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An aspartyl protease defines a novel pathway for export of Toxoplasma proteins into the host cell

ABSTRACT: Toxoplasma gondii is an obligate intracellular Apicomplexan parasite capable of invading and surviving within nucleated cells in most warm-blooded animals. This remarkable task is achieved through the delivery of effector proteins from the parasite into the parasitophorous vacuole and host cell cytosol that rewire host cellular pathways, facilitating parasite evasion of the immune system. Here, we have identified a novel export pathway in Toxoplasma that involves cleavage of effector proteins by the Golgi-resident aspartyl protease 5 (ASP5) prior to translocation into the host cell. We demonstrate that ASP5 cleaves a highly constrained amino acid motif that has some similarity to the PEXEL motif of Plasmodium parasites. We show that ASP5 can mature effectors at both the N- and C-terminal ends of proteins and is also required for the trafficking of proteins without this motif. Furthermore, we show that ASP5 controls establishment of the nanotubular network and is required for the efficient recruitment of host mitochondria to the parasitophorous vacuole membrane. Global assessment of host gene expression following infection reveals that ASP5-dependent pathways influence thousands of the transcriptional changes that Toxoplasma imparts on its host cell. This work characterizes the first identified machinery required for export of Toxoplasma effectors into the infected host cell.

ORGANISM(S): Homo sapiens

PROVIDER: GSE73986 | GEO | 2015/11/19

SECONDARY ACCESSION(S): PRJNA298673

REPOSITORIES: GEO

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Project description:The intracellular parasite Toxoplasma gondii uses a diverse repertoire of effector molecules to manipulate its host cell. The first effectors to be introduced into the host cell derive from rhoptries (ROPs) and are secreted exclusively during invasion. Once the parasite has established a parasitophorous vacuole, a subset of secreted dense granule effector proteins (GRAs) are then translocated across the parasitophorous vacuolar membrane in a MYR1-dependent manner to enter the host cell cytoplasm and nucleus. Translocation of these GRA effectors is also dependent on the activity of Toxoplasma Aspartyl protease 5 (ASP5) which either directly cleaves the effectors or potentiates translocation in some other way. To determine the totality of MYR1-dependent (predominantly GRA) and MYR1-independent (e.g., ROP) host responses as well as the degree of overlap between MYR1- and ASP5-dependent effectors, we analyzed the RNAseq expression profile of human foreskin fibroblasts infected with wild type (RH strain) Toxoplasma gondii tachyzoites, RHΔmyr1, RHΔasp5 and their respective complements. We show that a majority subset of the differentially regulated host gene responses during infection with wild type parasites are modulated in a MYR1-dependent manner. Gene set enrichment analysis of the MYR1-dependent host responses indicates that, among others, there is a clear up-regulation related to E2F transcription factors and the cell cycle and a clear down-regulation in expression related to interferon signaling. The ASP5-dependent host gene responses were less numerous and encompassed fewer gene sets than the MYR1-dependent responses, indicating that translocation and/or activity of a subset of effectors is MYR1- but not ASP5-dependent. Consistent with this, we show that even though MYR1 is normally cleaved by ASP5, such cleavage is not necessary for MYR1’s role in translocation. Interestingly, some responses were seen upon infection with RHΔmyr1 but not RH-WT; these previously “hidden” responses are likely due to the normally counter-balancing action of MYR1-dependent and -independent activities. The host genes and gene sets revealed here to be MYR1-dependent provide new insight into the relative contribution of GRA- vs. ROP-mediated effectors in co-opting host cell functions.

Project description:Toxoplasma gondii is an obligate intracellular parasite that must establish a favorable environment in the host cells in which it replicates. To do this, it controls key host cell processes by MYR-dependent translocation of dense granule proteins from the parasitophorous vacuole into the host cytosol. Using RNASeq, we previously reported that mutants lacking the MYR translocation apparatus fail to elicit a key set of host responses related to regulation of the cell cycle, including cyclin E and other targets of E2F transcription factors. As no previously reported effector was known to mediate such an effect, we report here our application of a set of criteria to proteins secreted by Toxoplasma leading to our discovery of a novel effector protein that induces the host to produce cyclin E. This inducer of Host Cyclin E (HCE1) is exported from the parasitophorous vacuole in a manner dependent on MYR1 and ASP5, another component of the export system, where it then localizes to the host’s nucleus. HCE1 expression is important for parasite growth as those lacking it produce plaques that are ~35% smaller in area than wild type parasites. RNASeq of host cells infected with parasites lacking HCE1 compared to those infected with wild type parasites shows a complete loss of the parasite’s ability to modulate key host cell cycle genes including those related to the E2F axis. Immunoprecipitation of HCE1 from infected host cells shows that HCE1 efficiently binds elements of the cyclin E regulatory complex: DP1 and its partners E2F3 and E2F4. Expression of HCE1 in Neospora caninum, which is not otherwise capable of up-regulating cyclin E, or in uninfected HFFs, shows nuclear localization of the expressed protein and results in strong cyclin E up-regulation. Thus, HCE1 is a novel, stand-alone effector protein that is necessary and sufficient to impact the E2F-axis of transcription resulting in co-opting of host functions to Toxoplasma’s advantage.

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An aspartyl protease defines a novel pathway for export of Toxoplasma proteins into the host cell

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