Cellular Microbiology (2014) 16(3), 355–363

doi:10.1111/cmi.12261 First published online 27 January 2014

Microreview Protein export in malaria parasites: an update Brendan Elsworth,1,2 Brendan S. Crabb1,2,3* and Paul R. Gilson1,2 1 Burnet Institute, 85 Commercial Road, Melbourne, Vic. 3004, Australia. 2 Monash University, Clayton, Vic., Australia. 3 University of Melbourne, Parkville, Vic. 3010, Australia. Summary Symptomatic malaria is caused by the infection of human red blood cells (RBCs) with Plasmodium parasites. The RBC is a peculiar environment for parasites to thrive in as they lack many of the normal cellular processes and resources present in other cells. Because of this, Plasmodium spp. have adapted to extensively remodel the host cell through the export of hundreds of proteins that have a range of functions, the best known of which are virulence-associated. Many exported parasite proteins are themselves involved in generating a novel trafficking system in the RBC that further promotes export. In this review we provide an overview of the parasite synthesized export machinery as well as recent developments in how different classes of exported proteins are recognized by this machinery.

Introduction Plasmodium species are principally intracellular parasites of hepatocytes and red blood cells (RBCs). Large-scale parasitism of the latter manifests itself as the devastating disease malaria, which until recently killed almost a million people annually and caused symptomatic disease in hundreds of millions more (WHO, 2011). On the face of it the RBC seems a poor choice of a host cell to parasitise. RBCs are small, nutrient poor, prone to lysis, lack vesicular trafficking pathways that many pathogens use to enter and exit cells and are constantly subject to quality control as they circulate through the spleen. Plasmodium parasites have, however, managed to overcome these Received 4 December, 2013; revised 4 January, 2014; accepted 6 January, 2014. *For correspondence. E-mail [email protected]; Tel. (+61) 3 9282 2111; Fax (+61) 3 9282 2100.

obstacles, to a large degree by modifying infected RBCs (iRBCs) through the export of effector proteins into them. By modifying the iRBC into a hospitable host the parasite can hide away for most of its cell cycle in a cell that through lack of an MHC system is virtually immunologically invisible. Protein export in Plasmodium is best understood in P. falciparum, which arguably modifies its host cell more than other Plasmodium spp., reviewed in Maier et al. (2009) and Prajapati and Singh (2013). Some exported proteins help permeabilize the RBC to allow nutrients and wastes to be exchanged with the blood plasma to facilitate rapid growth and parasite proliferation (Nguitragool et al., 2011). Other proteins strengthen the RBC cytoskeleton to reduce lysis associated with increased permeability and febrile episodes in the human host (Silva et al., 2005). A specialized family of proteins, known as PfEMP1, restricted to P. falciparum, are transported onto the surface of iRBC, where they localize to raised knob structures (Baruch et al., 1995; Smith et al., 1995; Su et al., 1995). These structures allow the iRBCs to adhere to vascular endothelium, thereby removing them from circulation and clearance by the spleen. Although protein export has been known about for many years it is only recently that we have discovered the specialized transport systems used to accurately dispatch hundreds of proteins into the iRBC. These proteins have a complex export pathway from the parasite endoplasmic reticulum (ER), across the parasitophorous vacuole membrane (PVM) surrounding the parasite and out into various destinations both within and on the outside of the iRBC.

Targeting requirements of exported proteins The majority of currently known exported proteins in P. falciparum contain the pentameric localization motif RxLxE/Q/D, termed the Plasmodium export element (PEXEL) or host-targeting signal (HT) (Hiller et al., 2004; Marti et al., 2004). The discovery of this motif allowed the predication of the parasite exportome, most proteins of which belong to large gene families or are hypothetical (Sargeant et al., 2006). While the discovery of this motif has vastly increased our knowledge of exported proteins it is not found in all exported proteins. These so-called PEXEL-negative exported proteins (PNEPs) represent a

© 2014 John Wiley & Sons Ltd

cellular microbiology

356 B. Elsworth, B. S. Crabb and P. R. Gilson growing portion of the exportome, although the export requirements and number of these proteins are less clearly understood (Heiber et al., 2013; Pasini et al., 2013). Protein sequence requirements for the export of PEXEL proteins In the ER of the parasite the PEXEL motif is cleaved after the leucine residue, resulting in an N-acetylated xE/Q/D N-terminus (Chang et al., 2008; Boddey et al., 2009; Osborne et al., 2010). Cleavage of the PEXEL is known to be necessary for the export of these proteins and is performed by Plasmepsin V, an essential ER resident aspartic protease (Fig. 1A) (Klemba and Goldberg, 2005; Boddey et al., 2010; Russo et al., 2010). The conserved arginine and leucine residues are required for cleavage of the PEXEL motif and mutation results in retention of the protein in the ER (Boddey et al., 2009; 2010; 2013; Russo et al., 2010). In contrast, mutation of the 5th position of the PEXEL (E/Q/D) does not block cleavage or N-acetylation but instead leads to inefficient export across the PVM, suggesting the N-terminus of the mature PEXEL might have an important recognition role (Boddey et al., 2009; Russo et al., 2010). It has previously been hypothesized that Plasmepsin V cleaves proteins containing non-canonical PEXEL motifs, with a lysine in place of arginine (KxLxE/Q/D), or a relaxed PEXEL (RxLxxE/Q/D) (Hiller et al., 2004; Marti et al., 2004). It is now known that only the relaxed PEXEL and not PEXEL-like lysine sequences are cleaved (Boddey et al., 2013). Thus, the PEXEL motif is now defined as RxLx(x)E/Q/D and the predicted PEXEL exportome has been recently expanded to contain 463 proteins (Boddey et al., 2013). The importance of the C-terminal region immediately downstream of the PEXEL motif remains unclear despite much recent study. Early on it was shown that green fluorescent protein (GFP) fused directly to a PEXEL motif was unable to be exported and required a spacer of 10 residues after the PEXEL (Knuepfer et al., 2005). While a similar construct containing an alanine spacer allowed

efficient export, other sequences were able to block export at the PVM (Gruring et al., 2012; Boddey et al., 2013; Tarr et al., 2013). These data, combined with information that will be discussed below, implicate complex recognition of the N-terminal parts of exported proteins that appears not dependent on conserved linear motifs. The role of the PEXEL motif in protein export The PEXEL motif has been shown to associate with the lipid, PI(3)P, in the ER (Bhattacharjee et al., 2012). It has been suggested that this ability of the PEXEL to bind PI(3)P (Bhattacharjee et al., 2012) or Plasmepsin V directly interacting with other chaperones in the ER (Boddey et al., 2010; Russo et al., 2010) may play a role in sorting exported proteins (into the iRBC) from secreted proteins (into the parasitophorous vacuole) (Fig. 1A). However, reporter proteins that utilize a viral protease domain to produce a mature N-terminus (xE) can also be efficiently exported (Tarr et al., 2013). The ability to bypass both PI(3)P binding and cleavage by Plasmepsin V suggests that neither are absolutely necessary for sorting proteins to be exported. However, under normal conditions Plasmepsin V is still required to efficiently release PEXEL proteins from the ER membrane and generate a mature N-terminus (Boddey et al., 2010; Russo et al., 2010). How cleaved PEXEL proteins progress beyond the ER to eventually be exported has not been entirely resolved. Recognition of exported protein could occur in the ER by chaperones and these complexes could then be guided to specific export competent zones at the parasite surface and PV (Fig. 1A). Alternatively, exported proteins may follow the default secretion pathway into the PV and then later be recognized by chaperones or directly by PVM spanning translocons (discussed below) (Fig. 1A). Interestingly, in the related parasite Toxoplasma, PEXEL processing appears to export proteins only as far as the PVM and not into the mammalian host (Hsiao et al., 2013). In another Plasmodium-related parasite Babesia bovis, over 100 proteins appear to be exported into their erythrocyte hosts (Gohil et al., 2013). All of these proteins however

Fig. 1. An overview of protein export in P. falciparum iRBCs. A. Exported proteins first enter the ER, where those with a PEXEL motif are cleaved by plasmepsin V. The exported proteins could then enter the default secretory pathway to the PV where they are recognized and exported, possibly by a member of the PTEX complex (right). Alternatively, the exported proteins may be recognized in the ER by chaperones and/or trafficked to specific export competent zones at the PPM/PV (left). B. Soluble exported proteins are delivered to the PV where they are translocated across the PVM into the iRBC by PTEX. Proteins that contain a transmembrane domain are delivered to the PPM. Either they are then translocated across the PPM by an unidentified translocon prior to translocation across the PVM by PTEX (top) or PTEX is able to span both membranes and translocates them directly from the PPM into the iRBC (bottom). C. In young parasites (top) exported proteins may then go on to associate with mobile MCs in the iRBC cytosol. These proteins are likely transported to the MCs as soluble complexes; however, some may use vesicular trafficking. In mature parasites (bottom) MCs tether to the iRBC membrane and protein complexes to be displayed at the iRBC surface may be pre-assembled here. Chaperone-rich J-dots may play role in helping to traffic and refold protein complexes en route or at their final destinations. © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 355–363

Protein export in malaria parasites Recognition of exported proteins in the ER and export to specific zones: Exported proteins are recognised by chaperones in the ER and are secreted to export competent zones in the PV Parasite cytoplasm

OR

Export by default secretion: Exported proteins enter the default secretory pathway and are recognised within the PV Parasite cytoplasm

ER Lumen

A

Nutrients

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ER Lumen

Wastes

Transmembrane Exported protein

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Uncleaved PEXEL protein Cleaved PEXEL protein

Parasite

PI(3)P Plasmepsin V PEXEL/PNEP recognition protein

Nucleus

PTEX

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ER

?

PVM

RBC cytosol RPM

Cytoadherence complex Chaperone complex

MC

JD

Unidentified Translcon

Maurer’s Cleft Vesicle

MC

Tether

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C

Two separate translocons exist: An unidentified translocon exists at the PPM to translocate proteins with a transmembrane domain RBC cytoplasm Parasitophorous vacuole Parasite cytoplasm

Maurer’s Clefts in young parasites: Proteins are transported to mobile MCs in the iRBC cytoplasm, possibly as a chaperone complex. Extracellular Transported by vesicles?

?

OR A single translocon spans the PPM and the PVM: PTEX spans the PV to translocate both soluble and transmembrane containing proteins RBC cytoplasm Parasitophorous vacuole Parasite cytoplasm

JD

RBC cytoplasm Parasitophorous vacuole Maurer’s Clefts in older parasites: Cytoadherence proteins are transported from tethered MCs to the iRBC membrane. Extracellular Transported by vesicles?

RBC cytoplasm Parasitophorous vacuole

© 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 355–363

Transported as soluble protein?

JD

Role of J-dots?

358 B. Elsworth, B. S. Crabb and P. R. Gilson lack PEXEL motifs probably because they do not need to traffic to and cross the PVM as the membrane breaks down soon after invasion.

(Fig. 1B). Alternatively, PTEX may span both the PPM and the PVM and play a role in the export of all proteins. The Plasmodium Translocon for EXport (PTEX)

Alternative requirements for protein export of PNEPs Of the small number of known PNEPs, most appear to be associated with the Maurer’s clefts (MCs, discussed below) and usually contain no N-terminal signal peptide but rather a single internal transmembrane domain (TM). A number of studies have shown the importance of the N-terminus and TM of PNEPs for correct export; however, there appears to be no single identifiable motif (Dixon et al., 2008; Haase et al., 2009; Saridaki et al., 2009; Pachlatko et al., 2010; Gruring et al., 2012). A cryptic export motif appears to exist between both PEXEL and PNEP proteins since replacing the N-terminus of a PNEP-TM construct with the N-terminus of PEXEL proteins was sufficient to facilitate export (Gruring et al., 2012). Recently a number of new PNEPs were revealed by identifying proteins with similar properties of known PNEPs (transcription timing, hydrophobic sequences and genomic positions in the sub-telomere regions) (Heiber et al., 2013). This new subset of exported proteins has not been extensively characterized and may represent a much larger portion of the exportome than previously thought, especially in non-falciparum species that contain fewer PEXEL proteins (Heiber et al., 2013; Pasini et al., 2013). These new PNEPs show a range of different protein structures that vary from the classical PNEP structure and include proteins with a standard signal peptide and/or either none, one or two transmembrane domains (Heiber et al., 2013).

Translocation of exported proteins across the PVM Proteins destined for export are first secreted into the PV and must then cross the PVM into the iRBC. The process of how proteins are recognized in the PV and then cross the PVM is still poorly understood. However, it is known that ATP and protein unfolding are required for export across the PVM (Ansorge et al., 1996; Gehde et al., 2009). These data suggested that a proteinaceous membrane channel or translocon was required and accordingly a Plasmodium Translocon for EXport (PTEX) was identified (de Koning-Ward et al., 2009). Interestingly, soluble PEXEL proteins and PNEPs are trapped in the PV when unfolding is inhibited, whereas similarly blocked TM-containing PNEPs are trapped at the parasite plasma membrane (PPM) (Gruring et al., 2012; Heiber et al., 2013). This suggests that a second translocon may be required for transfer of TM proteins across the PPM

PTEX is a > 1.2 MDa complex, with five known protein components, found on the vacuolar face of the PVM (Fig. 2) (de Koning-Ward et al., 2009; Bullen et al., 2012). Three of these proteins, EXP2, HSP101 and PTEX150, are essential core components, and two, Trx2 and PTEX88, are non-essential accessory components of the translocon since their genes can be deleted in Plasmodium berghei (de Koning-Ward et al., 2009; Matthews et al., 2013; Matz et al., 2013). All components are expressed in the asexual life cycle as well as in the late liver stage, gametocyte stages I/II and sporozoites, where export is known to occur (Matthews et al., 2013). PTEX is secreted from the dense granules into the PV within 10 min after invasion, when protein export commences (Bullen et al., 2012; Riglar et al., 2013). PTEX preferentially binds exported proteins over non-exported proteins and colocalizes with exported proteins as well as with a trapped exported reporter protein containing a conditionally highly stabilized mouse dihydrofolate reductase domain (mDHRF) (de Koning-Ward et al., 2009; Riglar et al., 2013). EXP2 forms a 600–700 kDa homo-oligomer that localizes to the PVM and is predicted to form the pore at the PVM (Fig. 2) (Bullen et al., 2012). HSP101 is a member of the AAA+ ATPase, HSP101/clpB family of ring-shaped hexamers that are typically involved in unfolding aggregated or mis-folded protein and is predicted to unfold proteins prior to translocation across the PVM (Fig. 2) (de Koning-Ward et al., 2009; El Bakkouri et al., 2010). PTEX150 has no homologues outside of Plasmodium but is known to bind both EXP2 and HSP101 strongly and may be fulfilling a structural role in the complex (Fig. 2) (Bullen et al., 2012). Deletion of trx2 leads to a reduced parasite growth rate due to a longer asexual cell cycle (Matthews et al., 2013). These parasites were less pathogenic because they caused less experimental cerebral malaria in P. berghei (Matthews et al., 2013). The crystal structure of Trx2 shows it to be a canonical thioredoxin with an additional basic N-terminus that may interact with the negatively charged regions of EXP2 or PTEX150; however, this remains to be shown experimentally (Sharma et al., 2011). The role of Trx2 in PTEX is still not understood but it may help to redox-regulate the complex or hydrolyse disulfide bonds to unfold cargo proteins containing them (de Koning-Ward et al., 2009; Matz et al., 2013). PTEX88 has no homologues outside of the Plasmodium genus and while its role in PTEX is not understood, PTEX88 deletion parasites have a severe growth defect of approximately © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 355–363

Protein export in malaria parasites Protein

Chaperone Complex RBC cytoplasm

PTEX88 TRX2

EXP2

EXP2

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PTEX150 HSP101

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Can gene Proposed role be deleted Forms the pore in the 600-700 kDa homo-oligomer. No Predicted structure similar to (Pf or Pb) PVM. haemolysin E. Key features

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AAA+ ATPase. HSP101/clpB family. Forms a hexamer. PTEX150 No homolgues found outside of Plasmodium.

No Unfolds proteins prior (Pf or Pb) to translocation.

TRX2

Thioredoxin. Positively charged. N-terminus. Deletion results in growth defect.

Yes (Pb only)

PTEX88

No homolgues found outside Yes of Plasmodium. (Pb only) Deletion results in growth defect.

Parasite cytoplasm

No Structural role. (Pf or Pb) Hydrolyses disulphide bonds of exported proteins OR helps to regulate PTEX. Unknown.

Fig. 2. The Plasmodium translocon of exported proteins (PTEX). A model of PTEX (left). Exported proteins are secreted into the parasitophorous vacuole, where they are unfolded and translocated across the parasitophorous vacuole membrane into the RBC cytoplasm by PTEX. The properties of the five known PTEX components are shown in the table (right). Pf, Plasmodium falciparum; Pb, Plasmodium berghei.

50%, compared to wild-type parasites (Matz et al., 2013). To date PTEX is the only known PV localized complex apparently capable of transporting proteins into the host compartment (de Koning-Ward et al., 2009) and although the indirect evidence supporting its role is strong, full characterization of how the complex functions remains to be completed.

Export beyond the PVM After gaining access to the host compartment, exported proteins need to be transported to their final destinations either within the RBC’s cytosol and cytoskeleton or to parasite manufactured membranous structures such as MCs (Fig. 1C). As erythrocytes lack a secretory system, many of the parasite’s exported proteins are dedicated to the trafficking of other exported proteins including chaperones that may refold the exported cargoes after they have passed through PTEX.

The role of chaperones in protein export In the PV and the iRBC cytoplasm chaperones most likely refold or maintain exported proteins in an unfolded state until they reach their final destination. A number of parasite chaperones, namely DnaJ or Hsp40 proteins, are exported into the PV and iRBC in P. falciparum (Hiller et al., 2004; Marti et al., 2004; Nyalwidhe and Lingelbach, 2006; Maier et al., 2009; Kulzer et al., 2010; 2012). At least two Hsp40 proteins have been shown to localize to © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 355–363

discrete iRBC compartments consequently known as J-dots in P. falciparum (Kulzer et al., 2010). J-dots contain PfEMP1 and may be transiently involved in its correct transport to or from MCs and/or for refolding of the cytoadherence protein (Fig. 1C) (Kulzer et al., 2012). Hsp40 proteins typically function cooperatively with Hsp70 ATPases, and since no parasite Hsp70s were known to be exported, human erythrocyte Hsp70s were thought to be recruited by the exported Hsp40s (Kulzer et al., 2010). Recently though, a parasite-derived exported Hsp70 protein (Hsp70-x) was discovered and found to localize to the J-dots and to the iRBC membrane (Kulzer et al., 2012). Interestingly, Hsp70-x is only found in Laverania parasites (P. falciparum and the closely related P. reichenowi), in which the number of exported Hsp40s has greatly expanded to nearly 20, up from one to two proteins in most other Plasmodium spp. It is possible the expanded repertoire of chaperones in the Laverania is dedicated to trafficking and refolding the greatly expanded PEXEL-exportome specific to this group (Kulzer et al., 2012).

The role of Maurer’s clefts Proteins exported to the iRBC membrane pass through MCs in the iRBC cytoplasm. In early stages of the cell cycle MCs are highly mobile before becoming stationary below the iRBC membrane by 20 h post invasion (Fig. 1C) (Gruring et al., 2011; McMillan et al., 2013). This immobilization is thought to be due to electron dense

360 B. Elsworth, B. S. Crabb and P. R. Gilson filaments known as tethers that form between the MCs and the iRBC membrane (Hanssen et al., 2008a; 2010). These tethers are membrane bound regions containing the protein MAHRP2 and possibly other unknown proteins (Pachlatko et al., 2010). Interestingly, MAHRP2 is found in structures adjacent to MCs in the early mobile phase, suggesting there is a later event that triggers rapid tether formation between the MCs and the iRBC membrane (McMillan et al., 2013). Host actin remodelling has also been investigated as playing a role in MC immobilization (Cyrklaff et al., 2011; McMillan et al., 2013).

in MC transport to the surface has not been settled (Cooke et al., 2006; Maier et al., 2007). A number of MC proteins are also necessary for correct localization of MCs at the iRBC and PfEMP1 surface expression. Both REX1 and Pf332 are associated with the cytoplasmic side of MCs and their deletion leads to abnormally stacked MCs at the iRBC membrane, compared to the normal single lamellae, as well as reduced PfEMP1 surface translocation (Hanssen et al., 2008b; Glenister et al., 2009; Nilsson et al., 2012). Conclusion

Trafficking from the PVM to the MCs and beyond PfEMP1, KAHRP and PfEMP3 all pass through the MCs and have been suggested to traffic in a cytoadherence complex to the iRBC (Wickham et al., 2001). Also, a number of PNEPs, Type A RIFINs, and some FIKK kinases have been shown to transiently localize to MCs (Haeggstrom et al., 2004; Nunes et al., 2007; 2010; Petter et al., 2007). Early studies suggested that MCs formed a continuous or vesicular membrane structure that allowed proteins with a TM domain, primarily PfEMP1, to be transported to the surface of the iRBC as integral membrane proteins (Wickert et al., 2003; 2004). However, recent studies have shown that MCs are formed within 2 h of invasion, have no membranous continuity with the PVM or the iRBC membrane and do not change in number during the life cycle (approximately 10–20 clefts cell−1 and stabilize in number by 8 h) (Gruring et al., 2011; McMillan et al., 2013). Supporting this model is the evidence that REX1, SBP1 and MAHRP1 are found in the MCs by 2 h after invasion, whereas MAHRP2 and Pf332 are not present until 4 h and 20 h respectively (Gruring et al., 2011; Nilsson et al., 2012; McMillan et al., 2013). Using photoswitchable fluorescent proteins it was shown that new proteins are likely to be transported from the parasite through the iRBC cytoplasm to pre-formed MCs (Gruring et al., 2011). This is further supported by the finding that PfEMP1 and other MC proteins are in a soluble state prior to membrane association at their final localizations, suggesting chaperones may maintain them in a soluble state in the iRBC (Papakrivos et al., 2005; Dixon et al., 2008; Gruring et al., 2011). A number of MC proteins are known to be necessary for correct PfEMP1 transport. These include the two integral membrane proteins SBP1 and MAHRP1 that are found in early MCs (Blisnick et al., 2000; Spycher et al., 2003; Gruring et al., 2011; McMillan et al., 2013). MAHRP1 is necessary for correct PfEMP1 entry into the MCs and also plays a structural role (Spycher et al., 2008). SBP knockout mutants cannot traffic PfEMP1 to the host cell surface but whether the block is in PfEMP1 entry into the MCs or

Plasmodium parasites have solved many problems inherent with living inside erythrocytes by extensively modifying the host cells through the export of effector proteins. A key event early in the evolution of this process is likely to have been the development of a means to selectively transfer proteins across the PVM encasing the parasite. PTEX appears to fulfil this function and is accordingly found in all Plasmodium spp. that have been examined to date. Also necessary was a mechanism to refold and traffic effector proteins to their final destinations within the host and this was achieved via the export of Hsp40 and possible exploitation of host Hsp70 chaperones. Once basic export was established, the range and function of exported proteins could be greatly expanded to exploit niches within the host and in particular to avoid host immune responses. This is particularly evident in the Laverania, which have the largest and most diverse exportomes as well as correspondingly expanded repertoire of exported Hsp40s and a Hsp70 for refolding and trafficking these proteins. Only a fraction of the known exportome has been characterized and there is likely a large number of yet unidentified exported proteins, particularly PNEPs. Establishing the functions and final destinations of these exported proteins will be fascinating along with fully understanding how the various PEXEL and PNEP proteins are trafficked and which pathways they have in common. Acknowledgements Our work is supported by grants from the Australian National Health and Medical Research Council. The authors gratefully acknowledge the contribution of the Victorian Operational Infrastructure Support Program and the NHMRC IRIISS scheme. BE was supported by an Australian Postgraduate Award.

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