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Structural characterization of apical membrane antigen 1 (AMA1) from Toxoplasma gondii.

ABSTRACT: Apical membrane antigen 1 (AMA1) is an essential component of the moving junction complex used by Apicomplexan parasites to invade host cells. We report the 2.0 A resolution x-ray crystal structure of the full ectodomain (domains I, II, and III) of AMA1 from the pervasive protozoan parasite Toxoplasma gondii. The structure of T. gondii AMA1 (TgAMA1) is the most complete of any AMA1 structure to date, with more than 97.5% of the ectodomain unambiguously modeled. Comparative sequence analysis reveals discrete segments of divergence in TgAMA1 that map to areas of established functional importance in AMA1 from Plasmodium vivax (PvAMA1) and Plasmodium falciparum (PfAMA1). Inspection of the TgAMA1 structure reveals a network of apical surface loops, reorganized in both size and chemistry relative to PvAMA1/PfAMA1, that appear to serve as structural filters restricting access to a central hydrophobic groove. The terminal portion of this groove is formed by an extended loop from DII that is 14 residues shorter in TgAMA1. A pair of tryptophan residues (Trp(353) and Trp(354)) anchor the DII loop in the hydrophobic groove and frame a conserved tyrosine (Tyr(230)), forming a contiguous surface that may be critical for moving junction assembly. The minimalist DIII structure folds into a cystine knot that probably stabilizes and orients the bulk of the ectodmain without providing excess surface area to which invasion-inhibitory antibodies can be generated. The detailed structural characterization of TgAMA1 provides valuable insight into the mechanism of host cell invasion by T. gondii.

SUBMITTER: Crawford J

PROVIDER: S-EPMC2865318 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Structural characterization of apical membrane antigen 1 (AMA1) from Toxoplasma gondii.

Crawford Joanna J Tonkin Michelle L ML Grujic Ognjen O Boulanger Martin J MJ

The Journal of biological chemistry 20100319 20

Apical membrane antigen 1 (AMA1) is an essential component of the moving junction complex used by Apicomplexan parasites to invade host cells. We report the 2.0 A resolution x-ray crystal structure of the full ectodomain (domains I, II, and III) of AMA1 from the pervasive protozoan parasite Toxoplasma gondii. The structure of T. gondii AMA1 (TgAMA1) is the most complete of any AMA1 structure to date, with more than 97.5% of the ectodomain unambiguously modeled. Comparative sequence analysis reve ...[more]

PMID: 20304917

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Project description:Apicomplexan parasites are the causative agents of globally prevalent diseases including malaria and toxoplasmosis. These obligate intracellular pathogens have evolved a sophisticated host cell invasion strategy that relies on a parasite-host cell junction anchored by interactions between apical membrane antigens (AMAs) on the parasite surface and rhoptry neck 2 (RON2) proteins discharged from the parasite and embedded in the host cell membrane. Key to formation of the AMA1-RON2 complex is displacement of an extended surface loop on AMA1 called the DII loop. While conformational flexibility of the DII loop is required to expose the mature RON2 binding groove, a definitive role of this substructure has not been elucidated. To establish a role of the DII loop in Toxoplasma gondii AMA1, we engineered a form of the protein where the mobile portion of the loop was replaced with a short Gly-Ser linker (TgAMA1ΔDIIloop). Isothermal titration calorimetry measurements with a panel of RON2 peptides revealed an influential role for the DII loop in governing selectivity. Most notably, an Eimeria tenella RON2 (EtRON2) peptide that showed only weak binding to TgAMA1 bound with high affinity to TgAMA1ΔDIIloop. To define the molecular basis for the differential binding, we determined the crystal structure of TgAMA1ΔDIIloop in complex with the EtRON2 peptide. When analyzed in the context of existing AMA1-RON2 structures, spatially distinct anchor points in the AMA1 groove were identified that, when engaged, appear to provide the necessary traction to outcompete the DII loop. Collectively, these data support a model where the AMA1 DII loop serves as a structural gatekeeper to selectively filter out ligands otherwise capable of binding with high affinity in the AMA1 apical groove. These data also highlight the importance of considering the functional implications of the DII loop in the ongoing development of therapeutic intervention strategies targeting the AMA1-RON2 invasion complex.

Project description:BackgroundCytauxzoon felis is a tick-transmitted apicomplexan that causes cytauxzoonosis in domestic cats (Felis catus). Even with intensive care, the mortality rate of acute cytauxzoonosis approaches 40% in domestic cats, while bobcats (Lynx rufus), the natural intermediate host of C. felis, remain clinically asymptomatic. However, multiple reports of domestic cats surviving acute disease without any treatment exist. One hypothesis for survival of these cats is infection with unique C. felis genotypes of lower pathogenicity. Prior studies have identified genetically distinct C. felis isolates containing polymorphisms within internal transcribed spacer regions (ITS) of the rRNA operon. However, these polymorphisms do not correlate with the clinical outcome of cytauxzoonosis, and so additional genetic markers are needed to test this hypothesis. We selected C. felis apical membrane antigen-1 (ama1) as a potential genetic marker of differential pathogenicity. AMA1 is a vaccine candidate for relatives of C. felis within Plasmodium spp.; however its historically high level of genetic polymorphism has resulted in escape from vaccine-induced immunity. While such diversity has hindered vaccine development, the expected polymorphism within the ama1 gene may be useful to evaluate population genetics.ResultsA 677 bp sequence of the C. felis ama1 gene was PCR-amplified from 84 domestic cats and 9 bobcats and demonstrated 99.9% sequence identity across all samples. A single nucleotide polymorphism (SNP) was identified in domestic cats and bobcats with evidence for co-infection with both genotypes identified in two domestic cats. The prevalence of the two genotypes varied with geographical distribution in domestic cats. Nucleotide diversity (π) and haplotype diversity (H) were calculated for C. felis ama1 and ama1 of related apicomplexans to assess genetic diversity. Based on these values (π = 0.00067 and H = 0.457), the diversity of the C. felis ama1 gene region analyzed is considerably lower than what is documented in related apicomplexans.ConclusionsIn surprising contrast to related apicomplexans, our results support that the sequence of the C. felis ama1 gene is highly conserved. While lack of genetic diversity limits utility of C. felis AMA1 as a genetic marker for clinical outcome, it supports further investigation as a vaccine candidate for cytauxzoonosis.

Project description:BackgroundApical membrane antigen 1 (AMA1) is one of the best-studied blood-stage malaria vaccine candidates. When an AMA1 vaccine was tested in a malaria naïve population, it induced functionally active antibodies judged by Growth Inhibition Assay (GIA). However, the same vaccine failed to induce higher growth-inhibitory activity in adults living in a malaria endemic area. Vaccination did induce functionally active antibodies in malaria-exposed children with less than 20% inhibition in GIA at baseline, but not in children with more than that level of baseline inhibition.MethodsTotal IgGs were purified from plasmas collected from the pediatric trial before and after immunization and pools of total IgGs were made. Another set of total IgGs was purified from U.S. adults immunized with AMA1 (US-total IgG). From these total IgGs, AMA1-specific and non-AMA1 IgGs were affinity purified and the functional activity of these IgGs was evaluated by GIA. Competition ELISA was performed with the U.S.-total IgG and non-AMA1 IgGs from malaria-exposed children.ResultsAMA1-specific IgGs from malaria-exposed children and U.S. vaccinees showed similar growth-inhibitory activity at the same concentrations. When mixed with U.S.-total IgG, non-AMA1 IgGs from children showed an interference effect in GIA. Interestingly, the interference effect was higher with non-AMA1 IgGs from higher titer pools. The non-AMA1 IgGs did not compete with anti-AMA1 antibody in U.S.-total IgG in the competition ELISA.ConclusionChildren living in a malaria endemic area have a fraction of IgGs that interferes with the biological activity of anti-AMA1 antibody as judged by GIA. While the mechanism of interference is not resolved in this study, these results suggest it is not caused by direct competition between non-AMA1 IgG and AMA1 protein. This study indicates that anti-malaria IgGs induced by natural exposure may interfere with the biological effect of antibody induced by an AMA1-based vaccine in the target population.

Dataset Information

Structural characterization of apical membrane antigen 1 (AMA1) from Toxoplasma gondii.

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Structural characterization of apical membrane antigen 1 (AMA1) from Toxoplasma gondii.

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