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Keywords:

  • dense granule;
  • falcipain;
  • invasion;
  • lysosome;
  • microneme;
  • plasmepsin;
  • rhoptry;
  • secretion;
  • subtilase;
  • toxopain

Abstract

  1. Top of page
  2. Abstract
  3. Secretory Pathway of the Apicomplexa
  4. Regulated Secretion in Apicomplexa
  5. Proteolytic Processing in The Apicomplexan Secretory Pathway
  6. Trafficking of T. gondii Proteases to Secretory Organelles
  7. Localization and Trafficking of Proteases in Plasmodium
  8. Conclusions
  9. Acknowledgments
  10. References

The Apicomplexan parasites Toxoplasma gondii and Plasmodium species are obligate intracellular parasites that rely upon unique secretory organelles for invasion and other specialized functions. Data is emerging that proteases are critical for the biogenesis of micronemes and rhoptries, regulated secretory organelles reminiscent of dense core granules and secretory lysosomes of higher eukaryotes. Proteases targeted to the Plasmodium food vacuole, a unique organelle dedicated to hemoglobin degradation, are also critical to parasite survival. Thus study of the targeting and function of the proteases of the Apicomplexa provides a fascinating model system to understand regulated secretion and secretory organelle biogenesis.

Toxoplasma gondii and Plasmodium falciparum are unicellular eukaryotes belonging to the Apicomplexa phylum. These obligate intracellular parasites are important human and animal pathogens. T. gondii has a broad host range and infects nearly all cell types. Although an estimated one-third of the world's population is infected, clinical toxoplasmosis usually manifests in immunocompromised individuals or children infected in utero. P. falciparum, the most devastating human malaria, is the cause of hundreds of millions of cases of malaria worldwide and 1–2 million deaths in sub-Saharan Africa.

Because the Apicomplexa are obligate intracellular parasites, their survival depends on their ability to invade host cells, avoid degradation by host cell machinery and propagate intracellularly. The Apicomplexa are named for the unique set of secretory organelles that are intimately associated with these functions. Several lines of evidence suggest that secreted proteases are important in the creation of a hospitable environment for these parasites. Although there are significant differences in host cell range among the Apicomplexa, it is thought that the basic molecular machinery responsible for host cell invasion is preserved throughout the phylum and that their secretory organelles are essential for invasion.

Secretory Pathway of the Apicomplexa

  1. Top of page
  2. Abstract
  3. Secretory Pathway of the Apicomplexa
  4. Regulated Secretion in Apicomplexa
  5. Proteolytic Processing in The Apicomplexan Secretory Pathway
  6. Trafficking of T. gondii Proteases to Secretory Organelles
  7. Localization and Trafficking of Proteases in Plasmodium
  8. Conclusions
  9. Acknowledgments
  10. References

Although much smaller than mammalian cells, T. gondii (2 × 8 μm) and P. falciparum (1.6 × 1 μm) have a well organized cellular structure (see Figure 1). The secretory pathway in these pathogens is highly polarized, imparting directionality to their invasion process, which occurs at the apical end of the parasite. Both T. gondii and Plasmodium species have complex life cycles with different life cycle stages that are specifically adapted for survival in a particular host.

image

Figure 1. Transmission electron micrograph of an RH strain tachyzoite of Toxoplasma gondii within a host cell.T. gondii tachyzoites are polarized cells with organelles common to all eukaryotes (mitochondria, nucleus and Golgi complex). The micronemes, rhoptries and dense granules are secretory organelles specific to the phylum Apicomplexa. Sequential release of organellar contents is thought to be essential for host cell invasion. The triple membrane surrounding the parasite is composed of a plasma membrane and two membranes that form vesicular structures called the inner membrane complex. The parasite lives within a parasitophorous vacuole that is lined with host mitochondria and ER. The vacuole is modified by secreted contents of rhoptries and dense granules. The conoid is a cytoskeletal structure at the apex of the tachyzoite.

T. gondii replicates by endodyogeny, a unique form of replication in which daughter cells are assembled within a mother cell. Invasive tachyzoites contain a triple membrane enclosing a centrally located nucleus (plasma membrane and double membrane inner membrane complex; see Figure 1). Proteins are synthesized in the endoplasmic reticulum (ER) before being trafficked to the Golgi complex. From the trans-Golgi network, the specialized secretory granules are formed.

In contrast, P. falciparum undergoes a complex 48-h developmental cycle within erythrocytes that is characterized by morphologically distinct forms: rings, trophozoites, and schizonts. Development culminates in lysis of the host erythrocyte and release of invasive merozoites. The food vacuole is a distinctive organelle of trophozoites that specializes in hemoglobin degradation. The specialized secretory organelles involved in invasion are assembled late in the erythrocyte life cycle in schizonts.

Post-Golgi trafficking in Apicomplexan parasites involves targeting to three distinctive secretory organelles: the micronemes, rhoptries and dense granules (Figure 2). Proteins are also sorted to the apicoplast (a unique chloroplast-like organelle) and, in Plasmodium, to the food vacuole (Figure 2).

image

Figure 2. Schematic of trafficking of proteases in T. gondii and Plasmodium.A) Trafficking in a T. gondii tachyzoite. It is assumed that trafficking in other Apicomplexa including malaria schizonts is similar. Microneme protein complexes are formed within the secretory pathway and targeted via cytoplasmic domains of type I integral membrane microneme proteins to micronemes. Complexes typically have a component whose proteolytic cleavage is required for transit through the secretory pathway. Multiple proteases are likely to be present in micronemes or the pathway to micronemes. Rhoptry proteins are also cleaved in transit in the secretory pathway, possibly in multivesicular bodies (MVB) or in the immature rhoptries (IR). Rhoptry protein trafficking occurs via the clathrin/AP1 pathway. Dense granules (DG) are the default secretory pathway. GPI-anchored surface antigens (SAGs) are also trafficked to the parasite plasma membrane. Organellar contents, including proteases, appear to be exclusively in either micronemes or rhoptries. Proteolytic processing of dense granule contents has not been described. B) Trafficking to the food vacuole of P. falciparum. The food vacuole of trophozoites contains many hydrolases including plasmepsins (aspartic), falcipains (cysteine), falcilysin (metallo) and DPAPI. Proplasmepsins and profalcipains are type II membrane proteins (yellow circles with black sticks) that are trafficked to the parasite plasma membrane and reach the food vacuole via cytostomes. Cytostomes are double membrane vesicles whose membranes are derived from the parasite plasma membrane (PPM) and the parasitophorous vacuole membrane (PVM). Hemoglobin (Hb) is taken up in cytostomes from the erythrocyte cytosol and trafficked to the food vacuole. There may also be a direct trafficking pathway to the food vacuole. ProDPAPI (red circle with purple triangle) is also trafficked to the parasitophorous vacuole (PV) and eventually resides in the food vacuole. Cleavage of the prodomains of the plasmepsins, falcipains (black stick attached to yellow circle) and DPAPI (purple triangle) occurs in an acidic environment either on route to or in the food vacuole resulting in release of mature plasmepsins/falcipains (yellow circle) or DPAPI (red circle). Falcipain maturation is probably autocatalytic, but removal of plasmepsin and DPAPI propeptides occurs in trans.

Malaria parasites (and perhaps T. gondii) also export proteins beyond their plasma membrane and extensively modify the erthrocyte cytosol and plasma membrane (1). Parasite-encoded proteases may well be involved in these processes, but study of these unusual aspects of the secretory pathway of the Apicomplexa are in their infancy and will not be a focus in this review.

Regulated Secretion in Apicomplexa

  1. Top of page
  2. Abstract
  3. Secretory Pathway of the Apicomplexa
  4. Regulated Secretion in Apicomplexa
  5. Proteolytic Processing in The Apicomplexan Secretory Pathway
  6. Trafficking of T. gondii Proteases to Secretory Organelles
  7. Localization and Trafficking of Proteases in Plasmodium
  8. Conclusions
  9. Acknowledgments
  10. References

Successful host cell invasion involves the sequential release of micronemes, rhoptries and dense granules. Micronemes have properties reminiscent of the dense core granules of the ciliates and neuroendocrine cells. Rhoptries are similar to lysosomal secretory organelles and are hypothesized to be derived from multivesicular bodies (2,3). As for other lysosome-related organelles, rhoptries appear to be derived from both the secretory and endocytic pathways but have features that distinguish them from classic lysosomes. Although morphologically similar to dense core granules, dense granules are the constitutive secretory pathway of T. gondii.

Biogenesis of regulated secretory organelles is not fully understood in any system. Aggregation of organellar contents in a calcium- and pH-dependent manner is thought to be the major mode of organellar assembly. Carboxypeptidase E has been implicated as a possible sorting receptor, but this remains controversial. Proteolysis appears to regulate biogenesis and maturation of both dense core granules (4–6) and lysosome-related secretory organelles (7). Biogenesis of micronemes and rhoptries appears to be regulated by proteolysis as well.

Of the Apicomplexa, T. gondii is the most genetically tractable and the most amenable to cell biology studies. Much of this review will therefore concentrate upon T. gondii, but it is believed that the function and biogenesis of secretory organelles of all the Apicomplexa is similar.

Proteolytic Processing in The Apicomplexan Secretory Pathway

  1. Top of page
  2. Abstract
  3. Secretory Pathway of the Apicomplexa
  4. Regulated Secretion in Apicomplexa
  5. Proteolytic Processing in The Apicomplexan Secretory Pathway
  6. Trafficking of T. gondii Proteases to Secretory Organelles
  7. Localization and Trafficking of Proteases in Plasmodium
  8. Conclusions
  9. Acknowledgments
  10. References

Although the exact molecular actors are still being defined, it is clear proteases play an essential role in host cell invasion by the Apicomplexa (reviewed in (8)). The serine protease inhibitors DCI and AEBSF (9) and cysteine protease inhibitors (10) prevent T. gondii tachyzoites from penetrating host cells. Inhibition is not caused by a change in morphology or an effect on gliding motility of the parasite (9). Invasion by Plasmodium is prevented by a variety of serine and cysteine protease inhibitors that probably affect different steps in invasion (reviewed in (11,12)). Proteolytic processing of microneme secretory proteins, PfAMA1 and TgMIC2, and malaria surface protein MSP-1 have been shown to be essential for host cell invasion (13).

Proteolysis during microneme formation and secretion

Micronemes are the smallest of the secretory organelles. Involved in the early stages of invasion, they contain many adhesins that help in the tight attachment of the parasite to the host cell. Microneme secretion is a calcium-dependent process and is enhanced by manipulations that increase intracellular calcium (14,15).

Microneme proteins assemble as macromolecular adhesion complexes. Microneme proteins that are type I integral membrane proteins contain microneme targeting signals in their C-terminal domains (16,17). The membrane proteins form a complex with soluble microneme proteins (17). Proper assembly of these complexes is required for exit from the ER/Golgi and targeting to micronemes (17,18). The MIC2/M2AP complex appears to be a major adhesive complex mediating invasion (18,19) but others such as the MIC1/4/6 complex are not essential (17).

Most microneme complexes have one member that is proteolytically cleaved during transit through the secretory pathway. Proteolysis is required for trafficking of M2AP and MIC5 (cited in (20)). Proteolytic cleavage of MIC3 is required for its adhesive properties (21) and deletion of its prodomain results in MIC3 that is retained within the secretory pathway (22). Proteolysis within the secretory pathway appears to regulate maturation of microneme contents and formation of micronemes, much as it appears to regulate formation of secretory granules in other eukaryotes (4–6).

As invasion occurs, T. gondii microneme proteins are secreted onto the parasite's surface and move towards the posterior end before being shed from the surface by a number of unidentified proteases (8). The interaction of the treadmilling proteins with the actin–myosin complex provides the force for host cell invasion. Mutation of sites cleaved during invasion does not affect trafficking to micronemes (23). The adhesive complexes are shed from the parasite surface as invasion proceeds and proteolysis at this phase is hypothesized to be a mechanism to inactivate the adhesins (24). Studies with protease inhibitors suggest that different proteases are involved in each of these steps and implicate both cysteine and serine proteases.

Proteolysis during rhoptry formation

Rhoptries are long club-shaped organelles that secrete proteins through their elongated necks at the apical tip of the parasite. In Toxoplasma, secretion of the micronemes precedes rhoptry exocytosis (25), but biological triggers of rhoptry secretion have not been identified. In Plasmodium, the sequence of secretory events is less firmly established, but probably proceeds similarly. Secretion coincides with formation of the parasitophorous vacuole (PV), where the parasite resides inside of host cells. The PV is mainly composed of host cell lipids (26). Rhoptry proteins and lipids, however, modify the vacuole, enabling T. gondii to subsist in the host cell without fusing with other organelles or acidifying, and prevent the parasite from being degraded by host cell lysosomes (27). Although rhoptries are thought to be similar in all the Apicomplexa, there is little homology among the characterized rhoptry proteins of T. gondii and malaria.

The rhoptries are the only known acidified organelle in T. gondii (28). Rhoptries are composed of lipids and protein and have a high cholesterol lipid content compared with the T. gondii plasma membrane (29). Rhoptries have internal membranes and type I transmembrane proteins ROP2/3/4 and TgSUB2 localize to the lumen of rhoptries rather than their periphery (2,30). Orthologs of the trafficking machinery for multivesicular body formation are present in T. gondii and malaria species and appear to be functionally conserved (3).

T. gondii ROP1 was originally identified as a component of ‘penetration enhancing factor’, but no definite function has been attributed to it (31). ROP1 is not essential, but rhoptries of T. gondii tachyzoites lacking ROP1 have a denser appearance (32,33). ROP2, the founding member of the ROP2/3/4/8 family, has been shown to mediate recruitment of host cell mitochondria and ER (34). Despite the existence of multiple other family members, ROP2 appears to be essential (34).

ROP2 targeting to rhoptries involves the clathrin/AP1 machinery and is mediated by YXXΦ and LL motifs in its cytoplasmic tail (35). There appear to be redundant rhoptry targeting signals as the prodomain of ROP4 (a ROP2 family member), as well as multiple segments of ROP1, are sufficient to target heterologous proteins to rhoptries (22,36,37).

Most characterized T. gondii rhoptry proteins are proteolytically cleaved during transit in the secretory pathway. In T. gondii only the cleavage site of ROP1 has been definitively mapped (38). Surprisingly, trafficking of ROP1 does not seem to be dependent on proteolytic processing since unprocessed ROP1 is correctly trafficked to rhoptries and secreted into the parasitophorous vacuole during invasion (37,39). It is unknown whether proteolytic processing of other rhoptry proteins affects their trafficking.

Many Plasmodium rhoptry proteins are associated in macromolecular complexes in immunoprecipitation studies, including the RAP1/2/3 complex and the RhopH1/H2/H3 complex (reviewed in (40)). Trafficking of RAP2/3 to the rhoptries is dependent upon association with RAP1 C-terminus, but the RAP1/2/3 proteins are not essential (41). Several Plasmodium rhoptry proteins including RAP1, RhopH 1 and RAMA are proteolytically cleaved, but the significance of processing has not been explored. RAP1 is proteolytically cleaved during maturation. The cleavage site for RAP1 (after A190 (40)) does not have homology with the T. gondii ROP1 cleavage site.

Once the parasite safely resides in the PV, the third secretory organelles, dense granules, secrete. T. gondii dense granule secretion is constitutive, but is also likely to have a regulated component (25,42). Dense granules represent the default pathway for secretion (43). There are no known signals for release. Components of the dense granules modify the PV and also associate with a vesicular network within the PV (42). Proteolytic processing of dense granule contents of T. gondii has not been described.

Proteolysis in the malaria food vacuole

The food vacuole is a specialized lysosome dedicated to hemoglobin degradation. Amino acids generated from the hydrolysis of hemoglobin are integrated into proteins of Plasmodium and heme moieties generated are detoxified by the formation of hemazoin crystals. Hemoglobin is taken up from the erythrocyte cytosol and traffics to the food vacuole. This acidic vacuole contains plasmepsins (aspartic), falcipains (cysteine), falcilysin (metallo) and aminopeptidases that systematically degrade hemoglobin into a usable form (reviewed in (44)).

Trafficking of T. gondii Proteases to Secretory Organelles

  1. Top of page
  2. Abstract
  3. Secretory Pathway of the Apicomplexa
  4. Regulated Secretion in Apicomplexa
  5. Proteolytic Processing in The Apicomplexan Secretory Pathway
  6. Trafficking of T. gondii Proteases to Secretory Organelles
  7. Localization and Trafficking of Proteases in Plasmodium
  8. Conclusions
  9. Acknowledgments
  10. References

Remarkably little is known about the targeting of proteases to secretory compartments. Many proteins are targeted to lysosomes via the mannose-6-phosphate receptor pathway, but glycosylation is unlikely to regulate intracellular trafficking in the Apicomplexa. A lipid-binding motif in the prodomain of the cathepsin orthologue cruzain mediates targeting to lysomes in Trypanosoma cruzi (45). Although targeting motifs for micronemes and rhoptries have been identified, these motifs are not present in TgSUB1, TgSUB2 or toxopain 1, proteases targeted to these organelles.

Subtilisin-like serine proteases (subtilases)

Serine proteases have been implicated to play an important part in invasion in Toxoplasma and Plasmodium. Rhomboids (46) and subtilases are likely to be important (8), but rhomboids of the Apicomplexa, though present in sequenced genomes, have not yet been characterized. Prohormone convertases (PCs) and furin are mammalian subtilases associated with regulated and constitutive secretory pathways, and these proteases are associated with maturation of both secretory granules (4,5,47) and melanosomes, lysosomal secretory organelles (7).

Subtilases are synthesized as preproproteins. Their N-terminal signal sequence directs them to the ER, where they undergo a primary cotranslational autocatalytic cleavage. The propeptide remains noncovalently attached to the subtilase and acts as an inhibitor as the protease traffics through the secretory pathway. Once it is in its correct environment, the propeptide dissociates leaving a mature enzyme. Many subtilases, including those of T. gondii and Plasmodium, undergo secondary processing that is blocked by BFA and low temperature, suggesting that it occurs after trafficking to the Golgi (48,49).

Three subtilases have been identified in P. falciparum, PfSUB-1, PfSUB-2 and PfSUB-3 (reviewed in (50)). In T. gondii, two subtilases have been cloned, TgSUB1 (49) and TgSUB2 (30). Eight other potential subtilases are present in the T. gondii genome, although preliminary RT-PCR experiments suggest that not all are expressed in tachyzoites, the form that is responsible for clinical disease (Squires & Kim, unpublished).

Microneme proteases

TgSUB1 is a microneme-associated subtilase (48,49) homologous to PfSUB1. TgSUB1 is secreted in a calcium-dependent manner from the parasite, like other T. gondii microneme proteins. TgSUB1 is unique in that it contains a GPI anchor (Binder & Kim, unpublished), yet is targeted to micronemes rather than the parasite's surface. Other known GPI-anchored proteins are expressed on the surface and substitution of a GPI anchor for the transmembrane domain and cytoplasmic tail redirects microneme proteins to the cell surface (17).

Chimeras between TgSUB1 and SAG1 (a GPI-anchored surface protein) demonstrate that neither the GPI anchor nor the proline-rich domain of TgSUB1 are necessary or sufficient for microneme targeting (Binder & Kim, unpublished). It appears that the lumenal portion of TgSUB1 containing the catalytic domain confers microneme targeting. TgSUB1 may also be part of a complex that is targeted to the micronemes but coimmunoprecipitation experiments do not reveal obvious interacting protein partners.

There is probably considerable redundancy in microneme proteases. A knockout of TgSUB1 is viable and has no obvious defect in invasion (Binder & Kim, unpublished). Catalytically inactive TgSUB1 Ser490Ala is partially processed (probably at different sites from active enzyme) (see Figure 3), whereas a similar mutant of the rhoptry protease, TgSUB2 Ser999Ala, accumulates as unprocessed precursor (30) (Figure 3). Thus it appears other proteases interact with TgSUB1 during its transit through the secretory pathway. Secretory granules typically have many proteases of differing function and specificity, and it is likely that TgSUB1 is one of many microneme proteases.

image

Figure 3. Expression of epitope-tagged TgSUB1 and TgSUB2 constructs and their catalytically inactive forms.A) Schematic of HA-tagged TgSUB1 and TgSUB2 expression constructs. TgSUB1 is under control of the M2AP promoter (and terminator) and TgSUB2 under control of the ROP1 promoter (and SAG1 terminator). Catalytically inactive mutants are the result of the catalytic Ser (of the catalytic triad of subtilases) being altered to an Ala residue. The cross-hatched box indicates the position of the HA tag. B) Western blots of the expression after transient transfection of RH strain tachyzoites. Wild-type TgSUB1-HA and TgSUB2-HA are correctly processed to their lower molecular weight forms. TgSUB1 S490A-HA shows accumulation of unprocessed precursor (dark arrow) and other forms (thin arrows) that do not comigrate with wild-type TgSUB1-HA. The major form of TgSUB2 S999A-HA is unprocessed precursor.

Although there is a qualitative defect in trafficking, with some retention in the secretory pathway, TgSUB1 Ser490Ala still localizes to the apical region (see Figure 4). Thus active TgSUB1 protease is not required for exit from the ER/Golgi, but active protease is more efficiently trafficked to the micronemes.

image

Figure 4. Localization of wild-type and the catalytically inactive mutant of the microneme protease TgSUB1.A) Immunofluorescence analysis (IFA) using antibodies specific for microneme proteins MIC2 (mouse monoclonal 6D10) and TgSUB1 (rabbit polyclonal) show an apical cap of labeling, a pattern characteristic of micronemal proteins. B) An IFA of transiently transfected TgSUB1-HA depicts apical microneme staining. The catalytically inactive mutant TgSUB1-S490A-HA localizes in the perinuclear area (Golgi) as well as apically. Both proteins were visualized using rat monoclonal antibody to the HA tag.

Proteases of rhoptries

Subtilases and cathepsins are hydrolases classically associated with lysosomal compartments and orthologs are present in T. gondii rhoptries (10,30). gp76, a GPI-anchored serine protease of malaria, apparently resides in rhoptries, but the gene corresponding to this protein has not been reported (51). Most of the proteases of malaria have not been localized and to date, genes encoding malaria proteases of micronemes or rhoptries have not been identified.

TgSUB2 localizes to the rhoptry secretory organelle. Furthermore, TgSUB2 shares a common cleavage site motif found among many rhoptry proteins, including the ROP1 and ROP2 family members (30). This motif, SΦXE, is present at three sites in TgSUB2 and site-directed mutagenesis of these sites alters TgSUB2 autoprocessing ((30); Thathy et al., unpublished). TgSUB2 coimmunoprecipitates with ROP1, ROP2 and ROP4 ((30); Thathy & Kim, unpublished data). A catalytically dead mutant of TgSUB2 (see Figure 3) is not processed but still associates with ROP1, suggesting that the interaction of TgSUB2 with rhoptry proteins does not require an active protease (30).

TgSUB2 is hypothesized to be a rhoptry protein maturase responsible for cleaving ROP1 and ROP2 family members. TgSUB2 is a predicted type I transmembrane protein but does not encode motifs for targeting to the rhoptries present in ROP2 and ROP2 family members. Studies are underway to test which domain or domains of TgSUB2 confer rhoptry targeting.

Genetic studies suggest that TgSUB2 is essential and may play an important role in biogenesis of the rhoptry secretory organelle (30). A knockout of TgSUB2 was unsuccessful. TgSUB2 antisense parasites displayed impaired rhoptry formation, accumulation of vesicular structures and impaired replication, leading us to hypothesize that TgSUB2 plays an important role in biogenesis (Thathy et al., unpublished). A similar but less pronounced effect was seen with antisense depletion of a cathepsin B homolog, toxopain-1, which localizes to the rhoptries (10). Cathepsin inhibitor III and subtilisin inhibitor III were reported to impair rhoptry biogenesis in a similar fashion (52). Intriguingly, antisense depletion of adapter protein μ1 or ROP2 also impair rhoptry biogenesis, suggesting that cleavage and trafficking of ROP2 is essential for rhoptry formation (53,54). This is also consistent with the model that rhoptry biogenesis occurs during every new round of replication as parasites undergo endodyogeny.

Localization and Trafficking of Proteases in Plasmodium

  1. Top of page
  2. Abstract
  3. Secretory Pathway of the Apicomplexa
  4. Regulated Secretion in Apicomplexa
  5. Proteolytic Processing in The Apicomplexan Secretory Pathway
  6. Trafficking of T. gondii Proteases to Secretory Organelles
  7. Localization and Trafficking of Proteases in Plasmodium
  8. Conclusions
  9. Acknowledgments
  10. References

Although the activity of malaria proteases has been more extensively characterized than those of Toxoplasma, definitive localization of most proteases found in the secretory organelles of Plasmodium is pending. Due to the technical difficulties associated with ImmunoEM, the small size of malaria merozoites, and lack of high quality antibodies very few double localization studies have been reported in malaria.

Malaria proteases implicated in invasion

The expression of subtilases PfSUB-1 and PfSUB-2 late in the intra-erythrocytic life cycle and in merozoites suggests that they play a role in invasion. Attempts to disrupt PfSUB-1 and PfSUB-2 have failed, suggesting that these are important, probably essential, proteases with nonredundant functions (cited in (12)). The substrate specificity of PfSUB1 has been characterized (after a VXXD motif) but the biologic substrate of PfSUB-1 is not known (55). PfSUB-2 is the most likely candidate for the ‘sheddase’ responsible for processing and shedding of MSP-1 and AMA-1 during merozoite invasion of erthrocytes (13), but this has not yet been proven.

Falcipain 1 is a cathepsin L-like cysteine protease related to hemoglobinases falcipain 2 and 3 (see below). The localization and timing of expression are consistent with a role for this falcipain in erythrocyte invasion (56). Falcipain 1 specific inhibitors were reported to inhibit invasion, but not other stages and did not affect rupture of the erythrocyte (56). Surprisingly, falcipain 1 knockouts invade erythrocytes normally (57) but have a defect in oocyte development (58). If falcipain 1 does play a role in invasion, it appears redundant proteases eventually compensate for the absence of falcipain 1 activity.

Malaria parasites may have different populations of dense granules. Falcipain 1, PfSUB-1 and PfSUB-2 all localize to apical dense granules. Although their secretion is apical (42), dense granules are normally dispersed throughout the cell, and they are usually released after invasion is complete. Double labeling studies have not been reported, and it is not known whether these proteases colocalize or have overlapping localization with other characterized apical antigens. Localization studies in Plasmodium are difficult, as illustrated by recent revised assignment of PfAMA1 to the micronemes rather than rhoptries as originally thought (59,60). For T. gondii, most secreted proteins with a likely role in invasion are released from micronemes. The inferred roles of falcipain 1, PfSUB-1 and PfSUB-2 in invasion suggest that their release from apical dense granules may be functionally equivalent to secretion of contents of T. gondii micronemes during invasion.

Malaria proteases involved in hemoglobin degradation

There are 10 plasmepsins (Plasmodium aspartic proteases) in the P. falciparum genome. Plasmepsins 1–4, 5, 9, 10 are expressed in intraerythrocytic stages (61); of these four, PM I, II, IV and HAP (histo-aspartic protease or PMIII), are the major aspartic acid proteases of the food vacuole (61).

How proteins are trafficked to the food vacuole is not understood (Figure 2B). The food vacuole plasmepsins and falcipains (see below) are predicted to be type II integral membrane proteins with large prodomains that are hypothesized (but not proven) to be involved in trafficking to the food vacuole. The T. gondii genome encodes plasmepsin, falcipain (cathepsin L) and cathepsin C orthologs, but neither conventional lysosomes nor a food vacuole equivalent have been identified.

Maturation of the plasmepsins is blocked by BFA (62,63). Localization studies (64) and studies with a PM II-GFP fusion (65) suggest a complex pathway for trafficking of plasmepsins that involves initial delivery to the plasma membrane and then to the food vacuole. ProPM I and proPM II are type II integral membrane proteins that are predicted to have their N-termini exposed to the parasite cytoplasm. ProPM II–GFP localizes to the membranes of cytostomal vacuoles (65). Cytostomes are openings on the Plasmodium parasite's surface that open into the cytoplasm of the erythrocyte. Double-membrane vesicles (originating in the parasite plasma membrane and the PV) detach from the cytostomes to carry hemoglobin to the food vacuole. Maturation is accompanied by removal of the prodomain, which encompasses the transmembrane domain. Activation is not autocatalytic and calpain inhibitors (ALLM, ALLN) prevent proprocessing but do not inhibit plasmepsin function (62,63). Proprotein processing occurs under acidic conditions, most likely in the food vacuole or in vesicles trafficking to the food vacuole.

The major cysteine proteases located in the food vacuole of P. falciparum are falcipain 2/2′ and falcipain 3, cathepsin L-like papain family members (66,67). Inhibitors of these proteases prevent hydrolysis of hemoglobin and cause an accumulation of undegraded red blood cell cytoplasm in the food vacuole. Falcipains are type II integral membrane proteins that probably traffic to the food vacuole in vesicles from the cytostome, similarly to plasmepsins, but their maturation is thought to be autocatalytic and stimulated by the acidic environment of the food vacuole.

Other enzymes involved in hemoglobin degradation are falcilysin, a metalloprotease (68), and dipeptidyl aminopeptidase I (69), or DPAP I, a cathepsin C homolog. Surprisingly, falcilysin does not contain a signal sequence to direct it into the secretory pathway or a propeptide that directs targeting (70). DPAP I is a food vacuole protease, but proDPAP I accumulates in the PV, suggesting that its trafficking involves a complex pathway (69).

The food vacuole hemoglobinases are synthesized earlier in the life cycle than proteases involved in invasion. It remains to be determined whether targeting domains or timing of synthesis (or both) govern the trafficking of Plasmodium proteases to the food vacuole or rhoptries, both of which have properties of lysosomes. Timing of synthesis is hypothesized to be important for proper targeting of malaria microneme protein AMA-1 (71).

Alternate functions and targeting of malaria hemoglobinases

Plasmepsins and falcipains have been implicated in parasite egress from the host cell (72–74). Both plasmepsin 2 and falcipain 2 cleave erythrocyte membrane skeletal proteins at neutral pH (72–74). Mature falcipain 2 localizes to vesicle-like structures in the host cytosol, trafficking from the PV to the erythrocyte membrane (73). Falcilysin may also have dual specificity with activity against some substrates at acidic pH (food vacuole) and others at a neutral pH (70). The trafficking of these proteases to locations other than the food vacuole has not been explored.

Conclusions

  1. Top of page
  2. Abstract
  3. Secretory Pathway of the Apicomplexa
  4. Regulated Secretion in Apicomplexa
  5. Proteolytic Processing in The Apicomplexan Secretory Pathway
  6. Trafficking of T. gondii Proteases to Secretory Organelles
  7. Localization and Trafficking of Proteases in Plasmodium
  8. Conclusions
  9. Acknowledgments
  10. References

During assembly of its organelles, the Apicomplexa must synthesize and target specific contents including proteases to appropriate and unique compartments. Several classes of proteases appear to play essential roles in the biogenesis of microneme and rhoptry secretory organelles and other critical processes such as hemoglobin degradation in the food vacuole. For TgSUB1, a microneme subtilase, a lumenal portion of TgSUB1 containing the catalytic domain is critical for targeting, but trafficking of other secreted proteases is not understood. The prodomains of plasmepsins and falcipains may regulate targeting to the food vacuole. Assembly of micronemes and rhoptries appears to be concurrent, implicating specific targeting pathways for these and other unique organelles of the Apicomplexa.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Secretory Pathway of the Apicomplexa
  4. Regulated Secretion in Apicomplexa
  5. Proteolytic Processing in The Apicomplexan Secretory Pathway
  6. Trafficking of T. gondii Proteases to Secretory Organelles
  7. Localization and Trafficking of Proteases in Plasmodium
  8. Conclusions
  9. Acknowledgments
  10. References

Supported by NIH R01 AI 46985 to K.K. We would like to thank Lloyd Fricker, Michael Blackman and Vern Carruthers for many stimulating discussions about this topic, and Steve Miller, Vandana Thathy, and Art Jongco for their contributions to the work discussed. E.B. was supported by NIH training grant T32 AI07506 awarded to the Albert Einstein College of Medicine.

References

  1. Top of page
  2. Abstract
  3. Secretory Pathway of the Apicomplexa
  4. Regulated Secretion in Apicomplexa
  5. Proteolytic Processing in The Apicomplexan Secretory Pathway
  6. Trafficking of T. gondii Proteases to Secretory Organelles
  7. Localization and Trafficking of Proteases in Plasmodium
  8. Conclusions
  9. Acknowledgments
  10. References