Phage-displayed peptides bind to the malarial protein apical membrane antigen-1 and inhibit the merozoite invasion of host erythrocytes

Apical membrane antigen-1 (AMA1) is a transmembrane protein present on the surface of merozoites that is thought to be involved in the process of parasite invasion of host erythrocytes. Although it is the target of a natural immune response that can inhibit invasion, little is known about the molecular mechanisms by which AMA1 facilitates the invasion process. In an attempt to identify peptides that specifically interact with and block the function of AMA1, a random peptide library displayed on the surface of filamentous phage was panned on recombinant AMA1 from Plasmodium falciparum. Three peptides with affinity for AMA1 were isolated, and characterization of their fine binding specificities indicated that they bind to a similar region on the surface of AMA1. One of these peptides was found to be a potent inhibitor of the invasion of P. falciparum merozoites into human erythrocytes. We propose that this peptide blocks interaction between AMA1 and a ligand on the erythrocyte surface that is involved in a critical step in malarial invasion. The identification and characterization of these peptide inhibitors now permit an evaluation of the essential requirements that are necessary for efficient neutralization of merozoite invasion by blocking AMA1 function.     

Structure and inter-domain interactions of domain II from the blood-stage malarial protein, apical membrane antigen 1

The malarial surface antigen apical membrane antigen (AMA1), from Plasmodium falciparum, is a leading candidate for inclusion in a vaccine against malaria. AMA1 is synthesised by mature blood-stages of the parasite and is located initially in the apical organelles of the merozoite. Prior to merozoite invasion of host erythrocytes, it is processed into a 66 kDa type 1 integral membrane protein on the merozoite surface. The pattern of disulphide bonds in AMA1 has been the basis for separation of the ectodomain into three domains, with three, two and three disulphide bonds, respectively. We have determined the solution structure of a 16kDa construct corresponding to the putative second domain of AMA1. While circular dichroism and hydrodynamic data were consistent with a folded structure for domain II, its NMR spectra were characterised by broad lines and significant peak overlap, more typical of a molten globule. Consistent with this, domain II bound the fluorescent dye 8-anilino-1-naphthalene sulphonate (ANS). We have nonetheless determined a structure, which defines the secondary structure elements and global fold. The two disulphide bonds link the N and C-terminal regions of the molecule, which come together to form a four-stranded beta-sheet linked to a short helix. A long loop linking the N and C-terminal regions contains four other alpha-helices, the locations of which are not fixed relative to the beta-sheet core, even though they are well-defined locally. Very recently this region of domain II has been shown to contain the epitope recognised by the invasion-inhibitory antibody 4G2, even though it does not contain any of the polymorphisms that are regarded as having arisen in response to the pressure of immune recognition.     

Identifying Plasmodium falciparum merozoite surface antigen 3 (MSP3) protein peptides that bind specifically to erythrocytes and inhibit merozoite invasion

Receptor-ligand interactions between synthetic peptides and normal human erythrocytes were studied to determine Plasmodium falciparum merozoite surface protein-3 (MSP-3) FC27 strain regions that specifically bind to membrane surface receptors on human erythrocytes. Three MSP-3 protein high activity binding peptides (HABPs) were identified; their binding to erythrocytes became saturable, had nanomolar affinity constants, and became sensitive on being treated with neuraminidase and trypsin but were resistant to chymotrypsin treatment. All of them specifically recognized 45-, 55-, and 72-kDa erythrocyte membrane proteins. They all presented alpha-helix structural elements. All HABPs inhibited in vitro P. falciparum merozoite invasion of erythrocytes by ~55%-85%, suggesting that MSP-3 protein's role in the invasion process probably functions by using mechanisms similar to those described for other MSP family antigens.     

Characterisation of Plasmodium falciparum RESA-like protein peptides that bind specifically to erythrocytes and inhibit invasion

The Plasmodium falciparum ring-erythrocyte surface antigen (RESA)-like putative protein was identified and characterised. PCR and RT-PCR assays revealed that the gene encoding this protein was both present and being transcribed in P. falciparum strain FCB-2 16 h after erythrocyte invasion. Indirect immunofluorescence studies detected this protein in infected erythrocyte (IE) cytosol in dense fluorescent granules similar to Maurer's clefts at 16-20 h (parasites in ring and trophozoite stages) and very strongly on IE membranes at 22 h, suggesting that it is synthesised during early ring stages (16 h) and transported to the infected red blood cell (RBC) membrane surface during the trophozoite stage (22 h). Western blotting showed that antisera produced against polymerised synthetic peptides of this protein recognised a 72-kDa band in P. falciparum schizont lysate. P. falciparum RESA-like peptides used in normal RBC binding assays revealed that peptides 30326 ((101)NAEKI LGFDD KNILE ALDLFY(120)), 30334 ((281)RVTWK KLRTK MIKAL KKSLTY(300)) and 30342 ((431)SSPQR LKFTA GGGFC GKLRNY(450)) bind with high activity and saturability, presenting nM affinity constants. These peptides contain alpha-helical structural elements, as determined by circular dichroism, and inhibit P. falciparum in vitro invasion of normal RBCs by up to 91%, suggesting that some RESA-like protein regions are involved in intra-erythrocyte stage P. falciparum invasion.     

Identification by the phage-display technique of peptides that bind to H7 flagellin of Escherichia coli

The four peptides interacting with H7 flagellin of Escherichia coli were selected from a phage display library. The library was selected four times, and the interacting phage peptides were competitively eluted with H7 flagellin. An enzyme-linked immunosorbent assay (ELISA) showed that these peptides were reactive with the H7 flagellin in a dose-dependent manner. Among them, a D1 phage clone showed the highest binding affinity to the H7 flagellin. We synthesized the D1 peptide (LHIHRPTLSIQG) corresponding to the peptide-encoding region of the D1 phage clone. The synthetic peptide showed micro-molar affinity (EC(50) value=1.9 microM) for the H7 flagellin. Furthermore, this D1 peptide interacted more specifically with the H7 flagellin than with the other flagellins (H1, H5, H12, or H23) of E. coli. In situ hybridization clearly showed that the peptide only detected those cells harboring the H7 flagellin gene (fliC). The peptide may specifically bind to the H7 flagellin on the cell surface. These results suggest that the phage-display technique could be used as a tool for identifying peptides as an alternative to using a ligand as a diagnostic reagent in food products or in clinical testing.     

Combinatorial evolution of high-affinity peptides that bind to the Thomsen-Friedenreich carcinoma antigen

Thomsen-Friedenreich (TF) antigen occurs on approximately 90% of human carcinomas, is likely involved in carcinoma cell homotypic aggregation, and has clinical value as a prognostic indicator and marker of metastasized cells. Previously, we isolated anti-TF antigen peptides from bacteriophage display libraries. These bound to TF antigen on carcinoma cells but were of low affinity and solubility. We hypothesized that peptide amino acid sequence changes would result in increased affinity and solubility, which would translate into improved carcinoma cell binding and increased inhibition of aggregation. The new peptides were more soluble and exhibited up to fivefold increase in affinity (K_d 60 nM). They bound cultured human breast and prostate carcinoma cells at low concentrations, whereas the earlier peptides did not. Moreover, the new peptides were potent inhibitors of homotypic aggregation. The maturated peptides will have expanded applications in basic studies of the TF antigen and particular utility as clinical carcinoma-targeting agents.     

Identification of Plasmodium falciparum RhopH3 protein peptides that specifically bind to erythrocytes and inhibit merozoite invasion

The identification of sequences involved in binding to erythrocytes is an important step for understanding the molecular basis of merozoite-erythrocyte interactions that take place during invasion of the Plasmodium falciparum malaria parasite into host cells. Several molecules located in the apical organelles (micronemes, rhoptry, dense granules) of the invasive-stage parasite are essential for erythrocyte recognition, invasion, and establishment of the nascent parasitophorous vacuole. Particularly, it has been demonstrated that rhoptry proteins play an important role in binding to erythrocyte surface receptors, among which is the PfRhopH3 protein, which triggers important immune responses in patients from endemic regions. It has also been reported that anti-RhopH3 antibodies inhibit in vitro invasion of erythrocytes, further supporting its direct involvement in erythrocyte invasion processes. In this study, PfRhopH3 consecutive peptides were synthesized and tested in erythrocyte binding assays for identifying those regions mediating binding to erythrocytes. Fourteen PfRhopH3 peptides presenting high specific binding activity were found, whose bindings were saturable and presented nanomolar dissociation constants. These high-activity binding peptides (HABPs) were characterized by having alpha-helical structural elements, as determined by circular dichroism, and having receptors of a possible sialic acid-dependent and/or glycoprotein-dependent nature, as evidenced in enzyme-treated erythrocyte binding assays and further corroborated by cross-linking assay results. Furthermore, these HABPs inhibited merozoite in vitro invasion of normal erythrocytes at 200 microM by up to 60% and 90%, suggesting that some RhopH3 protein regions are involved in the P. falciparum erythrocyte invasion.     

Peptide inhibitors of the malaria surface protein, apical membrane antigen 1: identification of key binding residues

Apical membrane antigen 1 (AMA1) is essential for malaria parasite invasion of erythrocytes and is therefore an attractive target for drug development. Peptides that bind AMA1 have been identified from random peptide libraries expressed on the surface of phage. Of these, R1, which binds to a hydrophobic ligand binding site on AMA1, was a particularly potent inhibitor of parasite invasion of erythrocytes in vitro. The solution structure of R1 contains a turn-like conformation between residues 5-10. Here the importance of residues in this turn-like structure for binding to AMA1 was examined by site-directed mutagenesis and NMR spectroscopy. The peptide was expressed as a fusion protein following replacement of Met16 by Leu in order to accommodate cyanogen bromide cleavage. This modified peptide (R2) displayed the same affinity for AMA1 as R1, showing that the identity of the side chain at position 16 was not critical for binding. Substitution of Phe5, Pro7, Leu8, and Phe9 with alanine led to significant (7.5- to >350-fold) decreases in affinity for AMA1. Comparison of backbone amide and C(α) H chemical shifts for these R2 analogues with corresponding values for R2 showed no significant changes, with the exception of R2(P7A), where slightly larger differences were observed, particularly for residues flanking position 7. The absence of significant changes in the secondary chemical shifts suggests that these mutations had little effect on the solution conformation of R2. The identification of a nonpolar region of these peptides containing residues essential for AMA1 binding establishes a basis for the design of anti-malarial drugs based on R1 mimetics.     

Trafficking of malarial proteins to the host cell cytoplasm and erythrocyte surface membrane involves multiple pathways

During the asexual stage of malaria infection, the intracellular parasite exports membranes into the erythrocyte cytoplasm and lipids and proteins to the host cell membrane, essentially "transforming" the erythrocyte. To investigate lipid and protein trafficking pathways within Plasmodium falciparum-infected erythrocytes, synchronous cultures are temporally analyzed by confocal fluorescence imaging microscopy for the production, location and morphology of exported membranes (vesicles) and parasite proteins. Highly mobile vesicles are observed as early as 4 h postinvasion in the erythrocyte cytoplasm of infected erythrocytes incubated in vitro with C6-NBD-labeled phospholipids. These vesicles are most prevalent in the trophozoite stage. An immunofluorescence technique is developed to simultaneously determine the morphology and distribution of the fluorescent membranes and a number of parasite proteins within a single parasitized erythrocyte. Parasite proteins are visualized with FITC- or Texas red-labeled monoclonal antibodies. Double-label immunofluorescence reveals that of the five parasite antigens examined, only one was predominantly associated with membranes in the erythrocyte cytoplasm. Two other parasite antigens localized only in part to these vesicles, with the majority of the exported antigens present in lipid-free aggregates in the host cell cytoplasm. Another parasite antigen transported into the erythrocyte cytoplasm is localized exclusively in lipid-free aggregates. A parasite plasma membrane (PPM) and/or parasitophorous vacuolar membrane (PVM) antigen which is not exported always colocalizes with fluorescent lipids in the PPM/PVM. Visualization of two parasite proteins simultaneously using FITC- and Texas red-labeled 2 degrees antibodies reveals that some parasite proteins are constitutively transported in the same vesicles, whereas other are segregated before export. Of the four exported antigens, only one appears to cross the barriers of the PPM and PVM through membrane-mediated events, whereas the others are exported across the PPM/PVM to the host cell cytoplasm and surface membrane through lipid (vesicle)-independent pathways.     

Plasmodium berghei circumvents immune responses induced by merozoite surface protein 1- and apical membrane antigen 1-based vaccines

Background

Two current leading malaria blood-stage vaccine candidate antigens for Plasmodium falciparum, the C-terminal region of merozoite surface protein 1 (MSP1₁₉) and apical membrane antigen 1 (AMA1), have been prioritized because of outstanding protective efficacies achieved in a rodent malaria Plasmodium yoelii model. However, P. falciparum vaccines based on these antigens have had disappointing outcomes in clinical trials. Discrepancies in the vaccine efficacies observed between the P. yoelii model and human clinical trials still remain problematic.

Methodology and Results

In this study, we assessed the protective efficacies of a series of MSP1₁₉- and AMA1-based vaccines using the P. berghei rodent malarial parasite and its transgenic models. Immunization of mice with a baculoviral-based vaccine (BBV) expressing P. falciparum MSP1₁₉ induced high titers of PfMSP1₁₉-specific antibodies that strongly reacted with P. falciparum blood-stage parasites. However, no protection was achieved following lethal challenge with transgenic P. berghei expressing PfMSP1₁₉ in place of native PbMSP1₁₉. Similarly, neither P. berghei MSP1₁₉- nor AMA1-BBV was effective against P. berghei. In contrast, immunization with P. yoelii MSP1₁₉- and AMA1-BBVs provided 100% and 40% protection, respectively, against P. yoelii lethal challenge. Mice that naturally acquired sterile immunity against P. berghei became cross-resistant to P. yoelii, but not vice versa.

Conclusion

This is the first study to address blood-stage vaccine efficacies using both P. berghei and P. yoelii models at the same time. P. berghei completely circumvents immune responses induced by MSP1₁₉- and AMA1-based vaccines, suggesting that P. berghei possesses additional molecules and/or mechanisms that circumvent the host''s immune responses to MSP1₁₉ and AMA1, which are lacking in P. yoelii. Although it is not known whether P. falciparum shares these escape mechanisms with P. berghei, P. berghei and its transgenic models may have potential as useful tools for identifying and evaluating new blood-stage vaccine candidate antigens for P. falciparum.     

The mechanism of erythrocyte invasion by the malarial parasite, Plasmodium falciparum

Plasmodium falciparum is the most virulent causative agent of malaria in man accounting for 80% of all malarial infections and 90% of the one million annual deaths attributed to malaria. P. falciparum is a unicellular, Apicomplexan parasite, that spends part of its lifecycle in the mosquito and part in man and it has evolved a special form of motility that enables it to burrow into animal cells, a process termed “host cell invasion”. The acute, life threatening, phase of malarial infection arises when the merozoite form of the parasite undergoes multiple cycles of red blood cell invasion and rapid proliferation. Here, we discuss the molecular machinery that enables malarial parasites to invade red blood cells and we focus particularly on the ATP-driven acto-myosin motor that powers invasion.     

Identification and characterization of peptides that bind to cyanovirin-N, a potent human immunodeficiency virus-inactivating protein

Cyanovirin-N (CV-N) exerts a potent human immunodeficiency virus (HIV)-inactivating activity against diverse strains of HIV by binding to the viral surface envelope glycoprotein gp120 and blocking its essential interactions with cellular receptors. Based on previous thermodynamic analyses, it has been speculated that discrete protein-protein interactions might play an important ancillary role in the CV-N/gp120 binding event, in addition to the interactions of CV-N with specific oligosaccharides present on gp120. Here, we report the identification and characterization of CV-N-binding peptides, which were isolated by screening of M13 phage-displayed peptide libraries. After performing three rounds of biopanning of the libraries against biotinylated CV-N, a CV-N-binding motif, X3CX6(W/F)(Y/F)CX2(Y/F), was evident. A vector was designed to express CV-N-binding peptides as a fusion with thioredoxin (Trx) containing a penta-His affinity tag. The CV-N-binding peptides fused with His-tagged Trx inhibited binding of the corresponding peptide-bearing phages to CV-N, confirming that the peptides possessed CV-N-binding activity. Optical biosensor binding studies showed that the one of the CV-N-binding peptide, TN10-1, bound to CV-N with a KD value of 1.9 microM. The results of alanine scanning mutagenesis of the peptide showed that aromatic residues at positions 11, 12, and 16, as well as the conformational structure of the peptide secured by a disulfide bond, were important for the binding interactions. A series of competitive binding assays confirmed that gp120 inhibited CV-N binding of the corresponding peptide-bearing phages, and suggested that TN10-1 peptides were mimicking the protein component of gp120 rather than mimicking specific oligosaccharides present on gp120.     

A role for apical membrane antigen 1 during invasion of hepatocytes by Plasmodium falciparum sporozoites

Plasmodium sporozoites are transmitted through the bite of infected mosquitoes and invade hepatocytes as a first and obligatory step of the parasite life cycle in man. Hepatocyte invasion involves proteins secreted from parasite vesicles called micronemes, the most characterized being the thrombospondin-related adhesive protein (TRAP). Here we investigated the expression and function of another microneme protein recently identified in Plasmodium falciparum sporozoites, apical membrane antigen 1 (AMA-1). P. falciparum AMA-1 is expressed in sporozoites and is lost after invasion of hepatocytes, and anti-AMA-1 antibodies inhibit sporozoite invasion, suggesting that the protein is involved during invasion of hepatocytes. As observed with TRAP, AMA-1 is initially mostly sequestered within the sporozoite. Upon microneme exocytosis, AMA-1 and TRAP relocate to the sporozoite surface, where they are proteolytically cleaved, resulting in the shedding of soluble fragments. A subset of serine protease inhibitors blocks the processing and shedding of both AMA-1 and TRAP and inhibits sporozoite infectivity, suggesting that interfering with sporozoite proteolytic processing may constitute a valuable strategy to prevent hepatocyte infection.     

MAEBL Plasmodium falciparum protein peptides bind specifically to erythrocytes and inhibit in vitro merozoite invasion

MAEBL is an erythrocyte binding protein located in the rhoptries and on the surface of mature merozoites, being expressed at the beginning of schizogony. The structure of MAEBL originally isolated from rodent malaria parasites suggested a molecule likely to be involved in invasion. We thus became interested in identifying possible MAEBL functional regions. Synthetic peptides spanning the MAEBL sequence were tested in erythrocyte binding assays to identify such possible MAEBL functional regions. Nine high activity binding peptides (HABPs) were identified: two were found in the M1 domain, one was found between the M1 and M2 regions, five in the erythrocyte binding domain (M2), and one in the protein's repeat region. The results showed that peptide binding was saturable; some HABPs inhibited in vitro merozoite invasion and specifically bound to a 33kDa protein on red blood cell membrane. HABPs' possible function in merozoite invasion of erythrocytes is also discussed.     

Independent roles of apical membrane antigen 1 and rhoptry neck proteins during host cell invasion by apicomplexa

During invasion, apicomplexan parasites form an intimate circumferential contact with the host cell, the tight junction (TJ), through which they actively glide. The TJ, which links the parasite motor to the host cell cytoskeleton, is thought to be composed of interacting apical membrane antigen 1 (AMA1) and rhoptry neck (RON) proteins. Here we find that, in Plasmodium berghei, while both AMA1 and RON4 are important for merozoite invasion of erythrocytes, only RON4 is required for sporozoite invasion of hepatocytes, indicating that RON4 acts independently of AMA1 in the sporozoite. Further, in the Toxoplasma gondii tachyzoite, AMA1 is dispensable for normal RON4 ring and functional TJ assembly but enhances tachyzoite apposition to the cell and internalization frequency. We propose that while the RON proteins act at the TJ, AMA1 mainly functions on the zoite surface to permit correct attachment to the cell, which may facilitate invasion depending on the zoite-cell combination.     

Plasmodium falciparum merozoite surface protein 6 (MSP-6) derived peptides bind erythrocytes and partially inhibit parasite invasion

This work shows that Plasmodium falciparum merozoite surface protein-6 (MSP-6) peptides specifically bind to membrane surface receptor on human erythrocytes. Three high activity binding peptides (HABPs) were found: peptides 31175 (41MYNNDKILSKNEVDTNIESN60) and 31178 (101YDIQATYQFPSTSGGNNVIP120) in the amino terminal region and 31191 (361EIDSTINNLVQEMIHLFSNNY380) at the carboxy terminal. Their binding to erythrocytes was saturable. HABPs 31191 and 31178 recognized 56 and 26 kDa receptors on erythrocyte membrane and inhibited in vitro Plasmodium falciparum merozoite invasion of erythrocytes by between 27% and 46% at 200 microg ml(-1) concentration, suggesting that these MSP-6 protein peptides play a possible role in the invasion process.     

Refolding, purification, and crystallization of apical membrane antigen 1 from Plasmodium falciparum

Extracellular domains of malaria antigens almost invariably contain disulphide linkages but lack N- and O-linked glycosylation. The best practical approach to generating recombinant extracellular Plasmodium proteins is not established and the problems encountered when using a bacterial expression/refolding approach are discussed in detail. Limited proteolysis experiments were used to identify a relatively non-flexible core region of the Plasmodium falciparum protein apical membrane antigen 1 (AMA1), and refolding/purification was used to generate two fragments of AMA1. Several chromatographically distinct AMA1 variants were identified that are presumably differentially refolded proteins. One of these AMA1 preparations proved to be crystallizable and generated two crystal forms that diffracted X-rays to 2 A resolution.     

Energetics and cooperativity of the hydrogen bonding and anchor interactions that bind peptides to MHC class II protein

The complexity of the interaction between major histocompatibility complex class II (MHC II) proteins and peptide ligands has been revealed through structural studies and crystallographic characterization. Peptides bind through side-chain "anchor" interactions with MHC II pockets and an extensive array of genetically conserved hydrogen bonds to the peptide backbone. Here we quantitatively investigate the kinetic hierarchy of these interactions. We present results detailing the impact of single side-chain mutations of peptide anchor residues on dissociation rates, utilizing two I-A(d)-restricted peptides, one of which has a known crystal structure, and 24 natural and non-natural amino acid mutant variants of these peptides. We find that the N-terminal P1, P4 and P6 anchor-pocket interactions can make significant contributions to binding stability. We also investigate the interactions of these peptides with four I-A(d) MHC II proteins, each mutated to disrupt conserved hydrogen bonds to the peptide backbone. These complexes exhibit kinetic behavior suggesting that binding energy is disproportionately invested near the peptide N terminus for backbone hydrogen bonds. We then evaluate the effects of simultaneously modifying both anchor and hydrogen bonding interactions. A quantitative analysis of 71 double mutant cycles reveals that there is little apparent cooperativity between anchor residue interactions and hydrogen bonds, even when they are directly adjacent (<5A).     

RNA aptamers that bind to and inhibit the ribosome-inactivating protein, pepocin

Pepocin, isolated from Cucurbita pepo, is a ribosome-inactivating protein (RIP). RIPs site-specifically recognize and depurinate an adenosine at position 4324 in rat 28 S rRNA, rendering the ribosome incapable of interacting with essential elongation factors. Aptamers that target pepocin were isolated from a degenerate RNA pool by in vitro selection. A conserved hairpin motif, quite different from the sequence of the toxin-substrate domain in rat 28 S rRNA, was identified in the aptamer sequences. The aptamers selectively bind to pepocin with dissociation constants between 20 and 30 nM and inhibit the N-glycosidase activity of pepocin on rat liver 28 S rRNA. Competitive binding experiments using aptamer variants suggest that the conserved hairpin region in the anti-pepocin aptamer binds near the catalytic site on pepocin and prevents the interaction of pepocin and 28 S rRNA. Anti-RIP aptamers have potential use in diagnostic systems for the detection of pepocin or could be used as therapy to prevent the action of pepocin in mammalian cells.