Abstract
The intracellular parasite Toxoplasma gondii employs a vast array of effector proteins from the rhoptry and dense granule organelles to modulate host cell biology; these effectors are known as ROPs and GRAs, respectively. To examine the individual impacts of ROPs and GRAs on host gene expression, we developed a robust, novel protocol to enrich for ultrapure populations of a naturally occurring and reproducible population of host cells called uninfected-injected (U-I) cells, which Toxoplasma injects with ROPs but subsequently fails to invade. We then performed single-cell transcriptomic analysis at 1 to 3 h postinfection on U-I cells (as well as on uninfected and infected controls) arising from infection with either wild-type parasites or parasites lacking the MYR1 protein, which is required for soluble GRAs to cross the parasitophorous vacuole membrane (PVM) and reach the host cell cytosol. Based on comparisons of infected and U-I cells, the host's earliest response to infection appears to be driven primarily by the injected ROPs, which appear to induce immune and cellular stress pathways. These ROP-dependent proinflammatory signatures appear to be counteracted by at least some of the MYR1-dependent GRAs and may be enhanced by the MYR-independent GRAs (which are found embedded within the PVM). Finally, signatures detected in uninfected bystander cells from the infected monolayers suggest that MYR1-dependent paracrine effects also counteract inflammatory ROP-dependent processes.IMPORTANCE This work performs transcriptomic analysis of U-I cells, captures the earliest stage of a host cell's interaction with Toxoplasma gondii, and dissects the effects of individual classes of parasite effectors on host cell biology.
Highlights
The intracellular parasite Toxoplasma gondii employs a vast array of effector proteins from the rhoptry and dense granule organelles to modulate host cell biology; these effectors are known as rhoptry organelle effectors (ROPs) and granule effectors (GRAs), respectively
According to the current model of parasite effector secretion (Fig. 1A), the host response to infection with Toxoplasma tachyzoites can be attributed to 5 parasite-dependent stimuli: (i) the secretion of paracrine effectors into the extracellular milieu, (ii) injection of ROPs, (iii) activity of MYR-independent GRAs (MIGs), (iv) secretion of MYR-dependent GRAs (MDGs), and (v) parasite invasion and establishment of the parasitophorous vacuole (PV)
We devised a novel pipeline in which (i) infections were designed to generate a heterogeneous pool of host cells, each impinged upon by one of several combinations of parasite-dependent stimuli; (ii) a fluorescence-activated cell sorting (FACS)-based protocol was employed to separate host cells of interest from infected monolayers based on the parasite-dependent stimuli by which they were affected; and (iii) the host cell transcriptomes were profiled by full-length scRNA-seq
Summary
The intracellular parasite Toxoplasma gondii employs a vast array of effector proteins from the rhoptry and dense granule organelles to modulate host cell biology; these effectors are known as ROPs and GRAs, respectively. Based on comparisons of infected and U-I cells, the host’s earliest response to infection appears to be driven primarily by the injected ROPs, which appear to induce immune and cellular stress pathways These ROP-dependent proinflammatory signatures appear to be counteracted by at least some of the MYR1-dependent GRAs and may be enhanced by the MYR-independent GRAs (which are found embedded within the PVM). During the acute phase of infection, this unicellular eukaryote rapidly expands within host tissues by penetrating host cells, establishing and replicating within an intracellular parasitophorous vacuole (PV), and simultaneously avoiding clearance by the host immune system (reviewed in reference 2) To orchestrate these events, Toxoplasma employs a vast repertoire of effector proteins housed primarily in two secretory organelles, the rhoptries and dense granules (Fig. 1A). The rhoptry organelle’s contribution is of particular interest given that ROP injection is essential to parasite invasion, survival, and virulence and that the functions of most ROPs are unknown
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