Intracellular pathogens have evolved a wide array of mechanisms to invade and co-opt their host cells for intracellular survival. Apicomplexan parasites such as Toxoplasma gondii employ the action of unique secretory organelles named rhoptries for internalization of the parasite and formation of a specialized niche within the host cell. We demonstrate that Toxoplasma gondii also uses secretion from the rhoptries during invasion to deliver a parasite-derived protein phosphatase 2C (PP2C-hn) into the host cell and direct it to the host nucleus. Delivery to the host nucleus does not require completion of invasion, as evidenced by the fact that parasites blocked in the initial stages of invasion with cytochalasin D are able to target PP2C-hn to the host nucleus. We have disrupted the gene encoding PP2C-hn and shown that PP2C-hn-knockout parasites exhibit a mild growth defect that can be rescued by complementation with the wild-type gene. The delivery of parasite effector proteins via the rhoptries provides a novel mechanism for Toxoplasma to directly access the command center of its host cell during infection by the parasite.Toxoplasma gondii is an obligate intracellular parasite in the phylum Apicomplexa that causes severe central nervous system disorders of immunocompromised (AIDS/transplant/lymphoma) individuals and birth defects in congenitally infected neonates worldwide (16). Toxoplasma infects a wide range of mammalian hosts and is capable of infecting virtually any nucleated cell type from these organisms. The parasite actively invades its host cell, establishing a specialized parasitophorous vacuole (PV) within the host cytoplasm (22). This vacuole fails to fuse with the host endocytic or exocytic pathways, thus avoiding lysosomal destruction, and provides a residence in which parasites can replicate within the host cell (29, 37). The processes of invasion and vacuole formation therefore establish an intimate yet separate association between the parasite and its host cell.Host cell invasion and PV formation are mediated in part by the action of the rhoptries, specialized secretory organelles that release their contents at the onset of invasion (32). The club-shaped rhoptries are composed of two suborganellar domains, the bulbous rhoptry bodies and the duct-like rhoptry necks. These domains appear to carry out very different roles in host cell invasion and establishment of the intracellular niche for survival. Proteins secreted from the rhoptry necks have recently been shown to be released into the moving junction, a ring-shaped structure that forms the intersection between the invading parasite and the host plasma membrane (1, 6). Rhoptry neck proteins in the moving junction likely serve to filter host transmembrane proteins from the nascent PV during invasion, a process that contributes to the nonfusogenic nature of the vacuole within the host cell. Rhoptry proteins from the other subcompartment, the rhoptry bodies, are secreted into the nascent PV, where they are destined to remain within the vacuole or a...
Oxidative stress is a key player in a variety of neurodegenerative disorders including Parkinson's disease. Widely used as a parkinsonian mimetic, 6-hydroxydopamine (6-OHDA) generates reactive oxygen species (ROS) as well as coordinated changes in gene transcription associated with the unfolded protein response (UPR) and apoptosis. Whether 6-OHDA-induced UPR activation is dependent on ROS has not yet been determined. The present study used molecular indicators of oxidative stress to place 6-OHDA-generated ROS upstream of the appearance of UPR markers such as activating transcription factor 3 (ATF3) and phosphorylated stress-activated protein kinase (SAPK/JNK) signaling molecules. Antioxidants completely blocked 6-OHDA-mediated UPR activation and rescued cells from toxicity. Moreover, cytochrome c release from mitochondria was observed after the appearance of early UPR markers, suggesting that cellular stress pathways are responsible for its release. Mechanistically, the 6-OHDA-induced UPR was independent of intracellular calcium changes. Rather, evidence of protein oxidation was observed before the expression of UPR markers, suggesting that the rapid accumulation of damaged proteins triggered cell stress/UPR. Taken together, 6-OHDAmediated cell death in dopaminergic cells proceeds via ROSdependent UPR up-regulation which leads to an interaction with the intrinsic mitochondrial pathway and downstream caspase activation.
Toxoplasma gondii is an obligate intracellular parasite that resides in the cytoplasm of its host in a unique membrane-bound vacuole known as the parasitophorous vacuole (PV). The membrane surrounding the parasite is remodeled by the dense granules, secretory organelles that release an array of proteins into the vacuole and to the PV membrane (PVM). Only a small portion of the protein constituents of the dense granules have been identified, and little is known regarding their roles in infection or how they are trafficked within the infected host cell. In this report, we identify a novel secreted dense granule protein, GRA14, and show that it is targeted to membranous structures within the vacuole known as the intravacuolar network and to the vacuolar membrane surrounding the parasite. We disrupted GRA14 and exploited the knockout strain to show that GRA14 can be transferred between vacuoles in a coinfection experiment with wild-type parasites. We also show that GRA14 has an unexpected topology in the PVM with its C terminus facing the host cytoplasm and its N terminus facing the vacuolar lumen. These findings have important implications both for the trafficking of GRA proteins to their ultimate destinations and for expectations of functional domains of GRA proteins at the host-parasite interface.Capable of infecting essentially any warm-blooded vertebrate, Toxoplasma gondii is one of the most successful pathogens on the planet (20, 39). Toxoplasma infects nearly onethird of the human population and causes potentially fatal disease in immunocompromised individuals and congenitally infected neonates (20). This protozoan parasite also causes ocular disease in immunocompetent individuals who are either congenitally or postnatally infected (46). As an obligate intracellular parasite, Toxoplasma enters the host cell into a nonfusogenic vacuole (the parasitophorous vacuole [PV]), in which the parasite replicates in the cytoplasm of its host. The PV membrane (PVM) is porous to small molecules (less than 1,300 Da) but otherwise serves as a boundary between the host and parasite during its intracellular survival (36).Toxoplasma invasion is mediated by a trio of specialized secretory organelles, named the micronemes, rhoptries, and dense granules, which contribute to the parasite's ability to initiate and sustain infection within its host. The first proteins secreted are from the micronemes, which release molecular adhesins that interact with the parasite's actin-myosin motor to provide the driving force for invasion (24). The rhoptries are then released and help to establish the nascent PV and modulate host cell processes (4). Lastly, proteins from the dense granules that are implicated in the remodeling and maintenance of the PV for intracellular survival are secreted (29).The precise role of dense granule proteins (GRAs) in the T. gondii life cycle is still largely unknown. To date, two groups of GRA proteins have been identified. The first group contains proteins that lack homology to organisms other than closely relat...
Toxoplasma gondii utilizes specialized secretory organelles called rhoptries to invade and hijack its host cell. Many rhoptry proteins are proteolytically processed at a highly conserved SΦXE site to remove organellar targeting sequences that may also affect protein activity. We have studied the trafficking and biogenesis of a secreted rhoptry metalloprotease with homology to insulysin that we named Toxolysin-1 (TLN1). Through genetic ablation and molecular dissection of TLN1 we have identified the smallest rhoptry targeting domain yet reported and expanded the consensus sequence of the rhopty pro-domain cleavage site. In addition to removal of its pro-domain, Toxolysin-1 undergoes a C-terminal cleavage event that occurs at a processing site not previously seen in Toxoplasma rhoptry proteins. While pro-domain cleavage occurs in the nascent rhoptries, processing of the C-terminal region precedes commitment to rhoptry targeting, suggesting that it is mediated by a different maturase, and we have identified residues critical for proteolysis. We have additionally shown that both pieces of TLN1 associate in a detergent resistant complex, formation of which is necessary for trafficking of the C-terminal portion to the rhoptries. Together, these studies reveal novel processing and trafficking events that are present in the protein constituents of this unusual secretory organelle.
Like most intracellular pathogens, Toxoplasma synthesizes and secretes an arsenal of proteins to successfully invade its host cell and hijack host functions for intracellular survival. The rhoptries are key secretory organelles that inject proteins into the host cell where they are positioned to coopt host processes, although little is known regarding how these proteins exert their functions. We show here that the rhoptry protein ROP13 is synthesized as a pre-pro-protein that is processed in the parasite. Processing occurs at a conserved SϕXE cleavage site as mutagenesis of glutamic acid to alanine at the P1 position disrupts ROP13 maturation. We also demonstrate that processing of the prodomain is not necessary for rhoptry targeting and secretion. While gene disruption reveals that ROP13 is not essential for growth in fibroblasts in vitro or for virulence in vivo, we find that ROP13 is a soluble effector protein that can access the cytoplasm of host cells. Exogenously expressed ROP13 in human cells remains cytosolic but also appears toxic, suggesting that overexpression of this effector protein is disrupting some function within the host cell.
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