Unknown,Transcriptomics,Genomics,Proteomics

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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. Three groups of human foreskin fibroblasts are compared. Each group has 3 replicates giving a total of 9 samples. The first group of samples are infected with wild type (GRA16HA) Toxoplasma gondii, the second group with Asp5 knock-out Toxoplasma gondii, and the final group remain uninfected. All fibroblasts are generated from one donor sample.

ORGANISM(S): Homo sapiens

SUBMITTER: Alexandra Garnham

PROVIDER: E-GEOD-73986 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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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