Functional Analysis of Toxoplasma gondii Protease Inhibitor 1

Abstract

We have characterized a Kazal family serine protease inhibitor, Toxoplasma gondii protease inhibitor 1 (TgPI-1), in the obligate intracellular parasite Toxoplasma gondii. TgPI-1 contains four inhibitor domains predicted to inhibit trypsin, chymotrypsin, and elastase. Antibodies against recombinant TgPI-1 detect two polypeptides, of 43 and 41 kDa, designated TgPI-1 43 and TgPI-1 41 , in tachyzoites, bradyzoites, and sporozoites. TgPI-1 43 and TgPI-1 41 are secreted constitutively from dense granules into the excreted/secreted antigen fraction as well as the parasitophorous vacuole that T. gondii occupies during intracellular replication. Recombinant TgPI-1 inhibits trypsin, chymotrypsin, pancreatic elastase, and neutrophil elastase. Immunoprecipitation studies with anti-rTgPI-1 antibodies reveal that recombinant TgPI-1 forms a complex with trypsin that is dependent on interactions with the active site of the protease. TgPI-1 is the first anti-trypsin/chymotrypsin inhibitor to be identified in bradyzoites and sporozoites, stages of the parasite that would be exposed to proteolytic enzymes in the digestive tract of the host.

A Kazal-like Extracellular Serine Protease Inhibitor from Phytophthora infestans Targets the Tomato Pathogenesis-related Protease P69B

“…Initial reaction velocities were measured by monitoring the absorbance change at 405 nm over reaction time using the HTS 7000 Bio Assay Reader (PerkinElmer Life Sciences). K i app was determined following the method described by Morris et al (15) Inhibition assays of plant proteases by rEPI1 were carried out with the QuantiCleave TM Protease Assay Kit (Pierce) and in-gel protease assays using the Bio-Rad zymogram buffer system. For the first method, 50 l of intercellular fluids were preincubated with or without 10 pmol of rEPI1 at 25°C for 30 min, and the protease activities were subsequently measured.…”

“…Some of these parasites secrete inhibitors that target host proteases and may aid in survival and colonization of the host. For instance, the apicomplexan obligate parasite Toxoplasma gondii secretes TgPI-1 and TgPI-2, four-domain serine protease inhibitors of the Kazal family (15)(16)(17)(18)(19), and the intestinal hookworm Ancylostoma ceylanicum secretes an 8-kDa broad spectrum serine protease inhibitor of the Kunitz family (14). In plants, the apoplast (intercellular fluid) forms a protease-rich environment that is colonized by many pathogens, including P. infestans and the fungus Cladosporium fulvum.…”

“…For example, parasites that colonize or transit through the mammalian digestive tract must adapt to the diverse and abundant array of proteases secreted in the gastric juices (13)(14)(15). Some of these parasites secrete inhibitors that target host proteases and may aid in survival and colonization of the host.…”

GRA Proteins of Toxoplasma gondii: Maintenance of Host-Parasite Interactions across the Parasitophorous Vacuolar Membrane

“…In the dense granule, there are 12 GRA proteins (GRA1-GRA14, but GRA11 and 13 are phantom-like) that have been previously identified in T. gondii tachyzoites [8][9][10][11][12] without sequence homology to each other in addition to 2 isoforms of nucleotide triphosphate hydrolase (NTPase I and II) [13] and 2 protease inhibitors (TgPI 1 and 2) [14,15]. All the GRA proteins are identified as excretory/secretory antigen (ESP).…”

Silencing of the hydra serine protease inhibitorKazal1gene mimics the humanSPINK1pancreatic phenotype

“…Amongst those, the sea anemone elastase inhibitor and the hydra antistatin act as serine protease inhibitors, the former one encoding a Kazal domain (KD), initially characterized in pancreatic tissue as trypsin inhibitor (Rawlings et al, 2004). KDs are 40 to 60 amino acids (aa) long and contain six cystein residues that form disulfide bridges; their protease inhibitor activity was documented in vertebrates (Kazal et al, 1948), blood-sucking insects (Mende et al, 1999), animal parasites as inhibitors of host digestive enzymes (Morris et al, 2002) and plant parasites (Tian et al, 2004). We isolated the hydra gene Kazal1, which encodes three KDs and exhibits a high level of expression in gland cells.…”