Abstract
Apicomplexans facilitate host cell invasion through formation of a tight-junction interface between parasite and host plasma membranes called the moving junction (MJ). A complex of the rhoptry neck proteins RONs 2/4/5/8 localize to the MJ during invasion where they are believed to provide a stable anchoring point for host penetration. During the initiation of invasion, the preformed MJ RON complex is injected into the host cell where RON2 spans the host plasma membrane while RONs 4/5/8 localize to its cytosolic face. While much attention has been directed toward an AMA1-RON2 interaction supposed to occur outside the cell, little is known about the functions of the MJ RONs positioned inside the host cell. Here we provide a detailed analysis of RON5 to resolve outstanding questions about MJ complex organization, assembly and function during invasion. Using a conditional knockdown approach, we show loss of RON5 results in complete degradation of RON2 and mistargeting of RON4 within the parasite secretory pathway, demonstrating that RON5 plays a key role in organization of the MJ RON complex. While RON8 is unaffected by knockdown of RON5, these parasites are unable to invade new host cells, providing the first genetic demonstration that RON5 plays a critical role in host cell penetration. Although invasion is not required for injection of rhoptry effectors into the host cytosol, parasites lacking RON5 also fail to form evacuoles suggesting an intact MJ complex is a prerequisite for secretion of rhoptry bulb contents. Additionally, while the MJ has been suggested to function in egress, disruption of the MJ complex by RON5 depletion does not impact this process. Finally, functional complementation of our conditional RON5 mutant reveals that while proteolytic separation of RON5 N- and C-terminal fragments is dispensable, a portion of the C-terminal domain is critical for RON2 stability and function in invasion.
Highlights
The Apicomplexa are a large phylum of eukaryotic pathogens comprised of,6,000 described species which cause extensive disease in humans and other animals [1,2]
Our findings demonstrate the key role of RON5 in facilitating apicomplexan host invasion and disease
Using a conditional knockdown approach, we show that depletion of RON5 results in the complete loss of RON2 and mistargeting of RON4, indicating RON5 is critical for organization of the moving junction (MJ) complex
Summary
The Apicomplexa are a large phylum of eukaryotic pathogens comprised of ,6,000 described species which cause extensive disease in humans and other animals [1,2]. The disease caused by these obligate intracellular parasites is dependent upon their ability to penetrate, form a specialized vacuole, and replicate within their host cells [5]. Invasion in apicomplexans is a highly coordinated process of attachment and penetration that depends on sequential protein secretion events from two different organelles, the micronemes and rhoptries [6]. Secretion from the micronemes releases molecular adhesins onto the parasite’s plasma membrane, facilitating attachment to the host cell surface [7]. Translocation of these adhesins in an apical to posterior direction via an actin-myosin motor within the parasite pellicle generates a unique gliding motility which is thought to provide the force for host cell penetration. The recent disruption of MIC2 and myosin A, key components of the gliding motility machinery previously considered essential to invasion, suggests the existence of alternative forces that can drive parasite penetration [8,9]
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