Not a Simple Tether: Binding of Toxoplasma gondii AMA1 to RON2 during Invasion Protects AMA1 from Rhomboid-Mediated Cleavage and Leads to Dephosphorylation of Its Cytosolic Tail.
Apical membrane antigen 1 (AMA1) is a receptor protein on the surface of Toxoplasma gondii that plays a critical role in host cell invasion. The ligand to which T gondii AMA1 (TgAMA1) binds, TgRON2, is secreted into the host cell membrane by the parasite during the early stages of invasion. The TgAMA1-TgRON2 complex forms the core of the "moving junction," a ring-shaped zone of tight contact between the parasite and host cell membranes, through which the parasite pushes itself during invasion. Paradoxically, the parasite also expresses rhomboid proteases that constitutively cleave the TgAMA1 transmembrane domain. How can TgAMA1 function effectively in host cell binding if its extracellular domain is constantly shed from the parasite surface? We show here that when TgAMA1 binds the domain 3 (D3) peptide of TgRON2, its susceptibility to cleavage by rhomboid protease(s) is greatly reduced. This likely serves to maintain parasite-host cell binding at the moving junction, a hypothesis supported by data showing that parasites expressing a hypercleavable version of TgAMA1 invade less efficiently than wild-type parasites do. Treatment of parasites with the D3 peptide was also found to reduce phosphorylation of S527 on the cytoplasmic tail of TgAMA1, and parasites expressing a phosphomimetic S527D allele of TgAMA1 showed an invasion defect. Taken together, these data suggest that TgAMA1-TgRON2 interaction at the moving junction protects TgAMA1 molecules that are actively engaged in host cell penetration from rhomboid-mediated cleavage and generates an outside-in signal that leads to dephosphorylation of the TgAMA1 cytosolic tail. Both of these effects are required for maximally efficient host cell invasion.



Nearly one-third of the world's population is infected with the protozoan parasite Toxoplasma gondii, which causes life-threatening disease in neonates and immunocompromised individuals. T. gondii is a member of the phylum Apicomplexa, which includes many other parasites of veterinary and medical importance, such as those that cause coccidiosis, babesiosis, and malaria. Apicomplexan parasites grow within their hosts through repeated cycles of host cell invasion, parasite replication, and host cell lysis. Parasites that cannot invade host cells cannot survive or cause disease. AMA1 is a highly conserved protein on the surface of apicomplexan parasites that is known to be important for invasion, and the work presented here reveals new and unexpected insights into AMA1 function. A more complete understanding of the role of AMA1 in invasion may ultimately contribute to the development of new chemotherapeutics designed to disrupt AMA1 function and invasion-related signaling in this important group of human pathogens.
Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont, USA.
Shruthi
S
Krishnamurthy
Department of Biology, University of Vermont, Burlington, Vermont, USA Vermont Genetics Network Proteomics Facility, University of Vermont, Burlington, Vermont, USA.
Bin
B
Deng
Department of Medicine, University of Vermont, Burlington, Vermont, USA.
Roxana
R
Del Rio
Department of Microbiology and Immunology, Stanford School of Medicine, Stanford, California, USA.
Kerry R
KR
Buchholz
Department of Microbiology and Immunology, Stanford School of Medicine, Stanford, California, USA.
Moritz
M
Treeck
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Siniša
S
Urban
Department of Microbiology and Immunology, Stanford School of Medicine, Stanford, California, USA.
John
J
Boothroyd
Department of Biology, University of Vermont, Burlington, Vermont, USA Vermont Genetics Network Proteomics Facility, University of Vermont, Burlington, Vermont, USA.
Ying-Wai
YW
Lam
Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont, USA Gary.Ward@uvm.edu.
Gary E
GE
Ward
D000953:Antigens, Protozoan / Q000378:metabolism*; D002478:Cells, Cultured; D005347:Fibroblasts / Q000469:parasitology; D006801:Humans; D010447:Peptide Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D015800:Protozoan Proteins / Q000378:metabolism*; D014122:Toxoplasma / Q000502:physiology*
C506564:AMA1 protein, Toxoplasma gondii; D000953:Antigens, Protozoan; D015800:Protozoan Proteins; C559757:rhoptry neck protein 2, Toxoplasma gondii; D010447:Peptide Hydrolases