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A druggable secretory protein maturase of Toxoplasma essential for invasion and egress.

ABSTRACT: Micronemes and rhoptries are specialized secretory organelles that deploy their contents at the apical tip of apicomplexan parasites in a regulated manner. The secretory proteins participate in motility, invasion, and egress and are subjected to proteolytic maturation prior to organellar storage and discharge. Here we establish that Toxoplasma gondii aspartyl protease 3 (ASP3) resides in the endosomal-like compartment and is crucially associated to rhoptry discharge during invasion and to host cell plasma membrane lysis during egress. A comparison of the N-terminome, by terminal amine isotopic labelling of substrates between wild type and ASP3 depleted parasites identified microneme and rhoptry proteins as repertoire of ASP3 substrates. The role of ASP3 as a maturase for previously described and newly identified secretory proteins is confirmed in vivo and in vitro. An antimalarial compound based on a hydroxyethylamine scaffold interrupts the lytic cycle of T. gondii at submicromolar concentration by targeting ASP3.

SUBMITTER: Dogga SK

PROVIDER: S-EPMC5595437 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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A druggable secretory protein maturase of Toxoplasma essential for invasion and egress.

Dogga Sunil Kumar SK Mukherjee Budhaditya B Jacot Damien D Kockmann Tobias T Molino Luca L Hammoudi Pierre-Mehdi PM Hartkoorn Ruben C RC Hehl Adrian B AB Soldati-Favre Dominique D

eLife 20170912

Micronemes and rhoptries are specialized secretory organelles that deploy their contents at the apical tip of apicomplexan parasites in a regulated manner. The secretory proteins participate in motility, invasion, and egress and are subjected to proteolytic maturation prior to organellar storage and discharge. Here we establish that <i>Toxoplasma gondii</i> aspartyl protease 3 (ASP3) resides in the endosomal-like compartment and is crucially associated to rhoptry discharge during invasion and to ...[more]

PMID: 28898199

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Project description:Microbial pathogens use proteases for their infections, such as digestion of proteins for nutrients and activation of their virulence factors. As an obligate intracellular parasite, Toxoplasma gondii must invade host cells to establish its intracellular propagation. To facilitate invasion, the parasites secrete invasion effectors from microneme and rhoptry, two unique organelles in apicomplexans. Previous work has shown that some micronemal invasion effectors experience a series of proteolytic cleavages within the parasite's secretion pathway for maturation, such as the aspartyl protease (TgASP3) and the cathepsin L-like protease (TgCPL), localized within the post-Golgi compartment (1) and the endolysosomal system (2), respectively. Furthermore, it has been shown that the precise maturation of micronemal effectors is critical for Toxoplasma invasion and egress (1). Here, we show that an endosome-like compartment (ELC)-residing cathepsin C-like protease (TgCPC1) mediates the final trimming of some micronemal effectors, and its loss further results in defects in the steps of invasion, egress, and migration throughout the parasite's lytic cycle. Notably, the deletion of TgCPC1 completely blocks the activation of subtilisin-like protease 1 (TgSUB1) in the parasites, which globally impairs the surface-trimming of many key micronemal invasion and egress effectors. Additionally, we found that TgCPC1 was not efficiently inhibited by the chemical inhibitor targeting its malarial ortholog, suggesting that these cathepsin C-like orthologs are structurally different within the apicomplexan phylum. Taken together, our findings identify a novel function of TgCPC1 in the processing of micronemal proteins within the secretory pathway of Toxoplasma parasites and expand the understanding of the roles of cathepsin C protease.ImportanceToxoplasma gondii is a microbial pathogen that is well adapted for disseminating infections. It can infect virtually all warm-blooded animals. Approximately one-third of the human population carries toxoplasmosis. During infection, the parasites sequentially secrete protein effectors from the microneme, rhoptry, and dense granule, three organelles exclusively found in apicomplexan parasites, to help establish their lytic cycle. Proteolytic cleavage of these secretory proteins is required for the parasite's optimal function. Previous work has revealed that two proteases residing within the parasite's secretory pathway cleave micronemal and rhoptry proteins, which mediate parasite invasion and egress. Here, we demonstrate that a cathepsin C-like protease (TgCPC1) is involved in processing several invasion and egress effectors. The genetic deletion of TgCPC1 prevented the complete maturation of some effectors in the parasites. Strikingly, the deletion led to a full inactivation of one surface-anchored protease, which globally impaired the trimming of some key micronemal proteins before secretion. Therefore, this finding represents a novel post-translational mechanism for the processing of virulence factors within microbial pathogens.

Project description:Microbial pathogens use proteases for their infections, such as digestion of proteins for nutrients and activation of their virulence factors. As an obligate intracellular parasite, Toxoplasma gondii must invade host cells to establish its intracellular propagation. To facilitate invasion, the parasites secrete invasion effectors from microneme and rhoptry, two unique organelles in apicomplexans. Previous work has shown that some micronemal invasion effectors experience a series of proteolytic cleavages within the parasite's secretion pathway for maturation, such as the aspartyl protease (TgASP3) and the cathepsin L-like protease (TgCPL), localized within the post-Golgi compartment and the endolysosomal system, respectively. Furthermore, it has been shown that the precise maturation of micronemal effectors is critical for Toxoplasma invasion and egress. Here, we show that an endosome-like compartment (ELC)-residing cathepsin C-like protease (TgCPC1) mediates the final trimming of some micronemal effectors, and its loss further results in defects in the steps of invasion, egress, and migration throughout the parasite's lytic cycle. Notably, the deletion of TgCPC1 completely blocks the activation of subtilisin-like protease 1 (TgSUB1) in the parasites, which globally impairs the surface-trimming of many key micronemal invasion and egress effectors. Additionally, we found that Toxoplasma is not efficiently inhibited by the chemical inhibitor targeting the malarial CPC ortholog, suggesting that these cathepsin C-like orthologs are structurally different within the apicomplexan phylum. Collectively, our findings identify a novel function of TgCPC1 in processing micronemal proteins within the Toxoplasma parasite's secretory pathway and expand the understanding of the roles of cathepsin C protease. IMPORTANCE Toxoplasma gondii is a microbial pathogen that is well adapted for disseminating infections. It can infect virtually all warm-blooded animals. Approximately one-third of the human population carries toxoplasmosis. During infection, the parasites sequentially secrete protein effectors from the microneme, rhoptry, and dense granule, three organelles exclusively found in apicomplexan parasites, to help establish their lytic cycle. Proteolytic cleavage of these secretory proteins is required for the parasite's optimal function. Previous work has revealed that two proteases residing within the parasite's secretory pathway cleave micronemal and rhoptry proteins, which mediate parasite invasion and egress. Here, we demonstrate that a cathepsin C-like protease (TgCPC1) is involved in processing several invasion and egress effectors. The genetic deletion of TgCPC1 prevented the complete maturation of some effectors in the parasites. Strikingly, the deletion led to a full inactivation of one surface-anchored protease, which globally impaired the trimming of some key micronemal proteins before secretion. Therefore, this finding represents a novel post-translational mechanism for the processing of virulence factors within microbial pathogens.

Project description:Apicomplexan parasites are dependent on an F-actin and myosin-based motility system for their invasion into and escape from animal host cells, as well as for their general motility. In Toxoplasma gondii and Plasmodium species, the actin filaments and myosin motor required for this process are located in a narrow space between the parasite plasma membrane and the underlying inner membrane complex, a set of flattened cisternae that covers most the cytoplasmic face of the plasma membrane. Here we show that the energy required for Toxoplasma motility is derived mostly, if not entirely, from glycolysis and lactic acid production. We also demonstrate that the glycolytic enzymes of Toxoplasma tachyzoites undergo a striking relocation from the parasites' cytoplasm to their pellicles upon Toxoplasma egress from host cells. Specifically, it appears that the glycolytic enzymes are translocated to the cytoplasmic face of the inner membrane complex as well as to the space between the plasma membrane and inner membrane complex. The glycolytic enzymes remain pellicle-associated during extended incubations of parasites in the extracellular milieu and do not revert to a cytoplasmic location until well after parasites have completed invasion of new host cells. Translocation of glycolytic enzymes to and from the Toxoplasma pellicle appears to occur in response to changes in extracellular [K(+)] experienced during egress and invasion, a signal that requires changes of [Ca(2+)](c) in the parasite during egress. Enzyme translocation is, however, not dependent on either F-actin or intact microtubules. Our observations indicate that Toxoplasma gondii is capable of relocating its main source of energy between its cytoplasm and pellicle in response to exit from or entry into host cells. We propose that this ability allows Toxoplasma to optimize ATP delivery to those cellular processes that are most critical for survival outside host cells and those required for growth and replication of intracellular parasites.

Dataset Information

A druggable secretory protein maturase of Toxoplasma essential for invasion and egress.

Publications

A druggable secretory protein maturase of Toxoplasma essential for invasion and egress.

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