A new class of apically confined, transmembrane micronemal proteins playing a pivotal role in host cell invasion by Toxoplasma gondii tachyzoites.

Transmembrane (TM) micronemal adhesins play crucial roles in the biology of Toxoplasma gondii invasive stages. Members of this protein class (e.g. MIC2, MIC6) are implicated to various extent in gliding motility and host cell attachment, whereas AMA1, which possesses three functional homologs (AMA2-4), physically connects the parasite and host cell membranes at the moving junction by binding to RON2, thus playing a key role in invasion. A common trait of these TM micronemal adhesins is their shedding from the parasite surface through the proteolytic cleavage by rhomboid proteases. The T. gondii genome encodes two thrombospondin-related paralogs, MIC14 (2344 aa) and MIC15 (2909 aa), showing the hallmarks of TM micronemal proteins, i.e., a large ectodomain with adhesive motifs, a C-terminal TM region and a short cytoplasmic tail potentially able to interact with the parasite actomyosin motors. While MIC14 is not expressed in tachyzoites, MIC15 has a micronemal localization and is relocated onto the parasite extreme apical surface, where it is confined during gliding, invasion and egress. Consistent with the lack of retrograde transport and a bona fide TM rhomboid cleavage site, the ectodomain of MIC15 is not recovered in the soluble fraction during secretion assays. Following several unsuccessful attempts to knockout the MIC15 gene, we generated a tetracycline-regulatable conditional knockdown (MIC15-cKD) and showed that MIC15-depleted tachyzoites are severely impaired in invasion (80%) and 50% less efficient during egress. In addition, the MIC15-cKD invasion defect is attenuated in parasites spontaneously upregulating MIC14 after prolonged growth in presence of tetracycline. Overall our results suggest that MIC14/MIC15 represent a novel class of early invasion factors.