IAI FigSearch
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Healer, J.
Right arrow Articles by Cowman, A. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Healer, J.
Right arrow Articles by Cowman, A. F.

 Previous Article  |  Next Article 

Infection and Immunity, April 2005, p. 2444-2451, Vol. 73, No. 4
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.4.2444-2451.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Functional Analysis of Plasmodium falciparum Apical Membrane Antigen 1 Utilizing Interspecies Domains

Julie Healer,1,2 Tony Triglia,1 Anthony N. Hodder,1 Alan W. Gemmill,3 and Alan F. Cowman1*

Walter and Eliza Hall Institute,1 Department of Medicine, University of Melbourne, Melbourne,2 Department of Clinical Psychology, Heidelberg Repatriation Hospital, Heidelberg, Victoria, Australia3

Received 27 April 2004/ Returned for modification 15 June 2004/ Accepted 21 December 2004


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Plasmodium falciparum apical membrane antigen 1 (AMA1) is a leading malaria vaccine candidate whose function has not been unequivocally defined. Partial complementation of function can be achieved by exchanging the AMA1 of P. falciparum (PfAMA1) with that of P. chabaudi (PcAMA1). In this study, parasites expressing chimeric AMA1 proteins were created to identify domains of PfAMA1 critical in erythrocyte invasion and which are important immune targets. We report that specific chimeric AMA1 proteins containing domains I to III from PfAMA1 and PcAMA1 were able to complement PfAMA1 function in erythrocyte invasion. We demonstrate that domain III does not contain dominant epitope targets of antibodies raised against Escherichia coli expressed and refolded PfAMA1 ectodomain. Furthermore, we generated a parasite line in which the N-terminal pro region of PfAMA1 does not undergo proteolytic cleavage and show that its removal is necessary for PfAMA1 function.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The invasion of erythrocytes by Plasmodium parasites is a prerequisite in the pathogenic blood-stage cycle of malaria. Interruption of this event by immune or chemotherapeutic means is a major focus designed to reduce the mortality and morbidity caused by malaria. Many of the putative or known invasion-related molecules are located on the merozoite surface prior to schizont rupture or are initially sequestered in specialized secretory organelles at the apical end of the merozoite and translocate onto the merozoite surface probably following a signaling event (2). Apical membrane antigen 1 (AMA1) is a low-abundance type I integral membrane protein synthesized in the mature blood stages (26) as an 83-kDa nascent polypeptide, which accumulates in the micronemes of developing merozoites (1, 11). Following N-terminal proteolytic cleavage, the mature form of the protein translocates to the merozoite surface (5, 25). Although merozoites are extracellular for only a short time, surface antigens are subject to immune attack and antibodies against AMA1 block merozoite invasion of erythrocytes (3, 6, 7, 14, 18-20, 24, 32, 33).

Unique among invasion-related proteins thus far identified, AMA1 has a homologue in another non-Plasmodium species, Toxoplasma gondii, indicating that it plays a fundamental role in the invasion process of all apicomplexan parasites (8, 13). Targeted gene disruption of AMA1 has been unsuccessful in both Plasmodium (36) and Toxoplasma (13), substantiating the essential role of AMA1 in parasite survival. Alignment of AMA1 from multiple Plasmodium species and Toxoplasma reveals conservation of all 16 cysteine residues and a considerable degree of sequence homology. Secondary-structure predictions have defined a conserved three-domain structure constrained by intradomain disulfide pairings (15), and transspecies complementation studies have determined functional conservation of AMA1 between P. falciparum and P. chabaudi (36).

Despite its low abundance, AMA1 is a highly immunogenic protein. Most individuals exposed to malaria develop anti-AMA1 antibodies after relatively few exposures (30). It has been calculated that 1% of the total immunoglobulin G in Papua New Guineans living with endemic malaria is against AMA1 (R. Anders, personal communication). Across P. falciparum strains, around 10% of the 622 amino acids are polymorphic, and diversity is thought to be crucial for evading neutralizing antibodies (4, 10, 12, 28). Since polymorphisms are found throughout the ectodomain of the molecule, it is difficult to predict exactly where protective epitopes are located, although a single monoclonal antibody is reported to display invasion inhibitory activity across different strains of P. falciparum as well as P. reichenowi (19). The target of this monoclonal antibody is thought to be a conformational epitope formed by domains I and II (17). More recently, monoclonal antibodies with specificity for P. falciparum AMA1 (PfAMA1) domain III were also shown to inhibit parasite invasion of erythrocytes, although the strain specificity of these was not tested (23).

Preceding domain I is an amino-terminal prosequence, which is longer in P. falciparum and the closely related P. reichenowi than in other plasmodia (19). N-terminal sequencing of PfAMA1 proteolytic fragments has specifically identified the cleavage site between the pro region and domain I (17). This sequence motif is conserved in P. reichenowi and all P. falciparum strains but is not present in other species, so the functional role of AMA1 N-terminal processing is unclear. We have attempted to assess the functional significance of different domains of AMA1 by constructing a series of transgenic parasites expressing chimeric AMA1 proteins composed of P. falciparum and P. chabaudi AMA1 (PcAMA1) domains. First, we confirm that domains I and II are important targets of polyclonal inhibitory antibodies. We also demonstrate that cleavage of the N-terminal PfAMA1 pro region is necessary for AMA1 function.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Parasites and transfection. P. falciparum clone D10, derived from FC27, an isolate from Papua New Guinea, was used. Parent and transfectant parasite lines were cultivated in vitro (34), synchronized (22) according to standard procedures and used in all assays described below. Parasites were transfected as described (37) with 100 µg of plasmid DNA and cultured for 48 h prior to selection with 5 nM WR99210. Parasites were cultured for 21 to 30 days with drug selection before numbers were high enough to be detected by light microscopy. Parasites were then cultured for 3 to 4 weeks in the absence of drug selection, followed by reselection on drug to select for homologous integration of the transfected plasmid.

Plasmid construction. The transfection plasmid for expression of AMA1 proteins has been described previously (36) but modified by replacement of the Toxoplasma gondii dhfr gene with the mutated human dhfr gene, allowing selection with the drug WR99210. Briefly, plasmid PP1-pGem, containing the AMA1 5' region, was digested with EcoRI and BamHI to release a 2.4 kb fragment containing the T. gondii dhfr gene. The insert containing the human dhfr gene was digested from the HH1 plasmid described previously (29) and inserted to generate plasmid pHAM. The chimeric PfAMA1 and PcAMA1 genes were constructed by PCR amplification of D10 (PfAMA1) and DS (PcAMA1) genomic DNA, respectively, using the conserved restriction enzyme sites NcoI (between the pro region and domain 1) and PacI (between domains 2 and 3) that allowed the domains to be shuffled. The full PfAMA1 and PcAMA1 genes were bounded by XhoI sites so that the chimeric genes could be subcloned into the pHAM vector such that the gene was placed 3' of the PfAMA1 promoter.

Nucleic acids and sequencing. Genomic DNA was extracted from parasites as described previously (35). Southern blotting was performed using standard procedures.

Briefly, transfectant parasite genomic DNAs were prepared from parasites following three cycles of selection on WR99210 to allow homologous recombination into the PfAMA1 5' untranslated region. These were subjected to digestion with restriction endonucleases EcoR1 or NsiI, transferred onto Hybond-N membranes (Amersham Pharmacia), and probed with the AMA1 fragments described in Fig. 2 to confirm that integration had occurred in the correct genomic locus. All plasmids were sequenced through the entire AMA1 gene prior to transfection, to confirm that there were no mutations.



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 2. Integration of plasmids into the PfAMA1 promoter region. A, Schematic representation of integration of plasmids PcAMA1, CHI1, and CHI2 into the AMA1 promoter region of P. falciparum. The plasmid, endogenous PfAMA1 gene, and integration event following homologous recombination via single crossover are shown. EcoRI (R) restriction sites are marked, and the position of the probe used for Southern blot analysis is indicated (-). Arrows indicate predicted sizes of bands expected by Southern blot analysis of parasite genomic DNA. B, Southern hybridization of EcoRI-digested genomic DNA from D10, D10-CHI 1, D10-PcAMA1, and D10-CHI2 parasites showing integration into the PfAMA1 locus. Filters were hybridized with a 700-bp probe from PfAMA1 5' region. The 2.5-kb band represents the endogenous PfAMA1 gene, which has been disrupted in all transgenic parasite lines; 4.1-kb and 7.4- to 7.6-kb bands represent integration of the plasmid via single recombination into PfAMA1 promoter region; 9.1-kb (D10-CHI1 and -CHI2) and 8.8-kb (D10-PcAMA1) bands indicate that two copies of plasmid have integrated into the PfAMA1 locus. C, Schematic representation of integration of plasmid CHI3 into the AMA1 locus of P. falciparum. The plasmid, endogenous PfAMA1 gene, and integration event following homologous recombination via single crossover are shown. NsiI (N) restriction sites are marked, and the position of the probe used for Southern blot analysis is indicated (-). Arrows indicate predicted band sizes expected by Southern blot analysis of parasite genomic DNA. D, Southern hybridization of NsiI-digested genomic DNA from D10 and D10-CHI3 parasite lines. A 700-bp probe from PfAMA1 domain 1 was used. The 5-kb band in the D10 track represents the PfAMA1 endogenous gene, which is absent from D10-CHI3. The 3.6-kb and 5.5-kb bands indicate homologous integration into the PfAMA1 promoter region, and the 4.1-kb band indicates that two copies of the plasmid have integrated into the PfAMA1 locus.

 
Antisera. Rabbit anti-PfAMA1 sera were generated against a high-pressure liquid chromatography-purified refolded fusion protein comprising amino acids 25 to 546 of the 3D7 AMA1 sequence (excludes the signal peptide, short N-terminal segment, and the transmembrane and cytoplasmic tail regions) with an N-terminal hexa-His tag. Antisera were purified on a Protein G-Sepharose column (Pharmacia), dialyzed against phosphate-buffered saline, diluted to 5 mg/ml, and sterilized by filtration through a 0.22-µM filter (Millipore). Mouse monoclonal antibodies with specificity for PcAMA1 were generated and screened for specificity using refolded domains of this protein (Tony Hodder, unpublished data). The monoclonal antibodies 2D3 and 4A12 were specific to domain 1, 3H9 was specific for domain 2, and 4G3 was specific for domain 3.

Immunoblot analysis. Proteins were extracted from late schizont preparations and separated on sodium dodecyl sulfate-8% polyacrylamide gel electrophoresis under nonreducing conditions and transferred to nitrocellulose membranes (Schleicher & Schuell). These filters were probed with either immunoglobulin G purified from polyclonal rabbit serum or monoclonal antibody supernatant followed by a goat anti-mouse or goat anti-rabbit HRP secondary antibody (Silenus) and developed with an enhanced chemiluminescence (ECL) kit (Amersham Pharmacia Biotech).

Immunofluorescence. Smears were fixed in methanol and rehydrated in phosphate-buffered saline prior to indirect immunofluorescence. Protein G-purified anti-AMA1 antibodies were used at 1:1,000. Monoclonal antibody supernatants were used at a 1:2 dilution in 1% fetal calf serum-phosphate-buffered saline.

Invasion-inhibition assays. Assays followed a protocol described previously (12). Briefly, parasites were synchronized twice with 5% sorbitol at 4-h intervals and then grown to trophozoite stage. Hematocrits and parasitemia were adjusted to 2% and 0.5%, respectively. Hypoxanthine-free RPMI with 10% human serum was used for assays. Parasites were cultured with antibody diluted to 0.5 mg/ml in phosphate-buffered saline or phosphate-buffered saline only in duplicate wells of 96-well flat-bottomed plates. 3H-labeled hypoxanthine (Amersham Pharmacia), 1 µCi/well, was added to each well diluted in hypoxanthine-free RPMI medium as above, and plates were incubated for a further 18 h. Parasites were then freeze-thawed prior to harvesting onto glass fiber filters and quantitated using a scintillation counter. Percentage invasion inhibition was calculated as [(mean cpm of control wells – mean cpm of test wells)/(mean cpm of control wells)]. Data generated were derived from six independent experiments.

Statistical analysis. Inhibition data were first tested for departure from normality using quantile-quantile plots followed by Kolmogorov-Smirnov tests for goodness-of-fit (31). Data were then subjected to one-way analysis of variance with parasite line and antibody fitted as a between subject factor, followed by sets of multiple pairwise comparisons using Fisher's least significant difference procedure to control the experimentwise type I error rate.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Chimeric AMA1 proteins are expressed in P. falciparum. Partial complementation of PfAMA1 function has been achieved by expression of P. chabaudi AMA1 in P. falciparum (36). To analyze the functional roles of the different AMA1 domains and their susceptibility to neutralizing antibodies, we constructed a series of transgenic P. falciparum lines expressing chimeric molecules consisting of AMA1 domains from P. chabaudi and P. falciparum (Fig. 1). The parasite D10-PcAMA1 was transfected with a construct that encoded the full PcAMA1 gene, whereas D10-CHI1 would express a chimeric AMA1 with the P. falciparum pro region followed by the coding region for the three PcAMA1 domains. D10-CHI2 was transfected with a plasmid with a chimeric AMA1 gene consisting of the PfAMA1 pro region and domain 3, while domains 1 and 2 were from PcAMA1. D10-CHI3 was transfected with a plasmid containing the PfAMA1 pro region and the coding region for domains 1 and 2, while domain 3 originated from PcAMA1. In parasites transfected with these constructs, the AMA1 chimeric genes would be transcribed under control of the PfAMA1 promoter.



View larger version (27K):
[in this window]
[in a new window]
  		FIG. 1. Schematic structure of AMA1 transgenes inserted into transfection constructs. NcoI and PacI: conserved restriction sites which divide the pro domain from domain 1 and domain 2 from domain 3 are shown. The solid arrow shows the site of N-terminal processing which results in a 66-kDa protein from the 83-kDa precursor form. Full-length PcAMA1 (DS strain) was used to generate parasite line D10-PcAMA1. CHI1 was synthesized by joining PfAMA1 pro domain to PcAMA1 domains 1, 2, and 3 transmembrane and cytoplasmic tail sequence. CHI2 comprised PfAMA1 pro-domain fused to PcAMA1 domains 1 and 2 followed by PfAMA1 domain 3 transmembrane and cytoplasmic tail sequence. CHI3 was synthesized with PfAMA1 pro-domain and domains 1 and 2 fused to PcAMA1 domain 3 transmembrane and cytoplasmic tail regions.

 
Following parasite culture, genomic DNA of transfected and parental wild-type parasite lines was obtained. Southern blotting of restriction enzyme digested genomic DNA revealed that, as in the previous study (36), the chimeric transgenes had integrated into the endogenous AMA1 locus via single crossover within the 5' untranslated region inserting the plasmid and reconstituting the endogenous PfAMA1 gene downstream of the crossover event (Fig. 2A and C). This would allow expression of the inserted transgene as well as the intact endogenous PfAMA1. Southern blotting of EcoRI-digested genomic DNA from D10, D10-CHI1, and D10-CHI2 (Fig. 2B) revealed that the 2.5 kb band, indicating endogenous PfAMA1 in the parental D10 strain, was absent from the tranfected parasite lines D10-CHI1, D10-PcAMA1, and D10-CHI2, and replaced with band sizes corresponding to an integration event in the 5'UTR of PfAMA1. Likewise, in NsiI-digested D10 and D10-CHI3 (Fig. 2D), the 5-kb band indicative of endogenous PfAMA1 seen in D10 disappeared in D10-CHI3, and is replaced with 3.6- and 5.5-kb bands corresponding to a similar integration event. In addition, Southern blots indicated that at least two copies of the plasmid were integrated into the PfAMA1 locus (CHI1/2 9.1 kb, PcAMA1 8.8 kb, and CHI3 4.1 kb).

To detect expression of the PcAMA1 domains in different parasite lines, Western blotting of schizont extracts was performed with polyclonal antibodies against refolded PfAMA1 ectodomain and monoclonal antibodies specific to domains of PcAMA1. This confirmed that both endogenous and transgenic AMA1 proteins were expressed correctly (Fig. 3). D10-PcAMA1 and D10-CHI1 parasites expressed both PfAMA1 and PcAMA1 domains 1 to 3 (Fig. 3A and B), whereas D10-CHI2 parasites expressed PfAMA1 and only PcAMA1 domains 1 and 2 (Fig. 3C).



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 3. Expression of chimeric Pf/PcAMA1 proteins in P. falciparum. Immunoblots of saponin-lysed schizonts of transfectant parasite lines expressing both full-length PfAMA1 and PcAMA1. Proteins were fractionated under nonreducing conditions on an 8% polyacrylamide gel and electroblotted onto nitrocellulose membrane. Nitrocellulose strips were incubated with a panel of antibodies: 1, rabbit polyclonal anti-PfAMA1(refolded 3D7 ectodomain); 2, monoclonal antibody 2D3 anti-PcAMA1 domain 1; 3, monoclonal antibody 3H9 anti-PcAMA1 domain 2, 4, monoclonal antibody 4G3 anti-PcAMA1 domain 3. A, Parasite line D10-PcAMA1; B, parasite line D10-CHI 1; C, parasite line D10-CHI 2.

 
Proteolytic processing of the N-terminal pro region of PfAMA1 is defective in one chimeric AMA1 line. Two forms of chimeric AMA1 were observed in Western blots of late schizont stage parasite lines D10-CHI1 and D10-CHI2, corresponding to full-length (83 kDa) and mature (66 kDa) forms of AMA1 (Fig. 3B and C). This indicated that normal proteolytic processing of the N-terminal pro region was occurring in these lines. PcAMA1 does not contain the N-terminal extension found in PfAMA1 and is detectable as a 68-kDa protein. In transgenic line D10-PcAMA1, only one isoform, a doublet of approximately 68 kDa, was detected, and no observable processing was expected since this AMA1 molecule was constructed with the PcAMA1 N-terminal sequence (Fig. 3A), however the presence of a visible doublet may indicate processing of a short N-terminal fragment.

In line D10-CHI3, the chimeric AMA1 molecule consisted of the N-terminal region of PfAMA1 up to the end of domain 2, fused to domain 3 from PcAMA1. Only the 83-kDa protein was detectable with a PcAMA1 domain 3-specific monoclonal antibody (4G3), indicating that cleavage of the N-terminal pro region of the chimeric protein had not occurred (Fig. 4A). Monoclonal antibody 4G3 can recognize PcAMA1 domain 3 as part of an N-terminally processed AMA1 fragment, as D10-CHI1 AMA1 is detectable as both unprocessed (83 kDa) and processed (66 kDa) forms in these Westerns (Fig. 4A). Samples of the same schizont preparations were run simultaneously and probed with a polyclonal anti-PfAMA1 antibody. This confirmed that endogenous PfAMA1 was present and processed normally in all parasite lines (Fig. 3 and 4A).



View larger version (43K):
[in this window]
[in a new window]
  		FIG. 4. Expression and processing of Pf/PcAMA1 chimeric proteins in P. falciparum. A, Immunoblots of saponin-lysed schizonts of parasite lines probed with rabbit polyclonal anti-PfAMA1 (panel 1) and monoclonal antibody 4G3 specific for PcAMA1 domain 3 (panel 2). D10, parent P. falciparum line; CHI3, comprising PfAMA1 with PcAMA1 domain 3 transmembrane and cytoplasmic tail regions; CHI1, comprising PfAMA1 N-terminal pro-domain with domains 1, 2, and 3, transmembrane and cytoplasmic tail of PcAMA1. B, Western blot showing time course of late trophozoite-schizont stages of two parasite lines expressing chimeric AMA1 proteins, detected with monoclonal antibody 4G3, specific to domain 3 of PcAMA1. Parasites were sampled at 28, 32, and 36 h following double sorbitol lysis of ring stage parasites. Approximately equal numbers of parasites were loaded for each time point shown. CHI3 is not proteolytically processed.

 
To rule out the possibility that schizont preparations used in this experiment were taken before proteolysis of CHI3 AMA1 could occur, schizonts were prepared for Western blotting from tightly synchronized cultures at 4-h intervals from 28 h postinvasion. As found in the previous experiment, only the unprocessed form of CHI3 AMA1 was detectable in contrast to D10-CHI1 parasites that showed normal processing (Fig. 4B).

Defective proteolytic processing of chimeric AMA1 results in abnormal subcellular localization. To examine whether chimeric forms of AMA1 were correctly localized, fluorescence imaging microscopy was performed on late stage schizonts and free merozoites. Using monoclonal antibody 4G3, which is specific to domain 3 of PcAMA1, the chimeric protein appeared to localize normally within D10-CHI1 and -CHI3 schizonts, to the micronemes within the apical region of the developing merozoites (Fig. 5A). However, in contrast to D10-CHI1, where PcAMA1 domain 3 is clearly visible on the surface of free merozoites, no chimeric AMA1 protein was detectable on the surface of D10-CHI3 parasites (Fig. 5B). Free merozoites of the other transgenic parasite lines (D10-CHI2 and -PcAMA1) showed normal apical and surface localization of chimeric AMA1 when visualized with fluorescence imaging microscopy using monoclonal antibody 4A12, which is specific to PcAMA1 domain 1 (Fig. 5C).



View larger version (29K):
[in this window]
[in a new window]
  		FIG. 5. N-terminal processing of PfAMA1 pro region is required for correct subcellular localization of AMA1. Immunofluorescence analysis of the subcellular localization of PcAMA1 in transfectant parasite lines D10-CHI3 and D10-CHI1 in methanol-fixed schizonts and free merozoites. A. Micronemal staining in schizonts of bothD10-CHI3 and -CHI1 transgenic parasite lines using PcAMA1 domain 3-specific monoclonal antibody 4G3. B. Transgenic AMA1 does not appear on the surface of free merozoites of D10-CHI3, but is normally distributed in D10-CHI1 parasites. C. Chimeric AMA1 is correctly localized in free merozoites of parasite lines D10-CHI2 and PcAMA1. Micronemal and surface localization of transgenic AMA1 was detected by monoclonal antibody 4A12 which is specific to domain 1 of PcAMA1. Lower images show bright field for comparison.

 
N-terminal proteolytic cleavage of AMA1 is a prerequisite For function. In order to analyze whether the chimeric AMA1 proteins expressed by these parasite lines were able to function in merozoite invasion of erythrocytes, it was necessary to functionally inhibit the endogenous PfAMA1 protein. Since neither a genetic knockout of PfAMA1 via targeted gene disruption nor allelic substitution of PfAMA1 for PcAMA1 was possible, we used the same approach that had been successful in a previous study (36), which was to test the ability of chimeric lines to undergo erythrocyte invasion in the presence of PfAMA1 antibodies which blocked invasion of the D10 parental line (Fig. 6).



View larger version (10K):
[in this window]
[in a new window]
  		FIG. 6. Function of chimeric AMA1 proteins in P. falciparum invasion of erythrocytes. Data represent the ability of parasites expressing chimeric AMA1 proteins to invade erythrocytes in the presence of antibodies that inhibit invasion via endogenous PfAMA1. Parasites were incubated with either anti-PfAMA1 antibodies (0.5 mg of protein G-purified immunoglobulin). Controls were parasites grown in the absence of purified immunoglobulin G. All data shown are growth relative to controls. Asterisks denote which parasite lines invaded at levels significantly higher than parental D10 parasites (P < 0.05). Error bars represent 95% confidence intervals of the mean of six experiments with each parasite line.

 
As found previously, D10 parasites expressing the full-length PcAMA1 transgene were able to invade human erythrocytes in the presence of anti-PfAMA1 antibodies which inhibited wild-type D10 invasion (P < 0.05). No significant differences were found in the invasion rates between parasite lines in the absence of antibody. Extrapolation of the difference in invasion efficiency corresponded to a functional complementation of 32%, a level consistent with the previous study. Two of the chimeric AMA1 proteins, in lines D10-CHI1 and -CHI2, were able to functionally complement PfAMA1 in merozoite invasion of erythrocytes (P < 0.05), albeit slightly less efficiently than PcAMA1 (although statistically, invasion of D10-PcAMA1, -CHI1, and -CHI2 were not significantly different from each other). This result indicates that antibodies that are inhibitory to D10 parasites are targeting epitopes within domains 1 and 2 of PfAMA1, which are not present in the CHI1 and CHI2 chimeric proteins. D10-CHI3 parasites were just as susceptible to the inhibitory effect of the anti-PfAMA1 antibodies as wild type D10 parasites, indicating that the chimeric AMA1 protein in which PcAMA1 domain III was substituted for PfAMA1 domain III was not functional in these parasites.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results show that distinct domains derived from the AMA1 of the distantly related Plasmodium species P. chabaudi can partially complement PfAMA1 function in merozoite invasion of human erythrocytes when expressed as fusion proteins with PfAMA1. Since targeted gene disruption and allelic replacement of PfAMA1 was not possible, antibodies that targeted the parental D10 strain AMA1 protein were used to functionally inactivate the endogenous PfAMA1 protein (36). This strategy was designed to reveal the location of inhibitory epitopes in the PfAMA1 molecule in a domain-specific manner. A more sophisticated approach allowing the dissection of antibody-mediated inhibition from nonfunctional chimeric proteins is the inducible knockout or knockdown of endogenous PfAMA1 in these parasite lines, however, this technology is not applicable to P. falciparum at this time.

Despite these limitations, the data presented here demonstrate that domains I and II of PfAMA1 contain important epitopes of neutralizing antibodies. Parasites which expressed either full-length PcAMA1 (D10-PcAMA1), a chimeric protein composed of the N-terminal pro region of PfAMA1 followed by the PcAMA1 sequence including transmembrane and cytoplasmic tail (D10-CHI1), or a chimeric protein composed of the N-terminal pro region of PfAMA1 followed by the PcAMA1 sequence for domains I and II, followed by PfAMA1 domain III, transmembrane and cytoplasmic tail regions (D10-CHI2; see Fig. 1 for transgene structure) were able to invade erythrocytes to a significantly higher degree than parental D10 parasites in the presence of growth-inhibitory anti-PfAMA1 antibodies.

The level of complementation achieved by PcAMA1 in this study reflects a real species-specific difference in the ability of PcAMA1 to mediate invasion into human erythrocytes and not an antigenic cross-reactivity in the susceptibility of PcAMA1 to anti-PfAMA1 antibodies. The reasoning behind this is that very little cross-protection is seen between diverse strains of P. falciparum such as 3D7 and FVO (18) and W2mef (12) in in vitro inhibition of invasion studies, and therefore virtually no cross-protection would be expected between two divergent species. This result confirms predictions based on multiallele comparisons that domain I is a major target of protective immunity (4, 27, 28) and is consistent with the reported epitope location of inhibitory monoclonal antibody 4G2 (17).

The finding that neutralizing epitopes are formed by domains I and II is interesting in the light of the results of our previous study, which found that a highly polymorphic region of domain I from W2mef expressed as part of a chimera with 3D7 AMA1 was not sufficient to confer resistance to a 3D7-specific inhibitory response or sensitivity to a W2mef-specific inhibitory response (12). Unfortunately, due to the processing defect of the chimera expressing PfAMA1 domains 1 and 2, it was impossible to demonstrate direct targeting of these epitopes by neutralizing antibodies in this study. However, results of the present study indicate that a more extensive area of domain I/II, encompassing and extending that included in the previous W2mef/3D7 chimera, forms the dominant epitopes of inhibitory antibodies. This result is consistent with a recently published study in which refolded domains I and II induced growth-inhibitory antibodies (21).

Since the parasites expressing a chimeric protein which included PfAMA1 domain III were relatively resistant to the inhibitory antibodies, we conclude that domain III in this context is not a major target of these inhibitory antibodies. Two studies have been published which specifically demonstrate the importance of domain III epitopes in protective immunity (23, 24). Our finding that parasites expressing an AMA1 chimera containing PfAMA1 domain III were relatively resistant to the inhibitory effects of the neutralizing antibody appears to contradict the assertion that domain III contains immunologically important residues. It is possible that antibody titer is crucial in the case of domain III targeting. Antibodies against domain III in the purified immunoglobulin G preparation used in this study have been estimated to comprise not more than 10% of the total reactivity (T. Hodder, unpublished observation). In the domain III studies mentioned, one used refolded domain III to affinity purify human antibodies (24) whereas the other used monoclonal antibodies (23) and thus both probably deployed a much higher titer per epitope than in the present study.

Our results demonstrate that even in the absence of a high-titer anti-domain III response, anti-AMA1 antibodies raised against the refolded ectodomain still confer protection against merozoite invasion of erythrocytes, at least in a strain-specific manner. This is important when considering the use of this particular preparation as an immunogen in human vaccine trials if domain I is as immunodominant in humans as it appears to be in rabbits. Further studies investigating human responses to AMA1 immunogens and the strain-specific nature of anti-AMA1 responses are urgently required.

The addition of the PfAMA1 pro region sequence onto PcAMA1 did not enhance the level of complementation of this protein in P. falciparum, and we conclude from a comparison of the performance of transgenic lines with and without this sequence that it is not necessary for AMA1 function in the D10 line. However, where the PfAMA1 pro region is present, its removal is necessary for AMA1 function, as the AMA1 chimera in which the pro region was not cleaved was unable to function in P. falciparum. Two alternative explanations for this finding are (i) that the processing defect results in retention of this chimeric molecule in the micronemes and (ii) that translocation from the micronemes occurs normally but proteolysis on the merozoite surface (as described for the C-terminal cleavage event (16) is prohibited. In the latter case, the protein would be subject to inhibition by antibodies, which possibly even act to inhibit this cleavage. By immunofluorescence, it was clear that no transgenic protein was detectable on the surface of CHI3 merozoites, in contrast to the other transgenic lines where chimeric AMA1 was clearly present.

From these data, and by virtue of the observation that none of these parasites showed any growth defect in normal culture, it appears that D10-CHI3 parasites only express the endogenous PfAMA1 on the merozoite surface and thus are subject to antibody inhibition in the invasion assay. Unfortunately, it was not possible to conclusively determine whether the processing defect affected the forward trafficking of this molecule from the micronemes to the merozoite surface. Previous studies have established that only N-terminally processed PfAMA1 is present on the merozoite surface (5, 25). Two studies have identified a micronemal location for PfAMA1 (1, 11) and immunofluorescence imaging studies using antibodies against AMA1 and EBA-175 found only the full-length protein containing the pro region to be present within the micronemes (11). The results of the present study are consistent with these findings, as defective N-terminal processing appears to result in the retention of chimeric AMA1 in the micronemes with no obvious merozoite surface localization, although electron microscopy would be required to prove this.

The nonprocessed (CHI3) chimeric AMA1 was sequenced through the cleavage site and no mutations were found. It is therefore pure conjecture as to what has caused this phenotype in the chimeric protein generated in this study. It was only observed in the molecule where domains I/II from P. falciparum-derived sequence were fused to PcAMA1 domain III; in the reciprocal chimeric line (CHI2, Pc domains I and II followed by Pf domain III) normal processing occurs, as it also does normally in the D10-CHI1 parasite line (composed of PcAMA1 domains I to III). One possibility is that misfolding of distal sites within the full-length protein makes the cleavage site inaccessible to the protease.

Despite much effort, the three-dimensional structure of AMA1 has not yet been solved, and no information exists on interactions between the different domains. Our data indicate that interactions are occurring between regions that are noncontiguous in sequence and have major implications for the structure-function relationship in AMA1. The importance of processing within domain III has been established in two studies, the first of which identified the cleavage site within PfAMA1 allowing its release from the merozoite surface (16), and second, determined that this processing was central to invasion success, since antibodies which inhibit invasion also inhibit this processing event (9). Although not directly addressed in this study, these findings are indicative that a C-terminal processing event is essential for the normal function of AMA1. It would be interesting to map the epitope targets of that inhibitory serum with respect to the domain structure of PfAMA1 to elucidate whether direct binding of the cleavage site was responsible for inhibition of processing or antibodies binding to another region caused structural change leading to inaccessibility of the protease.


   ACKNOWLEDGMENTS
 
We thank Robin Anders for provision of polyclonal and monoclonal antibodies against PfAMA1 and PcAMA1 and the Red Cross Blood Service (Melbourne, Australia) for erythrocytes and serum.

This work was supported by the NHMRC of Australia and the Wellcome Trust. J.H. was funded by a Fellowship from the Wellcome Trust, and A.F.C. is a Howard Hughes International Scholar.


   FOOTNOTES
 
* Corresponding author. Mailing address: 1G Royal Parade, Melbourne 3050, Australia. Phone: 61-3-9345 2555. Facsimile: 61-3-9347 0852. E-mail: cowman{at}wehi.edu.au. Back

Editor: J. F. Urban, Jr.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
1. Bannister, L. H., J. M. Hopkins, A. R. Dluzewski, G. Margos, I. T. Williams, M. J. Blackman, C. H. Kocken, A. W. Thomas, and G. H. Mitchell. 2003. Plasmodium falciparum apical membrane antigen 1 (PfAMA-1) is translocated within micronemes along subpellicular microtubules during merozoite development. J. Cell Sci. 116:3825-3834.[Abstract/Free Full Text]
2. Barnwell, J. W., and M. R. Galinski. 1998. Invasion of vertebrate cells: erythrocytes, p. 93-120. In I. W. Sherman (ed.), Malaria: parasite biology, pathogenesis and protection. ASM Press, Washington, D.C.
3. Collins, W. E., D. Pye, P. E. Crewther, K. L. Vandenberg, G. G. Galland, A. J. Sulzer, D. J. Kemp, S. J. Edwards, R. L. Coppel, J. S. Sullivan, C. L. Morris, and R. F. Anders. 1994. Protective immunity induced in squirrel monkeys with recombinant apical membrane antigen-1 of Plasmodium fragile. Am. J. Trop. Med. Hyg. 51:711-719.
4. Cortes, A., M. Mellombo, I. Mueller, A. Benet, J. C. Reeder, and R. F. Anders. 2003. Geographical structure of diversity and differences between symptomatic and asymptomatic infections for Plasmodium falciparum vaccine candidate AMA1. Infect. Immun. 71:1416-1426.[Abstract/Free Full Text]
5. Crewther, P. E., J. G. Culvenor, A. Silva, J. A. Cooper, and R. F. Anders. 1990. Plasmodium falciparum: Two antigens of similar size are located in different compartments of the rhoptry. Exp. Parasitol. 70:193-206.[CrossRef][Medline]
6. Crewther, P. E., M. L. S. M. Matthew, R. H. Flegg, and R. F. Anders. 1996. Protective immune responses to apical membrane antigen 1 of Plasmodium chabaudi involve recognition of strain-specific epitopes. Infect. Immun. 64:3310-3317.[Abstract]
7. Deans, J. A., T. Alderson, A. W. Thomas, G. H. Mitchell, E. S. Lennox, and S. Cohen. 1982. Rat monoclonal antibodies which inhibit the in vitro multiplication of Plasmodium knowlesi. Clin. Exp. Immunol. 49:297-309.[Medline]
8. Donahue, C. G., V. B. Carruthers, S. D. Gilk, and G. E. Ward. 2000. The Toxoplasma homolog of Plasmodium apical membrane antigen-1 (AMA-1) is a microneme protein secreted in response to elevated intracellular calcium levels. Mol. Biochem. Parasitol. 111:15-30.[CrossRef][Medline]
9. Dutta, S., J. D. Haynes, J. K. Moch, A. Barbosa, and D. E. Lanar. 2003. Invasion-inhibitory antibodies inhibit proteolytic processing of apical membrane antigen 1 of Plasmodium falciparum merozoites. Proc. Natl. Acad. Sci. USA 100:12295-12300.[Abstract/Free Full Text]
10. Escalante, A. A., H. M. Grebert, S. C. Chaiyaroj, M. Magris, S. Biswas, B. L. Nahlen, and A. A. Lal. 2001. Polymorphism in the gene encoding the apical membrane antigen-1 (AMA-1) of Plasmodium falciparum. X. Asembo Bay Cohort Project. Mol. Biochem. Parasitol. 113:279-287.[CrossRef][Medline]
11. Healer, J., S. Crawford, S. Ralph, G. McFadden, and A. F. Cowman. 2002. Independent translocation of two micronemal proteins in developing Plasmodium falciparum merozoites. Infect. Immun. 70:5751-5758.[Abstract/Free Full Text]
12. Healer, J., V. Murphy, R. Masciantonio, A. N. Hodder, A. W. Gemmill, R. Anders, A. F. Cowman, and A. H. Batchelor. 2004. Allelic polymorphisms in apical membrane antigen-1 are responsible for evasion of antibody-mediated inhibition in Plasmodium falciparum. Mol. Microbiol. 52:159-168.[CrossRef][Medline]
13. Hehl, A. B., C. Lekutis, M. E. Grigg, P. J. Bradley, J. F. Dubremetz, E. Ortega-Barria, and J. C. Boothroyd. 2000. Toxoplasma gondii homologue of plasmodium apical membrane antigen 1 is involved in invasion of host cells. Infect. Immun. 68:7078-7086.[Abstract/Free Full Text]
14. Hodder, A. N., P. E. Crewther, and R. F. Anders. 2001. Specificity of the protective antibody response to apical membrane Antigen 1. Infect. Immun. 69:3286-3294.[Abstract/Free Full Text]
15. Hodder, A. N., P. E. Crewther, M. L. S. M. Mattew, G. E. Reid, R. L. Moritz, R. J. Simpson, and R. F. Anders. 1996. The disulphide bond structure of Plasmodium apical membrane antigen-1. J. Biol. Chem. 271:29446-29452.[Abstract/Free Full Text]
16. Howell, S. A., I. Wells, S. L. Fleck, C. Kettleborough, C. R. Collins, and M. J. Blackman. 2003. A single malaria merozoite serine protease mediates shedding of multiple surface proteins by juxtamembrane cleavage. J. Biol. Chem. 278:23890-23898.[Abstract/Free Full Text]
17. Howell, S. A., C. Withers-Martinez, C. H. Kocken, A. W. Thomas, and M. J. Blackman. 2001. Proteolytic processing and primary structure of Plasmodium falciparum apical membrane antigen-1. J. Biol. Chem. 276:31311-31320.[Abstract/Free Full Text]
18. Kennedy, M. C., J. Wang, Y. Zhang, A. P. Miles, F. Chitsaz, A. Saul, C. A. Long, L. H. Miller, and A. W. Stowers. 2002. In vitro studies with recombinant Plasmodium falciparum apical membrane antigen 1 (AMA1): production and activity of an AMA1 vaccine and generation of A Multiallelic response. Infect. Immun. 70:6948-6960.[Abstract/Free Full Text]
19. Kocken, C. H., D. L. Narum, A. Massougbodji, B. Ayivi, M. A. Dubbeld, A. van der Wel, D. J. Conway, A. Sanni, and A. W. Thomas. 2000. Molecular characterisation of Plasmodium reichenowi apical membrane antigen-1 (AMA-1), comparison with P. falciparum AMA-1, and antibody-mediated inhibition of red cell invasion. Mol. Biochem. Parasitol. 109:147-156.[CrossRef][Medline]
20. Kocken, C. H., C. Withers-Martinez, M. A. Dubbeld, A. van der Wel, F. Hackett, M. J. Blackman, and A. W. Thomas. 2002. High level expression of the malaria blood-stage vaccine candidate Plasmodium falciparum apical membrane antigen 1 and induction of antibodies that inhibit erythrocyte invasion. Infect. Immun. 70:4471-4476.[Abstract/Free Full Text]
21. Lalitha, P. V., L. A. Ware, A. Barbosa, S. Dutta, J. K. Moch, J. D. Haynes, B. B. Fileta, C. E. White, and D. E. Lanar. 2004. Production of the subdomains of the Plasmodium falciparum apical membrane antigen 1 ectodomain and analysis of the immune response. Infect. Immun. 72:4464-4470.[Abstract/Free Full Text]
22. Lambros, C., and J. P. Vanderberg. 1979. Synchronization of Plasmodium falciparum erythrocytic stages in culture. J. Parasitol. 65:418-420.[CrossRef][Medline]
23. Mueller, M. S., A. Renard, F. Boato, D. Vogel, M. Naegeli, R. Zurbriggen, J. A. Robinson, and G. Pluschke. 2003. Induction of parasite growth-inhibitory antibodies by a virosomal formulation of a peptidomimetic of loop I from domain III of Plasmodium falciparum apical membrane antigen 1. Infect. Immun. 71:4749-4758.[Abstract/Free Full Text]
24. Nair, M., M. G. Hinds, A. M. Coley, A. N. Hodder, M. Foley, R. Anders, and R. S. Norton. 2002. Structure of Domain III of the Blood-stage malaria vaccine candidate, Plasmodium falciparum apical membrane antigen 1 (AMA1). J. Mol. Biol. 322:741.[CrossRef][Medline]
25. Narum, D. L., and A. W. Thomas. 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.[CrossRef][Medline]
26. Peterson, M. G., V. M. Marshall, J. A. Smythe, P. E. Crewther, A. Lew, A. Silva, R. F. Anders, and D. J. Kemp. 1989. Integral membrane protein located in the apical complex of Plasmodium falciparum. Mol. Cell. Biol. 9:3151-3154.[Abstract/Free Full Text]
27. Polley, S. D., W. Chokejindachai, and D. J. Conway. 2003. Allele frequency-based analyses robustly map sequence sites under balancing selection in a malaria vaccine candidate antigen. Genetics 165:555-561.[Abstract/Free Full Text]
28. Polley, S. D., and D. J. Conway. 2001. Strong diversifying selection on domains of the Plasmodium falciparum apical membrane antigen 1 gene. Genetics 158:1505-1512.[Abstract/Free Full Text]
29. Reed, M. B., K. J. Saliba, S. R. Caruana, K. Kirk, and A. F. Cowman. 2000. Pgh1 modulates sensitivity and resistance to multiple antimalarials in Plasmodium falciparum. Nature 403:906-909.[CrossRef][Medline]
30. Riley, E. M., G. E. Wagner, M. F. Ofori, J. G. Wheeler, B. D. Akanmori, K. Tetteh, D. McGuinness, S. Bennett, F. K. Nkrumah, R. F. Anders, and K. A. Koram. 2000. Lack of association between maternal antibody and protection of African infants from malaria infection. Infect. Immun. 68:5856-5863.[Abstract/Free Full Text]
31. Sokal, R., and F. Rohlf. 1995. Biometry, 3rd ed. Freeman, New York, N.Y.
32. Stowers, A. W., M. C. Kennedy, B. P. Keegan, A. Saul, C. A. Long, and L. H. Miller. 2002. Vaccination of monkeys with recombinant Plasmodium falciparum apical membrane antigen 1 confers protection against blood-stage malaria. Infect. Immun. 70:6961-6967.[Abstract/Free Full Text]
33. Thomas, A. W., J. A. Deans, G. H. Mitchell, T. Alderson, and S. Cohen. 1984. The Fab fragments of monoclonal IgG to a merozoite surface antigen inhibit Plasmodium knowlesi invasion of erythrocytes. Mol. Biochem. Parasitol. 13:187-199.[CrossRef][Medline]
34. Trager, W., and J. B. Jensen. 1976. Human malaria parasites in continuous culture. Science 193:673-675.[Abstract/Free Full Text]
35. Triglia, T., and A. F. Cowman. 1994. Primary structure and expression of the dihydropteroate synthetase gene of Plasmodium falciparum. Proc. Natl. Acad. Sci. USA 91:7149-7153.[Abstract/Free Full Text]
36. Triglia, T., J. Healer, S. R. Caruana, A. N. Hodder, R. F. Anders, B. S. Crabb, and A. F. Cowman. 2000. Apical membrane antigen 1 plays a central role in erythrocyte invasion by Plasmodium species. Mol. Microbiol. 38:706-718.[CrossRef][Medline]
37. Triglia, T., P. Wang, P. F. G. Sims, J. E. Hyde, and A. F. Cowman. 1998. Allelic exchange at the endogenous genomic locus in Plasmodium falciparum proves the role of dihydropteroate synthase in sulfadoxine-resistant malaria. EMBO J. 17:3807-3815.[CrossRef][Medline]

Infection and Immunity, April 2005, p. 2444-2451, Vol. 73, No. 4
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.4.2444-2451.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Healer, J.
Right arrow Articles by Cowman, A. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Healer, J.
Right arrow Articles by Cowman, A. F.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
J. Bacteriol. J. Virol. Eukaryot. Cell
Microbiol. Mol. Biol. Rev. Clin. Vaccine Immunol. All ASM Journals