Apicomplexan rhomboids have a potential role in microneme protein cleavage during host cell invasion.
ABSTRACT Apicomplexan parasites secrete transmembrane (TM) adhesive proteins as part of the process leading to host cell attachment and invasion. These microneme proteins are cleaved in their TM domains by an unidentified protease termed microneme protein protease 1 (MPP1). The cleavage site sequence (IA downward arrowGG), mapped in the Toxoplasma gondii microneme proteins TgMIC2 and TgMIC6, is conserved in microneme proteins of other apicomplexans including Plasmodium species. We report here the characterisation of novel T. gondii proteins belonging to the rhomboid family of intramembrane-cleaving serine proteases. T. gondii possesses six genes encoding rhomboid-like proteins. Four are localised along the secretory pathway and therefore constitute possible candidates for MPP1 activity. Toxoplasma rhomboids TgROM1, TgROM2 and TgROM5 cleave the TM domain of Drosophila Spitz, an established substrate for rhomboids from several species, demonstrating that they are active proteases. In addition, TgROM2 cleaves chimeric proteins that contain the TM domains of TgMIC2 and TgMIC12.
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ABSTRACT: Host cell invasion by Toxoplasma gondii and other apicomplexan parasites requires transmembrane adhesins that mediate binding to receptors on the substrate and host cell to facilitate motility and invasion. Rhomboid proteases (ROMs) are thought to cleave adhesins within their transmembrane segments, thus allowing the parasite to disengage from receptors and completely enter the host cell. To examine the specific roles of individual ROMs during invasion, we generated single, double, and triple knockouts for the three ROMs expressed in T. gondii tachyzoites. Analysis of these mutants demonstrated that ROM4 is the primary protease involved in adhesin processing and host cell invasion, whereas ROM1 or ROM5 plays negligible roles in these processes. Deletion of ROM4 blocked the shedding of adhesins such as MIC2 (microneme protein 2), causing them to accumulate on the surface of extracellular parasites. Increased surface adhesins led to nonproductive attachment, altered gliding motility, impaired moving junction formation, and reduced invasion efficiency. Despite the importance of ROM4 for efficient invasion, mutants lacking all three ROMs were viable and MIC2 was still efficiently removed from the surface of invaded mutant parasites, implying the existence of ROM-independent mechanisms for adhesin removal during invasion. Collectively, these results suggest that although ROM processing of adhesins is not absolutely essential, it is important for efficient host cell invasion by T. gondii.mBio 10/2014; 5(5):e01795-14. DOI:10.1128/mBio.01795-14 · 6.88 Impact Factor
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ABSTRACT: Invasion in several apicomplexan parasites, including Eimeria tenella, is accompanied by shedding of surface adhesins by intramembrane proteolysis mediated by rhomboid protease. We have previously identified E. tenella rhomboid 3 (EtROM3), but its precise role has not been elucidated. In this study, the interactions between EtROM3 and microneme (MIC) proteins were analyzed using the yeast two hybrid technique. The results showed that c-Myc-ROM3 fusion protein interacted with EtMIC4 protein in co-transformed AH109 yeasts, which was further confirmed by immunoprecipitation assay. Smaller EtMIC4 band from co-transformed cells suggested that EtROM3 was an active protease and involved in the cleavage of EtMIC4.Veterinary Parasitology 01/2014; DOI:10.1016/j.vetpar.2014.01.010 · 2.55 Impact Factor
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ABSTRACT: Rhomboid proteins are intramembrane serine proteases that are involved in a plethora of biological functions, but the evolutionary history of the rhomboid gene family is not clear.We performed a comprehensive molecular evolutionary analysis of the rhomboid gene family and also investigated the organization and sequence features of plant rhomboids in different subfamilies.Our results showed that eukaryotic rhomboids could be divided into five subfamilies (RhoA–RhoD and PARL). Most orthology groups appeared to be conserved only as single or low-copy genes in all lineages in RhoB–RhoD and PARL, whereas RhoA genes underwent several duplication events, resulting in multiple gene copies. These duplication events were due to whole genome duplications in plants and animals and the duplicates might have experienced functional divergence. We also identified a novel group of plant rhomboid (RhoB1) that might have lost their enzymatic activity; their existence suggests that they might have evolved new mechanisms.Plant and animal rhomboids have similar evolutionary patterns. In addition, there are mutations affecting key active sites in RBL8, RBL9 and one of the Brassicaceae PARL duplicates. This study delineates a possible evolutionary scheme for intramembrane proteins and illustrates distinct fates and a mechanism of evolution of gene duplicates.New Phytologist 11/2014; DOI:10.1111/nph.13174 · 6.55 Impact Factor