Calcium-dependent phosphorylation of Plasmodiumfalciparum serine repeat antigen 5 triggers merozoite egressReceived for publication, December 21, 2017, and in revised form, April 13, 2018 Published, Papers in Press, May 1, 2018, DOI 10.1074/jbc.RA117.001540

Gayatri R. Iyer‡1, Shailja Singh‡§1,2, Inderjeet Kaur‡1,3, Shalini Agarwal‡4, Mansoor A. Siddiqui‡, Abhisheka Bansal‡4,Gautam Kumar‡, Ekta Saini‡5, Gourab Paul‡, Asif Mohmmed‡6, Chetan E. Chitnis‡§7, and Pawan Malhotra‡8

From the ‡Malaria Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg,New Delhi 110 067, India and the §Department of Parasites and Insect Vectors, Institut Pasteur, 25–28 rue du Doctor Roux,75015 Paris, France

Edited by Luke O’Neill

The human malaria parasite Plasmodium falciparum prolif-erates in red blood cells following repeated cycles of invasion,multiplication, and egress. P. falciparum serine repeat antigen 5(PfSERA5), a putative serine protease, plays an important role inmerozoite egress. However, regulation of its activity leading tomerozoite egress is poorly understood. In this study, we showthat PfSERA5 undergoes phosphorylation prior to merozoiteegress. Immunoprecipitation of parasite lysates using anti-PfSERA5 serum followed by MS analysis identified calcium-de-pendent protein kinase 1 (PfCDPK1) as an interacting kinase.Association of PfSERA5 with PfCDPK1 was corroborated byco-sedimentation, co-immunoprecipitation, and co-immunolo-calization analyses. Interestingly, PfCDPK1 phosphorylatedPfSERA5 in vitro in the presence of Ca2� and enhanced its pro-teolytic activity. A PfCDPK1 inhibitor, purfalcamine, blockedthe phosphorylation and activation of PfSERA5 both in vitroas well as in schizonts, which, in turn, blocked merozoiteegress. Together, these results suggest that phosphorylation ofPfSERA5 by PfCDPK1 following a rise in cytosolic Ca2� levelsactivates its proteolytic activity to trigger merozoite egress.

Despite continued efforts to control malaria, it still remains amajor public health problem in the tropical world. The devel-opment and spread of artemisinin-resistant parasites in recentyears has raised serious concerns regarding the availability ofeffective tools to eliminate malaria (1, 2). Sustained efforts arethus required to understand parasite biology, identify noveldrug targets, and develop new therapeutic interventions against

malaria. The clinical manifestations of malaria are largelyattributed to its asexual stages, during which merozoitesrepeatedly invade, multiply within, and exit red blood cells fortheir propagation. The exit of invasive merozoites frominfected erythrocytes, a process referred to as parasite egress, isa critical step in blood-stage parasite growth. A cascade of pro-teolytic activities has been implicated in the release of merozo-ites from mature schizonts. However, an understanding of theregulation of the activities of such proteases leading to egressstill remains elusive (3–7).

A number of putative Plasmodium proteases, such as cys-teine proteases (falcipain-2, SERA9 proteins, dipeptidyl amino-peptidase 3 (DPAP 3)), aspartic protease (plasmepsin II) andserine protease (subtilisin-like protease 1 (SUB1)) are involvedin merozoite egress from P. falciparum schizonts (8 –12). Ofparticular interest are members of the SERA family of putativeproteases that possess a central papain-like protease domain(13). P. falciparum encodes nine SERA proteins, SERA 1–9(14). Among these, SERA 1–5 and SERA 9 have a serine residuein place of the conventional cysteine residue at the active site,whereas three SERA proteins, SERA 6 – 8, have cysteine at thekey catalytic position (15, 16). Such substitutions at the catalyticsites of enzyme homologs are well known in protozoan andmetazoan proteases, some of which play key regulatory roles(17). SERAs are proteins �100 –130 kDa in size and are mainlylocalized in the parasitophorous vacuole (13). Many membersof the PfSERA family are proteolytically processed by PfSUB1(11). Of the nine members of the PfSERA family, PfSERA5 andPfSERA6 are refractory to genetic deletion, suggesting that theyare essential for parasite growth and viability (18, 19). PfSERA5is the best studied PfSERA family member and is a promisingvaccine candidate as well as drug target (20, 21). Inhibitors ofPfSUB1, an enzyme involved in PfSERA5 processing, stallsmerozoite egress and parasite maturation, suggesting thatPfSERA5 and its processing enzyme PfSUB1 are importantdrug targets (11, 12). Although PfSERA5 is highly expressedat the late schizont stage, its proteolytic activity has been anissue of debate because of low proteolytic activity displayed

This work was supported by the Department of Biotechnology, Governmentof India (BT/01/CEIB/11/V/01). The authors declare that they have no con-flicts of interest with the contents of this article.

This article contains Figs. S1–S3 and Tables S1–S3.1 These authors contributed equally to this work.2 Present address: Special Center for Molecular Medicine, Jawaharlal Nehru

University, Delhi.3 Recipient of Innovative Young Biotechnologist Award BT/010/IYBA/2016/

03 from the Department of Biotechnology, Government of India.4 Present address: School of Life Sciences, Jawaharlal Nehru University, Delhi.5 Supported by a Department of Biotechnology junior research fellowship.6 Recipient of National Bioscience Award for Career Development BT/HRD/

NBA/34/01/2011(v) from the Department of Biotechnology, Governmentof India.

7 To whom correspondence may be addressed. E-mail: chetan.chitnis@pasteur.fr.

8 To whom correspondence may be addressed. E-mail: pawanmal@gmail.com.

9 The abbreviations used are: SERA, serine repeat antigen; Pf, Plasmodiumfalciparum; PTM, posttranslational modification; PV, parasitophorousvacuole; hpi, hour(s) post-invasion; IMAC, immobilized metal affinity chro-matography; LLVY-AMC, N-succinyl-Leu-Leu-Val-Tyr-AMC; AMC, 7-amino-4-methylcoumarin; Z, benzyloxycarbonyl; BAPTA-AM, 1,2-bis(2-aminophe-noxy) ethane-N,N,N�,N�-tetraacetic acid tetrakis (acetoxymethyl ester).

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by recombinant PfSERA5 expressed in Escherichia coli (13,22). A few recent studies have suggested that PfSERA5 servesas a pseudoprotease that regulates the kinetics/efficiencyof P. falciparum merozoite egress from mature schizonts(23, 24).

Posttranslational modifications (PTMs) of proteins, such asphosphorylation/dephosphorylation, palmitoylation, ubiquiti-nation, and sumoylation, are fast emerging as important mech-anisms for the regulation of functional activity in various organ-isms. Cross-talk between these PTMs may also play a role inregulating parasite development (25). However, little is knownabout the parasite proteases that undergo PTMs and about therole of PTMs in regulating their protease activities. In mostorganisms, including malaria parasites, of the many possiblePTMs, the role of phosphorylation in regulation of the cellcycle has been studied most extensively (26 –28). Given thatPfSERA5 is essential for parasite survival, we explored thepotential role of posttranslational modification, especially therole of phosphorylation of PfSERA5, in the regulation of itsprotease and merozoite egress activities. Immunoprecipitationusing anti-phosphoserine antibodies followed by MS analysisshowed that PfSERA5 is phosphorylated at the schizont stage.We identified PfCDPK1 as an interacting kinase that phosphor-ylates PfSERA5. Purfalcamine, a known chemical inhibitor ofPfCDPK1 (29), inhibited the native protease activity at the sch-izont stage and also blocked merozoite egress. These resultsprovide insight into the regulation of PfSERA5 protease activityand its link with merozoite egress.

Results

PfSERA5 exists in the phosphorylated form at schizont stagesof P. falciparum

Protein phosphorylation, in most eukaryotes, including Plas-modium spp., is one of the major regulatory processes that con-trols the functional activities of diverse proteins to modulatecellular processes (30). PfSERA5 expression in P. falciparum istightly regulated so that it is primarily expressed at the schizontstage. To determine whether phosphorylation plays a role in theregulation of PfSERA5 activity, we investigated the phosphor-ylation status of PfSERA5 in P. falciparum schizonts by immu-noprecipitation using anti-phosphoserine antibodies coupledto agarose. When eluates were probed using mouse antiserumraised against PfSERA5 by Western blotting, an �50-kDa bandcorresponding to the processed form of PfSERA5 was detected(Fig. 1A). When immunoprecipitates generated from P. falcip-arum schizont lysates with anti-phosphoserine antibodies weredigested with trypsin and analyzed by tandem MS (LC-MS/MS), peptides corresponding to a number of parasite proteins,including PfSERA5, were detected (Table S1). A peptide corre-sponding to the PfSERA5 sequence was identified with highconfidence (Fig. 1B). To identify the phosphorylation sites inPfSERA5, antibodies raised against PfSERA5 were used forimmunoprecipitation from P. falciparum schizonts lysates, andthe resulting immunoprecipitates were subjected to phospho-peptide analysis by LC-MS/MS. As shown in Fig. 1C, threephosphopeptides corresponding to PfSERA5 were detectedwith phosphorylation on Ser-183, Thr-549, and Ser-866 (the

Figure 1. PfSERA5 is phosphorylated at the schizont stage in P. falciparum. A, representative immunoblot showing a band of �50 kDa in eluates of the affinitypulldown from P. falciparum schizont extracts using anti-phosphoserine antibody when probed with anti-PfSERA5 mouse serum. WB, Western blot. B, identification ofPfSERA5 from the LC-MS/MS analysis of the above mentioned immunoprecipitates. Other proteins identified by this pulldown are summarized in Table S1. PSMs,peptide spectrum matches. C, the sites of phosphorylation in PfSERA5 as identified by LC-MS/MS analysis after immunoprecipitation using anti-PfSERA5 serum fromP. falciparum schizont extracts.

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spectra for these peptides are shown in Fig. S1). Together, theseresults suggest that PfSERA5 exists in the phosphorylated stateat the schizont stage.

PfCDPK1 associates with PfSERA5 in the parasitophorousvacuole of P. falciparum schizonts

Given that PfSERA5 is phosphorylated in late schizonts, wenext attempted to identify the kinase(s) that may associate withand phosphorylate PfSERA5. LC-MS/MS analysis of immuno-precipitates from P. falciparum schizont extract using anti-Pf-SERA5 serum revealed several kinases associated with PfSERA5(Table S2A). Among these kinases, PfCDPK1 is localized to theparasitophorous vacuole (PV) of P. falciparum-infected eryth-rocytes, similar to the localization observed for PfSERA5. Co-localization studies using immunofluorescence assays con-firmed co-localization of PfSERA5 and PfCDPK1 in the PV ofschizonts (Fig. 2A). Significant co-localization with a Pearson’scorrelation coefficient of �0.85 was observed with PfCDPK1and PfSERA5 (Fig. 2A).

Sedimentation analysis of P. falciparum schizont lysatesusing glycerol density gradient centrifugation was performed toascertain whether PfSERA5 and PfCDPK1 are present togetherin the same fraction(s). Western blotting using antibodiesraised against PfSERA5 and PfCDPK1 for 16 fractions andLC-MS/MS analysis showed that both proteins co-sedimenttogether in fractions 5 to 8 (Fig. 2B and Table S2B). Together,these results indicate a possible association of PfSERA5 andPfCDPK1.

To confirm the interaction between PfSERA5 and PfCDPK1,immunoprecipitation analysis was performed with P. falcipa-rum schizont lysates using either anti-PfSERA5 mouse serum

or anti-PfCDPK1 rat serum. Western blotting of immunopre-cipitates of parasite lysate generated with anti-PfSERA5 mouseserum showed the presence of PfCDPK1 (Fig. 3A). Similarly,immunoprecipitates generated with anti-PfCDPK1 rat se-rum from P. falciparum schizont lysates showed the pres-ence of full-length and proteolytically processed fragmentsof PfSERA5, as detected by Western blotting (Fig. 3A). Analysisby MS further confirmed the presence of PfCDPK1 andPfSERA5 in the immunoprecipitates of P. falciparum lysategenerated using anti-PfCDPK1 serum (Fig. 3B). The list ofunique peptides of both proteins identified by LC-MS/MS anal-ysis is summarized in Fig. 3B. A detailed list of all proteinsidentified in the immunoprecipitates is shown in Table S3.These results unequivocally suggest that PfCDPK1 interactswith PfSERA5 at the late schizont stage. Further, the recombi-nant PfCDPK1 and recombinant protease domain of PfSERA5(PfSERA550) were found to interact with each other in anELISA-based binding assay. We found that indeed both recom-binant proteins interact with each other in vitro (Fig. S2).

PfCDPK1 phosphorylates PfSERA5 and enhances its proteaseactivity

Given that PfSERA5 and PfCDPK1 co-localize in the PV, wetested whether PfCDPK1 can phosphorylate PfSERA5 in vitro.Recombinant PfCDPK1 was incubated with the recombinantprotease domain of PfSERA5 (PfSERA550) in the presence of[�-32P]ATP with or without Ca2�. Recombinant PfCDPK1phosphorylated PfSERA550 in the presence of Ca2� (Fig. 4A).Phosphorylation of PfSERA550 by PfCDPK1 was inhibited bypurfalcamine, a known inhibitor of PfCDPK1 (Fig. 4A). Subse-quently, the phosphorylation site(s) were mapped on in vitrophosphorylated PfSERA550 by MS. This analysis identifiedseven phosphorylated peptides corresponding to PfSERA5,each containing a single phosphorylated Ser/Thr residue (Fig. 4B).Among these phosphorylation sites, phosphorylation at Thr-549was also identified in PfSERA5 immunoprecipitated from parasiteextract using anti PfSERA5 sera, as shown in Fig. 1C.

Next we evaluated the possible role of phosphorylation in themodulation of protease activity of PfSERA5. RecombinantPfSERA550 has been shown to cleave LLVY-AMC, a syntheticfluorescent peptide substrate (13, 22). Using LLVY-AMC as asubstrate, we analyzed the activity of recombinant PfSERA550after incubation with recombinant PfCDPK1 in the presenceor absence of Ca2�. As shown in Fig. 4C, phosphorylatedPfSERA550 showed a considerable increase in protease activityfollowing incubation with PfCDPK1 in the presence of Ca2�

compared with nonphosphorylated PfSERA550. Addition ofpurfalcamine or EGTA inhibited the activation of PfSERA5protease activity (Fig. 4C), suggesting that PfCDPK1 phosphor-ylates PfSERA5 and modulates its activity.

PfCDPK1 activity regulates PfSERA5 function in P. falciparumschizonts to trigger egress

Purfalcamine, a PfCDPK1 inhibitor, or a specific PfSERA5inhibitory peptide, SE5 P2 (22), were examined next to probethe possible role of PfCDPK1 and PfSERA5 in merozoitesegress. We have shown previously that SE5 P2, a peptide fromthe C-terminal catalytic domain of PfSERA5, inhibits its activity

Figure 2. PfSERA5 and PfCDPK1 co-localize in the PV of P. falciparumschizonts. A, confocal microscopy analysis of P. falciparum schizonts demon-strates that PfSERA5 and PfCDPK1 co-localize in the PV. DAPI, 4�,6-diamidino-2-phenylindole. DIC, differential interference contrast. Scale bar, 2 �m. B,Western blotting (WB) shows that PfSERA5 and PfCDPK1 are concentrated infractions 6 – 8 when the protein extract of schizont-stage parasites was sub-jected to ultracentrifugation on a glycerol density gradient. This indicatesthat both of these proteins may be part of a complex within the parasite.

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(22). To analyze the activity of PfSERAs, especially PfSERA5,P. falciparum schizonts were incubated with the fluorogenicsubstrate LLVY-AMC. These assays were performed in thepresence or absence of either purfalcamine or SE5 P2 peptide.In the past, we have successfully used similar assay to analyzethe activity of cysteine protease(s) in intact parasitized erythro-cytes using a fluorogenic small peptide substrate, Z-Phe-Arg-AMC (Z-FR-AMC) (31). As shown in Fig. 5A, blue fluorescenceresulting from the cleavage of LLVY-AMC was detected in thecontrol schizonts. Purfalcamine- or SE5 P2-treated parasitesshowed little or no fluorescence signal, indicating a block inserine protease(s)–mediated proteolytic activity (Fig. 5B). Next,the biological effect of purfalcamine on parasite egress was eval-uated. P. falciparum schizonts treated with different concen-trations of the PfCDPK1 inhibitor purfalcamine were assayedfor egress �20 h after treatment using flow cytometry. A dose-

dependent block in merozoite egress was observed after purfal-camine treatment (Fig. 6). A similar block was also observedafter BAPTA-AM treatment (Fig. 6). To find out whether thisblock in merozoite egress is due to inhibition of phosphoryla-tion of PfSERA5, we tested the phosphorylation status ofPfSERA5 in purfalcamine-treated parasite extracts. As shownin Fig. 6, inset, purfalcamine significantly blocked the phos-phorylation of PfSERA5. Together, these results show thatinhibition of PfCDPK1 activity or PfSERA5 phosphorylationproduces a block in merozoite egress, suggesting that these twoproteins are critical players for merozoite egress.

Discussion

A role for the members of PfSERA family of putative pro-teases in merozoite egress from infected erythrocytes andsporozoite egress from oocysts has been suggested (11, 32).

Figure 3. PfSERA5 is associated with PfCDPK1 at the schizont stage of P. falciparum. A, representative immunoblots show an interaction between PfSERA5and PfCDPK1 in the parasite. Protein extracts of mature schizonts were independently used for immunoprecipitation (IP) using anti-PfSERA5 serum andanti-PfCDPK1 serum, and eluates were separated on SDS-PAGE and used for Western blotting (WB) with anti-CDPK1 and anti-PfSERA5 serum, respectively. B,the same eluates were trypsin-digested and analyzed by LC-MS/MS. The table shows the sequences of the unique peptides corresponding to PfCDPK1 andPfSERA5 identified by mass spectrometric analysis of the immunoprecipitate using anti-PfCDPK1 serum. A detailed list of all the proteins identified in thisanalysis is summarized in Table S3. PSMs, peptide spectrum matches.

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However, relatively weak in vitro catalytic activity and differ-ences in the crystal structure of PfSERA5 compared with papa-in-like cysteine proteases has raised questions about the role ofPfSERA5 as an actual protease during the egress process (13,23). A couple of genetic studies have recently describedPfSERA5 as a pseudoprotease that is essential for merozoitesegress from human erythrocytes, although its functional role isunclear (23, 24). However, the role of posttranslational modifi-cations in regulating the protease activity of SERA5 has neverbeen addressed. It is well known that phosphorylation by pro-tein kinases can regulate protease activities; a number of humanmetalloproteases, cysteine proteases, and threonine proteaseshave been shown to be activated/deactivated by kinases (33). Tofind out whether posttranslational modifications of proteasesor kinases in apicomplexan parasites play a role in regulating

biological processes, we investigated the phosphorylation sta-tus of PfSERA5 and its role in Plasmodium merozoite egressfrom infected erythrocytes.

To determine whether PfSERA5 is phosphorylated, P. falcip-arum schizont extracts were immunoprecipitated using anti-phosphoserine antibodies. Western blotting was performedusing anti-PfSERA5 serum, which identified a specific band ofPfSERA5 in immunoprecipitates. Mass spectrometry analysisof these immunoprecipitates and phosphopeptide analysisof immunoprecipitates made from P. falciparum extractsusing anti-PfSERA5 sera identified phosphorylation siteswithin PfSERA5, confirming the phosphorylation status ofPfSERA5 at the schizont stage. These results are in line withprevious reports where seven phosphorylation sites weredescribed within PfSERA5 at asexual blood stages (26, 28).

Figure 4. PfCDPK1 phosphorylates and enhances the enzymatic activity of recombinant PfSERA550. Recombinant PfCDPK1 phosphorylates PfSERA550 invitro. A, an autoradiograph showing the incorporation of the radiolabeled phosphate group in recombinant PfSERA550. B, the sites of phosphorylation inPfSERA550 as identified by LC-MS/MS analysis. The phosphorylation of Thr-549 is the common site, which was also detected in LC-MS/MS analysis followingimmunoprecipitation with anti-PfSERA5 serum from schizont-stage protein extracts (Fig. 1C). The asterisk denotes the common phosphorylation site(s)identified in in vitro phosphorylation studies as well as in schizont-stage immunoprecipitation experiments. C, preincubation of recombinant PfCDPK1 withrecombinant PfSERA550 significantly amplifies its protease activity, as measured by the hydrolysis of the fluorogenic synthetic peptide substrate LLVY-AMC.The fluorescence signal was completely abrogated by addition of inhibitors of PfCDPK1, purfalcamine or EGTA.

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We next looked for the kinase(s) that interact with and phos-phorylate PfSERA5. Because most of the SERAs are localizedin the PV, we focused on PfCDPK1, a kinase identified in theco-immunoprecipitates that is localized in the PV. A numberof experimental strategies, including density gradient sedi-mentation, co-localization by immunofluorescence assays,and co-immunoprecipitation studies with P. falciparum schi-zont extracts unequivocally suggested an interaction betweenPfSERA5 and PfCDPK1. The ability of PfCDPK1 to phosphor-ylate PfSERA5 was tested in vitro in the presence of Ca2�. Oneof the phosphorylation sites on PfSERA5 following in vitro phos-phorylation by PfCDPK1 was common to the phosphorylationsites found on PfSERA5 in the parasite at late schizont stage. Pur-falcamine,aknownPfCDPK1inhibitor(29),blockedthephosphor-ylation of recombinant PfSERA5 by recombinant PfCDPK1 invitro as well as phosphorylation of PfSERA5 in vivo in schizonts.

Having demonstrated that PfSERA5 exists in phosphorylatedform at the late schizont stage and that PfCDPK1 is possiblyinvolved in PfSERA5 phosphorylation, we next examined thesignificance of PfSERA5 phosphorylation and the conse-quences of blocking this phosphorylation using a PfCDPK1inhibitor. Phosphorylation of PfSERA5 by PfCDPK1 in vitrogreatly enhanced the proteolytic activity of PfSERA5, as mea-sured using the fluorescent peptide substrate LLVY-AMC (Fig.

4). Purfalcamine blocked phosphorylation of PfSERA5 byPfCDPK1 and inhibited the rise in proteolytic activity ofPfSERA5 (Fig. 4). We also examined these activities in vivo inthe presence of purfalcamine and a PfSERA5-specific peptidethat has been shown previously to inhibit PfSERA5 activity invitro as well as in cultured parasites (22). Fluorescence measure-ments of parasites incubated with LLVY-AMC substrate andtreated with purfalcamine or the PfSERA5 inhibitory peptideSE5 P2 showed a considerable drop in fluorescence signal fromAMC release compared with untreated parasites. These resultsthus demonstrated that purfalcamine or SE5 P2 peptideblocked the PfSERA5 activity in vivo, confirming the need forPfSERA5 and its phosphorylation by PfCDPK1 for activationof its protease activity. We further analyzed the effect of pur-falcamine on parasite development, especially in merozoiteegress, as a role for PfSERAs, in particularly PfSERA5, has beensuggested earlier in merozoite egress (11, 12, 19). Incubation ofschizont-stage parasites with purfalcamine blocked meroziteegress, demonstrating that both PfCDPK1 and PfSERA5 activ-ities are required for merozoite egress from infected erythro-cytes. Further, we found that phosphorylation of PfSERA5 wasblocked in purfalcamine-treated schizont extracts (Fig. 6, inset).Overall, these results demonstrate that PfCDPK1 phosphory-lates PfSERA5 following a rise in Ca2� in mature schizonts and

Figure 5. Purfalcamine and SE5 P2 peptide block protease activity. A, i, a synthetic fluorescent peptide substrate, LLVY-AMC, was added to P. falciparumcultures at early schizont stage, and its hydrolysis was visualized by blue fluorescence. DIC, differential interference contrast. ii, the fluorescence signal wasinhibited by addition of the PfSERA5 inhibitory peptide SE5 P2 (500 �M), and this change was quantified spectrophotometrically. B, i and ii, hydrolysis of thesynthetic substrate peptide was also inhibited by purfalcamine, a known inhibitor of PfCDPK1. This inhibition was captured and quantified using confocalmicroscopy. Together, these results indicate possible regulation of PfSERA5 activity by phosphorylation.

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that this phosphorylation step activates the proteolyticactivity of PfSERA5 to trigger merozoite egress. BecauseCollins et al. (24) have proposed a role of PfSERA5 in timelyrupture of invasive merozoites, we speculate that its phos-phorylation is a key regulator in the later stages of egress,which needs to be explored further. The process of egress isa complicated and multiproteolytic cascade of events; mul-tiple phosphorylation events are expected to take place,involving multiple kinases and their substrates. A role forPfCDPK1 and PfCDPK5 in merozoite egress has been sug-gested earlier (29, 34, 35). PfCDPK1 has also been shown tobe essential for merozoite invasion and plays a role in thephosphorylation of PfMTIP and PfGAP45, components ofthe glideosome complex, which provides gliding motility tothe merozoite during invasion (36 –38). These datasets sug-gest that multiple kinases and proteases participate in theprocesses of egress and invasion during blood-stage growthof malaria parasites.

In conclusion, the results of this study demonstrate thatPfSERA5 is phosphorylated in P. falciparum schizonts byPfCDPK1 following a rise in intracellular Ca2�, which hasbeen shown previously to trigger egress. Phosphorylationenhances the protease activity of PfSERA5. Although this isone of the first reports to show that the activity of a parasiteprotease can be regulated by a kinase, many human proteaseshave been shown to be regulated by different kinases (33).The evidence discussed above thus clearly provides dataabout a link between PfSERA5 protease activity and its phos-phorylation status, which is essential for the timely egress ofmerozoites from infected erythrocytes. Understanding thekinase–protease cross-talk may lead to the introduction ofcombined therapies targeting specific components of bothfamilies as part of the new generation of anti-malarialtherapies.

Experimental procedures

In vitro parasite culture

P. falciparum 3D7 was cultured in 4% hematocrit with RPMI1640 with L-glutamine (Invitrogen) supplemented with 0.5%Albumax I (Invitrogen), 2.1 mg ml�1 sodium bicarbonate (Sig-ma-Aldrich), and 25 �g ml�1 gentamycin reagent solution(Invitrogen) according to a protocol described previously (39).Parasite cultures were maintained in a mixed-gas environment(5% O2, 5% CO2, and 90% N2) at 37 °C. Parasites were synchro-nized by two consecutive sorbitol treatments 4 h apart at earlyring stage and allowed to mature into schizonts (44 – 46 h post-invasion (hpi)) for further experiments.

Cloning, expression, and purification of PfSERA5 andgeneration of PfSERA5 antiserum

The plasmid expressing the catalytically active region ofPfSERA550 (�50-kDa domain) was obtained from AnthonyN. Hodder (Walter and Eliza Hall Institute, Australia). Pro-tein was expressed and purified by a protocol described pre-viously (13, 22). Polyclonal antibodies against recombinantPfSERA550 were generated in mice and used for furtherexperiments.

Glycerol density gradient centrifugation

Late schizonts (44 – 46 hpi) from a P. falciparum 3D7 culturewere lysed in 0.5% NP-40 in HEPES-buffered saline (10 mM

HEPES, 2 mM MgCl2, 10 mM KCl, 0.5 mM EDTA, and 150 mM

NaCl (pH 7.0)) containing protease and phosphatase inhibitormixtures (Roche Diagnostics Corp.) for 15–20 min at 4 °C.Lysates were cleared by centrifugation at 13,000 rpm for 10min, and �500 �l of lysate was layered on top of an 11-mlultracentrifuge tube containing a 45–5% glycerol density gradi-ent. Tubes were centrifuged at 38,000 rpm for 19 h at 4 °C in anSW41 rotor (Beckman Coulter Life Sciences). The molecular

Figure 6. Effect of the PfCDPK1 inhibitor purfalcamine on merozoites egress. Schizont-stage parasites (40 – 42 hpi) were treated with DMSO control,purfalcamine (500 nm, 1 �M, and 10 �M), or BAPTA-AM at 50 �M in duplicates and assayed for egress by flow cytometer. Data from two independentexperiments (mean � S.E.) showed a dose-dependent effect of purfalcamine on parasite egress. P, purfalcamine; B-AM, BAPTA-AM. Egress in the DMSO controlwas considered 100% for all experiments. The representative FACS raw data file shows loss of schizonts in the DMSO control, whereas the presence of stalledschizonts is seen in the presence of purfalcamine at 10 �M concentration. Inset, purfalcamine treatment abrogated the phosphorylation of PfSERA5 inschizont-stage parasites. Western blotting (WB) using anti-PfSERA5 serum shows a band at �50 kDa in DMSO-treated schizont extracts after pulldown usinganti-phosphoserine–agarose, whereas no such band was visible in purfalcamine-treated parasite extracts.

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mass standards BSA (66 kDa), aldolase, catalase (220 kDa), fer-ritin (440 kDa), and thyroglobulin (660 kDa) were also preparedin the same buffer and run in parallel. Twenty fractions of 500�l were collected from each gradient, and equal volumes ofeach fraction were mixed with Laemmli buffer and analyzedby Western blotting with antibodies against PfSERA5 andPfCDPK1.

Indirect immunofluorescence assay

Synchronized P. falciparum 3D7 cultures at late schizontstage (44 – 46 hpi) were smeared on glass slides, air-dried, andfixed in chilled absolute methanol for 30 min. Nonspecific bind-ing sites on the cells were blocked in 3% BSA prepared in PBS.Smears were probed with anti-PfSERA5 mouse serum and anti-PfCDPK1 rat serum at dilutions of 1:50 and 1:100, respectively,for 1 h at 37 °C. The slides were then washed once with PBST(PBS � 0.02% Tween 20) and three times with PBS. Smearswere probed with Alexa Fluor 594 – conjugated goat anti-mouse IgG antibody (Invitrogen) and Alexa Fluor 488–conjugated donkey anti-rat IgG antibody (Invitrogen) at dilu-tions of 1:500 and 1:200, respectively, for 1 h at 37 °C. Allantibodies were diluted in 1% BSA prepared in PBS. Slides werewashed, mounted, and sealed. Nuclear DNA was visualizedwith 4�,6-diamidino-2-phenylindole (Invitrogen). Images wereacquired using a Nikon A1R confocal microscope using the NISElements software.

Co-immunoprecipitation

Immunoprecipitation experiments were performed usingthe Pierce Cross-link Immunoprecipitation Kit (product no.26147; Pierce Biotechnology Inc., Rockford, IL) according tothe instructions provided by the manufacturer. Briefly, eryth-rocytes infected with synchronized P. falciparum 3D7 late schi-zonts (44 – 46 hpi) were pelleted at 1000 � g for 5 min, treatedwith 0.15% saponin prepared in PBS for 10 min at 4 °C, andcentrifuged at 2500 � g for 20 min. The parasite pellet obtainedwas washed two to three times with PBS by centrifugation at15,000 � g for 1 min. The parasites were subsequently lysedusing lysis buffer (250 mM Tris, 150 mM NaCl, 1 mM EDTA, 1%NP-40, and 5% glycerol (pH 7.4)) containing protease and phos-phatase inhibitor cocktails (Roche Diagnostics) for 15–20 minat 4 °C with intermittent mixing. Lysates were clarified by cen-trifugation at 15,000 � g for 30 min. The protein concentrationof the supernatants was determined by the BCA protein esti-mation assay kit (Pierce Biotechnology Inc., Rockford, IL).Approximately 1 mg of total protein was incubated overnight at4 °C with about 10 �g of anti-PfSERA5 and/or anti-PfCDPK1serum cross-linked to 10 �l of protein A/G–Sepharose resinusing disuccinimidyl suberate as a cross-linker with constantmixing. An equal amount of protein was allowed to bind to theresin conjugated to normal mouse or rat serum as a control.Following binding, resins were washed with wash buffer andbound proteins were eluted using the elution buffer (Tris-gly-cine (pH 2.8)). The eluates were separated on SDS-PAGE andanalyzed by Western blotting using antisera against PfSERA5and PfCDPK1.

Immunoprecipitation assay for the identification ofphosphorylated PfSERA5 in schizont extracts

For the detection of native phosphorylated PfSERA5, celllysates prepared from late schizonts (44 – 46 hpi) of the P. fal-ciparum 3D7 strain (as mentioned earlier) were incubated withmouse monoclonal anti-phosphoserine antibodies coupled toagarose (product no. A8076, Sigma-Aldrich) for 12–15 h (orovernight) at 4 °C to allow binding. About 2 mg of total cellularprotein was used for binding to either anti-phosphoserine aga-rose or control agarose beads. The bound phosphoproteinswere eluted with Tris-glycine buffer (pH 2.8) and analyzed byWestern blotting using anti-PfSERA5 mouse serum. The elu-ates were also digested with trypsin for identification of pro-teins by MS as described below.

Immunoprecipitation using anti-phosphoserine–agarosewas also performed using schizont (44 – 46 hpi) lysates treatedwith DMSO or purfalcamine. The eluates were separated bySDS-PAGE and transferred onto polyvinylidene difluoridemembranes. The membranes were probed with anti-PfSERA5serum.

Protein digestion and identification by LC-MS/MS

Proteins in the immunoprecipitated samples were proteoly-sed by in-solution trypsin digestion. Samples were brought to afinal volume of 100 �l in 50 mM ammonium bicarbonate bufferto adjust the pH to 8.0, reduced with 10 mM DTT (final concen-tration) for 1 h at room temperature, and alkylated with 40 mM

iodoacetamide (Sigma-Aldrich) for 1 h at room temperature inthe dark. Proteins were digested by the addition of trypsin-Gold(Promega) at a ratio of 1:50 (w/w) of trypsin:protein. For com-plete digestion, samples were placed in a water bath at 37 °C for18 h. The digestion was stopped by acidification using formicacid (0.1% final concentration). The peptide mixtures were con-centrated in a SpeedVac and analyzed by LC-MS/MS.

Enrichment of phosphopeptides

For phosphorylation studies of PfSERA5 by LC-MS/MS(with both recombinant PfSERA5 and PfSERA5 from parasitelysates), the trypsin-digested peptide mixtures were firstenriched for phosphopeptides before mass spectrometric anal-ysis. Phosphopeptides from the digested peptide pool wereenriched using immobilized metal affinity chromatography(IMAC) as described by Villén and Gygi (40). Briefly, vacuum-dried peptides were resuspended in 120 �l of IMAC bindingbuffer (40% acetonitrile and 25 mM formic acid) and allowed tobind to 10 �l of pre-equilibrated iron–nitrilotriacetic acidIMAC beads (Sigma-Aldrich). The peptides were incubated for1 h at room temperature with constant mixing. Following theincubation, the flow-through containing nonphosphorylatedpeptides was collected separately. The resin was washed threetimes with 120 �l of IMAC binding buffer. The bound phos-phopeptides were eluted three times with 40 �l of IMAC elu-tion buffer (50 mM K2HPO4 in NH4OH (pH 10.0)). The eluateswere pooled, and the pH was neutralized with 40 �l of 10%formic acid. The flow-through and eluates were vacuum-dried,desalted, and analyzed by LC-MS/MS.

Mass spectrometry analysis was performed on an OrbitrapVelos Pro MS coupled to Easy n-LC 1000 (Thermo Fisher Sci-

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entific). Tryptically digested peptide mixtures were loaded onto a reverse phase C-18 precolumn (Acclaim PepMap, 75 �m �2 cm, 3 �m, 100 Å, Thermo Fisher Scientific) that was in linewith an analytical column (Acclaim PepMap, 50 �m � 15 cm, 2�m, 100 Å). The peptides were separated using a gradient of 5%solvent B (0.1% formic acid in 95/5 acetonitrile/water) to 50%solvent B over 60 min. For recombinant proteins, a relativelysmaller gradient was used. The eluted peptides were injectedinto the Orbitrap Velos Pro MS, and MS1 data were acquiredusing a mass range from 350 –2000 Da in full scan mode at60,000 resolution. The top 20 precursors were allowed to frag-ment using collision-induced dissociation in an ion trap with acollision energy of 35. For phosphopeptide analysis, precursorswere fragmented using high-energy collision dissociation anddetected in an Orbitrap at a resolution of 7500. The raw datawere analyzed using Proteome Discoverer 1.4 (Thermo FisherScientific) with the SEQUEST algorithm against the P. falcipa-rum database downloaded from PlasmoDB and UniProt. Theidentification of peptides was validated using Percolator. Thesites of phosphorylation were scored using PhosphoRS 3.0.

In vitro kinase assay

Phosphorylation assays were performed as described previ-ously (38). Briefly, recombinant PfCDPK1 (40 nM) was allowedto phosphorylate 1 �g of recombinant PfSERA5 in kinase assaybuffer (50 mM MgCl2, 50 mM Tris (pH 8.0), 20 mM sodiumorthovanadate, 20 mM sodium fluoride, 1 mM DTT, and 2.5 mM

CaCl2) for 1 h at 30 °C using radioactive [�-33P]ATP as thesource of the phosphate group. Syntide-2 was used as a controlsubstrate for PfCDPK1. The reaction was stopped by adding 4�SDS-PAGE Laemmli buffer. The reaction mixture was separatedon a polyacrylamide gel, and phosphate incorporation wasdetected using Storage Phosphor Screen (GE Healthcare). Purfal-camine, a known inhibitor of PfCDPK1 (29), was used to confirmthe role of PfCDPK1 in phosphorylating PfSERA5.

Protease activity assay with recombinant PfSERA5 followingphosphorylation by recombinant PfCDPK1

To evaluate the effect of PfCDPK1-mediated phosphoryla-tion on the proteolytic activity of recombinant PfSERA5, 1 �gof PfSERA5 was incubated with recombinant PfCDPK1 (40 nM)in a kinase assay buffer in a 50-�l reaction volume as describedpreviously (38). Here, the radioactive [�-33P]ATP was replacedwith nonradioactive ATP as the source of the phosphate group.The reaction was allowed to proceed for 1 h at 30 °C. The reactionmixture was diluted to 400 �l in PfSERA5 activity assay buffer (0.1M NaHCO3 (pH 7.5), 5 mM CaCl2, and 0.1% Tween 20) and incu-bated with 20 �M 7-amino-4-methyl coumarin (LLVY-AMC)(Sigma-Aldrich) at room temperature. Released AMC wasdetected and measured using a LS50B PerkinElmer Life Sciencesfluorimeter (excitation 355 nm, emission 460 nm) over a period of7–8 h, with readings taken every 30 min. The values at which thefluorescence plateaued were considered for representation.

In situ protease activity assay

Erythrocytes infected with P. falciparum 3D7 schizonts(44 – 46 hpi) were separated from uninfected erythrocytes bycentrifugation on 65% Percoll solution to achieve 85–90%

purity (as described previously). Purified schizonts were addedto individual wells of a 96-well plate to 1% hematocrit in a200-�l medium volume. The culture was treated with 1 �M

purfalcamine for 2 h at 37 °C. Appropriate control wells wereset up simultaneously. After the treatment, the wells wereloaded with a fluorogenic serine protease substrate (20 �M

LLVY-AMC, Sigma-Aldrich) for 20 –30 min. Cells werescraped from each well and mounted on glass slides, and AMCfluorescence was observed using the 405 filter of a Nikon A1confocal microscope. Image analysis was performed using theImagePro analyzer (v6.2). An alternative method of quantifyingthe released AMC was employed while studying the effect ofSE5 P2 peptide (final concentration of 500 �M) on serine pro-tease activity in the schizonts. Here, the release of AMC wasmonitored using a LS50B PerkinElmer Life Sciences fluorime-ter (excitation 355 nm, emission 460 nm) after incubation ofSE5 P2 with mature schizonts for 2 h at 37 °C.

Merozoite egress inhibition assay

To determine the effect of inhibition of PfSERA5 phosphor-ylation by inhibiting PfCDPK1 on P. falciparum merozoiteegress, egress inhibition assays were performed as describedpreviously (41). Briefly, late-stage schizonts (42– 44 hpi) weretreated with different concentrations of purfalcamine (0.5–10�M), 50 �M BAPTA-AM, and appropriate solvent controls for8 –10 h in duplicate wells. Following incubation, parasites fromtreated and control wells were collected, washed, and stainedwith ethidium bromide (10 mg/ml) and ethidium bromide–positive cells were scored by flow cytometry on a FACSCalibur(BD Biosciences) using CellQuest software. The fluorescencesignal (FL-2) was detected with a 90-nm band pass filter usingan excitation laser of 488 nm collecting typically 100,000 cellsper sample. Post-acquisition data were analyzed using FlowJosoftware (Tree Star, Inc.) by determining the proportion ofFL-2–positive cells representing schizonts. Inhibition of egressof merozoites was calculated as follows: % inhibition of mero-zoite egress 100 ((T-C)/(I-C)), where I is the frequency ofschizonts (percent) at the start of the assay, T is the frequencyof schizonts (percent) after treatment, and C is the frequency ofschizonts (percent) in the control.

Sequences of SE5 P2 and P3 peptides

The sequences of SE5 P2 and P3 peptides were as follows: SE5P2, Ac-KASPEFYHNLYFKNF-NH2; SE5 P3, Ac-NVGKKN-LFSEKEDN-NH2.

Author contributions—G. R. I., I. K., C. E. C., and P. M. conceptual-ization; G. R. I., A. B., G. K., I. K., and P. M. resources; G. R. I., S. S.,I. K., S. A., M. A. S., A. B., E. S., and A. M. data curation; G. R. I., S. S.,I. K., S. A., C. E. C., and P. M. formal analysis; C. E. C., I. K., and P. M.supervision; C. E. C. and P. M. funding acquisition; G. R. I., S. S., I. K.,S. A., M. A. S., A. B., E. S., G. P., and A. M. validation; G. R. I., S. S.,I. K., S. A., M. A. S., E. S., and A. M. investigation; G. R. I., I. K., S. A.,and P. M. visualization; G. R. I., S. S., I. K., S. A., M. A. S., A. B., E. S.,G. P., and A. M. methodology; G. R. I., I. K., S. A., C. E. C., and P. M.writing-original draft; G. R. I., I. K., C. E. C., and P. M. project admin-istration; P. M., G. R. I., I. K., S. A., E. S., A. M., and C. E. C. writing-review and editing.

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Acknowledgments—We thank Dr. Brendan Crabb and Dr. AnthonyN. Hodder for kindly providing the PfSERA5P50 expression clone. Wealso thank the Rotary Blood Bank for providing human red blood cellsand Surbhi Dabral for help with confocal microscopy.

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Chitnis and Pawan MalhotraE.Abhisheka Bansal, Gautam Kumar, Ekta Saini, Gourab Paul, Asif Mohmmed, Chetan

Gayatri R. Iyer, Shailja Singh, Inderjeet Kaur, Shalini Agarwal, Mansoor A. Siddiqui,antigen 5 triggers merozoite egress

serine repeatPlasmodium falciparumCalcium-dependent phosphorylation of

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