXEL cleavage, andthis is the first step in a pathway for export of proteins (7, 76). The PEXEL arginine and leucineresidues are required for recognition and cleavage by plasmepsin V, whereas the fifth amino acidis important in export postcleavage (7, 8, 38, 56). Consequently, the function of the PEXELmotif is twofold: to enable identification of PEXEL proteins for export and cleavage to releasethe N terminus from the ER membrane, and to uncover the export signal Ac-xE/Q/D at the newN terminus, which directs the mature protein to the host cell (7, 76).

The importance of the processed Ac-xE/Q/D N terminus in trafficking events after plasmepsinV cleavage has been demonstrated by engineering protein reporters cleaved in the ER either bysignal peptidase or by plasmepsin V to produce identical proteins. The reporter protein cleavedby signal peptidase to produce Ac-xQ at the N terminus was secreted to the PV, not exported(7). In contrast, the reporter protein processed by plasmepsin V to produce the same N terminus(Ac-xQ) was efficiently exported. Therefore, plasmepsin V recognition and cleavage reveals asignal that is essential for progression into the export pathway. This finding has been confirmedby a recent study fusing the protein sequence from a mature PEXEL protein (i.e., starting xE)onto a domain from a viral capsid protease that self-cleaves upon folding in the ER to reveal themature PEXEL protein, and this processed protein was also efficiently exported (92). This showedthat plasmepsin V function could be replaced, analogous to PEXEL-negative protein export (seebelow). Nevertheless, it was clear that processing of PEXEL proteins reveals the export signal andthis is essential. The precise mechanism(s) after cleavage that directs mature PEXEL proteins tothe PVM remains unknown; however, like most mechanisms of cargo selection in the ER, it mayinvolve specific proteins interacting directly or indirectly with plasmepsin V, be it a chaperone orcargo receptor, that escort the protein after PEXEL processing (Figure 4).

How Are PNEPs Exported?

Plasmodium spp. express a second class of exported proteins, called PEXEL-negative exported pro-teins (PNEPs) (reviewed in 83), that do not contain an N-terminal hydrophobic signal sequence,a PEXEL motif, or other conserved export sequences. PNEPs are a relatively small group inP. falciparum and include proteins such as ring-exported proteins 1 and 2 (REX1, REX2) (21, 32)and skeleton binding protein 1 (SBP1) (see Table 1). The major virulence protein PfEMP1 isalso defined as a PNEP as this family has no hydrophobic signal sequence and does not possessa PEXEL at the N terminus; PfEMP1 and other PNEPs cannot be cleaved by plasmepsin V (6).


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Therefore, PNEPs may use a trafficking pathway in the ER distinct from that used by PEXELproteins. Recent studies have shown that the first 10 amino acids of the PNEP (i.e., REX2), as wellas the transmembrane domain, are required for export, which suggested the N terminus containsan export signal functionally equivalent to that in plasmepsin V–cleaved PEXEL proteins (30, 32).The ability to complement the N terminus of a PNEP with the N terminus of a cleaved PEXEL

C Y T O P L A S M

E R L U M E N

C Y T O P L A S M

E R L U M E N

C Y T O P L A S M

E R L U M E N

C Y T O P L A S M

E R L U M E N

PT

EX

PT

EX

PT

EX

PT

EX

PT

EX

PT

EX

PT

EX

PT

EX

PT

EX

PT

EX

PT

EX

PT

EX

PT

EX

6

Unfolding andtranslocation

N

1 2 3 4 5

Model 1

Model 2

N

Model 3

N

RBC

PV

PVMH

SP

HSP

N-ATase

PM5

PM5 N-ATase

RBC

PV

PVM

RBC

PV

PVM

HSPPM5

AT

Pa

rasi

teP

ara

site

Pa

rasi

te

PM5

PM5

1 2 3

PNEPmodel

N

N-terminalprocessing?

Loading ofexport vesicle

RBC

PV

PVM

5

Unfolding andtranslocationPI3P binding?

PTEX spansPM and PVM?

4b4a

PMtranslocon?

PM

Ribosome Signal sequence

PEXEL

PI3P

PEXEL receptor

Acetyl-CoA

HSP HSP101

Export translocon

PM5 AT

N-ATase TMD

PM translocon

PM5

HSP

HSP

OuterER leafletreceptor

model

InnerER leafletreceptor

model

PI3P bindingPEXEL

cleavage N-acetylation ER transitLoading of

export vesicle

InnerER leaflet

chaperonemodel

Pa

rasi

te

N-ATase

AT

a

bor


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protein strongly supports this hypothesis (30). Trafficking of PNEPs is also brefeldin A sensitive,suggesting that it involves vesicular trafficking from the ER to the parasite membrane (30). Thisindicates that although there are distinct differences between trafficking of PNEPs and traffickingof PEXEL proteins, there may be some overlap in their export mechanism. While we do notunderstand the molecular basis for PEXEL and PNEP export, it is likely that the differences be-tween them may be important with respect to timing of expression, subcellular localization, andtopology of the proteins in the erythrocyte.

Another difference between PEXEL proteins and PNEPs is acetylation of the N terminus;however, the importance of this feature in export is uncertain. Whereas virtually all exportedPEXEL proteins are N-acetylated after cleavage (8, 12), the PNEP REX2 is not N-acetylated afterN-terminal processing in parasites (32). N-acetylation is common in eukaryotes and generallyoccurs in the cytoplasm. In Plasmodium spp. this would require PEXEL proteins to face theoutside of the ER membrane rather than the ER lumen during transport, and this has beensuggested as a potential route for PEXEL protein export (51, 75) (Figure 4). However, eukaryoticcells possess ER acetylation machinery (41, 45, 104). BLAST searches of Plasmodium genomesequences (25) using this human machinery readily identified homologues of these components(PF3D7_1437000, an N-acetyltransferase homologue, and PF3D7_1036800, a putative acetyl-CoA transporter) in all Plasmodium species. Although the role of N-acetylation in export is unclear,it may enhance the avidity of protein-protein interactions between the plasmepsin V–processedN terminus of exported proteins and potential PEXEL receptors or chaperones (80) (Figure 4).

How Is PfEMP1 Exported?

Export of the PNEP protein PfEMP1 is a complex and multistep process as it is large with avariable number of folded domains (Figure 5) and must traverse the parasite membrane, PVM,Maurer’s cleft membrane, and erythrocyte membrane to display the adhesive ectodomain on theoutside surface. The first step in export requires the transmembrane domain for ER entry, as is the

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 4Models of PEXEL and PNEP export. (a) Three models of PEXEL protein export. In Model 1, PEXEL proteins are targeted to the ERby their signal sequence anchored in the ER membrane. The PEXEL binds PI3P present on the cytoplasmic ER leaflet ( gray), possiblytargeting the protein toward PM5 (Step 1). In Step 2, PEXEL processing occurs and the matured protein is selected by an ER receptor.The new N terminus of the PEXEL protein is acetylated by a cytoplasmic N-ATase using cytoplasmic acetyl-CoA (Step 3). Cargoproteins transit the ER membrane (Step 4) and are loaded onto vesicle membranes via an ER receptor (Step 5). Vesicles dock at regionsof the PV containing the PTEX translocon in the PVM. Cargo proteins are released, unfolded, and translocated across the PVM intothe RBC (Step 6). In Model 2, PEXEL proteins are targeted to the ER, where PI3P binding, PEXEL cleavage, and cargo recruitmentoccur inside the ER lumen ( green) (Steps 1 and 2). However, Step 3 involves N-acetylation of the new N terminus in the ER by anER-resident N-ATase that utilizes acetyl-CoA imported into the ER by an AT. Cargo proteins transit the ER and traffic to the PV viavesicles (Steps 4 and 5). Step 6 occurs as in Model 1. In Model 3, Steps 1 to 6 occur as in Model 2, except PEXEL-cleaved cargo arerecruited by Hsp101 (Hsp) after cleavage by PM5 (Step 2) and escort cargo to PTEX (Step 6). (b) Model of PNEP export. In Step 1,proteins are targeted to the ER by a TMD. PNEP-PI3P binding at the parasite ER is unknown. In Step 2, the N terminus of PNEPsmay be processed (e.g., REX2 is cleaved by an unknown protease, not PM5). Also of note, REX2 was not N-acetylated after processing(32). In Step 3, proteins transit the ER and are loaded into export vesicles for transport to the PM. In Step 4, protein unfolding isrequired at the PM, suggesting a translocon mechanism (30). This could be a unique PM translocon (Step 4a) or indicate that PTEXspans both the PM and the PVM (Step 4b). The protein is translocated into the RBC (Step 5) and associates with Maurer’s clefts.Abbreviations: AT, acetyl-CoA transporter; ER, endoplasmic reticulum; N-ATase, N-acetyltransferase; PEXEL, Plasmodium exportelement; PI3P, phosphatidylinositol-3-phosphate; PM, parasite membrane; PM5, plasmepsin V; PNEP, PEXEL-negative exportedprotein; PTEX, Plasmodium translocon of exported proteins; PV, parasitophorous vacuole; PVM, parasitophorous vacuole membrane;REX, ring-exported protein 2; RBC, red blood cell; TMD, transmembrane domain.


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Tab

le1

Pro

pert

ies

ofPl

asm

odiu

mfa

lcip

arum

prot

eins

expo

rted

toer

ythr

ocyt

e

Nam

eA

cces

sion

num

ber

Size

ofge

nefa

mily

Gen

etic

knoc

kout

Fina

lloc

aliz

atio

nFu

ncti

onFe

atur

e/st

ruct

ure

Ref

eren

ce(s

)Pl

asm

odiu

mfa

lcip

arum

prot

eins

Pro

tein

sw

ith

PE

XE

L/V

TSa

KA

HR

P/H

RP

1P

F3D

7_02

0200

01

Yes

Und

ersi

deof

eryt

hroc

yte

mem

bran

e

Form

atio

nof

knob

stru

ctur

esH

is-r

ich

dom

ains

18

HR

P2/

3P

F3D

7_08

3180

0PF3

D7_

1372

200

2N

otat

-te

mpt

ed;

som

est

rain

sla

ckge

ne

Ery

thro

cyte

cyto

plas

mU

nkno

wn

His

-ric

hpr

otei

ns10

1

GB

P13

0e.

g.,P

F3D

7_10

3520

03

Yes

Ery

thro

cyte

cyto

plas

mIn

crea

sed

eryt

hroc

yte

rigi

dity

whe

nfu

nctio

nre

mov

edU

niqu

e46

,54

PfE

MP

2/M

ESA

PF3

D7_

0500

800

1N

otat

-te

mpt

ed;

som

est

rain

sla

ckge

ne

Und

ersi

deof

eryt

hroc

yte

mem

bran

e

Bin

dsto

host

prot

ein

4.1

Lar

ge,h

ighl

yre

petit

ive

17

PfE

MP

3P

F3D

7_02

0190

01

Yes

Und

ersi

deof

eryt

hroc

yte

mem

bran

e

Tru

ncat

ion

acts

asdo

min

ant

nega

tive

bloc

king

PfE

MP

1tr

affic

king

Lar

ge,h

ighl

yre

petit

ive

98

PfP

TP

1P

F3D

7_02

0220

01

Yes

Mau

rer’

scl

eft

PfE

MP

1re

crui

tmen

tint

oM

aure

r’s

clef

tsSi

ngle

TM

D54

PfP

TP

3P

F3D

7_14

7860

01

Yes

Ery

thro

cyte

cyto

plas

mP

fEM

P1

disp

lay

onth

eer

ythr

ocyt

esu

rfac

e,in

crea

sed

rigi

dity

ofer

ythr

ocyt

e

Lar

gere

petit

ive

prot

ein

54

PfP

TP

4P

F3D

7_07

3090

01

Yes

ND

Dec

reas

edP

fEM

P1

disp

lay

onth

eer

ythr

ocyt

esu

rfac

eR

epet

itive

prot

ein

54

PfP

TP

5P

F3D

7_10

0210

01

Yes

ND

Dec

reas

edP

fEM

P1

disp

lay

onth

eer

ythr

ocyt

esu

rfac

eR

epet

itive

prot

ein

54


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PfP

TP

6P

F3D

7_13

0200

0Y

esN

DD

ecre

ased

PfE

MP

1di

spla

yon

the

eryt

hroc

yte

surf

ace

Uni

que

54

MA

L8P

1.15

4P

F3D

7_08

0190

01

Yes

ND

Dec

reas

edri

gidi

tyof

eryt

hroc

yte

for

knoc

kout

Uni

que

54

PF1

3_00

73P

F3D

7_13

0140

01

Yes

ND

Incr

ease

dri

gidi

tyof

eryt

hroc

yte

for

knoc

kout

Uni

que

54

RIF

INe.

g.,P

F3D

7_03

2480

0∼2

00Y

esE

ryth

rocy

tesu

rfac

e/M

aure

r’s

clef

t

Not

know

nM

embe

rsof

2TM

Dsu

perf

amily

15

STE

VO

Re.

g.,P

F3D

7_02

2140

0∼3

0Y

esE

ryth

rocy

tem

em-

bran

e/M

aure

r’s

clef

t

Incr

ease

dri

gidi

tyof

eryt

hroc

yte

inga

met

ocyt

ecl

ones

havi

ngin

crea

sed

expr

essi

on

Mem

bers

of2T

MD

supe

rfam

ily

15,7

7

FIK

Ke.

g.,P

F3D

7_01

0260

0∼1

8Y

esE

ryth

rocy

tecy

topl

asm

Pot

entia

lkin

ases

Kin

ase

dom

ain

66,7

8

PF1

0_03

81P

F3D

7_10

3910

01

Yes

ND

Req

uire

dfo

rkn

obas

sem

bly

Hsp

40-l

ike

(Dna

Jty

peIV

)54

PFB

0920

wP

F3D

7_02

2010

01

Yes

ND

Incr

ease

dri

gidi

tyof

eryt

hroc

yte

for

knoc

kout

Hsp

40-l

ike

(Dna

Jty

peIV

)54

PfG

EC

OP

F3D

7_12

5300

01

Yes

Ery

thro

cyte

cyto

plas

mG

amet

ocyt

epr

otei

nno

tkno

wn

Hsp

40-l

ike

(Dna

Jty

peIV

)62

Dna

Je.

g.,P

F3D

7_11

3340

0∼1

9Y

esE

ryth

rocy

tecy

topl

asm

,som

ein

J-do

ts

Lik

ely

coch

aper

ones

Hsp

40-l

ike

(Dna

Jty

peII

/III

/IV

)49

,56,

78

PH

IST

ae.

g.,P

F3D

7_01

0200

∼17

Not atte

mpt

edN

DP

HIS

Tdo

mai

nfu

nctio

nun

know

nP

HIS

Ta

dom

ain

56,7

8

PH

IST

be.

g.,P

F3D

7_02

0160

0∼2

3N

ot atte

mpt

edN

DP

HIS

Tdo

mai

nfu

nctio

nun

know

nP

HIS

Tb

dom

ain

56,7

8

PFD

1170

cP

F3D

7_04

2460

01

Yes

ND

Req

uire

dfo

rkn

obas

sem

bly

PH

IST

bdo

mai

n54

PF1

4_00

18P

F3D

7_14

0160

01

Yes

ND

Dec

reas

edri

gidi

tyof

eryt

hroc

yte

for

knoc

kout

PH

IST

bdo

mai

n54

(Con

tinue

d)


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onl

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Tab

le1

(Con

tinu

ed)

Nam

eA

cces

sion

num

ber

Size

ofge

nefa

mily

Gen

etic

knoc

kout

Fina

lloc

aliz

atio

nFu

ncti

onFe

atur

e/st

ruct

ure

Ref

eren

ce(s

)P

HIS

Tb

(Dna

J)e.

g.,P

F3D

7_01

0220

0∼7

Yes

Ery

thro

cyte

cyto

plas

mL

ikel

yco

chap

eron

esH

sp40

-lik

e(D

naJ

type

IV)

56,7

8

RE

SAP

F3D

7_01

0220

01

Yes

Und

ersi

deof

eryt

hroc

yte

mem

bran

e

Bin

dsan

dst

abili

zes

spec

trin

tetr

amer

enha

ncin

gre

sist

ance

toth

erm

alan

dm

echa

nica

lin

sults

,dec

reas

edri

gidi

tyof

eryt

hroc

yte

for

knoc

kout

Hsp

40-l

ike

(Dna

Jty

peIV

),P

HIS

Tb

dom

ain

20,5

4

PH

IST

ce.

g.,P

F3D

7_11

3340

0∼1

4N

ot atte

mpt

edN

DP

HIS

Tdo

mai

nfu

nctio

nun

know

nP

HIS

Tc

dom

ain

56,7

8

PfP

TP

2P

F3D

7_07

3110

01

Yes

Ves

icle

sin

eryt

hroc

yte

cyto

plas

m

PfE

MP

1di

spla

yon

the

eryt

hroc

yte

surf

ace

Rep

etiti

vedo

mai

n,P

HIS

Tc

dom

ain

54

PfM

C-2

TM

e.g.

,PF3

D7_

0101

300

∼12

Not atte

mpt

edM

aure

r’s

clef

tand

unde

rer

ythr

ocyt

em

embr

ane

Unk

now

nM

embe

rsof

2TM

Dsu

perf

amily

50

Pro

tein

sla

ckin

gP

EX

EL

:PN

EP

sP

fEM

P1

fam

ilye.

g.,V

ar2C

SAP

F3D

7_12

0060

0∼6

0Y

es;e

.g.,

var2

csaE

ryth

rocy

tesu

rfac

eA

dher

ence

toho

stre

cept

ors,

antig

enic

vari

atio

n,e.

g.,

Var

2CSA

bind

sto

CSA

c

Lar

gem

embr

ane

prot

ein

with

DB

Ldo

mai

ns

3,81

,90

SBP

1P

F3D

7_05

0130

01

Yes

Mau

rer’

scl

eft

Tra

ffick

ing

ofP

fEM

P1

toM

aure

r’s

clef

t/er

ythr

ocyt

esu

rfac

e

Sing

leT

MD

16,5

3

MA

HR

P1

PF3

D7_

0207

000

1Y

esM

aure

r’s

clef

tT

raffi

ckin

gof

PfE

MP

1to

Mau

rer’

scl

eft

Sing

leT

MD

85,8

7

MA

HR

P2

PF3

D7_

0206

900.

11

No

Mau

rer’

scl

eft

Com

pone

ntof

Mau

rer’

scl

eft

teth

ers

Sing

lehy

drop

hobi

cre

gion

68


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onl

y.


RE

X1

PF3

D7_

0935

900

1Y

es;s

ome

stra

ins

lack

gene

Mau

rer’

scl

eft

Req

uire

dfo

rM

aure

r’s

clef

tar

chite

ctur

ean

dlin

ked

toP

fEM

P1

disp

lay

Sign

alse

quen

ce,

coile

d-co

ildo

mai

n

21,3

4,37

RE

X2

PF3

D7_

0936

000

1N

ot at-

tem

pted

;so

me

stra

ins

lack

gene

Mau

rer’

scl

eft

Unk

now

nSi

ngle

TM

D32

,84

Pf3

32P

F3D

7_11

4900

01

Yes

Mau

rer’

scl

eft

Pot

entia

ladh

esiv

epr

otei

n,po

ssib

lyM

aure

r’s

clef

tar

chite

ctur

ean

dho

stce

llri

gidi

ty

Lar

gepr

otei

nw

ithT

MD

and

sing

leD

BL

dom

ain

28,3

9

CL

AG

3P

F3D

7_03

0250

0PF3

D7_

0302

200

2N

o;so

me

stra

ins

lack

eith

erge

ne

Ery

thro

cyte

surf

ace

Surf

ace

anio

nch

anne

lP

ossi

bly

two

TM

Ds

65

Hsp

70-x

PF3

D7_

0831

700

1N

ot atte

mpt

edJ-

dots

Unk

now

nA

TP

ase

and

Hsp

70do

mai

ns48

SUR

FIN

e.g.

,PF3

D7_

0402

200

∼11

Not atte

mpt

edM

aure

r’s

clef

tN

otkn

own

Cys

-ric

hdo

mai

nTw

ova

riab

ledo

mai

nsSi

ngle

TM

D

63

a All

PE

XE

Lpr

otei

nsco

ntai

na

sign

alse

quen

ce.

Abb

revi

atio

ns:P

fEM

P1,

Plas

mod

ium

falci

paru

mer

ythr

ocyt

em

embr

ane

prot

ein

1;C

SA,c

hond

roiti

nsu

lfate

A;T

MD

,tra

nsm

embr

ane

dom

ain;

Hsp

,hea

tsho

ckpr

otei

n;P

HIS

T,P

lasm

odiu

mhe

lical

inte

rspe

rsed

subt

elom

eric

supe

rfam

ily;D

BL

,Duf

fybi

ndin

g-lik

e;C

ys,c

yste

ine.


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PHIST(>60)

GBP(~3)

R R R R R

KAHRP RIFIN(~200)

STEVOR(~30)

PfMC-2TM(~12)

PEXEL PEXEL

FIKK(~18)

PfEMP1(~60)

Ectodomain

SURFIN(~11)

PNEPs

RESA(~7)

DnaJHsp40-like

(~19)

1 TMDfamily

(~30)

2 or moreTMD family

(~25)

OtherPNEPs

(~8)

REX1(~1)

HPD motif(or type IV variant)

SignalSignalHis Knob

PHIST

PHIST DnaJ

DnaJ

Domain ofunknown function R45 kinase

TMD

TMD* TMD

NTSDBLn CIDRn ATS

Cysteine-richdomain

Var1 Var2

Coiled coil

Domain ofunknown function

Figure 5Domain architecture of exported protein families in Plasmodium falciparum. Schematic of exported protein families containing anN-terminal signal sequence and PEXEL motif is shown. Gray dashed lines indicate the position of PEXEL cleavage. Protein domainsare indicated at their predicted positions, and the number/position of TMDs is also depicted. Some RIFINs lack the first TMD. Not allP. falciparum PEXEL proteins are shown. Schematic of exported proteins that lack a PEXEL motif (i.e., PNEPs) is shown at thebottom. Variability of PfEMP1 domains is depicted by DBLn and CIDRn, where n represents different variations and combinations ofeach domain. Other PNEPs include SBP1, MAHRP1, MAHRP2, REX2, Pf332, CLAG3.1, CLAG3.2, and Hsp70-x. REX1 has anN-terminal signal sequence but no TMD. The predicted number of proteins in each family is shown in parentheses. Abbreviations:ATS, acidic terminal segment; CIDRn, cysteine-rich interdomain region; DBLn, Duffy binding-like; NTS, N-terminal segment;PEXEL, Plasmodium export element; PfEMP1, Plasmodium falciparum erythrocyte membrane protein 1; PNEP, PEXEL-negativeexport protein; R, glycophorin-binding repeat; RIFIN, repetitive interspersed family; TMD, transmembrane domain; Var1, Var2,Variable domains.


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case for other PNEPs, confirming important trafficking information is encoded in this region (44).For export and display of PfEMP1 on the host cell surface, the semiconserved head region (NTS,DBL1, and CIDR domains) and the transmembrane domain and cytoplasmic tail are required (59).The N terminus of PfEMP1 is also predicted to be myristoylated in the parasite cytoplasm, whichmay facilitate protein trafficking as well as anchoring the protein in the erythrocyte membrane(72).

PfEMP1 expression starts 4 h postinvasion and does not appear on the surface of theP. falciparum–infected erythrocyte until 16 h; however, it is trafficked to and stored in Maurer’sclefts (47, 69, 105). Whether PfEMP1 is translocated across the PVM using the PTEX translocon(see below) is unknown (19). It has been proposed that PfEMP1 is incorporated into Maurer’sclefts as they form at the PVM; however, it was recently shown that Maurer’s clefts are gener-ated early after invasion, before PfEMP1 would reach the PVM, and that the number of cleftsdoes not increase over time (see Timing of Events in Export, below) (31). This finding suggestsPfEMP1 may be trafficked to Maurer’s clefts after they have formed, involving transit as a solublecomplex (44, 69). The next step in transport of PfEMP1 is transfer from Maurer’s clefts onto theerythrocyte surface, and this process is poorly defined (44, 59).

Role of Phosphatidylinositol-3-Phosphate

Another mechanism for export has been proposed, involving binding of the PEXEL tophosphatidylinositol-3-phosphate (PI3P) on the lumenal side of the ER membrane, prior to plas-mepsin V cleavage (5) (Figure 4). PI3P binding of PEXEL proteins upon ER targeting couldrecruit cargo to export competent or plasmepsin V–rich zones in the ER, but binding wouldattach the PEXEL to the membrane, away from the plasmepsin V active site. The PI3P hy-pothesis originated from studies on Phytophthora infestans, a pathogenic oomycete that expressesvirulence effectors with an N-terminal RxLR motif required for host cell entry (42). However,there is considerable uncertainty of a role for PI3P in the export of effector proteins in oomycetes(99, 107). The PEXEL motif of Plasmodium spp. may be functionally equivalent to the RxLRmotif in oomycetes, and this was shown by complementation of each signal for export (4, 29).Although this suggested complementarity between the RxLR from oomycetes and PEXEL fromPlasmodium spp., several other laboratories have so far not been able to demonstrate export ofoomycete proteins in P. falciparum (30).

In vitro experiments have shown that PI3P binds to the PEXEL and the key amino acidinvolved was the conserved arginine, providing evidence that PI3P may be involved in export(5). This hypothesis suggests that proteins destined for export bind to PI3P in the ER lumenand are trafficked via a vesicular pathway and released from the membrane by plasmepsin Vcleavage (Figure 4). It is unclear how PI3P binding contributes to export as plasmepsin V veryrapidly cleaves the PEXEL motif in the parasite ER, which would release the proteins from PI3Pbinding. In addition, efficient export was observed when the normal N-terminal export signal(i.e., xE) was revealed by cleavage of a viral capsid protease (92). Because these constructs lack thePEXEL arginine and leucine (required for plasmepsin V cleavage), they would not bind PI3P, andthese results are therefore inconsistent with ER lipid binding as the export mechanism. A potentialrole for PI3P binding in the export of PfEMP1 and other PNEPs has also been suggested and arecombinant PfEMP1 N terminus reportedly binds this lipid via a conserved lysine residue (i.e.,KxLxD). The role of lysine binding to PI3P in export is unclear as substitution of the PEXELarginine for lysine in KAHRP (i.e., KxLxQ) completely blocked export; the chimeric motif couldnot be cleaved by plasmepsin V and the protein accumulated in the ER (6). Other PNEPs have


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Plasmodiumtranslocon ofexported proteins(PTEX translocon):a parasite-derivedcomplex that enablesexport across the PVM

not been shown to bind PI3P, and at this stage there is no convincing evidence that PI3P plays arole in the export of PfEMP1, or other PNEPs, in infected erythrocytes.

In P. falciparum PI3P was localized to the lumenal side of the ER membrane by transgenicexpression of FYVE domains that bind specifically to this lipid, which was important for proteinexport (5). The presence of PI3P in this compartment of the ER is unusual in eukaryotic cellsand only occurs under certain circumstances, such as transient recruitment of PI3-kinase to thelumenal side of the ER for autophagosome formation, where PI3P is absent. There is no obviousPI3-kinase residing within the ER or secretory pathway, and autophagosomal-like structures havenot been observed in P. falciparum blood stages (22, 43). Moreover, ER recruitment of PI3-kinase requires a three-member complex, and orthologues cannot be identified in the genomesof different Plasmodium spp. (43). Thus, the characterized machinery that recruits PI3-kinase tothe ER may not be present in protozoa. If Plasmodium spp. produce ER-resident PI3P in a steadystate, as would be required for continuous protein export, it would be the first eukaryotic organismidentified to do so.

PROTEIN EXPORT MACHINERY

For export, PEXEL proteins and PNEPs must traverse the parasite membrane and PVM, and atranslocation machine is hypothesized to be responsible for this process (56). Proteomic analysis ofdetergent-resistant membranes identified a complex with properties expected of a translocon; it wasnamed Plasmodium translocon of exported proteins (PTEX) (19). PTEX is a >1.2-MDa multimericcomplex (11) composed of at least five members: exported protein 2 (EXP2) (40), thioredoxin 2(Trx2) (10), heat shock protein 101 (Hsp101), PTEX150, and PTEX88. PTEX components areconserved in Plasmodium spp., as expected for a protein export translocon. In addition, they areexpressed in dense granules of merozoites and deposited into the PVM after invasion when proteinexport commences (11). Furthermore, PTEX specifically binds PEXEL proteins, consistent withtheir translocation by this protein machine; however, a direct interaction between PTEX andPNEPs has not been demonstrated (19). The properties of PTEX in Plasmodium spp. is consistentwith a potential role in export across the PVM.

Biochemical analysis and homology with proteins of known function have suggested possi-ble roles for PTEX components. Hsp101 is a ClpA/B-like AAA+ ATPase of a type commonlyassociated with translocons and is expected to function in protein unfolding for translocationthrough the translocon pore (19) (Figure 6). EXP2 forms ∼600-kDa homo-oligomers and ismore tightly membrane associated than other components, consistent with its hypothesized roleas the membrane-spanning pore (11). Solubility studies show the PTEX complex is arranged inthe order EXP2-PTEX150-Hsp101, with EXP2 inserting into the PVM (11). Presumably, Trx2is involved in chemical reduction of cysteine-linked proteins for unfolding by Hsp101 beforethreading through the channel. PTEX150 and PTEX88 have no homology to other proteins andtheir function is unknown.

There is circumstantial evidence that PTEX is the export translocon in Plasmodium spp.; how-ever, lack of a good system for conditional gene expression has made it difficult to provide directevidence. Studies using three-dimensional structured illumination microscopy have provided di-rect evidence that PTEX is indeed the export translocon (74). This finding was based on previousstudies with dihydrofolate reductase fused to a signal sequence and PEXEL, which demonstratedthat unfolding is required for export, as antifolate drugs binding dihydrofolate reductase preventprotein unfolding and block export (26). Blocked translocation intermediates spatially associatewith PTEX clusters, providing direct evidence that export of PEXEL proteins occurs in PTEX-rich domains of the PVM (74).


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PVMPVM

PMPM

PTEX150

Hsp101PTEX88

Trx2

Protein for exportis unfolded

Parasite cytoplasm

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ATP

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Figure 6Model of translocation by the Plasmodium translocon of exported proteins (PTEX). Proteins destined forexport arrive at the parasitophorous vacuole (PV) by fusion of an export vesicle with the parasite membrane(PM). The protein is recognized in the vacuolar space and unfolded perhaps by Hsp101, which contains anAAA+ ATPase domain. The protein is fed through the Hsp101-PTEX150 complex to EXP2, which is theputative pore in the parasitophorous vacuole membrane (PVM). Once in the erythrocyte cytoplasm theprotein is refolded, which may involve parasite and/or host chaperones.

The current translocation model is that proteins for export arrive at the vacuolar space byvesicular trafficking and are unfolded. Hsp101 tightly binds a PTEX150 multimer and they as-sociate with oligomerized EXP2 present in the PVM (Figure 6). Cargo proteins would transitthe translocon pore and be refolded into the erythrocyte by exported parasite proteins such asHSP70-x/HSP40-like (see next section) and possibly by host chaperones.

EXPORTED PROTEINS AND THEIR FUNCTIONS

The Exportome of Plasmodium Spp.

The PEXEL motif is highly conserved and its identification in P. falciparum provided the meansto predict the exportome for Plasmodium spp. (38, 56). This revealed P. falciparum encodes thelargest exportome, with over 6% of its genome devoted to >350 exported proteins (78, 94). Thelarge catalogue of PEXEL proteins unique to P. falciparum is due partly to expansion and radi-ation of genes encoding protein families, including the PHIST (Plasmodium helical interspersedsubtelomeric) superfamily and heat shock protein 40-like (Hsp40-like) families (78) (Table 1). Alarge-scale investigation revealed that some P. falciparum PEXEL proteins are involved in cellularremodeling, cytoadherence, and parasite survival (54) (Table 1). The smaller PEXEL exportomefor parasites such as P. berghei and P. yoelii suggests they require fewer exported proteins to establishtheir intracellular niche, or deploy a larger catalogue of PNEPs yet to be identified.

It has not been possible to predict the full repertoire of exported proteins in Plasmodium spp.using bioinformatics, as PNEPs lack a conserved sequence element. The number of PNEPs inP. falciparum is presently relatively small, with the PfEMP1 and SURFIN families (106), as wellas nine others, currently known (Table 1; Figure 5). Because P. falciparum uniquely expressesthe large adhesive PfEMP1 family (as does P. reichenowi ) and extrudes Maurer’s clefts into theerythrocyte to transport and sort proteins, it might be expected that a proportion of exportedP. falciparum proteins are unique and required for the trafficking and function of PfEMP1. Theexpression of 19 exported Hsp40-like proteins (9, 78) and at least one exported Hsp70 protein


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(48) is consistent with this possibility (see below). Functional analysis of exported P. falciparumproteins has shown that many are involved in PfEMP1 trafficking and surface display, as well asmaintaining Maurer’s clefts architecture (Table 1). Examples are discussed below.

Cytoadherence Complex

PfEMP1-mediated cytoadherence to host receptors requires the cytoadherence complex, which isessential for structurally reinforcing PfEMP1 in the cell membrane and is composed of ancillaryexported proteins (18, 105). The best-studied example is KAHRP, which is exported soon aftermerozoite invasion. After export, KAHRP concentrates around the cytoplasmic face of Maurer’sclefts (105), possibly associating with the acidic terminal sequence (ATS) of PfEMP1 (67, 95,96). Recent in vitro binding studies suggest that KAHRP may not directly interact with the ATSbut that another exported protein, having a PHISTc domain (PF3D7_0936800), may bind (58).After interacting with Maurer’s clefts, KAHRP then traffics to and binds host spectrin in thecytoskeleton (67, 70) to generate one of the most prominent features of the P. falciparum–infectederythrocyte, knobs, which are a principal component of the cytoadherence complex (Figure 3).

Maurer’s clefts migrate to the erythrocyte periphery, transporting PfEMP1 and other pro-teins/complexes (see below) to beneath the erythrocyte membrane. Maurer’s clefts eventu-ally tether to the host cell membrane before another remarkable remodeling step ensues—translocation of PfEMP1 from Maurer’s clefts onto the erythrocyte surface. This process beginsat approximately 16 h post merozoite invasion (Figure 3) by mechanism(s) that are poorly under-stood (see below for a description of exported proteins involved) (47, 69). It may involve vesicularbudding from Maurer’s clefts and fusion with the erythrocyte membrane, presenting PfEMP1on the surface, where it concentrates within knobs, with the adherent ectodomain facing externalmilieu.

KAHRP is the main constituent of knobs, and disruption of its expression causes loss of knobs onthe cell surface (18). However, although this does not reduce PfEMP1 trafficking to the erythrocytesurface, consistent with the suggestion that it does not directly bind the ATS domain, it doesdramatically reduce cytoadherence under physiological flow conditions. This demonstrates thatknobs are important for PfEMP1-mediated adherence. Knob formation does not rely solely onKAHRP. The exported PEXEL proteins, PFD1170c and PF10_0381, play key roles as disruptionof the corresponding genes gave a knobless phenotype (PfEMP1 surface localization was againnot affected), similar to that observed when KAHRP expression was disrupted (54).

PFD1170c has a PHISTb domain that is likely involved in protein-protein interactions; how-ever, its specific function in knob assembly is currently unknown (54). PF10_0381 contains a typeIV DnaJ domain and is classified as Hsp40-like (Table 1; Figure 5). Hsp40 proteins are cochap-erones that regulate Hsp70 function; they are divided into four classes based on the presence orabsence of four domains. These include the DnaJ domain with a conserved His-Pro-Asp (HPD)motif (Figure 5), a Gly/Phe-rich region, a cysteine-rich zinc-binding domain, and a C-terminalsubstrate-binding domain (14). Type I Hsp40 proteins have all four domains; type II proteins lackthe zinc-binding domain; and type III and IV proteins have a signature DnaJ domain, with typeIV exhibiting variations in the HPD motif (9, 14, 97). The HPD motif is essential for stimulatingHsp70 ATPase activity, and its mutation abolishes the Hsp40-Hsp70 interaction. P. falciparum ispredicted to encode 19 exported Hsp40-like proteins (type II/III/IV), with 11 possessing the varianttype IV HPD motif (Table 1; Figure 5). This suggests they perform important cochaperone-likefunctions in erythrocytes, independently of Hsp70, and the role of PF10_0381 in knob assemblyis a good example. Recently, an exported parasite Hsp70 was identified (Hsp70-x) and it may bea target of one or more exported type II/III Hsp40-like proteins (48) (Table 1).


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PfEMP1 Trafficking and Display on the Erythrocyte Surface

Following formation of the cytoadherence complex, PfEMP1 is exposed on the erythrocyte sur-face, a process requiring the function of a number of PNEPs and PEXEL proteins. Several PNEPscharacterized in P. falciparum are required for trafficking of PfEMP1. Disruption of the genes en-coding membrane-associated histidine-rich protein 1 (MAHRP1) (87) and SBP1 (16, 53) abolisheddisplay of PfEMP1 on the infected erythrocyte surface. MAHRP1 and SBP1 are membrane pro-teins and localize in Maurer’s clefts. They are thought to facilitate PfEMP1 transfer to Maurer’sclefts during, or after, their genesis from the PVM. Loss of MAHRP1 function destabilizes Mau-rer’s clefts and it may play a structural role rather than a direct role in PfEMP1 transfer. The PNEPREX1 is also localized in Maurer’s cleft by a coiled-coil domain (21) (Figure 5). REX1 mutationdecreased PfEMP1 trafficking to the erythrocyte surface; however, these mutations destabilizedMaurer’s clefts architecture, suggesting it may be a structural protein.

Six PEXEL proteins identified in P. falciparum play roles in PfEMP1 trafficking and are namedPTP1–6 (PfEMP1-trafficking proteins 1–6) (54) (Table 1). Disruption of PTP3, PTP4, and PTP6expression in P. falciparum resulted in decreased levels of exported PfEMP1, whereas inactivationof PTP1, PTP2, and PTP3 expression abolished PfEMP1 display on the erythrocyte surface.PTP1 localizes in Maurer’s clefts and may help load or traffic PfEMP1 from the PVM into thesestructures. PTP2 and PTP3 are localized in Maurer’s clefts and also in vesicles in the erythrocytecytosol; their function is essential for the translocation of PfEMP1 from Maurer’s clefts onto theerythrocyte surface.

Enhancing Erythrocyte Rigidity

A key consequence of P. falciparum infection is that erythrocytes lose their deformability, be-coming increasingly spherocytic and rigid. Although this improves the ability to cytoadhere tohost receptors, it can block microcapillaries in the vasculature of major organs, contributing tomalaria pathogenesis (64). Uninfected erythrocytes are highly deformable, due largely to the elas-tic properties of the underlying cytoskeleton; however, exported parasite proteins that bind theerythrocyte cytoskeleton dramatically decrease its elasticity. The ring-infected erythrocyte surfaceantigen (RESA) is one such protein, and it is deployed into the erythrocyte soon after merozoiteinvasion, where it binds spectrin (24). This stabilizes the spectrin tetramer against dissociation,increasing cellular rigidity and sensitivity to thermal degradation (61, 71). RESA also slows thetransit velocity of parasitized erythrocytes, especially at febrile temperature (20). RESA is impor-tant for protecting the integrity of parasitized erythrocytes during febrile attack. Other proteinsassociated with modulation of parasite-infected erythrocyte rigidity include Pf332, a megadaltonprotein (28, 39), two exported FIKK kinases that appear to phosphorylate the cytoskeleton (66),members of the STEVOR family (77), and several PEXEL proteins examined in a large-scalegene knockout screen (54) (Table 1).

New Permeability Pathways

Parasite expansion through intraerythrocytic development requires nutrient uptake and wasteexchange. Parasites rapidly establish a new permeability pathway (NPP) in the infected erythrocytemembrane, conferring significant permeability to the membrane (1, 27, 88). The NPP ensures anoverall shift in the intracellular ionic environment so the cell does not lyse from osmotic stress ascontents change. The NPP is also called the plasmodial surface anion channel and is conferred byexpression of either of two exported PNEPs (CLAG3.1/3.2) that are conserved across Plasmodium


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spp. (65). Either CLAG3.1 or CLAG3.2 is inserted into the membrane of the host cell duringmerozoite invasion or soon after by an unknown mechanism. The two CLAG3 proteins undergoexpression switching, a property that may assist in evading host immune responses, as a portionof these proteins may be exposed on the erythrocyte surface.

TIMING OF EVENTS IN EXPORT

The ability to perform live-cell imaging and super-resolution microscopy has provided new toolsto follow progression of P. falciparum parasites through the life cycle as well as new insights intomerozoite invasion of erythrocytes and cellular remodeling events (31, 74). Merozoite invasion isan ordered process with the final stages involving release of apical organelles for the formationof the PVM and PV from parasite lipid and proteins (73). After invasion is completed, the ringstage is surprisingly dynamic, going through actin-dependent changes in shape that include elon-gated amoeboid-like extensions (31). Visualization of proteins via super-resolution microscopyhas shown that some of the shape changes observed in the newly invaded ring may result fromfusion of dense granules with the parasite membrane and release of contents (74). This importantstep establishes the structure of the PV and PVM by insertion of proteins and complexes such asthe PTEX translocon machinery from dense granules (11). Rapid deployment of the transloconto the PVM would enable export of proteins such as RESA within minutes of merozoite invasion,where these proteins traffic to the underside of erythrocyte membranes associating with spectrin.

The Maurer’s clefts are formed during ring-stage development of parasites by budding fromthe PVM. Previously, Maurer’s clefts were thought to form throughout ring-stage developmentof P. falciparum; however, a recent study has suggested they are synthesized in early ring stages andtheir numbers remain stable throughout blood-stage development (31). This has raised questionsas to how proteins that localize to Maurer’s clefts are trafficked to this organelle when they areexpressed after Maurer’s clefts are released from the PV. It recalled previous models that suggestedmembrane-associated proteins such as PfEMP1 are trafficked to Maurer’s clefts from the PV invesicles or as a soluble complex (69). Certainly, soluble PEXEL-containing proteins, such asKAHRP and PfEMP3, are expressed and exported approximately 4 h postinvasion through theparasite membrane and PVM into the host cell cytosol, where they bind to the outside of Maurer’sclefts, a step that is essential for assembly in complexes under the parasitized erythrocyte (98, 105).Regardless of the timing of Maurer’s cleft formation and release, they play an essential role in hostcell remodeling.

CONCLUSIONS AND OUTLOOK

Many viruses and bacteria commandeer host cell machinery to subvert cellular processes andensure their survival and proliferation. In contrast, the malaria parasite invades and renovatesthe erythrocyte, turning a terminally differentiated cell, lacking most of the machinery and func-tions of a typical cell, into a dynamic and privileged niche in which it can grow, transport manyhundreds of proteins, scavenge nutrients, sequester in the presence of a circulating bloodstream,and facilitate transmission back into mosquitoes. Our understanding of this process has greatlyincreased; however, there is much to understand. Most exported proteins have been identifiedthrough bioinformatic searches of Plasmodium genome sequences using the PEXEL motif—howmany exported proteins remain undiscovered? Of the more than 540 PNEPs and PEXEL proteinsso far identified in P. falciparum, only a fraction have been characterized functionally.

The PTEX complex has been identified at the PVM and it has the features expected of atranslocation machine, but proof of its role in this process is lacking. Although it is membrane


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associated, it is not clear whether it is associated only with the PVM or whether it spans the PV toconnect directly to the parasite membrane. The latter model would be similar to several bacterialsecretion systems that deploy both soluble and membrane-associated proteins across two or moremembranes in one translocation step.

Plasmodium spp. have developed two modes of protein export—one for PEXEL proteins andanother for PNEPs—and the reasons are not obvious, but different routes may be required forparticular subcellular locations and membrane topologies in the host cell. Plasmepsin V cleavageof the PEXEL motif in the ER is a key event that identifies these proteins for export, but therole of PI3P in exporting either PEXEL proteins or PNEPs is presently not clear. Currentdata suggest that the PTEX translocon is involved in exporting PEXEL-containing proteins;however, it is also likely involved in trafficking of PNEPs. Once these proteins are traffickedacross the parasite membrane and PVM, are they loaded directly into Maurer’s clefts or is therea vesicular system, or do they transfer as chaperoned soluble complexes? It is likely that all thesepossibilities function in Plasmodium spp., and the exact trafficking route they take depends ontheir final localization in the infected erythrocyte.

SUMMARY POINTS

1. A large number of proteins are exported to the P. falciparum–infected erythrocyte andthese proteins play an important role in remodeling the host cell.

2. PfEMP1 is a major virulence protein in P. falciparum responsible for cytoadherence andantigenic variation.

3. The presence of a PEXEL/VTS motif, and its processing by plasmepsin V, is requiredfor the export of several hundred proteins, and this is conserved in Plasmodium spp.

4. There are two classes of exported proteins, PEXEL and PNEP, and they appear to beexported by different trafficking routes, at least through the ER.

5. The PTEX complex located at the PVM has been implicated as a translocation machinefor protein export.

FUTURE ISSUES

1. The location and role of PI3P binding by PEXEL proteins prior to cleavage by plasmepsinV need to be clarified.

2. The mechanism of PEXEL export immediately following plasmepsin V requires exam-ination. Is there a PEXEL receptor or chaperone?

3. The trafficking route for PEXEL proteins and PNEPs from the ER to the parasitemembrane needs to be defined.

4. Functional evidence that the PTEX complex is involved in exporting PEXEL proteinsand PNEPs is required.

5. The mechanism of protein transfer from the PVM to Maurer’s clefts and to the erythro-cyte membrane requires detailed investigation.


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DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

We thank all past and present members of the laboratory for their contributions. T. Spielmannand J. Przyborski are thanked for advice on the manuscript. Funding by the National Health andMedical Research Council of Australia, the Australian Research Council, and the Howard HughesMedical Institute is acknowledged. We apologize to colleagues whose work we could not cite dueto page limitations.

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MI67-FrontMatter ARI 12 August 2013 12:26

Annual Review ofMicrobiology

Volume 67, 2013 Contents

Fifty Years Fused to LacJonathan Beckwith � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

3′ Cap-Independent Translation Enhancers of Plant VirusesAnne E. Simon and W. Allen Miller � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �21

Acyl-Homoserine Lactone Quorum Sensing: From Evolutionto ApplicationMartin Schuster, D. Joseph Sexton, Stephen P. Diggle, and E. Peter Greenberg � � � � � � � � �43

Mechanisms of Acid Resistance in Escherichia coliUsheer Kanjee and Walid A. Houry � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �65

The Biology of the PmrA/PmrB Two-Component System: The MajorRegulator of Lipopolysaccharide ModificationsH. Deborah Chen and Eduardo A. Groisman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �83

Transcription Regulation at the Core: Similarities Among Bacterial,Archaeal, and Eukaryotic RNA PolymerasesKimberly B. Decker and Deborah M. Hinton � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 113

Bacterial Responses to Reactive Chlorine SpeciesMichael J. Gray, Wei-Yun Wholey, and Ursula Jakob � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 141

It Takes a Village: Ecological and Fitness Impactsof Multipartite MutualismElizabeth A. Hussa and Heidi Goodrich-Blair � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 161

Electrophysiology of BacteriaAnne H. Delcour � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 179

Microbial Contributions to Phosphorus Cycling in Eutrophic Lakesand WastewaterKatherine D. McMahon and Emily K. Read � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 199

Structure and Operation of Bacterial Tripartite PumpsPhilip Hinchliffe, Martyn F. Symmons, Colin Hughes, and Vassilis Koronakis � � � � � � � � � 221

Plasmodium Nesting: Remaking the Erythrocyte from the Inside OutJustin A. Boddey and Alan F. Cowman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 243

vi

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The Algal Past and Parasite Present of the ApicoplastGiel G. van Dooren and Boris Striepen � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 271

Hypoxia and Gene Expression in Eukaryotic MicrobesGeraldine Butler � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 291

Wall Teichoic Acids of Gram-Positive BacteriaStephanie Brown, John P. Santa Maria Jr., and Suzanne Walker � � � � � � � � � � � � � � � � � � � � � � 313

Archaeal Biofilms: The Great UnexploredAlvaro Orell, Sabrina Frols, and Sonja-Verena Albers � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 337

An Inquiry into the Molecular Basis of HSV Latency and ReactivationBernard Roizman and Richard J. Whitley � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 355

Molecular Bacteria-Fungi Interactions: Effects on Environment, Food,and MedicineKirstin Scherlach, Katharina Graupner, and Christian Hertweck � � � � � � � � � � � � � � � � � � � � � � � 375

Fusarium PathogenomicsLi-Jun Ma, David M. Geiser, Robert H. Proctor, Alejandro P. Rooney,

Kerry O’Donnell, Frances Trail, Donald M. Gardiner, John M. Manners,and Kemal Kazan � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 399

Biological Consequences and Advantages of AsymmetricBacterial GrowthDavid T. Kysela, Pamela J.B. Brown, Kerwyn Casey Huang, and Yves V. Brun � � � � � � � 417

Archaea in Biogeochemical CyclesPierre Offre, Anja Spang, and Christa Schleper � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 437

Experimental Approaches for Defining Functional Roles of Microbesin the Human GutGautam Dantas, Morten O.A. Sommer, Patrick H. Degnan,

and Andrew L. Goodman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 459

Plant Cell Wall Deconstruction by Ascomycete FungiN. Louise Glass, Monika Schmoll, Jamie H.D. Cate, and Samuel Coradetti � � � � � � � � � � � � 477

Cnidarian-Microbe Interactions and the Origin of InnateImmunity in MetazoansThomas C.G. Bosch � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 499

On the Biological Success of VirusesBrian R. Wasik and Paul E. Turner � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 519

Prions and the Potential Transmissibility of ProteinMisfolding DiseasesAllison Kraus, Bradley R. Groveman, and Byron Caughey � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 543

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The Wonderful World of Archaeal VirusesDavid Prangishvili � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 565

Tip Growth in Filamentous Fungi: A Road Trip to the ApexMeritxell Riquelme � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 587

A Paradigm for Endosymbiotic Life: Cell Differentiation of RhizobiumBacteria Provoked by Host Plant FactorsEva Kondorosi, Peter Mergaert, and Attila Kereszt � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 611

Neutrophils Versus Staphylococcus aureus: A Biological Tug of WarAndras N. Spaan, Bas G.J. Surewaard, Reindert Nijland,

and Jos A.G. van Strijp � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 629

Index

Cumulative Index of Contributing Authors, Volumes 63–67 � � � � � � � � � � � � � � � � � � � � � � � � � � � 651

Errata

An online log of corrections to Annual Review of Microbiology articles may be found athttp://micro.annualreviews.org/

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Documents

Plasmodium Nesting: Remaking the Erythrocyte from the Inside Out