Martin J. Boulanger

The RON2-AMA1 Interaction is a Critical Step in Moving Junction-Dependent Invasion by Apicomplexan Parasites

Obligate intracellular Apicomplexa parasites share a unique invasion mechanism involving a tight interaction between the host cell and the parasite surfaces called the moving junction (MJ). The MJ, which is the anchoring structure for the invasion process, is formed by secretion of a macromolecular complex (RON2/4/5/8), derived from secretory organelles called rhoptries, into the host cell membrane. AMA1, a protein secreted from micronemes and associated with the parasite surface during invasion, has been shown in vitro to bind the MJ complex through a direct association with RON2. Here we show that RON2 is inserted as an integral membrane protein in the host cell and, using several interaction assays with native or recombinant proteins, we define the region that binds AMA1. Our studies were performed both in Toxoplasma gondii and Plasmodium falciparum and although AMA1 and RON2 proteins have diverged between Apicomplexa species, we show an intra-species conservation of their interaction. More importantly, invasion inhibition assays using recombinant proteins demonstrate that the RON2-AMA1 interaction is crucial for both T. gondii and P. falciparum entry into their host cells. This work provides the first evidence that AMA1 uses the rhoptry neck protein RON2 as a receptor to promote invasion by Apicomplexa parasites.

Host Cell Invasion by Apicomplexan Parasites: Insights from the Co-Structure of Ama1 with a Ron2 Peptide

The structure of a eukaryotic pathogen adhesin bound to its receptor provides a basis for design of therapeutics. Apicomplexan parasites such as Toxoplasma gondii and Plasmodium species actively invade host cells through a moving junction (MJ) complex assembled at the parasite–host cell interface. MJ assembly is initiated by injection of parasite rhoptry neck proteins (RONs) into the host cell, where RON2 spans the membrane and functions as a receptor for apical membrane antigen 1 (AMA1) on the parasite. We have determined the structure of TgAMA1 complexed with a RON2 peptide at 1.95 angstrom resolution. A stepwise assembly mechanism results in an extensive buried surface area, enabling the MJ complex to resist the mechanical forces encountered during host cell invasion. Besides providing insights into host cell invasion by apicomplexan parasites, the structure offers a basis for designing therapeutics targeting these global pathogens.

Catalytic Roles for Two Water Bridged Residues (Asp-98 and His-255) in the Active Site of Copper-containing Nitrite Reductase

Two active site residues, Asp-98 and His-255, of copper-containing nitrite reductase (NIR) from Alcaligenes faecalis have been mutated to probe the catalytic mechanism. Three mutations at these two sites (D98N, H255D, and H255N) result in large reductions in activity relative to native NIR, suggesting that both residues are involved intimately in the reaction mechanism. Crystal structures of these mutants have been determined using data collected to better than 1.9-Å resolution. In the native structure, His-255 Nε2 forms a hydrogen bond through a bridging water molecule to the side chain of Asp-98, which also forms a hydrogen bond to a water or nitrite oxygen ligated to the active site copper. In the D98N mutant, reorientation of the Asn-98 side chain results in the loss of the hydrogen bond to the copper ligand water, consistent with a negatively charged Asp-98 directing the binding and protonation of nitrite in the native enzyme. An additional solvent molecule is situated between residues 255 and the bridging water in the H255N and H255D mutants and likely inhibits nitrite binding. The interaction of His-255 with the bridging water appears to be necessary for catalysis and may donate a proton to reaction intermediates in addition to Asp-98.

Structural and functional insights into the malaria parasite moving junction complex.

Members of the phylum Apicomplexa, which include the malaria parasite Plasmodium, share many features in their invasion mechanism in spite of their diverse host cell specificities and life cycle characteristics. The formation of a moving junction (MJ) between the membranes of the invading apicomplexan parasite and the host cell is common to these intracellular pathogens. The MJ contains two key parasite components: the surface protein Apical Membrane Antigen 1 (AMA1) and its receptor, the Rhoptry Neck Protein (RON) complex, which is targeted to the host cell membrane during invasion. In particular, RON2, a transmembrane component of the RON complex, interacts directly with AMA1. Here, we report the crystal structure of AMA1 from Plasmodium falciparum in complex with a peptide derived from the extracellular region of PfRON2, highlighting clear specificities of the P. falciparum RON2-AMA1 interaction. The receptor-binding site of PfAMA1 comprises the hydrophobic groove and a region that becomes exposed by displacement of the flexible Domain II loop. Mutations of key contact residues of PfRON2 and PfAMA1 abrogate binding between the recombinant proteins. Although PfRON2 contacts some polymorphic residues, binding studies with PfAMA1 from different strains show that these have little effect on affinity. Moreover, we demonstrate that the PfRON2 peptide inhibits erythrocyte invasion by P. falciparum merozoites and that this strong inhibitory potency is not affected by AMA1 polymorphisms. In parallel, we have determined the crystal structure of PfAMA1 in complex with the invasion-inhibitory peptide R1 derived by phage display, revealing an unexpected structural mimicry of the PfRON2 peptide. These results identify the key residues governing the interactions between AMA1 and RON2 in P. falciparum and suggest novel approaches to antimalarial therapeutics.

Plasticity and redundancy among AMA–RON pairs ensure host cell entry of Toxoplasma parasites

Malaria and toxoplasmosis are infectious diseases caused by the apicomplexan parasites Plasmodium and Toxoplasma gondii, respectively. These parasites have developed an invasion mechanism involving the formation of a moving junction (MJ) that anchors the parasite to the host cell and forms a ring through which the parasite penetrates. The composition and the assembly of the MJ, and in particular the presence of protein AMA1 and its interaction with protein RON2 at the MJ, have been the subject of intense controversy. Here, using reverse genetics, we show that AMA1, a vaccine candidate, interacts with RON2 to maintain the MJ structural integrity in T. gondii and is subsequently required for parasite internalization. Moreover, we show that disruption of the AMA1 gene results in upregulation of AMA1 and RON2 homologues that cooperate to support residual invasion. Our study highlights a considerable complexity and molecular plasticity in the architecture of the MJ.

Chromodomain Antagonists That Target the Polycomb-Group Methyllysine Reader Protein Chromobox Homolog 7 (CBX7)

We report here a peptide-driven approach to create first inhibitors of the chromobox homolog 7 (CBX7), a methyllysine reader protein. CBX7 uses its chromodomain to bind histone 3, lysine 27 trimethylated (H3K27me3), and this recognition event is implicated in silencing multiple tumor suppressors. Small trimethyllysine containing peptides were used as the basic scaffold from which potent ligands for disruption of CBX7-H3K27me3 complex were developed. Potency of ligands was determined by fluorescence polarization and/or isothermal titration calorimetry. Binding of one ligand was characterized in detail using 2D NMR and X-ray crystallography, revealing a structural motif unique among human CBX proteins. Inhibitors with a ∼200 nM potency for CBX7 binding and 10-fold/400-fold selectivity over related CBX8/CBX1 proteins were identified. These are the first reported inhibitors of any chromodomain.

Improving Biophysical Properties of a Bispecific Antibody Scaffold to Aid Developability: Quality by Molecular Design.

While the concept of Quality-by-Design is addressed at the upstream and downstream process development stages, we questioned whether there are advantages to addressing the issues of biologics quality early in the design of the molecule based on fundamental biophysical characterization, and thereby reduce complexities in the product development stages. Although limited number of bispecific therapeutics are in clinic, these developments have been plagued with difficulty in producing materials of sufficient quality and quantity for both preclinical and clinical studies. The engineered heterodimeric Fc is an industry-wide favorite scaffold for the design of bispecific protein therapeutics because of its structural, and potentially pharmacokinetic, similarity to the natural antibody. Development of molecules based on this concept, however, is challenged by the presence of potential homodimer contamination and stability loss relative to the natural Fc. We engineered a heterodimeric Fc with high heterodimeric specificity that also retains natural Fc-like biophysical properties, and demonstrate here that use of engineered Fc domains that mirror the natural system translates into an efficient and robust upstream stable cell line selection process as a first step toward a more developable therapeutic.

Immunization with a functional protein complex required for erythrocyte invasion protects against lethal malaria

Significance Apical membrane antigen 1 (AMA1) is a leading blood-stage vaccine candidate. Despite the vaccine’s ability to elicit high-titer AMA1-specific antibodies, it showed little efficacy in clinical trials against a homologous parasite. AMA1 interacts with a 49-aa region of rhoptry neck protein 2 (RON2), another parasite protein, during merozoite invasion. In this study, we demonstrate that immunization with a functional complex of AMA1-RON2 peptide (RON2L) induces antibody-mediated complete protection against lethal Plasmodium yoelli challenge. Interestingly, the qualitative increase in efficacy appears to be related in part to a switch in the proportion of antibodies targeting the RON2-binding site in AMA1. Our data suggest that a multiallele AMA1 (to overcome polymorphisms) in complex with RON2L should be effective in protecting against all Plasmodium falciparum parasites. An essential step in the invasion of red blood cells (RBCs) by Plasmodium falciparum (Pf) merozoites is the binding of rhoptry neck protein 2 (RON2) to the hydrophobic groove of apical membrane antigen 1 (AMA1), triggering junction formation between the apical end of the merozoite and the RBC surface to initiate invasion. Vaccination with AMA1 provided protection against homologous parasites in one of two phase 2 clinical trials; however, despite its ability to induce high-titer invasion-blocking antibodies in a controlled human challenge trial, the vaccine conferred little protection even against the homologous parasite. Here we provide evidence that immunization with an AMA1-RON2 peptide complex, but not with AMA1 alone, provided complete protection against a lethal Plasmodium yoelii challenge in mice. Significantly, IgG from mice immunized with the complex transferred protection. Furthermore, IgG from PfAMA1-RON2–immunized animals showed enhanced invasion inhibition compared with IgG elicited by AMA1 alone. Interestingly, this qualitative increase in inhibitory activity appears to be related, at least in part, to a switch in the proportion of IgG specific for certain loop regions in AMA1 surrounding the binding site of RON2. Antibodies induced by the complex were not sufficient to block the FVO strain heterologous parasite, however, reinforcing the need to include multiallele AMA1 to cover polymorphisms. Our results suggest that AMA1 subunit vaccines may be highly effective when presented to the immune system as an invasion complex with RON2.

Integrated Bioinformatic and Targeted Deletion Analyses of the SRS Gene Superfamily Identify SRS29C as a Negative Regulator of Toxoplasma Virulence

ABSTRACT The Toxoplasma gondii SRS gene superfamily is structurally related to SRS29B (formerly SAG1), a surface adhesin that binds host cells and stimulates host immunity. Comparative genomic analyses of three Toxoplasma strains identified 182 SRS genes distributed across 14 chromosomes at 57 genomic loci. Eight distinct SRS subfamilies were resolved. A core 69 functional gene orthologs were identified, and strain-specific expansions and pseudogenization were common. Gene expression profiling demonstrated differential expression of SRS genes in a developmental-stage- and strain-specific fashion and identified nine SRS genes as priority targets for gene deletion among the tissue-encysting coccidia. A Δsag1 ∆sag2A mutant was significantly attenuated in murine acute virulence and showed upregulated SRS29C (formerly SRS2) expression. Transgenic overexpression of SRS29C in the virulent RH parent was similarly attenuated. Together, these findings reveal SRS29C to be an important regulator of acute virulence in mice and demonstrate the power of integrated genomic analysis to guide experimental investigations. IMPORTANCE Parasitic species employ large gene families to subvert host immunity to enable pathogen colonization and cause disease. Toxoplasma gondii contains a large surface coat gene superfamily that encodes adhesins and virulence factors that facilitate infection in susceptible hosts. We generated an integrated bioinformatic resource to predict which genes from within this 182-gene superfamily of adhesin-encoding genes play an essential role in the host-pathogen interaction. Targeted gene deletion experiments with predicted candidate surface antigens identified SRS29C as an important negative regulator of acute virulence in murine models of Toxoplasma infection. Our integrated computational and experimental approach provides a comprehensive framework, or road map, for the assembly and discovery of additional key pathogenesis genes contained within other large surface coat gene superfamilies from a broad array of eukaryotic pathogens. Parasitic species employ large gene families to subvert host immunity to enable pathogen colonization and cause disease. Toxoplasma gondii contains a large surface coat gene superfamily that encodes adhesins and virulence factors that facilitate infection in susceptible hosts. We generated an integrated bioinformatic resource to predict which genes from within this 182-gene superfamily of adhesin-encoding genes play an essential role in the host-pathogen interaction. Targeted gene deletion experiments with predicted candidate surface antigens identified SRS29C as an important negative regulator of acute virulence in murine models of Toxoplasma infection. Our integrated computational and experimental approach provides a comprehensive framework, or road map, for the assembly and discovery of additional key pathogenesis genes contained within other large surface coat gene superfamilies from a broad array of eukaryotic pathogens.

Structural and Biochemical Characterization of Plasmodium falciparum 12 (Pf12) Reveals a Unique Interdomain Organization and the Potential for an Antiparallel Arrangement with Pf41

Background: Pf12 is the archetypal member of the 6-Cys protein family, members of which are important Plasmodium vaccine targets. Results: Purifying selection and apical localization of Pf12, crystal structure of tandem 6-Cys domains, and mass spectrometry of cross-linked Pf12-Pf41 heterodimer are shown. Conclusion: A functionally important role for Pf12 and potential for antiparallel heterodimer is provided. Significance: First full-length 6-Cys protein structure and first details of heterodimer organization are revealed. Plasmodium falciparum is the most devastating agent of human malaria. A major contributor to its virulence is a complex lifecycle with multiple parasite forms, each presenting a different repertoire of surface antigens. Importantly, members of the 6-Cys s48/45 family of proteins are found on the surface of P. falciparum in every stage, and several of these antigens have been investigated as vaccine targets. Pf12 is the archetypal member of the 6-Cys protein family, containing just two s48/45 domains, whereas other members have up to 14 of these domains. Pf12 is strongly recognized by immune sera from naturally infected patients. Here we show that Pf12 is highly conserved and under purifying selection. Immunofluorescence data reveals a punctate staining pattern with an apical organization in late schizonts. Together, these data are consistent with an important functional role for Pf12 in parasite-host cell attachment or invasion. To infer the structural and functional diversity between Pf12 and the other 11 6-Cys domain proteins, we solved the 1.90 Å resolution crystal structure of the Pf12 ectodomain. Structural analysis reveals a unique organization between the membrane proximal and membrane distal domains and clear homology with the SRS-domain containing proteins of Toxoplasma gondii. Cross-linking and mass spectrometry confirm the previously identified Pf12-Pf41 heterodimeric complex, and analysis of individual cross-links supports an unexpected antiparallel organization. Collectively, the localization and structure of Pf12 and details of its interaction with Pf41 reveal important insight into the structural and functional properties of this archetypal member of the 6-Cys protein family.