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Molecular & Biochemical Parasitology 174 (2010) 66–69

Contents lists available at ScienceDirect

Molecular & Biochemical Parasitology

hort communication

ocalization of apical sushi protein in Plasmodium falciparum merozoites

nand Srivastava1, Shailja Singh, Shikha Dhawan, M. Mahmood Alam,sif Mohmmed, Chetan E. Chitnis ∗

alaria Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi 110067, India

r t i c l e i n f o

rticle history:eceived 4 March 2010

a b s t r a c t

Plasmodium falciparum belongs to the Apicomplexan group of parasites and is characterised by presenceof specialized secretory organelles at the apical end. These apical organelles, referred to as microneme and

eceived in revised form 1 June 2010ccepted 2 June 2010vailable online 9 June 2010

eywords:lasmodium falciparum

rhoptries, contain proteins that play important roles during host cell invasion by mediating specific func-tions such as initial attachment, apical reorientation and junction formation. Recently, a protein referredto as P. falciparum apical sushi protein (PfASP), which is expressed at late schizont stage, was localized tomicronemes of P. falciparum merozoites. In the present study, we have used indirect immunofluorescenceassays and immunoelectron microscopy to demonstrate that PfASP is localized in the neck of rhoptriesand not in micronemes as previously described.

pical sushi protein

hoptry neck protein

Malaria remains one of the important and most widespread par-sitic diseases. It is responsible for over one million deaths per year,ostly in sub-Saharan Africa [1]. Malaria parasites belong to Api-

omplexan group of parasites and harbour specialized secretoryrganelles referred to as rhoptries and micronemes at the apicalnd. During development of blood stage schizonts various pro-eins needed for initial attachment, reorientation and invasion intorythrocyte get compartmentalized into these apical organelles2]. Previous studies have shown that micronemes contain sev-ral proteins including apical membrane antigen-1 (AMA-1) [3]nd erythrocyte binding antigens such as EBA-175, EBA-140 andBA-180 [4,5]. The rhoptries consist of two distinct parts: an elec-ron dense rounded basal bulb and a less dense rhoptry neckhat ends just beneath the plasma membrane at the apical promi-ence [6]. Plasmodium falciparum rhoptries contain different setsf proteins in the basal bulb and neck. The P. falciparum higholecular mass rhoptry protein (PfRhopH) complex comprising of

hree distinct gene products namely, RhopH1(CLAG3.1), RhopH2nd RhopH3 is present in the rhoptry bulb [7,8]. Rhoptry neckrotein (PfRON4), a homologue of Toxoplasma gondii rhoptry neckrotein TgRON4, is shown to be present at the apical end of the

hoptries [9]. Recently, a GPI anchored merozoite protein, Pf34,nd apical asparagine rich protein (PfAARP), were also localizedo the rhoptry neck [10,11]. After the release of merozoites fromchizonts contents of both rhoptries and micronemes are secreted

∗ Corresponding author. Tel.: +91 11 2674 2895; fax: +91 11 2674 2895.E-mail address: cchitnis@icgeb.res.in (C.E. Chitnis).

1 Current address: Institut Pasteur, Unité de Biologie des Interactions Hôte-arasite, CNRS URA2581, 25, rue du Docteur Roux, F-75724 Paris Cedex 15, France.

166-6851/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.molbiopara.2010.06.003

© 2010 Elsevier B.V. All rights reserved.

to the merozoite surface prior to entry of host cell. We haverecently shown that exposure of P. falciparum merozoite to lowpotassium ion concentrations as found in blood plasma leads tosecretion of microneme proteins such as EBA-175 and AMA-1 [12].Subsequently, interaction of EBA-175 with glycophorin A on theRBC surface provides a signal for release of rhoptry proteins [12].These data suggest that contents of apical organelles are releasedin a well defined sequence. Correct localization of proteins inapical organelles is thus important for effective host cell inva-sion.

Recently, a protein named as P. falciparum apical sushi protein(PfASP) was identified [13]. Orthologs of PfASP are also found inmurine (Plasmodium yoelii and Plasmodium berghei), simian (Plas-modium knowlesi) and human (Plasmodium vivax) malaria parasites[13]. The gene encoding PfASP is 2639 bp long, consists of fourexons and is highly up-regulated in schizont stage parasites. PfASPcontains an N-terminal signal sequence and a C-terminal trans-membrane domain. The extracellular region of PfASP contains twodistinct cysteine rich regions which are conserved in orthologsof PfASP. Based on conserved cysteine-rich regions, PfASP proteinwas divided into four different regions labelled as regions I–IV(Fig. 1A). Region IV contains a domain that shares homology with“sushi domains” which are characterised by a consensus sequencethat spans approximately 60 residues and contains four invariantcysteine residues [14,15]. The sushi domains present in variouscomplement regulator proteins found in mammals and viruses are

known to interact with complement components C3b and C4b,interferon-� and interleukin-15 [14]. Analysis of the transcriptionprofile of the gene encoding PfASP shows that the PfASP gene istranscribed at late schizont stage [16]. Detection using specific anti-sera suggested that PfASP was localized to micronemes at the apical

A. Srivastava et al. / Molecular & Biochem

Fig. 1. (A) Classification of various regions of PfASP based on the presence of cys-teines (vertical lines). ASP contains two cysteine rich regions: region II and regionIV. The N-terminus of ASP contains signal sequence (SS) while C-terminus containshydrophobic regions which is GPI anchored (TM). Region IV contains sushi domain.(B) SDS-PAGE profile of purified recombinant PfASP-RIV and mobility shift underreduced and non-reduced conditions. Difference in mobility of PfASP-RIV proteinunder reduced and non-reduced conditions indicates the presence of disulphidebtsi

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onds. (C) Western blotting of P. falciparum schizont extracts under reducing condi-ion with mouse sera raised against recombinant PfASP-RIV. Anti-PfASP-RIV mouseera react specifically with a protein of expected size (50 kDa) in P. falciparum sch-zont extracts.

nds merozoites [13]. Here we have re-examined the localizationf PfASP in P. falciparum merozoites.

Specific polyclonal sera were raised in mice against PfASP toollow its expression in P. falciparum blood stage parasites. A DNAragment encoding region IV of PfASP (PfASP-RIV; 1863–2100 bp)ith 6 × His-tag was cloned in pET28a (+) (Novagen) vector andas used for expression of recombinant protein in Escherichia coliLR (DE3) (Novagen) cells. The recombinant protein (PfASP-RIV;21–700 amino acid) was purified from cell lysates using Ni-NTAffinity chromatography (GE Healthcare) followed by gel perme-tion chromatography using Superdex 75 column (GE Healthcare).urified recombinant PfASP-RIV migrated on SDS-PAGE with thexpected molecular weight of 8.9 kDa (Fig. 1B). PfASP-RIV showedifferential migration when separated under reducing and non-educing conditions, suggesting presence of disulfide linkages.

Polyclonal sera were raised in BALB/c mice against recombi-ant PfASP-RIV (rPfASP-RIV). Purified rPfASP-RIV protein (25 �g)as formulated in complete/incomplete Freund’s adjuvant and

njected intra-peritoneally in mice. Sera from mice immunized

ith rPfASP-RIV formulated with complete and incomplete Fre-nd’s adjuvant showed ELISA endpoint titres of approximately:700,000 for recognition of the recombinant antigen follow-

ng one primary and two booster immunization. PfASP has beenreviously shown to be processed into an N-terminal 30 kDa frag-

ical Parasitology 174 (2010) 66–69 67

ment and a C-terminal 50 kDa fragment that remain attached bydisulphide bonds [13]. Antisera raised against PfASP-RIV shouldrecognize the C-terminal 50 kDa fragment. Anti-PfASP-RIV mousesera reacted with a 50 kDa protein from P. falciparum 3D7 sch-izont extracts separated under reducing conditions by Westernblotting (Fig. 1C) confirming that the sera recognize PfASP withspecificity.

Anti-PfASP-RIV mouse sera were used to localize PfASP in P. fal-ciparum 3D7 strain merozoites by immunofluorescence assays asdescribed earlier [17]. Briefly, thin smears of P. falciparum infectederythrocytes were made on glass slides and fixed with a mixture ofmethanol/acetone. Slides were blocked in blocking buffer (1 × PBS,10% FCS) for 2 h at RT. After blocking, slides were incubated withanti-PfASP mouse sera diluted in blocking buffer (1:300) and rabbitserum against EBA-175 (1:300) [18], CLAG3.1 (1:300) [8] or PfAARP(1:300) [10] for 1 h at RT. Slides were washed with 1 × PBS for 1 hand incubated with Alexafluor 488 labelled goat anti-mouse IgGand Alexafluor 594 labelled goat anti-rabbit IgG (Molecular Probes)at dilutions of 1:100, for 1 h. DAPI was used for nuclear DNA stain-ing. The slides were subsequently washed twice in 1 × PBS-Tween0.05%, once in 1 × PBS and cover slip was mounted in the presence ofantifade mounting media (Bio-Rad). The parasites were visualizedusing Nikon TE 2000-U fluorescence microscope.

Immunofluorescence assays carried out using anti-PfASP mousesera at various erythrocytic stages showed fluorescence labellingin mature schizonts with segmented merozoites. PfASP was testedfor co-localization in schizonts using sera against microneme pro-tein (EBA-175), rhoptry bulb protein (CLAG3.1) and rhoptry neckprotein (PfAARP). As expected anti-EBA-175 sera recognised EBA-175 in a punctate pattern near apical end of merozoites withinschizonts [19]. Anti-PfASP sera also stained the apical end of mero-zoites. However, PfASP did not co-localize with EBA-175 (Fig. 2A).Sera against other microneme proteins, AMA-1 and EBA-140, alsodid not co-localize with PfASP (data not shown).

PfASP was further tested for co-localization with rhoptry bulbprotein, CLAG3.1. Anti-CLAG3.1 sera showed punctate staining atthe apical end of merozoite (Fig. 2B) but did not co-localize withPfASP. The staining pattern suggested that PfASP is localized api-cal to CLAG3.1 suggesting that it may be localized in rhoptry neck.Sera against the rhoptry neck protein, PfAARP, were used to con-firm localization of PfASP in rhoptry neck. PfASP co-localized withPfAARP at the apical ends of merozoites (Fig. 2C).

In order to further validate the results obtained fromimmunofluorescence co-localization studies, we performedimmune-electron microscopy (IEM) with late-stage P. falciparum3D7 schizonts containing segmented merozoites. Parasites werefixed in 4% para-formaldehyde, 0.04% glutaraldehyde in PBS at 4 ◦Cfor 1 h and subsequently embedded in gelatin, and infiltrated witha cryo-preservative and plasticizer (2.3 M sucrose/20% polyvinylpyrrolidone). After freezing in liquid nitrogen, samples weresectioned with a Leica Ultracut UCT cryo-ultramicrotome (LeicaMicrosystems Inc., Bannockburn, IL) at −60 ◦C. Ultrathin sectionswere blocked with 5% fetal bovine serum and 5% normal goat serumin PBS for 30 min and subsequently stained with anti-PfASP mouseantibody (1:100 dilution in blocking buffer), washed thoroughlyand incubated with 18 nm colloidal gold-conjugated anti-mouseIgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA)for 1 h. Sections were stained with 0.3% uranyl acetate/1.7% methylcellulose and visualized under JEOL 1200EX transmission electronmicroscope (JEOL USA Inc., Peabody, MA). Pre-immune serumwhich was used as a control in these experiments showed no

reactivity. IEM studies using anti-PfASP mouse sera indicated thatPfASP is localized in the rhoptry neck at the apical end of rhoptriesin segmented merozoites within mature schizonts (Fig. 3A and B).No staining was detected in the rhoptry bulb or any other organellewithin the merozoites.

68 A. Srivastava et al. / Molecular & Biochemical Parasitology 174 (2010) 66–69

Fig. 2. Co-localization of PfASP in late stage P. falciparum schizonts with microneme protein, EBA-175 (A); rhoptry bulb protein, Clag3.1 (B); and rhoptry neck protein, PfAARP(C). P. falciparum schizonts were stained with anti-PfASP-RIV mouse sera (green) and either anti-EBA-175 (red), anti-CLAG3.1 (red) or anti-PfAARP (red) rabbit sera. Parasitenuclei were stained with DAPI (blue). PfASP did not co-localize with microneme protein EBA-175 or rhoptry bulb protein CLAG3.1 but co-localized with rhoptry neck proteinPfAARP in segmented P. falciparum merozoites within late-stage schizonts.

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ig. 3. Localization of PfASP in segmented merozoites within late stage schizonts bontaining segmented merozoites were labelled with anti-PfASP-RIV mouse sera aegmented merozoites of late-stage P. falciparum schizonts. Scale bar = 250 nm.

Previously researchers have used apical membrane antigen-(AMA-1) as marker for micronemes to study co-localization withfASP. AMA-1 is highly expressed in schizont stage parasites andn free merozoites it is secreted and rapidly translocates over the

erozoite surface. Co-localization studies using anti-AMA-1 seras microneme marker may have yielded misleading results.

Taken together, the observations reported here suggest thatfASP is a rhoptry neck protein which is expressed at late schizonttage in segmented merozoites. Stage specific expression and local-

zation of PfASP in rhoptry neck suggest that it could be involvedn the process of erythrocyte invasion. Presence of PfASP homologsn other Plasmodium species as well as in Apicomplexans such as T.ondii and Cryptosporidium parvum, indicates that it is evolutionaryonserved and may play an important role in erythrocyte inva-

uno-electron microscopy. Ultra thin sections of late-stage P. falciparum schizontsld-conjugated anti-mouse IgG. Labelling was observed in the neck of rhoptries in

sion. Studies to identify the functional role of PfASP in erythrocyteinvasion are needed.

Acknowledgements

We thank Wandy Beatty for helping us with the immunoelec-tron microscopic studies and Shahid Jameel, ICGEB, New Delhi forproviding access to confocal microscope. We thank Osamu Kaneko,University of Nagasaki, for providing anti-CLAG3.1 antibodies.

Anand Srivastava received financial support from the Council forScientific and Industrial Research, India in the form of Junior andSenior Research fellowships. This work is partly supported by Mal-sig grant and EVIMalaR Network of Excellence grant from theEuropean Commission.

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