Abstract
The endoplasmic reticulum is thought to play an essential role during egress of malaria parasites because the ER is assumed to be the calcium (Ca2+) signaling hub and required for biogenesis of egress organelles. However, no proteins localized to the parasite ER have been shown to play a role in egress of malaria parasites. In this study, we generated conditional mutants of the Plasmodium falciparum Endoplasmic Reticulum-resident Calcium-binding protein (PfERC), a member of the CREC family. Knockdown of PfERC shows that this gene is essential for asexual growth of P. falciparum. Analysis of the intraerythocytic lifecycle revealed that PfERC is required for parasite egress and invasion. We found that PfERC knockdown prevents the rupture of the parasitophorous vacuole membrane. This is because PfERC knockdown inhibited the proteolytic maturation of the subtilisin-like serine protease, SUB1, and the essential SUB1 substrate, the merozoite surface protein 1. PfERC knockdown further inhibited the proteolytic maturation of the essential invasion ligand, Apical Membrane Antigen 1 (AMA1), which occurs during egress. These data establish the ER-resident CREC family protein, PfERC, as a key early regulator of the egress proteolytic cascade of malaria parasites.
Introduction
Members of the phylum Apicomplexa are responsible for severe human diseases such as malaria, toxoplasmosis, and cryptosporidiosis. Together, this group of obligate intracellular parasites causes several hundred million infections every year and remains one of the major drivers of infant mortality (1-4). In fact, malaria results in nearly half a million deaths each year and most of the mortality is attributed to one species, Plasmodium falciparum. All the clinical symptoms of malaria are directly correlated to the asexual lifecycle of malaria parasites within the host red blood cells.
The egress and subsequent invasion of daughter parasites into the host cells are essential for the propagation of apicomplexan parasites. Both egress and invasion are ordered and essential processes which are regulated by signaling pathways dependent upon the second messengers, cGMP and Ca2+ (5-9). Upon invading the host cell, the parasites create and reside within a host-derived vacuole called the parasitophorous vacuole (PV). Within this vacuole, the parasites grow and divide into daughter cells, which must egress from the host cell to complete the life cycle. This event is triggered by the activation of a cGMP dependent protein kinase (PKG) and the inhibition of PKG activity blocks egress (6, 10, 11). Ca2+-signaling also induces egress although it is uncertain whether this pathway works downstream (7) or synergistically with cGMP signaling (6, 12, 13). For example, studies have shown that blocking the release of Ca2+ from intracellular stores using cell permeable Ca2+ chelators blocks egress in malaria parasites (13-15). It has been suggested that this release of Ca2+ into the cytoplasm comes from the parasite ER; however, the parasite genome lacks identifiable orthologs of ligand-gated Ca2+ channels such as the inositol 1,4,5,-triphosphate or ryanodine receptors (16). Increase in cytoplasmic Ca2+ is then thought to activate calcium dependent protein kinases (CDPKs) resulting in release of egress-related vesicles (12, 17).
In malaria parasites, these egress-related vesicles contain specific proteases that require proteolytic processing to be activated (18-20). For example, one such pivotal enzyme is the serine protease, Subtilisin 1 (SUB1), which undergoes two cleavage events. First, the zymogen undergoes Ca2+ dependent autoprocessing in the ER (21, 22) and then, it is cleaved again by the aspartic protease, Plasmepsin X (PMX) (19, 20). The release of the processed form of SUB1 into the parasitophorous vacuole (PV) commits the parasites for egress resulting in the rapid (∼10 minutes) breakdown of the parasitophorous vacuole membrane (PVM) (18, 23). Then, substrates of SUB1 such as merozoite surface protein 1 (MSP1) and serine-repeat antigen 6 (SERA6) help breakdown the RBC cytoskeleton and the RBC membrane (RBCM) (24, 25). Once egress is completed, the merozoites subsequently invade fresh RBCs to start the 48-hour asexual cycle again. Like egress, invasion requires specific secretory events such as fusion of micronemes to the merozoite membrane and secretion of rhoptry contents into the host cell, which provides the ligand-receptor pair essential for driving the parasite into the host cell (26, 27).
The parasite endoplasmic reticulum (ER) is thought to play a key role in egress and invasion of daughter merozoites. The putative functions of the parasite ER during these lifecycle stages, include biogenesis of the specific egress and invasion related organelles, transporting proteins to these organelles, and propagating Ca2+ signals essential for egress and invasion (28, 29). However, none of the proteins responsible for these ER-related functions during egress and invasion of apicomplexan parasites have been identified. One potential candidate is the ER-resident calcium binding protein PfERC (PF3D7_1108600). In malaria parasites, PfERC is the only protein with identifiable Ca2+-binding domains localized to the ER and it is capable of binding Ca2+ (30). However, the biological function of PfERC is unknown. To address this, we used CRISPR/Cas9 based gene editing approach to generate conditional mutants of PfERC. The conditional mutants allowed us to determine that this ER-resident protein controls the nested proteolytic cascade in P. falciparum that regulates the egress of malaria parasites from human RBCs.
Results
PfERC is an CREC family protein localized in the ER
PfERC is a protein related to the CREC (Calumenin, Reticulocalbin 1 and 3, ERC-55, Cab-45) family of proteins, which are characterized by the presence of multiple EF-hands and localization in various parts of the secretory pathway (31, 32) (Figure 1A and Supplementary Figure 1). PfERC contains a signal peptide, multiple EF-hands, and an ER-retention signal (Figure 1A). The domain structure of PfERC is homologous to other members of the CREC family of proteins (Figure 1A and Supplementary Figure 1). However, PfERC differs from its mammalian homologs in that it only contains 5 predicted EF-hands although a 6th degenerate EF-hand (residues 314-325) may be present in its extended C-terminus (Supplementary Figure 1) (30). Various roles have been attributed to CREC members including Ca2+ signaling and homeostasis, and one member, RCN3, has been shown to interact with the subtilisin-like peptidase, PACE4, though the functional significance of this interaction is unknown (31, 33). As PfERC expression peaks during early schizont stage parasites, we hypothesized that PfERC is required for egress of daughter parasites during this terminal stage of the asexual lifecycle (30, 34) (Figure 1A).
Generating conditional mutants of PfERC
In order to determine the biological role of PfERC, we used CRISPR/Cas9 gene editing to generate conditional mutants of PfERC. In these parasite lines, the endogenous locus of PfERC was tagged with the inducible ribozyme, glmS or the inactive version of the ribozyme, M9 (termed PfERC-glmS and PfERC-M9 respectively) (Figure 1B and 1C) (35). PCR analysis of DNA isolated from PfERC-glmS and PfERC-M9 parasite clones from two independent transfections demonstrate the correct insertion of the hemagglutinin (HA) tag and the glmS/M9 ribozymes at the endogenous PfERC locus (Figure 1D). We detected expression of PfERC fused to the HA tag in the PfERC-glmS and PfERC-M9 clones at the expected size and but not in the parental line (Figure 1E). Immunofluorescence microscopy confirmed that PfERC localized to the ER by co-staining with anti-HA and anti-BiP antibodies (Figure 1F).
To determine if PfERC was essential for intraerythrocytic survival, we grew asynchronous PfERC-glmS and PfERC-M9 parasites in the presence of glucosamine (GlcN), which activates the glmS ribozyme leading to mRNA cleavage (Figure 1B). We observed a reproducible reduction of PfERC expression in PfERC-glmS parasites while there was no reduction in PfERC expression in PfERC-M9 parasites grown under identical conditions (Figure 2A and 2B). Importantly, this reduction in PfERC levels inhibited the asexual expansion of PfERC-glmS parasites, while the PfERC-M9 parasites were able to grow normally under the same conditions (Figure 2C). This inhibition of the asexual growth of PfERC-glmS parasites was dose-dependent upon GlcN (Supplementary figure 2A).
PfERC is essential for schizont to ring transition
Since our data show that PfERC was essential for growth within the host RBC, we used synchronous parasites to determine which asexual stage was affected by knockdown. We added GlcN to synchronized schizonts and observed the morphological development of the asexual stages at regular intervals during the intraerythrocytic life cycle (Figure 2D). All intracellular stages were morphologically normal in both PfERC-glmS and PfERC-M9 parasites grown with GlcN (Figure 2D and Supplementary Figure 2B). However, 55hrs after addition of GlcN, the PfERC-glmS parasites remained either as morphologically normal schizonts or were observed as daughter merozoites in the extracellular space as well as some that were attached to RBCs (Figure 2D). On the other hand, PfERC-M9 parasites were able to egress and re-invade fresh RBCs and developed into ring stage parasites (Figure 2D and Supplementary figure 2B).
These data suggest that knockdown of PfERC resulted in a defect in the conversion of schizonts into rings. To test this, we induced knockdown and observed the conversion of schizonts into rings via flow cytometry at 44, 48, and 56 h post-addition of GlcN. We found that over the course of 12 hours, PfERC-M9 parasites transitioned from schizonts to rings as determined by the ring:schizont ratio while PfERC-glmS parasites were unable to convert from schizonts into rings resulting in a drastically reduced ratio (Figure 2E). Using synchronized PfERC-glmS and PfERC-M9 parasites, treated as in Figure 2D, we observed the final hours of the asexual lifecycle using thin blood smears and quantified parasites using flow cytometry (Supplementary Figure 2B and Figure 2F-H). These data show that there was a delay in the disappearance of the morphologically normal PfERC-glmS schizonts over the final few hours of the asexual life cycle compared to PfERC-M9 schizonts, suggesting that knockdown of PfERC led to a defect in egress (Figure 2F and 2G). Consequently, the delayed egress lead to reduced numbers of ring stage parasites in PfERC-glmS parasites unlike PfERC-M9 parasites (Figure 2F and 2H).
PfERC is not required for calcium storage
Since PfERC resides in the ER and possesses Ca2+ binding domains, we hypothesized that PfERC is required for egress because it plays a role in Ca2+ homeostasis in the ER. To test this model, synchronized PfERC-glmS and PfERC-M9 schizonts were incubated with GlcN and allowed to proceed through one asexual cycle until they formed schizonts again. The second cycle schizonts were isolated using saponin lysis and loaded with Fluo-4AM to measure cytosolic Ca2+ (Supplementary Figure 3A). To assess if the storage of Ca2+ in the ER of the parasite was affected by knockdown of PfERC, we added the SERCA-Ca2+ ATPase inhibitor, Cyclopiazonic acid (CPA), to these saponin-isolated parasites (Supplementary Figure 3A and 3B) (36). Inhibiting the SERCA-Ca2+ ATPase allows Ca2+ stored in the ER to leak into the cytoplasm, which results in a detectable change in the fluorescence of Fluo-4AM (Supplementary Figure 3B). Our measurements show that there was no difference in the amount of Ca2+ that leaked from the parasite ER, upon SERCA-Ca2+ ATPase inhibition, between PfERC-glmS and PfERC-M9 schizonts (Supplementary Figure 3B).
To test if there was a defect in Ca2+ storage in neutral stores, we used the ionophore, Ionomycin, which releases Ca2+ from all neutral stores in the cell and measured the release of Ca2+ into the cytoplasm of PfERC-glmS and PfERC-M9 schizonts. The parasites were isolated as described above and the changes in cytoplasmic Ca2+ were measured using Fluo-4AM (Supplementary Figure 3A and 3C). Again, we did not observe any difference in the amount of Ca2+ released into the cytoplasm of PfERC-glmS and PfERC-M9 schizonts treated with ionomycin (Supplementary Figure 3C). These data suggest that the availability of free Ca2+ in the ER (or other neutral Ca2+ stores) of P. falciparum is not affected by knockdown of PfERC. Furthermore, these data suggest that the observed egress defect upon PfERC knockdown was not a result of disequilibrium of Ca2+ in the parasite ER.
PfERC is required for PVM breakdown
Since we could not observe a defect in ER Ca2+ storage upon knockdown, we further analyzed how egress of PfERC-glmS parasites was failing during knockdown. Egress of daughter merozoites from the infected RBC is an ordered and rapid process where the PVM breakdown precedes the disruption of RBCM (Figure 3A) (11). Therefore, we took synchronized PfERC-glmS and PfERC-M9 schizonts and initiated knockdown with addition of GlcN. These schizonts were allowed to reinvade fresh RBCs and proceed through the asexual stages for 48 hours until they developed into schizonts again. Then, these second cycle schizonts were incubated with inhibitors that block key steps during egress of P. falciparum (Figure 3A). To ensure synchronized egress, we used reversible inhibitors of PKG, Compound 1 (C1) or Compound 2 (C2), because inhibition of PKG allows merozoites to develop normally but prevents them from initiating egress (Figure 3A) (6, 11). We used flow cytometry to observe PfERC-glmS and PfERC-M9 schizonts after washing off C1 and saw that there was a delay in the egress of PfERC-glmS schizonts while the majority (>60%) of the PfERC-M9 schizonts were able to complete egress within two hours after washout of C1 (Figure 3B). Removal of C1 initiates the breakdown of the PVM followed by RBCM rupture (Figure 3A), suggesting that PfERC-glmS parasites fail to breach one of these membranes down despite removal of the PKG inhibitor.
Therefore, we tested whether PfERC knockdown prevented rupture of PVM or if PfERC was required for RBCM breakdown (Figure 3A). PfERC-glmS and PfERC-M9 schizonts (where knockdown had been initiated in the previous cycle) were incubated with C2 (6, 11) and observed by scanning electron microscopy (SEM) (Figure 3A and 3C). We observed that parasites treated with C2 were morphologically identical and had developed into mature schizonts within the PVM inside the RBC (Figure 3C). Then, we washed C2 from the parasites and observed these schizonts after 30 mins by SEM (Figure 3C). During this time period, the majority of PfERC-M9 schizonts were able to initiate egress after removal of C2 and we observed free merozoites attached to the RBC as well as clusters of merozoites that had broken out of the PVM but were contained by a collapsed RBCM wrapped around them (Figure 3C and Supplementary Figure 4). In contrast, the majority of PfERC-glmS schizonts were still stuck within the RBC and looked identical to the C2 arrested schizonts, suggesting that they had not initiated egress even though PKG was no longer inhibited (Figure 3C and Supplementary Figure 4). These data suggest that knockdown of PfERC blocks egress at an early step, perhaps blocking the rupture of the PVM (Figure 3C).
We directly observed if breakdown of the PVM was impacted by knockdown of PfERC using transmission electron microscopy (TEM) (Figure 3D). Knockdown was induced by adding GlcN to PfERC-glmS and PfERC-M9 schizonts and these parasites were allowed to go through one asexual cycle and develop into schizonts again 48hrs later. These schizonts were prevented from completing egress using the irreversible cysteine protease inhibitor, E-64 (Figure 3A). This inhibitor blocks the breakdown of the RBCM but allows both the breakdown of PVM and poration of the RBCM, which results in the loss of the electron dense contents of the infected RBC (Figure 3A) (11, 25, 37). Our results show that the PfERC-M9 schizonts were able to break down the PVM as well as proceed with the poration of the RBCM after an 8-hour incubation with E-64, while the PfERC-glmS mutants were unable to proceed through the first step of egress and failed to rupture the PVM (Figure 3D). Overall, these data demonstrate that PfERC function is critical for the breakdown of the PVM (Figure 3C and 3D).
SUB1 maturation requires PfERC
Electron microscopy data show that knockdown of PfERC prevents the breakdown of the PVM (Figure 3). A key event required for PVM breakdown is the proteolytic processing of SUB1, which is required to start a proteolytic cascade that ends in the release of merozoites from the infected RBC (18, 25). Therefore, we tested if knockdown of PfERC affects processing of PfSUB1. This protease is processed twice, once in the ER, where it undergoes a Ca2+-dependent autocatalytic processing from its zymogen form (83-kDa) into a 54-kDa semi-proenzyme form (p54) (21, 22, 38). From the ER, SUB1 is transported to the egress-related secretory vesicles, the exonemes, which are secreted into the PV to initiate breakdown of the PV membrane. It is proposed that during trafficking of SUB1 to exonemes, it is processed by PMX from its semi-proenzyme form (p54) to its mature form (p47) (19, 20). The secretion of mature p47 form of SUB1 initiates the breakdown of the PVM (18, 38). Given that one CREC family member has been shown to transiently interact with a subtilisin like protease in mammalian cells (33), we hypothesized that PfERC is required for one of the proteolytic maturation steps of SUB1, most likely the first Ca2+-dependent autocatalytic processing step in the ER.
To test this hypothesis, PfERC-glmS and PfERC-M9 schizonts were incubated with GlcN and allowed to progress through one asexual growth cycle (48 hours) to develop into schizonts again. Lysates from these synchronized schizonts were separated on a Western blot and probed with antibodies against SUB1 (Figure 4A and Supplementary Figure 5A). No change was observed in the Ca2+-dependent autoprocessing of the zymogen-form of SUB1 into the semi-proenzyme (p54) form (Figure 5A and Supplementary Figure 5A). Surprisingly, we observed a reproducible and significant decrease in the processing of SUB1 from p54 to the p47 form in PfERC-glmS parasites (Figure 4A and 4B). Compared to PfERC-M9 parasites, there was a >50% decrease in the amount of processed SUB1 (p47) in PfERC-glmS parasites (Figure 4B). This effect was also observed in cells treated with Compound 1, suggesting that PfERC is required for SUB1 processing prior to secretion of exonemes (Figure 5C and 5D). Taken together, our data suggest that PfERC is essential for the proteolytic maturation of SUB1.
Since we observed the presence of some mature SUB1 in PfERC-glmS parasites (Figure 4A), we tested if the activity of SUB1 was affected upon knockdown of PfERC by assaying for the processing of a known SUB1 substrate, the merozoite surface protein 1 (MSP1). MSP1 is required for the initial attachment of merozoites onto RBCs and it has been shown that correct processing of MSP1 by SUB1, is also required for efficient egress as it plays a role in breakdown of the RBC cytoskeleton after release from the PVM (24, 39-41). Lysates from synchronized second-cycle PfERC-glmS and PfERC-M9 schizonts, treated as above, were separated on a Western blot and probed using anti-MSP1 antibodies (Figure 4E and Supplementary Figure 5B and 5C). Our data show that there was significant inhibition of MSP1 processing in PfERC-glmS parasites as compared to PfERC-M9 parasites after knockdown (Figure 4F and Supplementary Figure 5C). These data reveal that knockdown of PfERC leads to defects in SUB1 processing and activity, and consequently, MSP1 processing (Figure 4 and Supplementary Figure 5B and 5C).
PfERC is not required for protein trafficking or organelle biogenesis
MSP1 is a GPI-anchored merozoite membrane protein that is presumably processed by SUB1 once the protease is secreted into the PV (24, 25). Therefore, we wanted to verify that knockdown of PfERC led to a specific defect in the egress cascade and is not due to a block in protein trafficking via the ER or defects in the biogenesis of egress and invasion related organelles. To address this, we used super resolution structured illumination microscopy (SR-SIM) to observe if there was a difference in the surface expression of MSP1 between PfERC-glmS and PfERC-M9 schizonts upon knockdown of PfERC (Figure 5A). As before, knockdown was initiated in synchronized PfERC-glmS and PfERC-M9 schizonts and after 48 hours, these schizonts were stained with anti-MSP1 antibodies. Our data shows that there was no difference in the trafficking of MSP1 to the surface of developing PfERC-glmS or PfERC-M9 merozoites after knockdown (Figure 5A and 5B). Further, the localization of the rhoptry protein, RAP1, which also traffics through the ER, was observed in these schizonts using SR-SIM. Our data show that there was no difference in the localization of RAP1 in schizonts between PfERC-glmS and PfERC-M9 parasites suggesting that the knockdown of PfERC does not cause a generalized defect in the secretory pathway (Figure 5C and 5D).
As the ER produces the lipid membranes required for generating organelles essential for egress and invasion, we observed if organelle biogenesis was inhibited upon PfERC knockdown. To test this, knockdown was initiated in synchronized PfERC-glmS and PfERC-M9 schizonts and after 48 hours, these schizonts were treated with Compound 1 for 4hrs. Then, we observed these C1-treated schizonts using TEM. In these PfERC-glmS and PfERC-M9 parasites both micronemes and rhoptries remain morphologically intact (Figure 5E). Likewise, we observed morphologically intact micronemes and rhoptries in PfERC-glmS and PfERC-M9 schizonts that were further incubated with E-64 for 8 hours (Supplementary Figure 6). Together, these data suggest that the knockdown of PfERC does not lead to defects in organelle biogenesis (Figure 3D, Figure 5E, and Supplementary Figure 6).
PfERC is required for invasion of merozoites
The synchronized growth assays suggest that knockdown of PfERC inhibits the invasion of merozoites into RBCs (Figure 2D, H and Figure 5C, D). To assess if invasion was inhibited upon knockdown, PfERC-glmS and PfERC-M9 schizonts in the second cycle after 48 hours in GlcN, were incubated with the PKG inhibitor, C1, for four hours (Figure 3A). The inhibitor was then washed off and the formation of ring stages was observed over two hours by flow cytometry (Figure 6A). We observed that there was a delay in the formation of ring stages as well as a drastic decrease in the numbers of ring stage parasites formed in PfERC-glmS parasites compared to the PfERC-M9 control (Figure 6A). This could be due to inhibition of egress or could be a combination of defects in egress and invasion due to PfERC knockdown.
To decouple the egress and invasion phenotypes, we directly measured the efficiency of merozoite invasion (Figure 6B). This was accomplished by incubating second cycle PfERC-glmS and PfERC-M9 schizonts with E-64 and then mechanically releasing the merozoites (Figure 6B) (42). Incubation with E-64 for 8 hours allows for the completion of schizogony and formation of invasion-competent merozoites (Supplementary Figure 6). These purified merozoites were then allowed to invade fresh RBCs and the invasion rate was quantified using flow cytometry as described previously (Figure 6C and Supplementary Figure 7) (42). These data show that there was a drastic reduction in the invasion efficiency of PfERC-glmS merozoites as compared to the control PfERC-M9 merozoites, thus demonstrating that knockdown of PfERC led to a defect in invasion as well (Figure 6C).
The reduced invasion of PfERC-glmS merozoites could be explained by the reduction in processing of MSP1, which is required for the initial attachment of merozoites to the RBC (39-41, 43). Invasion of RBCs by P. falciparum merozoites requires secretion of contents from another apical organelle, the rhoptries, into the RBC (44-46). Proteins in the rhoptries, like the rhoptry-bulb protein, RAP1, require proteolytic processing for their activity (19, 20). Once in the rhoptry, RAP1 is processed by the aspartic protease, Plasmepsin IX (PMIX), from a pro-form (p83) to a mature form (p67) (19, 20, 47, 48). Therefore, we tested if RAP1 processing was inhibited by knockdown of PfERC using Western blot analysis (Figure 6D and Supplementary Figure 5D). Our data show that the proteolytic processing of RAP1 was not inhibited by the knockdown of PfERC (Figure 6E), showing that knockdown does not lead to a generalized defect in the processing of all proteins that traverse through the secretory pathway.
PfERC is required for AMA1 processing but not secretion
Another key and essential step in invasion of merozoites is the formation of a tight junction between the parasite and the RBC and AMA1 is critical for the formation of this tight junction (26, 27). AMA1 is trafficked from micronemes to the merozoite surface and there it is processed from its pro-form (p83) to its mature form (p66) by an unknown protease (49-51). Studies have shown that secretion of micronemes require Ca2+ signaling pathways (9) and, although our data suggest that PfERC is not required for Ca2+ storage in the ER, we could not rule out a role for PfERC in Ca2+ signaling. Since the translocation of AMA1 is dependent upon this Ca2+ signaling pathway, we observed if AMA1 exocytosis was inhibited upon PfERC knockdown (6, 12). Synchronized PfERC-M9 or PfERC-glmS schizonts where knockdown had been initiated the previous cycle were incubated with C1 to achieve tight synchronization. Then, C1 was washed off and the parasites were incubated with E-64 to trap merozoites that had initiated egress within the RBC membrane (12). Using immunofluorescence microscopy, we observed AMA1 localization in the either micronemes or on the surface of merozoites (Figure 7A). Our data show that there was no difference in the localization of AMA1 between PfERC-M9 or PfERC-glmS parasites, suggesting that PfERC is not required for the signaling necessary for vesicle secretion (Figure 7A and B).
The proteolytic processing of AMA1 is critical for its function during invasion (52). Therefore, we tested if the processing of the AMA1 was affected upon knockdown of PfERC. As before, after initiating knockdown in synchronized schizonts, we isolated lysates from second cycle PfERC-glmS and PfERC-M9 schizonts on a Western blot and probed it with anti-AMA1 antibodies (Figure 7C and Supplementary Figure 5E). We observed a significant reduction in the proteolytic processing of AMA1 upon knockdown (Figure 7C). Indeed, there was a >40% decrease in the processing of AMA1 in PfERC-glmS mutants compared to the PfERC-M9 control (Figure 7D). These data suggest that PfERC is required for the correct processing of AMA1 and therefore, essential for invasion of merozoites into the host RBC.
Discussion
In this study, we revealed the biological role of a conserved Ca2+-binding protein that resides in the lumen of the ER of Plasmodium falciparum. Our data show that PfERC is essential for asexual replication of malaria parasites. Knockdown of PfERC did not affect the ring and trophozoite development but clearly inhibited the subsequent schizont-to-ring transition. Specifically, these data show that PfERC is required for both egress from infected RBCs and invasion into host erythrocytes. This is consistent with data that suggest PfERC may be transcriptionally controlled by the invasion-specific transcription factor PfAP2-I (53). Knockdown of PfERC leads to defects in the processing of proteins critical for invasion of merozoites into the host RBC, namely, MSP1 and AMA1. However, the observed invasion defect is likely a secondary effect because several proteins critical for invasion are processed during egress (19, 20, 24, 25). Given the kinetic limitations of the conditional knockdown system, we cannot tease out a specific role for PfERC in invasion. As invasion occurs rapidly (<2mins), a potential specific invasion-related function of PfERC could be tested using a small molecule that specifically targets PfERC (54). Overall, these data show that PfERC is essential for egress of merozoites from the infected RBC and for invasion of merozoites into the host erythrocyte.
During the formation of daughter merozoites in schizonts, several key egress and invasion related organelles essential for propagation of the infection are generated. The ER is thought to play a key role in the biogenesis of these organelles and the ER is responsible for transporting the essential proteins to these organelles (28, 29). As an ER-resident protein, knockdown of PfERC could affect several ER functions such as protein trafficking, organellar biogenesis, and Ca2+ signaling. Therefore, we tested if PfERC functions in the trafficking of proteins required for schizont to ring transition such as MSP1, AMA1, and RAP1. A defect in the secretory pathway would explain the observed deficiencies in the proteolytic processing of SUB1, MSP1 and AMA1, as transport out of the ER is required for their maturation (6, 22, 48). However, super-resolution and electron microscopy experiments show that proteins on the merozoite surface, micronemes, and rhoptries are trafficked normally and biogenesis of egress and invasion organelles is normal. Likewise, Western blot analysis showed that the proteolytic processing of a rhoptry protein, RAP1, which is processed after transport to the organelle, occurs normally upon knockdown of PfERC (19, 20). These data show that knockdown of PfERC does not result in a generalized defect in protein trafficking via the ER, or in organelle biogenesis. Instead, these data show that PfERC knockdown specifically inhibits the proteolytic maturation of a subset of proteins essential for egress and invasion.
A key enzyme that is required for initiating egress is the protease SUB1 as the exocytosis of this serine protease into the PV results in the rupture of both the PVM and the RBCM (18, 25). It is produced as an 82-kDa zymogen in the ER, where it rapidly self-processes into a 54-kDa semi-proenzyme in the ER (38). If PfERC was needed for this autoprocessing event, then this would explain the observed knockdown phenotypes as they are similar to that seen when SUB1 is conditionally knocked out (25). To test if PfERC interacts directly with SUB1, we performed co-IP experiments but failed to detect any interaction between these two proteins. This is not surprising since any such interaction is likely to be very transient as has been shown for other CREC family members (33). Instead, PfERC was essential for the second processing step of SUB1 that produces the mature, active form of the protease (p54 to p47). This processing event occurs once is trafficked out of the ER suggesting a role for PfERC in SUB1 maturation once it leaves the ER (6, 13, 20, 21). One possibility is that a fraction of PfERC does leave the ER to enable SUB1 maturation as a subset of ER-resident proteins are known to be secreted beyond the organelle (55). Another model is that PfERC is required for the maturation of an unknown protease in this pathway that works upstream of SUB1. The recently discovered aspartic protease, PMX, is likely responsible for the maturation of SUB1 from p54 to p47 (19, 20). In turn, PMX itself is proteolytically matured from a 67kDa zymogen to a 45kDa active protease (19, 20). But unlike most aspartyl proteases, PMX does not autoprocess because inhibitors that block PMX activity do not inhibit its maturation (19). The maturase responsible for PMX cleavage is unknown and therefore, if this unknown protease requires PfERC for its activity, then knockdown of PfERC would lead to the observed defects in egress.
CREC family members are known to regulate the function of Ca2+ pumps and channels such as the Ryanodine and IP3 recepors (56, 57). Therefore, one interesting possibility we considered was that PfERC may play a role in the signal-dependent release of Ca2+ from the ER. This is difficult to test in Plasmodium since there are no clear orthologs for a ligand-dependent Ca2+ channel in its genome (16). Intracellular Ca2+ stores are required for egress and invasion of malaria parasites since cell permeable Ca2+chelators block egress of Plasmodium parasites from host RBCs (13-15, 58, 59). Further, Ca2+ binding proteins in the parasite cytoplasm are essential for egress of malaria parasites, for example, the Ca2+ dependent protein kinase, PfCDPK5, is required for secretion of egress specific organelles, such as those containing AMA1 (12, 60). As PfCDPK5 is thought to be activated upon the signal-dependent release of intracellular Ca2+ into the cytoplasm (12), we tested if PfERC was required for exocytosis of AMA1-containing vesicles. The data suggest that PfERC is not required for the PfCDPK5-dependent translocation of AMA1 onto merozoite membrane. However, PfERC is required for the essential proteolytic maturation of AMA1, suggesting that this CREC family member regulates (directly or indirectly) the unknown protease that processes AMA1.
The release of the mature SUB1 into the PV kickstarts the egress cascade (25)and the cGMP signaling pathway is thought to be essential for vesicle exocytosis via the release of intracellular Ca2+ stores (58). We independently tested whether PfERC knockdown inhibited this signaling pathway, using the PKG inhibitor Compound 1, and show that PfERC knockdown inhibited SUB1 maturation even when PKG activity was inhibited. This data together with the experiments testing AMA1 translocation onto the merozoite membrane suggest that PfERC does not play a role in the Ca2+-dependent exocytosis of egress-specific organelles. Instead, our data suggest a model where PfERC plays a key role in the maturation of SUB1 prior to its secretion into the PV. In the absence of PfERC, exocytosis of immature SUB1 fails to breakdown the PVM as well as prevents proteolytic maturation of key invasion ligands on the merozoite surface, such as MSP1 and AMA1.
A principal finding of these studies is the identification of an early regulator in the ER of P. falciparum with a specific role in egress of malaria parasites from RBCs and potentially in the invasion of parasites into the RBC. These data help build a model where PfERC modulates the maturation of the egress proteolytic cascade. These studies lay the foundation for understanding the vital and key role that ER-resident proteins play in the egress of human malaria parasites from the infected RBC and their re-entry into the host cell. Some studies have suggested that a key class of antimalarials containing endoperoxides, which includes the frontline antimalarial artemisinin, may target PfERC (54) and one of the transcriptomic responses of artemisinin-resistant parasites is the overexpression of PfERC (61). These data suggest that targeting PfERC, and thus egress, is a viable strategy for antimalarial drug development.
Material and Methods
Cell culture and transfections
Plasmodium parasites were cultured in RPMI 1640 medium supplemented with Albumax I (Gibco) and transfected as described earlier (62-65). To generate PfERC-glmS and PfERC-M9 parasites, a mix of two plasmids (50µg of each) was transfected in duplicate into 3D7 parasites. The plasmid mix contained pUF1-Cas9-guide (66) which contains the DHOD resistance gene, and pPfERC-HA-SDEL-glmS or pPfERC-HA-SDEL-M9, which are marker-free. Drug pressure was applied 48hrs after transfection, using 1µM DSM1 (67), selecting for Cas9 expression. DSM1 was removed from the culturing medium once the parasites were detected in the culture, around 3 weeks post-transfection.
Construction of PfERC plasmids
Genomic DNA was isolated from P. falciparum cultures using the QIAamp DNA blood kit (Qiagen). Constructs utilized in this study were confirmed by sequencing. PCR products were inserted into the respective plasmids using the In-Fusion cloning system (Clontech), the sequence- and ligation-independent cloning (SLIC) method (64, 65), T4-ligation (New England BioLabs), or site-directed mutagenesis using QuickChange (Agilent). To generate the pHA-SDEL-glmS/M9 plasmid, primers 1+2 were used to add an SDEL sequence at the end of the HA tag in pHA-glmS and pHA-M9 plasmids (64, 65).
For generating the PfERC-glmS/M9 conditional mutants, pHA-SDEL-glmS/M9 plasmid, consisting of two homology regions flanking the HA-SDEL tag and the glmS or M9 sequence, was used as a donor DNA template. To allow efficient genomic integration of the pHA-SDEL-glmS and pHA-SDEL-M9 donor plasmids, 800-bp sequences were used for each homology region. The C-terminus of the pferc coding region was PCR amplified from genomic DNA using primers 3+4 (containing the shield mutation) and was inserted into pHA-SDEL-glmS and pHA-SDEL-M9 using restriction sites SacII and AfeI. The 3’UTR of pferc was PCR amplified from genomic DNA using primers 5+6 and was inserted into pHA-SDEL-glmS and pHA-SDEL-M9 (already containing the C-terminus region) using restriction sites HindIII and NheI. For expression of PfERC guide RNA, oligos 7+8 were inserted into pUF1-Cas9-guide as previously described (64, 65). Briefly, pUF1-Cas9-guide was digested with BtgZI and annealed oligos were inserted using SLIC. Primers 3+6 and primers 3+9 (which recognizes the glmS/M9 sequence) were used for clone verification.
Plasmodium growth assays
Asynchronous growth assays were done as described previously (71, 72). Briefly, 5mM glucosamine (GlcN) (Sigma) was added to the growth medium and parasitemia was monitored every 24hrs using a CyAn ADP (Beckman Coulter) or CytoFLEX (Beckman Coulter) flow cytometers and analyzed by FlowJo software (Treestar, Inc.). As required, parasites were subcultured to avoid high parasite density, and relative parasitemia at each time point was back-calculated based on actual parasitemia multiplied by the relevant dilution factors. One hundred percent parasitemia was determined as the highest relative parasitemia and was used to normalize parasite growth. Data were fit to exponential growth equations using Prism (GraphPad Software, Inc.).
To determine the ring:schizont ratio of PfERC-glmS and PfERC-M9 parasites, 7.5mM GlcN was added to percoll isolated schizont-stage parasites and parasites were allowed to egress and reinvade fresh RBCs. Two hours later, 5% sorbitol +7.5mM GlcN was added to the invaded culture to lyse any remaining schizonts and isolate two-hour rings. The ring-stage parasites were grown again in media supplemented with GlcN. Then samples were taken at 44hrs, 48hrs, and 56hrs, and read by flow cytometry to determine the population of rings and schizonts present at those times using FlowJo software (Treestar, Inc.). To determine the development of each life cycle stage during the asexual lifecycle of PfERC-glmS and PfERC-M9 parasites, 7.5mM was added to percoll isolated schizont-stage parasites and parasites were allowed to egress and reinvade fresh RBCs. At specific times Hema-3 stained blood smears were used to count parasite stages and the percentage of the specific lifecycle stage was calculated as: Time 0hr is when GlcN was added.
To determine the % amount of rings or schizonts, samples of synchronized schizonts grown with 7.5mM GlcN for about 48hrs were taken and fixed with 8% paraformaldehyde and 0.3% glutarladehyde. Samples were read by flow cytometry. For growth assays using Compound 1 (4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl] pyridine), synchronized schizonts were grown with 7.5mM GlcN for about 48hrs. Then, schizonts were percoll isolated and incubated with Compound 1 for 4hrs and then removed by gently washing parasites twice with 1mL of warm, complete RPMI + 7.5mM GlcN. Parasites were resuspended with fresh media and RBCs and fixed samples (as above) were read by flow cytometry. DNA content was determined using Hoechst 33342 staining (ThermoFisher).
Western blotting
Western blotting for Plasmodium parasites was performed as described previously (64, 65). Briefly, parasites were permeabilized selectively by treatment with ice-cold 0.04% saponin in PBS for 10 min and pellets were collected for detection of proteins with the parasite. For detection of MSP1, schizonts were isolated on a Percoll gradient (Genesee Scientific) and whole-cell lysates were generated by sonication. The antibodies used in this study were rat anti-HA (3F10; Roche, 1:3,000), rabbit anti-HA (715500; Invitrogen, 1:100), rabbit anti-PfEF1α (from D. Goldberg, 1:2,000), mouse anti-plasmepsin V (from D. Goldberg, 1:400), rabbit anti-SUB1 (from Z. Dou and M. Blackman, 1:10,000), rat anti-AMA1 (28G2; Alan Thomas via BEI Resources, NIAID, NIH 1:500), mouse anti-MSP1 (12.4; European Malaria Reagent Repository, 1:500) and mouse anti-RAP1 (2.29; European Malaria Reagent Repository, 1:500). The secondary antibodies that were used are IRDye 680CW goat anti-rabbit IgG and IRDye 800CW goat anti-mouse IgG (LICOR Biosciences) (1:20,000). The Western blot images were processed using the Odyssey Clx LICOR infrared imaging system software (LICOR Biosciences). Calculation of knockdown and processing ratios was determined by both the Odyssey infrared imaging system software and ImageJ 1.8 (NIH).
Immunofluorescence microscopy
For IFAs, cells were fixed as described previously (64, 65). The antibodies used for IFA were: rat anti-HA antibody (clone 3F10; Roche, 1:100), mouse anti-AMA1 (1F9 from Alan Cowman), rat anti-PfGRP78 (MRA-1247; BEI Resources, NIAID, NIH 1:100), mouse anti-MSP1 (12.4; European Malaria Reagent Repository, 1:500), rat anti-AMA1 (28G2; Alan Thomas via BEI Resources, NIAID, NIH 1:500), and mouse anti-RAP1 (2.29; European Malaria Reagent Repository, 1:500). Secondary antibodies used were anti-rat antibody conjugated to Alexa Fluor 488 or 546 and anti-rabbit antibody conjugated to Alexa Fluor 488, (Life Technologies, 1:100). Cells were mounted on ProLong diamond with 4’,6’-diamidino-2-phenylindole (DAPI) (Invitrogen) and imaged using a Delta-Vision II microscope system with an Olympus IX-71 inverted microscope using a 100x objective or an Elyra S1 SR-SIM microscope (Zeiss). Image processing, analysis, and display were performed using SoftWorx or Axiovision and Adobe Photoshop. Adjustments to brightness and contrast were made for display purposes.
AMA1 Translocation Assays
To observe AMA1 translocation in our mutants, 7.5mM GlcN was added to percoll isolated schizont-stage parasites and parasites were allowed to egress and reinvade fresh RBCs. 44-48hrs later, schizonts were percoll purified and incubated with 1.5µM Compound 1 for 4 hours at 37°C. Then, Compound 1 removed by washing parasites twice with 1mL of warm complete RMPI +7.5mM GlcN. These parasites were immediately resuspended in media plus 7.5mM GlcN and 20 µM E-64 (Sigma) and incubated at 37°C in a still incubator for 6hrs. Parasites were then fixed as in (64, 65) and probed with anti-AMA1 (1F9) antibodies. Images were taken using a Delta-Vision II microscope system with an Olympus IX-71 inverted microscope using a 100x objective and using an Elyra S1 SR-SIM microscope (Zeiss).
Invasion Rate Quantification
To calculate the invasion rate, parasites were treated as described previously (42). Briefly, 7.5mM GlcN was added to percoll isolated schizont-stage parasites and parasites were allowed to egress and reinvade fresh RBCs. 48hrs later, schizonts were percoll purified and incubated with 20µM E-64 (Sigma) at 37°C in an incubator for 7-8hrs. Once incubation was done, merozoites were isolated by gently resuspending and passing the schizonts through a 1.2µm Acrodisic Syringe Filter (PALL). Merozoites were spun at 2000xg for 5min, and then resuspended in 100µL of complete RMPI medium and added to a 1mL culture of uninfected RBCs at 2% hematocrit. Cultures were grown in a FluoroDish cell culture dish (World Precision Instruments) and gassed in a chamber for 20-24hrs. Invasion rate was then measured by the following equation: where “iRBC” is the parasitemia 20-24hrs later, “RBC/µL” are the free RBCs used before addition of merozoites and “Mz/µL” are the merozoites found in the 100µL suspension used before adding to fresh RBCs. Values for these variables were acquired by flow cytometry (CytoFLEX Beckman Coulter) with cells stained with acridine orange. The data were normalized using the IR values for PfERC-M9 merozoites as 100%.
Transmission Electron Microscopy
7.5mM GlcN was added to percoll isolated schizont-stage parasites and parasites were allowed to egress and reinvade fresh RBCs. 48hrs later, parasites were percoll-isolated and then incubated with 20µM E-64 for 8hrs. After incubation, parasites were washed with 1X PBS and gently resuspended in 2.5% glutaraldehyde in 0.1M sodium cacodylate-HCl (Sigma) buffer pH 7.2 for 1hr at room temperature. Parasites were then rinsed in 0.1M Cacodylate-HCl buffer before agar-enrobing the cells in 3% Noble agar. Parasites were post fixed in 1% osmium tetroxide/0.1M Cacodylate-HCl buffer for 1 hour and rinsed in buffer and deionized water. Dehydration of the parasite samples was done with an ethanol series and then exposed to Propylene oxide before infiltration with Epon-Araldite. The blocks of parasites were trimmed, and sections were obtained using a Reichert Ultracut S ultramicrotome (Leica, Inc., Deerfield, IL). 60-70nm sections were placed on 200-mesh copper grids and post-stained with ethanolic uranyl acetate and Reynolds Lead Citrate. Grids were viewed with a JEOL JEM-1011 Transmission Electron Microscope (JEOL USA, Inc., Peabody, MA) using an accelerating voltage of 80 KeV. Images were acquired using an AMT XR80M Wide-Angle Multi-Discipline Mid-Mount CCD Camera (Advanced Microscopy Techniques, Woburn, MA).
Scanning Electron Microscopy
7.5mM GlcN was added to percoll isolated schizont-stage parasites and parasites were allowed to egress and reinvade fresh RBCs. 48hrs later, parasites were percoll-isolated and then incubated with 2µM Compound 2 (4-[7-[(dimethylamino)methyl]-2-(4-fluorphenyl)imidazo[1,2-a]pyridine-3-yl]pyrimidin-2-amine) for 4 hours without shaking at 37°C in an incubator. After incubation, parasites were washed twice with warm, complete RPMI + 7.5mM GlcN. Samples were taken immediately after washing off Compound 2 and then 30min after and fixed as with TEM samples. Parasites were rinsed with 0.1M Cacodylate-HCl buffer before placing on glass coverslips prepared with 0.1% Poly-L-lysine. Parasites were allowed to settle onto the glass coverslips in a moist chamber overnight and then post fixed in 1% osmium tetroxide/0.1M Cacodylate-HCl buffer for 30 minutes. Cells on coverslips were rinsed three times in deionized water and then dehydrated with an ethanol series. The glass coverslips were critical point dried in an Autosamdri-814 Critical Point Dryer (Tousimis Research Corporation, Rockville, MD), mounted onto aluminum pin stubs with colloidal paint, and sputter coated with gold-palladium with a Leica EM ACE600 Coater (Leica Microsystems Inc., Buffalo Grove, IL). Stubs were examined with the FE-SEM FEI Teneo (FEI, Inc., Hillsboro, OR) using the secondary electron detector to obtain digital images.
Calcium Measurements
To measure Ca2+ in PfERC mutants, knockdown was induced on synchronized schizonts. After 48hrs, schizonts were percoll purified and permeabilized selectively by treatment with ice-cold 0.04% saponin in PBS for 10 min. Isolated parasites were then washed 2X with BAG Buffer (116mM NaCl, 5.4mM KCl, 0.8mM MgSO4·7H2O, 50mM HEPES, 5.5mM Glucose) + 7.5mM GlcN and incubated with 10µM Fluo-4AM (ThermoFisher) for 45min while rocking at 37°C. After incubation, cells were washed 2X with BAG buffer + 7.5mM GlcN and immediately taken for fluorimetric measurements. Fluorescence measurements were carried out in a cuvette (Sarstedt) containing parasites suspended in 2.5 ml of BAG buffer and 100uM EGTA (Sigma). The cuvette was placed in a Hitachi F-4500 fluorescence spectrophotometer and Fluo-4AM excitation was done at 505 nm with emission read at 530 nm (68). Drugs and reagents were added via a Hamilton syringe. Final concentration of CPA (Sigma) was 3 µM, and Ionomycin (Sigma) at 2µM.
Acknowledgments
We thank Michael Reese and Meg Phillips for comments on the manuscript; Dan Goldberg for comments on the manuscript, anti Ef1α and anti-PMV antibodies; The European Malaria Reagent Repository for anti-MSP1 12.4 and anti-RAP1 2.29, antibodies; Alan Thomas and BEI Resources NIAID, NIH for anti-AMA1 28G2 and anti-BiP antibodies; Zhicheng Dou and Michael Blackman for anti-SUB1 antibody; Alan Cowman for anti-AMA1 (1F9); Purnima Bhanot for Compound 1 and Compound 2; Muthugapatti Kandasamy at the University of Georgia Biomedical Microscopy Core, Julie Nelson at the CTEGD Cytomtetry Shared Resource Lab for technical assistance; Mary Ard from the Georgia Electron Microscopy core at the University of Gerogia for assistance with SEM; Michael Cipriano for assistance with protein alignment. This work was supported by UGA Startup funds and UGA Faculty Research Grant (FRG-SE0031) to V.M., and the US National Institutes of Health (R21AI133322) to V.M. and S.N.J.M., and (T32AI060546) to M.A.F.