Michelle L. Tonkin

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.

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.

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.

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.

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

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 Å 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 (Trp353 and Trp354) anchor the DII loop in the hydrophobic groove and frame a conserved tyrosine (Tyr230), 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.

Systems-Based Analysis of the Sarcocystis neurona Genome Identifies Pathways That Contribute to a Heteroxenous Life Cycle

ABSTRACT Sarcocystis neurona is a member of the coccidia, a clade of single-celled parasites of medical and veterinary importance including Eimeria, Sarcocystis, Neospora, and Toxoplasma. Unlike Eimeria, a single-host enteric pathogen, Sarcocystis, Neospora, and Toxoplasma are two-host parasites that infect and produce infectious tissue cysts in a wide range of intermediate hosts. As a genus, Sarcocystis is one of the most successful protozoan parasites; all vertebrates, including birds, reptiles, fish, and mammals are hosts to at least one Sarcocystis species. Here we sequenced Sarcocystis neurona, the causal agent of fatal equine protozoal myeloencephalitis. The S. neurona genome is 127 Mbp, more than twice the size of other sequenced coccidian genomes. Comparative analyses identified conservation of the invasion machinery among the coccidia. However, many dense-granule and rhoptry kinase genes, responsible for altering host effector pathways in Toxoplasma and Neospora, are absent from S. neurona. Further, S. neurona has a divergent repertoire of SRS proteins, previously implicated in tissue cyst formation in Toxoplasma. Systems-based analyses identified a series of metabolic innovations, including the ability to exploit alternative sources of energy. Finally, we present an S. neurona model detailing conserved molecular innovations that promote the transition from a purely enteric lifestyle (Eimeria) to a heteroxenous parasite capable of infecting a wide range of intermediate hosts. IMPORTANCE Sarcocystis neurona is a member of the coccidia, a clade of single-celled apicomplexan parasites responsible for major economic and health care burdens worldwide. A cousin of Plasmodium, Cryptosporidium, Theileria, and Eimeria, Sarcocystis is one of the most successful parasite genera; it is capable of infecting all vertebrates (fish, reptiles, birds, and mammals—including humans). The past decade has witnessed an increasing number of human outbreaks of clinical significance associated with acute sarcocystosis. Among Sarcocystis species, S. neurona has a wide host range and causes fatal encephalitis in horses, marine mammals, and several other mammals. To provide insights into the transition from a purely enteric parasite (e.g., Eimeria) to one that forms tissue cysts (Toxoplasma), we present the first genome sequence of S. neurona. Comparisons with other coccidian genomes highlight the molecular innovations that drive its distinct life cycle strategies. Sarcocystis neurona is a member of the coccidia, a clade of single-celled apicomplexan parasites responsible for major economic and health care burdens worldwide. A cousin of Plasmodium, Cryptosporidium, Theileria, and Eimeria, Sarcocystis is one of the most successful parasite genera; it is capable of infecting all vertebrates (fish, reptiles, birds, and mammals—including humans). The past decade has witnessed an increasing number of human outbreaks of clinical significance associated with acute sarcocystosis. Among Sarcocystis species, S. neurona has a wide host range and causes fatal encephalitis in horses, marine mammals, and several other mammals. To provide insights into the transition from a purely enteric parasite (e.g., Eimeria) to one that forms tissue cysts (Toxoplasma), we present the first genome sequence of S. neurona. Comparisons with other coccidian genomes highlight the molecular innovations that drive its distinct life cycle strategies.

Babesia divergens and Neospora caninum apical membrane antigen 1 structures reveal selectivity and plasticity in apicomplexan parasite host cell invasion

Host cell invasion by the obligate intracellular apicomplexan parasites, including Plasmodium (malaria) and Toxoplasma (toxoplasmosis), requires a step‐wise mechanism unique among known host–pathogen interactions. A key step is the formation of the moving junction (MJ) complex, a circumferential constriction between the apical tip of the parasite and the host cell membrane that traverses in a posterior direction to enclose the parasite in a protective vacuole essential for intracellular survival. The leading model of MJ assembly proposes that Rhoptry Neck Protein 2 (RON2) is secreted into the host cell and integrated into the membrane where it serves as the receptor for apical membrane antigen 1 (AMA1) on the parasite surface. We have previously demonstrated that the AMA1‐RON2 interaction is an effective target for inhibiting apicomplexan invasion. To better understand the AMA1‐dependant molecular recognition events that promote invasion, including the significant AMA1‐RON2 interaction, we present the structural characterization of AMA1 from the apicomplexan parasites Babesia divergens (BdAMA1) and Neospora caninum (NcAMA1) by X‐ray crystallography. These studies offer intriguing structural insight into the RON2‐binding surface groove in the AMA1 apical domain, which shows clear evidence for receptor–ligand co‐evolution, and the hyper variability of the membrane proximal domain, which in Plasmodium is responsible for direct binding to erythrocytes. By incorporating the structural analysis of BdAMA1 and NcAMA1 with existing AMA1 structures and complexes we were able to define conserved pockets in the AMA1 apical groove that could be targeted for the design of broadly reactive therapeutics.

Toxoplasma gondii Sporozoites Invade Host Cells Using Two Novel Paralogues of RON2 and AMA1

Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa. The interaction of two well-studied proteins, Apical Membrane Antigen 1 (AMA1) and Rhoptry Neck protein 2 (RON2), has been shown to be critical for invasion by the asexual tachyzoite stage. Recently, two paralogues of these proteins, dubbed sporoAMA1 and sporoRON2 (or RON2L2), respectively, have been identified but not further characterized in proteomic and transcriptomic analyses of Toxoplasma sporozoites. Here, we show that sporoAMA1 and sporoRON2 localize to the apical region of sporozoites and that, in vitro, they interact specifically and exclusively, with no detectable interaction of sporoAMA1 with generic RON2 or sporoRON2 with generic AMA1. Structural studies of the interacting domains of sporoRON2 and sporoAMA1 indicate a novel pairing that is similar in overall form but distinct in detail from the previously described interaction of the generic pairing. Most notably, binding of sporoRON2 domain 3 to domains I/II of sporoAMA1 results in major alterations in the latter protein at the site of binding and allosterically in the membrane-proximal domain III of sporoAMA1 suggesting a possible role in signaling. Lastly, pretreatment of sporozoites with domain 3 of sporoRON2 substantially impedes their invasion into host cells while having no effect on tachyzoites, and vice versa for domain 3 of generic RON2 (which inhibits tachyzoite but not sporozoite invasion). These data indicate that sporozoites and tachyzoites each use a distinct pair of paralogous AMA1 and RON2 proteins for invasion into host cells, possibly due to the very different environment in which they each must function.

The multifunctional role of the pallilysin-associated Treponema pallidum protein, Tp0750, in promoting fibrinolysis and extracellular matrix component degradation.

The mechanisms that facilitate dissemination of the highly invasive spirochaete, Treponema pallidum, are incompletely understood. Previous studies showed the treponemal metalloprotease pallilysin (Tp0751) possesses fibrin clot degradation capability, suggesting a role in treponemal dissemination. In the current study we report characterization of the functionally linked protein Tp0750. Structural modelling predicts Tp0750 contains a von Willebrand factor type A (vWFA) domain, a protein‐protein interaction domain commonly observed in extracellular matrix (ECM)‐binding proteins. We report Tp0750 is a serine protease that degrades the major clot components fibrinogen and fibronectin. We also demonstrate Tp0750 cleaves a matrix metalloprotease (MMP) peptide substrate that is targeted by several MMPs, enzymes central to ECM remodelling. Through proteomic analyses we show Tp0750 binds the endothelial fibrinolytic receptor, annexin A2, in a specific and dose‐dependent manner. These results suggest Tp0750 constitutes a multifunctional protein that is able to (1) degrade infection‐limiting clots by both inhibiting clot formation through degradation of host coagulation cascade proteins and promoting clot dissolution by complexing with host proteins involved in the fibrinolytic cascade and (2) facilitate ECM degradation via MMP‐like proteolysis of host components. We propose that through these activities Tp0750 functions in concert with pallilysin to enable T. pallidum dissemination.