Functional investigation of rhomboid proteases and their substrates in Toxoplasma gondii

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Toxoplasma gondii is a member of the phylum Apicomplexa, which groups important human and animal pathogens, including Plasmodium, the causative agent of malaria. Apicomplexan parasites invade host cells in an active manner, which critically relies on an actomyosin system and the regulated secretion of proteins from specialized organelles, named micronemes (Carruthers and Sibley, 1997). These micronemal proteins (MICs) are released onto the parasite's surface as complexes, containing both soluble and transmembrane proteins. In Toxoplasma gondii a large repertoire of functionally non-redundant MICs participates in gliding motility, host cell attachment, moving junction formation, rhoptry secretion and invasion. Some transmembrane microneme proteins (TM-MICs) also function as escorters, assuring trafficking of their complexes to the micronemes. The MICs present a modular design, possessing an ectodomain, capable of interacting with host cell receptors, and a short cytoplasmic tail, shown in micronemal protein -2 and -6 (TgMIC2 and TgMIC6) to connect to the actomyosin system via binding to aldolase (Jewett and Sibley, 2003). Within the ectodomain a variety of domains have been shown to contribute to host cell adhesion and recognition. Among these, a new structural module termed Microneme Adhesive Repeat (MAR) present on micronemal protein 1 (TgMIC1) was shown to be responsible for the recognition of sialyated oligosaccharides on the host cell surface (Blumenschein et al., 2007). Sialic acids serve as key determinant for invasion by the Apicomplexa, in general, and by T. gondii, in particular. During invasion the adhesive complexes are shed from the parasite's surface by the action of the micronemal protein protease 1 (MPP1), which cleaves the TM-MICs in the transmembrane spanning domain. The MPP1 activity is presumably important during invasion and is likely mediated by a rhomboid serine protease constitutively active at the plasma membrane of the parasite. In T. gondii, the plasma membrane rhomboid proteases -4 and -5 (TgROM4 and TgROM5) are the primary candidates for the MPP1 activity (Brossier et al., 2005; Dowse et al., 2005). In this study we aimed to better understand the function of the micronemal proteins during host cell invasion and identify the rhomboid-like protease responsible for the MPP1 activity in Toxoplasma. Apicomplexan parasites are obligatory intracellular parasites, which need to invade host cells in order to survive and propagate, and any knowledge regarding host cell invasion may provide new tools in the fight against this deadly pathogens.