Mononeme: a new secretory organelle in merozoites recognized by localization of rhomboid-1 protease

Mononeme: a new secretory organelle in merozoites recognized by localization of rhomboid-1 protease. and asexual phases consisting of dormant, slow-growing forms called bradyzoites and fast-growing forms called tachyzoites, which are extremely efficient at invading a variety of cells and disseminating within the sponsor (13). contains several units of secretory organelles including micronemes, rhoptries, and dense granules whose sequential secretion is responsible for attachment, penetration, and survival in the infected cell, respectively (8, 14). Attachment to sponsor cells is initiated by secretion of microneme (MIC) proteins, which comprise a family of adhesive molecules (3, 6, 9). MIC proteins are typically found in complexes consisting of a transmembrane protein and a soluble partner(s) (10). MIC protein complexes also form higher-order complexes: for example, BI-671800 MIC2/M2AP is definitely a heterohexamer composed of three molecules of MIC2 associated with three molecules of M2AP (21). Apicomplexans use a unique form of locomotion called gliding motility, which relies on apical secretion and trafficking of MICs along the surface of the parasite (32). Translocation is definitely mediated by an connection between the cytoplasmic tail of MIC2 and aldolase, which bridges to the actin-myosin engine of the parasite (20). Upon contact with sponsor cells, MIC2 and its connected binding partner M2AP are released at the tip of the parasite, where they participate in binding to the sponsor cell and then translocated toward the posterior end of the parasite during penetration into the sponsor cell (6, 31). In the posterior end, the complex is definitely released from the surface by proteolysis within the transmembrane website of MIC2 (7, 30, 40) and this cleavage is required for efficient invasion of sponsor cells (4). MIC6 and TgAMA1 have also been shown to be cleaved within their transmembrane domains (18, 30), and AMA1 provides a vital function in formation of the moving junction (1, 24). Recent data strongly suggest that parasite-derived, rhomboid-like proteases cleave MIC proteins IFNA7 in order to launch them from your parasite membrane (5, 12, 36). Rhomboids are ubiquitous serine proteases that are able to recognize and cleave BI-671800 their substrates within their transmembrane domains (35, 37). Rhomboids have been extensively analyzed in contains six rhomboid-like genes (was conditionally suppressed BI-671800 using the tetracycline-repressible system explained previously (25, 26). MATERIALS AND METHODS Growth of sponsor cells and strains. strain tTa (26) and transformants BI-671800 were maintained by growth in monolayers of human being foreskin fibroblast (HFFs), propagated in Dulbecco’s revised Eagle’s medium comprising 10% fetal bovine serum, 2 mM glutamine, 20 mM HEPES (pH 7.5), and 20 g ml?1 gentamicin. Chloramphenicol (20 g/ml) (Sigma-Aldrich, St. Louis, MO), phleomycin (5 g/ml) (Invitrogen, San Diego, CA), and anhydrotetracycline (Atc) (1.5 g/ml) (Clontech, Palo Alto, CA) were added to the media as indicated. Antibodies. The HA9 epitope (YPYDVPDYA) was recognized using rabbit polyclonal antisera (Zymed, CA), monoclonal antibody (MAb) 16B12 (Covance, CA), or MAb HA-7 (Sigma-Aldrich, St. Louis, MO) for immunofluorescence (IF), Western blotting, and immunoelectron microscopy (immuno-EM) experiments, respectively. MIC2 was recognized with MAb 6D10 and rabbit polyclonal anti-C-domain antibodies for IF and immuno-EM experiments, respectively (39). MIC4 was recognized with rabbit polyclonal antibodies, generously provided by Dominique Soldati, and MAb 5B1. TgAMA1 was recognized with the MAb B3.90, generously provided by Gary Ward (University or college of Vermont). SAG1 was recognized using MAb DG52 directly coupled to Alexa 488 or Alexa 594 (Molecular Probes). RON4 was recognized with mouse polyclonal antisera, generously provided by Peter Bradley (University or college of California). GRA1 was recognized with mouse MAb Tg-17-43, a kind gift of Marie France Cesbron-Delauw. Generation of open reading framework (NCBI accession no. “type”:”entrez-protein”,”attrs”:”text”:”AAT84608″,”term_id”:”50845222″,”term_text”:”AAT84608″AAT84608) with an N-terminal HA9 tag was put in the previously explained vectors p7TetOS1 and p7TetOS4, downstream of the inducible promoters that consist of seven TetO sequences fused with the upstream areas from your and genes, respectively (25), to generate the plasmids pS1HA9-ROM1 and pS4HA9-ROM1. Transactivator-expressing parasites, referred to as tTa.