BIOLOGYBARTICLE SUMMARY

Protein Kinase A Dependent Phosphorylation of Apical Membrane Antigen 1 Plays an Important Role in Erythrocyte Invasion by the Malaria Parasite Kerstin Leykauf1., Moritz Treeck2.¤, Paul R. Gilson1, Thomas Nebl3, Thomas Braulke4, Alan F. Cowman3,

Tim W. Gilberger2,5*, Brendan S. Crabb1,6,7*

1 Macfarlane Burnet Institute for Medical Research & Public Health, Melbourne, Victoria, Australia, 2 Bernhard Nocht Institute for Tropical Medicine, Department of

Molecular Parasitology, Hamburg, Germany, 3 Walter & Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia, 4 Department of Biochemistry, Children’s

Hospital, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 5 M.G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton,

Ontario, Canada, 6 The University of Melbourne, Victoria, Australia, 7 Monash University, Victoria, Australia

Abstract

Apicomplexan parasites are obligate intracellular parasites that infect a variety of hosts, causing significant diseases in livestock and humans. The invasive forms of the parasites invade their host cells by gliding motility, an active process driven by parasite adhesion proteins and molecular motors. A crucial point during host cell invasion is the formation of a ring- shaped area of intimate contact between the parasite and the host known as a tight junction. As the invasive zoite propels itself into the host-cell, the junction moves down the length of the parasite. This process must be tightly regulated and signalling is likely to play a role in this event. One crucial protein for tight-junction formation is the apical membrane antigen 1 (AMA1). Here we have investigated the phosphorylation status of this key player in the invasion process in the human malaria parasite Plasmodium falciparum. We show that the cytoplasmic tail of P. falciparum AMA1 is phosphorylated at serine 610. We provide evidence that the enzyme responsible for serine 610 phosphorylation is the cAMP regulated protein kinase A (PfPKA). Importantly, mutation of AMA1 serine 610 to alanine abrogates phosphorylation of AMA1 in vivo and dramatically impedes invasion. In addition to shedding unexpected new light on AMA1 function, this work represents the first time PKA has been implicated in merozoite invasion.

Citation: Leykauf K, Treeck M, Gilson PR, Nebl T, Braulke T, et al. (2010) Protein Kinase A Dependent Phosphorylation of Apical Membrane Antigen 1 Plays an Important Role in Erythrocyte Invasion by the Malaria Parasite. PLoS Pathog 6(6): e1000941. doi:10.1371/journal.ppat.1000941

Editor: Kami Kim, Albert Einstein College of Medicine, United States of America

Received November 9, 2009; Accepted May 5, 2010; Published June 3, 2010

Copyright: � 2010 Leykauf et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by NHMRC of Australia and Deutsche Forschungsgemeinschaft grants (GRK1459 and GI312), by a grant from the NIH (RO1 AI 43906-06A1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected] (BSC); [email protected] (TWG)

. These authors contributed equally to this work.

¤ Current address: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, United States of America

Introduction

Malaria is one of the most devastating infectious diseases of

mankind and is a leading cause of morbidity and mortality in

tropical and sub-tropical regions where 40% of the world’s

population live. The most pathogenic species that infects humans

is Plasmodium falciparum and in 2002 it was estimated that out a total

of 515 million clinical cases, 2–3 million were fatal [1]. Central to

malarial pathogenesis is the large-scale invasion of red blood cells

(RBCs) by Plasmodium parasites. The invasive merozoite forms of

the parasite infect RBCs via a complex multi-step process

involving sequential receptor-ligand interactions and signal

transduction events (reviewed in [2]). Merozoite invasion is an

intense area of investigation by many groups as it is a point in the

parasite lifecycle that is particularly vulnerable to immune and

drug intervention. While signalling within the parasite, particularly

that triggered by calcium, is known to be involved in RBC

invasion, the specific nature of this process including the identity of

the key molecular players remains largely a mystery. To address

this we have been studying an essential transmembrane protein

present on the invasive merozoite surface, apical membrane

antigen 1 (AMA1). AMA1 is one of the most promising blood-

stage malaria vaccine candidates and is amongst the best studied of

the ,5000 Plasmodium proteins.

In P. falciparum, AMA1 is synthesised during merozoite

development towards the end of the blood stage cell cycle and is

stored in apical secretory organelles called micronemes [3,4]. It is

a type I integral membrane protein of 83 kDa with a large N-

terminal ectodomain, a single transmembrane domain near the C-

terminus and a small cytoplasmic tail of 56 amino acids

(PfAMA183) [5,6]. Before schizont rupture the N-terminal

prosequence is cleaved resulting in a 66-kDa form (PfAMA166)

that translocates from the micronemes to the merozoites surface

[7]. In a second proteolytic processing step during invasion the

bulk of the ectodomain is shed quantitatively as 44-kDa and 48-

kDa fragments by the membrane bound subtilisin-like protease

PfSUB2 leaving a 22-kDa transmembrane fragment that is taken

into the newly invaded RBC [8,9]. AMA1-specific antibodies and

peptides block invasion at a step after the long-distant primary

contacts between parasite and host cell have occurred but prior to

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the close interactions seen during tight junction formation [10–

12]. Some functional insights of antibody-based inhibition were

gained from using the monoclonal antibody 4G2 that targets an

epitope adjacent to the conserved hydrophobic trough in the

ectodomain [13–15]. This antibody binding led to the abrogation

of AMA1 interaction with rhoptry neck proteins (RONs, [14]) that

was previously established as important part of the tight junction

[16,17]. Apart from the critical interactions of the ectodomain the

cytoplasmic tail of AMA1 has also been shown to be essential for

invasion [18]. Mutational analysis hinted an important role for tail

phosphorylation in the invasion process.

In this paper we show that the PfAMA1 tail is phosphorylated at

a specific serine residue (S610) and that the enzyme responsible for

this event is the parasite-encoded protein kinase A (PfPKA).

Moreover, we show that S610 plays a crucial role for AMA1

function and parasite invasion. This phosphorylation event has

implications for understanding the regulation of invasion, for the

function of AMA1 and for the development of new therapeutic

approaches.

Results

The cytoplasmic domain of AMA1 is phosphorylated by a cAMP dependent kinase present in schizont and merozoite lysates

To investigate if the cytoplasmic domain of AMA1 is

phosphorylated by parasite kinases we generated a glutathione

S-transferase (GST) fusion protein of the AMA1 cytoplasmic tail

(Figure 1A) and performed in vitro phosphorylation assays with P.

falciparum 3D7 lysates. Autoradiography of the AMA1 tail resolved

by SDS-PAGE indicated it was specifically phosphorylated to

comparable amounts by schizont and merozoite lysates (Figure 1B).

Control reactions with non-infected RBC lysate gave only a

background signal, indicating that the AMA1 tail was phosphor-

ylated by parasite kinases rather than RBC kinases (Figure 1B). As

loading controls the membrane was probed with an anti-AMA1

antibody that specifically recognised the AMA1 tail. No signal can

be detected for the GST proteins because the antibody used was

specific for the AMA1 tail only (Figure 1).

Invasion of RBCs by Plasmodium is known to involve calcium ion

(Ca2+) fluxes [19] which might trigger AMA1 phosphorylation. To

address this, calcium ions in parasite lysates were chelated by

EGTA/EDTA before incubation with the AMA1 tail. Conversely,

to increase the calcium concentration in the assay 2mM CaCl2 was

added to the buffer. Whereas EGTA/EDTA had little effect

on the intensity of the phosphorylation signal compared to the

untreated sample (Figure 1C, left panel and Figure 1D),

the addition of Ca2+ to the buffer resulted in a 3 fold increase of

the phosphorylation signal (Figure 1C, left panel and Figure 1D).

In addition to Ca2+, cAMP is also a common second messenger

and has been shown to be involved in parasite development during

the asexual blood stages [20,21]. Thus, we tested if cAMP had an

effect on AMA1 tail phosphorylation. As shown in Figure 1C (right

panel) the addition of cAMP to the in vitro phosphorylation assay

led to a dramatic 17-fold increase of AMA1 tail phosphorylation

(Figure 1D), which indicated an involvement of the protein kinase

A (PKA) in the phosphorylation event.

The recombinant AMA1 tail is phosphorylated on residue S610

A multiple alignment of 13 AMA1 protein sequences using the

software PRALINE (www.ibi.vu.nl) showed that the C-terminal

cytoplasmic domain of apicomplexan AMA1 is highly conserved

among different Plasmodium species as well as other apicomplexans

(Toxoplasma gondii, Babesia bovis, Theileria parva, Theileria annulata)

suggesting a common function during host cell invasion.

Additionally, the AMA1 tails contain several potential phosphor-

ylation sites with six amino acids being predicted as phosphory-

lation sites in PfAMA1 (www.cbs.dtu.dk/services/NetPhos) (Y576,

Y585, S590, S610, T612, Y622, Figure 2A).

The dependence on cAMP suggested that parasite-encoded

PKA (PFI1685w), the only apparently recognisable cAMP

dependent kinase expressed at this stage of the life cycle [22],

was responsible for the observed phosphorylation. Consistent

with that, the NetPhosK program (www.cbs.dtu.dk/services/

NetPhosK) predicted that residue S610 is phosphorylated by

PKA, showing the highest score when compared to all the other

serines, threonines and tyrosines in the AMA1 tail. To establish if

the prediction of S610 phosphorylation by PfPKA was correct,

site-directed mutagenesis was performed. Firstly, a GST-fusion

protein mutant containing a stop codon at position S610 was

generated (Figure 2A) that lacks the highly conserved C-terminus

with its putative phosphorylation sites including the S610. In vitro

phosphorylation assays with 3D7 schizont lysates indicated this

mutant was not phosphorylated in the presence of cAMP

(Figure 2B), suggesting that none of the less conserved proximal

predicted phosphorylation sites Y576, Y585, S590 were involved

in this phosphorylation event or that if these sites are phosphor-

ylated their phosphorylation is dependent on the presence of serine

610 or residues downstream of S610. Although tyrosine kinases are

apparently absent in the parasite’s genome [23] two predicted

tyrosines were included in the mutagenic analyses as controls. The

band for the stop mutant is missing in the loading control since the

anti-AMA1 antibody detects a peptide C-terminally of S610stop

(Figure 2B). Secondly, to verify that S610A is responsible for

protein phosphorylation, we exchanged the remaining phosphor-

ylation sites (including S601) and subjected these mutant AMA1s

to in vitro phosphorylation. Only the stop mutant and S610 lacked

a phosphorylation signal suggesting that S610 is either the only

residue being phosphorylated under the given conditions or S610

Author Summary

The invasion of host cells by zoites of the phylum apicomplexa is an active event that is powered by the parasite invasion machinery. It can be divided in several distinct steps that involve binding to the host cell, reorientation and tight junction formation that are accompanied by sequential secretion of specialised organelles that store proteins involved in these events. A great number of proteins are now known to be involved in invasion but how the invasion process is regulated remains obscure. Recently, phosphorylation of some proteins with a defined function in invasion like GAP45, MTIP and AMA1 were reported and provided the first insight into putative regulation mechanism of invasion. Using mutational analysis we now demonstrate that AMA1 is phosphorylat- ed in the cytoplasmic domain at serine 610 in a cAMP dependent manner and that mutation of S610 dramatically reduces the efficiency of invasion into erythrocytes. We identified protein kinase A (PfPKA) as a late transcribed kinase that is responsible for phosphorylating AMA1 at this specific residue. This work describes for the first time PKA signalling being implicated in merozoite invasion, provid- ing a new avenue for understanding the initiation and regulation of invasion. Significantly also, the PKA-AMA1 pathway defines a promising new and validated drug target for therapeutic intervention.

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phosphorylation enables the phosphorylation of other sites in the

AMA1 tail (Figure 2B).

PfPKA phosphorylates AMA1 S610 To confirm the involvement of PfPKA as the phosphorylating

kinase, two PKA inhibitors, H89 and KT 5720, were tested for

their ability to block phosphorylation of the AMA1 cytoplasmic

domain. These compounds are competitive antagonists of ATP’s

access to a binding pocket on the catalytic subunit of PKA and

H89 has been used in Plasmodium parasites previously where it

appears to affect the cell cycle and arrest proliferation during

schizogany [20,21]. To determine biologically relevant concen-

trations of PKA inhibitors to use in the in vitro phosphorylation

assays, the growth inhibitory effects of the drugs were measured in

live cultures. These indicated that the IC90 for H89 and KT 5720

was about 50 mM and 10 mM, respectively (data not shown). In the

in vitro phosphorylation assays, both compounds completely

blocked the stimulatory effect of cAMP upon the phosphorylation

of the recombinant AMA1 tail indicating potent inhibition of PKA

stimulation (Figure 2C and 2D). In the absence of additional

cAMP, H89 and KT 5720 decreased AMA1 tail phosphorylation

by 70% and 55% respectively in in vitro phosphorylation assays

using 3D7 schizont lysates (Figure 2C and 2E). Although we can’t

completely rule out that the inhibition of cAMP stimulated kinase

activity is due to methanol, the solvent of KT5720, rather than

KT5720 itself, it is very unlikely for two reasons: firstly, both

inhibitors show similar inhibitions, even if only KT5720 is

dissolved in methanol, whereas H89 is dissolved in H2O.

Secondly, methanol per se had no effect on the outcome of the

in vitro phosphorylation assay in the absence of additional cAMP as

shown in Figure 2C and 2E. Although H89 and KT5720 have

been used extensively in animal cell PKA studies for many years

some doubts about the specificity of these compounds for PKA,

especially at the used concentrations, remain and they may have

effects on other kinases such as PKB [24]. We can therefore not be

entirely sure that the background (unstimulated) level of AMA1

tail phosphorylation is completely due to PKA since other kinases

may also be inhibited.

To further validate PfPKA as an AMA1 phosphorylating kinase,

we over-expressed HA-tagged PfPKA catalytic subunit in late

Figure 1. Recombinant AMA1 C-terminal tail is phosphorylated in vitro by P. falciparum (3D7 line) parasite lysates in a calcium and cAMP dependent manner. (A) Schematic representation of AMA1 and the GST-fusion protein used. Signal peptide (blue), prosequence (PS), ectodomains I, II & III, transmembrane domain (grey), cytoplasmic tail (C) and thrombin cleavage site are indicated. (B, C) Auto-radiographs showing phosphorylation of recombinant AMA1 tail by parasite lysates in the presence of 1.5 mM EGTA/1 mM EDTA or 2 mM CaCl2 or 1 mM cAMP. The AMA1 tail was incubated with schizont (S), merozoite (M) or red blood cell (RBC) lysates and 32[P]-c -ATP. After washing the GST part was cleaved off with thrombin. As a loading control the membrane was probed with an anti-AMA1 antibody detecting the AMA1 tail. Molecular sizes are indicated on the left. (D) Quantitation of signal intensities in panel C with Image Gauge software. In the absence of additional EGTA/EDTA or cAMP the strength of the phosphorylation signal in untreated schizont lysate was set to 100% and all other signals are relative to that. The numbers of experimental replicates in in vitro phosphorylation assays are found in the Supplementary data (Text S1). Error bars correspond to standard deviation. doi:10.1371/journal.ppat.1000941.g001

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Figure 2. PfPKA phosphorylates the AMA1 tail exclusively on residue S610. (A) Multiple alignment of AMA1 C-terminal cytoplasmic domains including protein sequences of nine different Plasmodium species, T. gondii, B. bovis and two Theileria species. The conservation is scored and colour coded by PRALINE (www.ibi.vu.nl). Amino acids predicted to be phosphorylated in P. falciparum AMA1 by NetPhos (www.cbs.dtu.dk/ services/NetPhos) are scaled up in font size according to their relative predicted probabilities above the alignment and numbered regarding the protein sequence. As shown below the alignment the GST-fusion protein of the AMA1 tail was mutated at residues S601, S610, T612, T613, Y621 and Y622, respectively. (B, C) SDS-PAGE and radiograph of phosphorylation samples. GST-fusion proteins of the AMA1 tail and mutants were incubated with schizont lysate and 32[P]- c -ATP. As a loading control the membrane was probed with an anti-AMA1-C antibody. (C) H89 (50 mM) and KT5720 (10 mM), two inhibitors to PKA reverse the effect of cAMP (1 mM) on AMA1 tail phosphorylation. (D, E) Quantitation of signal intensities in (C) with Image Gauge software. The phosphorylation signal strength in untreated schizont lysate was set to 100% and all other signals were relative to that. The numbers of experimental replicates in in vitro phosphorylation assays are found in the Supplementary data (Text S1). Error bars correspond to standard deviation. (F) PfPKA-HA was immuno-precipitated from lysate of 3D7PfPKA-HA transgenic parasites using protein-G-sepharose. PfPKA-HA was detected by Western Blot analysis using anti-HA antibodies and parasite lysate, washed fraction or sepharose beads. (G) SDS-PAGE and radiograph of in vitro phosphorylation of AMA1-GST using either protein-G-sepharose incubated with 3D7PfPKA-HA or with wild type (WT) parasites in the presence of 32[P]- c -ATP. doi:10.1371/journal.ppat.1000941.g002

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blood stages. Transgenic expression was verified by either Western

Blotting or IFA using anti-HA antibodies (Figure S1). The

antibodies detected a double band at around 43 kDa correspond-

ing well with the theoretical molecular weight of the fusion-protein

of 42.5 kDa. The doublet might represent a phosphorylated form

of PfPKA as has been described for other PKA proteins [25]. In

vivo activation of PKA in Schizosaccharomyces pombe requires

threonine phosphorylation at its activation loop and is dependent

on PDK1 [26]. Subsequent immuno-precipitation of the HA-

tagged PfPKA from 3D7PfPKA-HA parasites allowed the purifica-

tion of the PfPKA-HA (Figure 2F). Late stage specific over-

expression of the catalytic subunit of PfPKA had no effect on

parasite growth rate (data not shown). This purified PfPKA readily

phosphorylated the recombinant AMA1 tail (Figure 2G). Taken

together, the in vitro data suggest that cAMP triggers AMA1 tail

phosphorylation on residue S610 by PfPKA.

Phosphorylation of native AMA1 and the importance of S610 for RBC invasion

To investigate the phosphorylation status of native AMA1 we

analysed mature schizont stage parasite extracts from 3D7

parental parasites by 2DE (Figure 3A-C). When analysed by

Western Blotting using antibodies that recognise the C-terminus of

AMA1, the 66 kDa AMA1 species was shown to be comprised of 5

distinct spots (termed ‘‘a-e’’ respectively with spot ‘‘a’’ being the

most negatively charged and ‘‘e’’ the least) that separated on a

basis of their isoelectric point. Three of these spots (a, c and d)

incorporated radiolabelled phosphate indicating that these spots

represent phosphorylated forms of AMA1 (Figure 3B).

To determine if S610 was phosphorylated in vivo the

phosphorylation patterns of wild type AMA1 and the non-

phosphorylatable S610A and PM (all potential sites mutated)

mutants were compared by 2DE. Because previous studies had

indicated these phosphorylation sites were essential for AMA1

function we used a complementation approach [18]. We

generated 3D7 parasites episomally expressing the W2mef allelic

form of AMA1 tagged at the C-terminus with the TY1 epitope to

distinguish it from the endogenous 3D7 AMA1 protein. Three

lines were created expressing either the W2mef wild type form

(AMA1-WT-TY1), a form with the S610A mutation (AMA1-

S610A-TY1) or another form with each potential phosphorylation

site mutated (AMA1-PM-TY1) (Figure 3D-I). Samples were

analysed by 2DE and Western Blot which were probed with

either TY1 antibodies or polyclonal AMA1-C-terminal antibodies

where indicated. It was apparent that the 66 kDa species in the

AMA1-WT-TY1 line separates into 8 visible spots (Figure 3E)

consistent with the expression of two different forms of AMA1 in

this parasite line. The W2mef TY1 tagged spots, termed a’-e’,

were generally of slightly higher molecular weight than the

untagged 3D7 species and their PIs were a little greater

(Figure 3D,E). We assume 10 spots are present in this line but

that 2 spots (b and b’) are masked by other spots or are below

detectable levels. Consistent with the masking of b’, anti-TY1

antibodies detect spots a’, c’, d’ and e’ in AMA1-WT-TY1

parasites (Figure 3D). Strikingly, the banding pattern in AMA1-

S610A-TY1 is much simpler with only 2 TY1-tagged spots

observed, b’ and e’ (Figure 3F and G). Mutation of all potential

phosphorylation sites in the AMA1 tail to alanine (including S610)

gave an identical pattern to the S610A-only mutant (Figure 3H

and I). The mutation of S610 to an alanine residue appears to

have eliminated the presence of all phosphorylated species of

AMA1. One possible scenario might be that spots a, c & d

represent 3 separate phosphorylation sites on the protein with the

modification of S610 as prerequisite for the phosphorylation of the

other sites. Alternatively, all three spots could contain phosphor-

ylated S610, but two of the three contain additional charge-

modifying posttranslational modifications depending on S610

phosphorylation. Indeed, our data suggest that there are at least

two forms that differ in charge for reasons other than

phosphorylation (b & e). This data confirms a crucial role for

S610 in the phosphorylation of AMA1.

Due to the inability to obtain perfectly synchronous parasites it is

difficult to precisely determine when S610 phosphorylation is

occurring. However, to address this issue to some extent we

prepared a time course experiment examining AMA1 species

present in schizonts, merozoites and ring-stages (Figure 4). By

Western Blotting 2DE gels with an antibody that recognises the C-

terminal tail of AMA1 we show that the 66 kDa AMA1 species (a

form that appears late in schizogony) has a more complex banding

pattern in merozoites than it does in schizonts, most notably the

appearance of a more negatively charged, apparently phosphory-

lated, species (arrowhead in Figure 4). This is in contrast to the 83

kDa precursor form of AMA1 that appears to change little in

pattern between schizonts and merozoites. While, all phosphory-

lated species of the 66 kDa fragment can be detected in some

schizont preparations (see Figure 3A-C for example), the time-

course data shown in Figure 4 suggests that the bulk of secondary

modifications of the AMA1 tail, including phosphorylation, occurs

very late in blood stage development, perhaps even in the short-

lived merozoites themselves. Interestingly, the cleaved C-terminal

form of AMA1 in newly invaded ring-stage parasites reveals a much

simpler pattern of secondary modifications, which could point

towards - among other possibilities - a de-phosphorylation event of

S610 during this stage (Figure 4). We caution that precise knowledge

about the timing of AMA1 phosphorylation and its potential de-

phosphorylation will require the generation of new reagents, most

probably a functional S610 phospho-specific antibody.

We next tested the functional consequences of S610A mutation

in vivo. To do this we used a similar complementation approach as

previously described [18,27]. Briefly, full length AMA1 bearing a

C-terminal TY1 epitope tag for immuno-detection was ectopically

expressed in the 3D7 parasite line under the AMA1 promoter

(Figure 5A). To test the invasion capability of phosphorylation

defective mutants, a S610A mutation (AMA1-S610A-TY1) or

mutations of all six predicted phosphorylation sites in the AMA1

tail to alanines (AMA1-PM-TY1) were introduced by site-directed

mutagenesis (Figure 5B). Importantly, all the AMA1-TY1

proteins, wild type and mutants, were derived from the W2mef

parasite strain. In this strain the AMA1 protein bears crucial

amino acid changes that makes it resistant to the invasion blocking

effects of the R1 peptide that binds to the 3D7 AMA1 protein

[28]. This strain specific binding blocks RBC invasion by 3D7

parasites though it does not prevent initial interaction steps

between the merozoite and RBC [18]. All chimeric proteins were

correctly expressed as TY1 fusion proteins (Figure 5C, left panel).

Proteolytic cleavage of the HA-fusion was indistinguishable from

the endogenous protein as shown with the 3D7 specific

monoclonal AMA1 antibody 1F9 [29] (Figure 5C, right panel).

As previously reported, over-expression of AMA1-WT-TY1

(derived from W2mef) functionally complements the endogenous

AMA1 (,78.5% (+/2 5.1% s.d.) invasion) while both mutants

AMA1-S610A-TY1 and AMA1-PM-TY1 revealed a drastically

decreased invasion capability in the presence of R1 peptide

(,21.2% (+/25.9% s.d.) and ,20.3% (+/2 5.2% s.d.) invasion,

respectively) (Figure 5D). In comparison, invasion is blocked up to

,96% (+/2 1.3% s.d.) in the parental 3D7 parasite line with the

R1 peptide. This comparable failure of the PM and the S610A

mutant to complement AMA1 function demonstrates an impor-

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tant role for AMA1 residue S610 and, together with the above in

vitro data, strongly implicates PKA-mediated phosphorylation of

S610 as a vital step in promoting efficient invasion. Direct

comparison of the temporal aspect of invasion blockade induced

by either R1 peptide binding or S610A mutation using video-

microscopy revealed no apparent differences. Both parasites are

arrested after the re-orientation of the merozoite on the surface of

the host cell (Video S1, Figure S2). In summary, this data

demonstrates that the cytoplasmic tail of AMA1 is phosphorylated

by parasite-encoded PKA, and that this is crucial to AMA1

function during the invasion. The vital role of S610 was further

investigated by targeting two other single amino acids that are

predicted and could be putatively involved in phosphorylation of

AMA1: Y576 and Y585. The substitution of these amino acids

with alanine and its subsequent ectopic expression (AMA1-

Y576A-TY1 and AMA1-Y585A-TY1) did not impair their

function in complementation assays (Figure 5D). Additionally,

we have tried to mimic the phosphorylation state of AMA1 by

Figure 3. Two dimensional gel analyses confirm that P. falciparum AMA1 is phosphorylated at S610 in vivo. P. falciparum proteins were metabolically labelled with 32[P], detergent extracted and resulting 2DE blots developed using Deep Purple stain to visualize total protein (A). Autoradiography detected in vivo phosphorylated proteins (B), and Western Blotting with an anti-AMA1 antibody detected the AMA1 tail (C). Isoelectric point and molecular sizes are indicated on the top and left. 2D spots corresponding to the 83 kDa precursor (AMA183) or post- translationally processed ,66 kDa AMA1 fragment (AMA66) are highlighted by bounding boxes, respectively. Magnification of the AMA166 region reveals a series of five discrete 32[P]-labelled (a, c, d) or -unlabelled (b, e) protein spots recognized by anti-AMA1 antibody (arrows). (D-I) all show ,66 kDa species of AMA1 separated by 2DE and analysed by Western Blot. 3D7 parasites either expressed a transgenic TY1-tagged wild type W2mef AMA1 (AMA1-WT-TY1; D, E), or mutant forms where the W2mef transgene carried the AMA1-S610A mutation (AMA1-S610A-TY1; F, G) or mutations at all putative phosphorylation sites in the cytoplasmic tail AMA1-PM-TY1; H, I). Blots were probed with a mouse monoclonal antibody against TY1 to detect transgenic epitope-tagged AMA1 followed and by an anti-AMA1 antibody against the AMA1 tail to detect both endogenously expressed 3D7 AMA1 as well as the W2mef transgenic species. doi:10.1371/journal.ppat.1000941.g003

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expressing AMA1-S610E-TY1 and AMA1-S610D-TY1 mutants

in the presence of R1 peptide, however they failed to complement

the invasion efficiency of the AMA1-WT-TY1 allele and

performed little better than the non-phosphorylatable S610A

allele (Figure 5D). This could be because the negatively charged

amino acids fail to fully substitute the biological activity of a

phosphate group or because they cannot lose their charge through

the action of phosphatases. The latter seems a distinct possibility

since the 2D pattern of AMA1 spots in ring-stage parasites is

greatly simplified compared to merozoites suggesting the activity

of phosphatases.

Discussion

Merozoite invasion of RBCs is a rapid yet complex multi-step

process. It can be broadly separated into a ,11 sec pre-invasion

stage which is characterised by long-distance interactions and

extensive deformation of the host cell and a ,17 second invasion

step where the parasite has formed a moving tight junction [30].

The pre-invasion stage is particularly poorly understood both in

terms of the receptor-ligand interactions involved and in the

communication events that link the subsequent stages of invasion.

However, it appears likely that during the pre-invasion stage a

signalling cascade occurs to promote subsequent invasion steps,

such as apical organelle secretion and activation of the actin-

myosin motor. Most notably in this regard calcium dependent

kinase 1 (CDPK1) has been implicated to be involved in

phosphorylating motor components [31].

In the present work, we have shown that the major

transmembrane protein known to be present on the surface of

free merozoites AMA1, appears to be phosphorylated at a specific

residue, S610, in its cytoplasmic tail. We show that this occurs

both in in vitro assays using recombinant AMA1 with parasite

extracts and also in vivo in whole parasites where S610 appears to

be a dominant site of phosphorylation. Given that we observed 3

phosphorylated isoforms of AMA1 it remains possible that another

site(s) of the AMA1 tail is/are also phosphorylated. However, if

this is the case, these sites must be subsequent to and dependent of

serine 610 phosphorylation as all isoforms are absent when this

residue is mutated. Several lines of evidence indicate that the

enzyme responsible for this event is parasite-encoded PfPKA, an

enzyme previously unknown to be involved in invasion. Firstly, in

vitro S610 is phosphorylated strongly in a cAMP dependent

manner and PfPKA is the only known cAMP dependent kinase

expressed in the blood stages of P. falciparum. In fact, PfPKA

transcription is co-regulated with that of AMA1 late in the blood-

stage cycle and S610 is strongly predicted by publicly available

software to constitute a PKA site. Secondly, two different PKA

inhibitors H89 and KT5720 block this phosphorylation in vitro.

Thirdly, we show that immuno-precipitated PfPKA phosphory-

lates the tail of AMA1 in vitro. Finally we use a complementation

approach to show that S610 is important for efficient red blood

cell invasion. In this experiment, mutant parasites that express an

AMA1 with an alanine in this position are unable to efficiently

invade host cells. Taken together, we conclude that PfPKA

mediated phosphorylation of AMA1 at S610 is vital to its

functioning and hence to the invasion process.

So what is the role of this process in invasion and what can we

learn from host cell entry at other parasite stages? With respect to

the latter, PfPKA is also expressed in the hepatocyte invasive

sporozoite stages and indeed PKA has been implicated in invasion

via genetic deletion of P. berghei adenylyl cyclase alpha (ACa; [32]).

ACa is responsible for rapidly generating cAMP from ATP in

response to a stimulus, in this case uracil derivates [32]. The

membrane associated ACa is not expressed in blood-stages

however another adenylyl cyclase, ACb, is expressed during the

blood-stages [33]. In fact, the gene encoding ACb is co-transcribed

with genes encoding PKA and AMA1 late in the blood-stage cycle.

Hence, ACb is a strong candidate to produce the cAMP required

to activate PKA during invasion. Experimental confirmation of

this is required but if true the identification of the activation signals

of ACb should shed considerable light on the primary trigger for

merozoite invasion of red blood cells.

It will be interesting to discover how the phosphorylation of

AMA1 renders the merozoites competent to invade. Phosphory-

lation does not appear to influence AMA1 trafficking since

expression of a tail deletion mutant in transgenic parasites does not

appear to effect localisation [18]. Previous experiments have

indicated that the interaction of AMA1 with RON proteins is

essential for tight junction formation [14,34]. Does phosphoryla-

tion of the AMA1 interfere with RON/AMA1 complex

formation? This appears unlikely as AMA1 without the cytoplas-

mic tail (like wild type AMA1) remains capable of interacting with

RON4 [18]. It remains to be determined whether the phosphor-

ylation of the AMA1 tail by PKA has a direct effect on an AMA1

binding function or whether this event is a key step in signal

transduction pathway. It is interesting to note that the AMA1-

S610A-TY1 mutant invades at 20% of AMA1-WT-TY1 levels in

Figure 4. Maturation of phosphorylated AMA1 species. Schizont, merozoite and ring-stage parental 3D7 parasites were analysed from a sequential time-course experiment by two dimensional gel electrophoresis and Western Blotting. Blots were probed with C-terminal AMA1 tail antibodies. Bands representing 83 kDa, 66 kDa and 22 kDa AMA1 species as well as presumed cross-reacting contaminating species (cont) are indicated on the right. Isoelectric points and molecular weight markers are indicated on the top and left of the autoradiographs, respectively. doi:10.1371/journal.ppat.1000941.g004

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the presence of R1 peptide whereas in the absence of a

complementing W2mef protein the 3D7 parasites only invade

with ,4% efficiency. This indicates that there is not an absolute

need for S610 phosphorylation for every successful invasion or that

there is some cross-talk between the native 3D7 protein whose tail

can presumably still be phosphorylated and the W2mef AMA1-

S610A-TY1 mutant that cannot be phosphorylated but whose

ectodomains probably remain functional.

Figure 5. The Pf PKA phosphorylated residue S610 is required for efficient merozoite invasion. (A) Schematic representation of the ectopically expressed TY1-tagged AMA1. Signal peptide (blue), prosequence (PS), ectodomains I, II & III, transmembrane domain (grey), cytoplasmic tail (C) and TY1-tag (red) are indicated. (B) Mutations introduced into the cytoplasmic tail of W2mef-derived AMA1 are shown in red colour. (C) Expression of W2mef-derived AMA1-TY1 and native 3D7 AMA1 detected by Western Blot analysis using an anti-TY1 and anti-AMA1 (1F9) antibody, respectively (C, left panel). Whereas no AMA1-TY1 protein can be detected in 3D7 wild type parasites, a double band corresponding to AMA183-TY1 and processed AMA166-TY1 can be detected in AMA1-TY1-expressing parasites. (C, right panel) The endogenous 3D7 AMA1 is recognised by the anti- AMA1-1F9 antibody and shows identical proteolytic forms of AMA1 (AMA183 and AMA166). (D) Invasion inhibition assays using AMA1-TY1 expressing parasite strains. Assays were performed in the presence of 100 mg/mL R1 peptide and were performed in triplicate in three independent experiments. Error bars correspond to standard deviation. 3D7 and AMA1-WT-TY1 served as control. doi:10.1371/journal.ppat.1000941.g005

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It is interesting to consider the likelihood of a link between cAMP

signalling and calcium signalling during merozoite invasion. Both the

Ono et al. study in sporozoites [32] and earlier work in P. falciparum

blood-stages [20] have demonstrated an inter-relationship between

these two pathways and indeed in higher order eukaryotes this

relationship is now well established (reviewed in [35]). How these are

involved in invasion by merozoites and sporozoites is unclear

although it appears likely that cAMP signalling operates upstream

of intracellular calcium release [20,32].

In summary, this study opens up a new area of investigation for

those interested in understanding P. falciparum merozoite invasion.

Knowledge of the molecular events that trigger PKA signalling as

well as those that follow as a consequence of AMA1 phosphory-

lation will be essential to a full understanding of merozoite invasion.

Materials and Methods

Ethics statement The culture of malaria parasites using donated blood and serum

from the Australian Red Cross Society has been approved by The

Walter and Eliza Hall Institute Human Research Ethics

Committee and by the Bernhard Nocht Institute. The use of this

material follows long-standing protocols and has not been

associated with any adverse or other unforeseen events and no

data of relevance to specific patients has been generated. A Supply

Agreement has been executed between the Australian Red Cross

Society and the Walter and Eliza Hall Institute and between the

Australian Red Cross Society and the Burnet Institute covering the

provision of blood and blood products for non-clinical use.

In vitro phosphorylation assay Magnetic cell-sorted P. falciparum 3D7 parasites were cultured in

the absence of RBCs until about half of the schizonts had ruptured

to release their merozoites. A schizont enriched fraction was

prepared by centrifuging the parasite culture at 15006g for 5 min

to pellet the schizonts. To harvest the merozoites, the supernatant

was then spun at 30006g for 15 mins and enrichment process was

checked by microscopy of Giemsa-stained smears of the schizonts

and merozoites. Schizonts were then released from host cells by

saponin lysis. To make parasite lysates with kinase activity, the

parasite pellets were lysed in 10 volumes of ice cold buffer B (50

mM b-glycerophosphate pH 7.3, 1% Triton X-100, 1 mM DTT,

Complete Protease Inhibitor Cocktail [Roche] and phosphatase

inhibitors, 50 mM Na3VO4 and 50 mM NaF,) and incubated on a

rotating wheel overnight at 4uC. Control extracts were made from

similar amounts of whole uninfected red blood cells under

identical conditions. To clear the lysates they were centrifuged

at 160006g and the protein concentration of the supernatant was

determined by BCA protein assay (Pierce). 3 mg total protein lysate

(or 1 mL of immuno-precipitated PfPKA-HA-sepharose) was

mixed with ,10 mg of the GST or GST-AMA1-tail fusion

proteins immobilized on glutathione-sepharose beads. To the

beads buffer B containing different compounds was added to a

final volume of 100 mL. The compounds used were 1.5 mM

EGTA/1 mM EDTA, 2 mM CaCl2, 1 mM cAMP, 10 mM KT

5720, 50 mM H89 and 1% methanol for a control. The

concentration of stock solutions, solvents and replicate numbers

are indicated in the supplementary data (Text S1). The reaction

was initiated by the addition of 3 mL ATP mixture composed of

100 mL 1 M MgCl2 and 150 mCi 32[P]ATP (10 mCi/mL). After 30

minutes at 30uC, the immobilised fusion proteins were pelleted

and washed in buffer B. The AMA1-tail was removed from the

beads as whole GST-AMA1-tail fusion protein with 10 mM

glutathione in PBS or the tail only was cleaved from the GST with

thrombin. Eluted proteins were immediately suspended in SDS gel

sample buffer and were boiled for 5 min, resolved on a 4–12%

gradient SDS-PAGE gel, blotted onto a PVDF membrane.

Imaging of 32[P]-labelled protein bands was achieved by direct

autoradiography (1–2 day exposure) of dry blots using FUJIFILM

BAS-TR2040 tritium imaging plates and a FLA-3000 lumino-

metric detection system.

PfPKA-HA immunoprecipitation PKA-HA was immunoprecipitated using anti-HA antibodies

(5 mg/mL) and protein-G-sepharose (Invitrogen). 20 mL of a 10%

synchronized schizont culture (PfPKA-HA expressing and wild

type parasite strain) were saponin lysed. The resulting parasite

pellet was washed in icecold PBS and hypotonically lysed in 10

volumes of ice-cold buffer B without detergent by several passages

through a needle and 2 hours incubation on a rotating wheel at

4uC. The membrane fraction was separated from the soluble

proteins by centrifugation at 13.0006g for 30 minutes. The

resulting supernatant was precleared with protein-G-sepharose

beads. The precleared lysate was incubated with anti-HA

antibodies with a final concentration of 5 mg/mL for 3 hours.

20 mL of protein-G-sepharose was used to precipitate the PKA-

HA antibody complexes. It was washed 5x with 10 volumes of cold

PBS and subsequently stored in buffer B at 280uC until used. For

each experiment, 3 mL of PKA-HA coupled beads were used.

Erythrocyte invasion assays Parasite erythrocyte invasion assays were performed using 3D7

and transgenic parasites AMA1-WT-TY1, AMA1-S610A-TY1

and AMA1-PM-TY1 (3D7 background). Parasitemia of sorbitol

synchronized parasite culture was measured using the FACS. For

the experiment a parasite culture with 0.5–1% parasitemia of late

trophozoites (4% hematocrit) was incubated in a 96-well Plate

(100 mL per well) under standard culturing conditions for 48 hours

to allow re-infection in the presence or absence (control) of

100 mg/mL R1. After reinvasion occurred, parasites were stained

with 1 mg/mL ethidium bromide for 30 minutes at 37uC, washed

three times with media and then counted using the FACS. Assays

were performed in triplicates on three independent occasions.

Metabolic labelling of phosphorylated P. falciparum protein

Metabolic labelling of phosphorylated parasite protein was

achieved by incubating ,161010 sorbitol-synchronized P. falci-

parum 3D7 parasites with 100 mCi/mL of 32[P]-monosodium

phosphate (Perkin-Elmer) in 50 mL phosphate-free RPMI medi-

um (Gibco) supplemented with 25 mM HEPES (pH 7.2), 0.5%

(w/v) Albumax, 0.4% (w/v) glucose, 0.2% (w/v) Na2HCO3 at

37uC overnight. Late blood-stage parasites were released from host

cells by saponin lysis, extensively washed in TBS, and parasite

pellets extracted in 10 volumes of HNET (25 mM HEPES,

pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100) for one

hour with vortexing. Detergent-soluble schizont extract was

clarified by ultracentrifugation at 75,0006g for 30 mins at 4uC.

All procedures were carried out in the presence of protease and

phosphatase inhibitors on ice, unless otherwise stated. 32[P]-

labelled parasite extracts were snap-frozen in liquid nitrogen and

stored at -80uC until required.

2D gel electrophoresis (2DE) Two-dimensional gel electrophoresis was performed using

conditions required for optimal extraction and separation of P.

falciparum-infected erythrocyte proteins [36]. Frozen parasite

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PLoS Pathogens | www.plospathogens.org 9 June 2010 | Volume 6 | Issue 6 | e1000941

extracts were processed using 2-D Clean-Up Kit (GE Healthcare).

The resulting precipitates (,100 mg protein) were redissolved in

300 mL 2-DE sample buffer (7 M urea, 2 M thiourea, 2% ASB-14,

1% DTT, 1% ampholytes), loaded onto 13 cm pI 4–7 IPG strips

by passive re-hydration for 12 hours, and focussed using a fast

voltage gradient (8000V max, 24,000 Vh) at 15uC, using an Ettan

IPGphor 3 system (GE Healthcare). The second dimension was

carried out on 7.5% polyacrylamide gels using a Hoefer SE 600

system (GE Healthcare) at 75V overnight.

Protein staining, autoradiography and Western analyses of 2DE blots

2-DE gels were electrophoretically transferred onto Immobilon-

PSQ PVDF membranes (Millipore) in Towbin’s transfer buffer

containing 20% methanol and 0.01% SDS. Complete transfer of

total protein was confirmed using Deep Purple protein stain (GE

Healthcare). Imaging of 32[P]-labelled parasite phospho-protein

spots was achieved by direct autoradiography of dry blots using

FUJIFILM BAS-TR2040 tritium imaging plates and a FLA-3000

luminometric detection system (14 day exposure). 2DE Western

blot analyses of protein extracts of 3D7 parasites expressing wild

type and/or transgenic AMA1-WT-TY1 or AMA1-S610A-TY1

was carried out as detailed in the figure legend.

Nucleic acids and DNA constructs The ama1 gene was either amplified from 3D7 or W2mef P.

falciparum gDNA (Table S1). For the generation of GST fusion

proteins the DNA sequence of the PfAMA1 C-terminal tail was

cloned into BamHI and EcoRI restriction sites of the bacterial

expression vector pGEX-4T-3 (Pharmacia Biotech). This con-

struct produces fusion proteins of PfAMA1 tail C-terminally bound

to glutathione S-transferase (GST). Different mutants of the GST-

PfAMA1 tail fusion protein were achieved by using a site-directed

mutagenesis kit (Stratagene) and sequences were confirmed by

sequencing (Table S1).

For transfecting 3D7 parasites ama1 was cloned into the KpnI

and AvrII restriction sites of the pARL-AMA1-GFP Vector and

sequences were confirmed by sequencing. To ensure correct

timing of transcription, expression of the AMA1 transgenes were

controlled by the AMA1 promoter. In vitro mutagenesis of ama1

was achieved by using a two-step primer directed PCR

mutagenesis method (Table S1) with proof reading Vent

polymerase (NEB).

Parasites strains and transfection P. falciparum asexual stages were cultured in human 0+

erythrocytes according to standard procedures. W2mef is derived

from the Indochina III/CDC strain. 3D7 parasites were

transfected with 100 mg of purified plasmid DNA. Positive

selection for transfectants was achieved using 10 nM WR99210,

an antifolate that selects for the presence of the human dhfr gene.

For further and more detailed information see Text S1.

Supporting Information

Figure S1 (A) Western Blot of ectopically expressed PfPKA-HA

using anti-HA antibodies. Whereas in wild type parasite material

(WT) no fusion protein was detectable two protein bands with the

predicted size of approximately 43 kDa were visualized in the

transgenic parasite line 3D7PfPKA-HA. (B) Immuno-fluorescence

images of PfPKA-HA expressing parasites 3D7PfPKA-HA

revealed cytosolic distribution (green) using anti-HA antibodies.

Blue: DAPI stained nucleus.

Found at: doi:10.1371/journal.ppat.1000941.s001 (0.14 MB

DOC)

Figure S2 M1 video file depicted in time-lapse micrographs.

After schizont rupture (t = 20s) free merozoites attack an

erythrocyte (black arrows (t = 28s). Around 40 seconds after

primary contact (t = 63s) the erythrocyte looses its shape -

culminating in a spiked round structure with at least three

merozoites apically attached to it (red arrows).

Found at: doi:10.1371/journal.ppat.1000941.s002 (0.22 MB

DOC)

Table S1 Oligonucleotides used in this study.

Found at: doi:10.1371/journal.ppat.1000941.s003 (0.05 MB

DOC)

Text S1 Supplemental materials and methods.

Found at: doi:10.1371/journal.ppat.1000941.s004 (0.06 MB

DOC)

Video S1 Video-microscopy of attempted invasion event of

AMA1-S610A-TY1 expressing parasites in the presence of the R1

peptide. Although the merozoites are able to attack and induce

echinocytosis in the erythrocyte, invasion is blocked.

Found at: doi:10.1371/journal.ppat.1000941.s005 (1.3 MB MPG)

Acknowledgments

We thank the Australian Red Cross Blood Bank for the provision of human

blood and serum. We thank Mick Foley for providing the mAB 1F9 and

Jacobus Pharmaceuticals for providing WR99210.

Author Contributions

Conceived and designed the experiments: KL MT PRG TN TB AFC

TWG BSC. Performed the experiments: KL MT TN. Analyzed the data:

KL MT PRG TN TB TWG BSC. Contributed reagents/materials/

analysis tools: MT TB AFC TWG BSC. Wrote the paper: KL PRG TN

TWG BSC.

References

1. Snow RW, Guerra CA, Noor AM, Myint HY, Hay SI (2005) The global distribution of clinical episodes of Plasmodium falciparum malaria. Nature 434:

214–217.

2. Cowman AF, Crabb BS (2006) Invasion of red blood cells by malaria parasites. Cell 124: 755–766.

3. Bannister L, Mitchell G (2003) The ins, outs and roundabouts of malaria. Trends Parasitol 19: 209–213.

4. Healer J, Crawford S, Ralph S, McFadden G, Cowman AF (2002) Independent translocation of two micronemal proteins in developing Plasmodium falciparum

merozoites. Infect Immun 70: 5751–5758.

5. Hodder A, Crewther P, Matthew M, Reid G, Moritz R, et al. (1996) The disulfide bond structure of Plasmodium apical membrane antigen-1. J Biol Chem

271: 29446–29452. 6. Peterson MG, Marshall VM, Smythe JA, Crewther PE, Lew A, et al. (1989)

Integral membrane protein located in the apical complex of Plasmodium

falciparum. Mol Cell Biol 9: 3151–3154.

7. Narum DL, Thomas AW (1994) Differential localization of full-length and processed forms of PF83/AMA-1 an apical membrane antigen of Plasmodium

falciparum merozoites. Mol Biochem Parasitol 67: 59–68.

8. Harris PK, Yeoh S, Dluzewski AR, O’Donnell RA, Withers-Martinez C, et al. (2005) Molecular identification of a malaria merozoite surface sheddase. PLoS

Pathog 1: 241–251. 9. Howell SA, Well I, Fleck SL, Kettleborough C, Collins CR, et al. (2003) A single

malaria merozoite serine protease mediates shedding of multiple surface proteins by juxtamembrane cleavage. J Biol Chem 278: 23890–23898.

10. Mital J, Meissner M, Soldati D, Ward GE (2005) Conditional expression of

Toxoplasma gondii apical membrane antigen-1 (TgAMA1) demonstrates that TgAMA1 plays a critical role in host cell invasion. Mol Biol Cell 16: 4341–4349.

11. Mitchell GH, Thomas AW, Margos G, Dluzewski AR, Bannister LH (2004) Apical membrane antigen 1, a major malaria vaccine candidate, mediates the

close attachment of invasive merozoites to host red blood cells. Infect Immun 72:

154–158.

A Role for PKA in Merozoite Invasion

PLoS Pathogens | www.plospathogens.org 10 June 2010 | Volume 6 | Issue 6 | e1000941

12. Urquiza M, Suarez JE, Cardenas C, Lopez R, Puentes A, et al. (2000) Plasmodium

falciparum AMA-1 erythrocyte binding peptides implicate AMA-1 as erythrocyte binding protein. Vaccine 19: 508–513.

13. Bai T, Becker M, Gupta A, Strike P, Murphy VJ, et al. (2005) Structure of

AMA1 from Plasmodium falciparum reveals a clustering of polymorphisms that surround a conserved hydrophobic pocket. Proc Natl Acad Sci U S A 102:

12736–12741. 14. Collins CR, Withers-Martinez C, Hackett F, Blackman MJ (2009) An inhibitory

antibody blocks interactions between components of the malarial invasion

machinery. PLoS Pathog 5: e1000273. 15. Kocken CH, van der Wel AM, Dubbeld MA, Narum DL, van de Rijke FM,

et al. (1998) Precise timing of expression of a Plasmodium falciparum-derived transgene in Plasmodium berghei is a critical determinant of subsequent subcellular

localization. J Biol Chem 273: 15119–15124. 16. Alexander DL, Arastu-Kapur S, Dubremetz JF, Boothroyd JC (2006)

Plasmodium falciparum AMA1 binds a rhoptry neck protein homologous to

TgRON4, a component of the moving junction in Toxoplasma gondii. Eukaryot Cell 5: 1169–1173.

17. Alexander DL, Mital J, Ward GE, Bradley P, Boothroyd JC (2005) Identification of the moving junction complex of Toxoplasma gondii: a collaboration between

distinct secretory organelles. PLoS Pathog 1: e17.

18. Treeck M, Zacherl S, Herrmann S, Cabrera A, Kono M, et al. (2009) Functional analysis of the leading malaria vaccine candidate AMA-1 reveals an essential

role for the cytoplasmic domain in the invasion process. PLoS Pathog 5: e1000322.

19. Nagamune K, Moreno SN, Chini EN, Sibley LD (2008) Calcium regulation and signaling in apicomplexan parasites. Subcell Biochem 47: 70–81.

20. Beraldo FH, Almeida FM, da Silva AM, Garcia CR (2005) Cyclic AMP and

calcium interplay as second messengers in melatonin-dependent regulation of Plasmodium falciparum cell cycle. J Cell Biol 170: 551–557.

21. Syin C, Parzy D, Traincard F, Boccaccio I, Joshi MB, et al. (2001) The H89 cAMP-dependent protein kinase inhibitor blocks Plasmodium falciparum develop-

ment in infected erythrocytes. Eur J Biochem 268: 4842–4849.

22. Bozdech Z, Zhu J, Joachimiak MP, Cohen FE, Pulliam B, et al. (2003) Expression profiling of the schizont and trophozoite stages of Plasmodium

falciparum with a long-oligonucleotide microarray. Genome Biol 4: R9. 23. Ward P, Equinet L, Packer J, Doerig C (2004) Protein kinases of the human

malaria parasite Plasmodium falciparum: the kinome of a divergent eukaryote. BMC Genomics 5: 79.

24. Murray AJ (2008) Pharmacological PKA inhibition: all may not be what it

seems. Sci Signal 1: re4.

25. Steinberg RA, Cauthron RD, Symcox MM, Shuntoh H (1993) Autoactivation of

catalytic (C alpha) subunit of cyclic AMP-dependent protein kinase by

phosphorylation of threonine 197. Mol Cell Biol 13: 2332–2341.

26. Tang Y, McLeod M (2004) In vivo activation of protein kinase A in

Schizosaccharomyces pombe requires threonine phosphorylation at its activation

loop and is dependent on PDK1. Genetics 168: 1843–1853.

27. Healer J, Triglia T, Hodder AN, Gemmill AW, Cowman AF (2005) Functional

analysis of Plasmodium falciparum apical membrane antigen 1 utilizing interspecies

domains. Infect Immun 73: 2444–2451.

28. Harris KS, Casey JL, Coley AM, Masciantonio R, Sabo JK, et al. (2005) Binding

hot spot for invasion inhibitory molecules on Plasmodium falciparum apical

membrane antigen 1. Infect Immun 73: 6981–6989.

29. Coley AM, Parisi K, Masciantonio R, Hoeck J, Casey JL, et al. (2006) The most

polymorphic residue on Plasmodium falciparum apical membrane antigen 1

determines binding of an invasion-inhibitory antibody. Infect Immun 74:

2628–2636.

30. Gilson PR, Crabb BS (2009) Morphology and kinetics of the three distinct

phases of red blood cell invasion by Plasmodium falciparum merozoites.

Int J Parasitol 39: 91–96.

31. Green JL, Rees-Channer RR, Howell SA, Martin SR, Knuepfer E, et al. (2008)

The motor complex of Plasmodium falciparum: phosphorylation by a calcium-

dependent protein kinase. J Biol Chem 283: 30980–30989.

32. Ono T, Cabrita-Santos L, Leitao R, Bettiol E, Purcell LA, et al. (2008) Adenylyl

cyclase alpha and cAMP signaling mediate Plasmodium sporozoite apical

regulated exocytosis and hepatocyte infection. PLoS Pathog 4: e1000008.

33. Baker DA (2004) Adenylyl and guanylyl cyclases from the malaria parasite

Plasmodium falciparum. IUBMB Life 56: 535–540.

34. Besteiro S, Michelin A, Poncet J, Dubremetz JF, Lebrun M (2009) Export of a

Toxoplasma gondii rhoptry neck protein complex at the host cell membrane to

form the moving junction during invasion. PLoS Pathog 5: e1000309.

35. Borodinsky LN, Spitzer NC (2006) Second messenger pas de deux: the

coordinated dance between calcium and cAMP. Sci STKE 2006: pe22.

36. Rabilloud T, Blisnick T, Heller M, Luche S, Aebersold R, et al. (1999) Analysis

of membrane proteins by two-dimensional electrophoresis: comparison of the

proteins extracted from normal or Plasmodium falciparum-infected erythrocyte

ghosts. Electrophoresis 20: 3603–3610.

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PLoS Pathogens | www.plospathogens.org 11 June 2010 | Volume 6 | Issue 6 | e1000941