Research Article: RON5 Is Critical for Organization and Function of the Toxoplasma Moving Junction Complex

Date Published: March 20, 2014

Publisher: Public Library of Science

Author(s): Josh R. Beck, Allan L. Chen, Elliot W. Kim, Peter J. Bradley, Michael J. Blackman.


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.

Partial Text

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]. Species of particular interest include Toxoplasma gondii, which chronically infects approximately one-third of all humans and causes neurological disorders in immunocompromised individuals as well as the human malarial agent, Plasmodium falciparum, which is the cause of nearly a million deaths annually [3], [4]. 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]. Thus, a better understanding of the parasite molecules and processes that facilitate host cell invasion is needed to aid in development of better therapeutics and control strategies.

The establishment of a tight-junction interface between invading apicomplexan parasites and their host cells was first observed by electron microscopy over 30 years ago [41]. More recently, the exciting discovery that a complex of rhoptry neck proteins is secreted into this tight-junction provided candidates for understanding the molecular basis for this unique mechanism of host cell penetration [11]. While a relatively thorough characterization of RON protein topology within the MJ has been carried out, a hydrophobic stretch of residues in the N-terminus of RON5 has been noted as a potential transmembrane region, which would impact the positioning of RON5 in this model (see [14], [29]). We show here that the RON5 N-terminal domain in which this hydrophobic region is contained is not a part of the mature MJ complex. Instead, this pro domain likely plays roles in RON5 folding or trafficking as deletion of proRON5 resulted in gross mistargeting of the remainder of the protein. While the RON5 pro region is necessary for trafficking, it does not appear to be sufficient as C-terminal truncations of RON5N also result in mistargeting. The C-terminal region of RON5N is the most highly conserved portion of the protein, potentially suggesting that this region is critical for complex assembly in addition to trafficking. Importantly, a version of RON5 lacking the entire RON5C domain (RON5Δ1258-1702) targets to the rhoptry necks but cannot rescue RON2 stability (see below), showing that RON5 contains the necessary rhoptry neck targeting information independent of RON2.




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