Differential Impacts on Host Transcription by ROP and GRA Effectors from the Intracellular ParasiteToxoplasma gondii

Abstract

words 16Importance: 36 words ABSTRACT 30The intracellular parasite Toxoplasma gondii employs a vast array of effector 31 proteins from the rhoptry and dense granule organelles to modulate host cell biology; 32 these effectors are known as ROPs and GRAs, respectively. To examine the individual 33 impacts of ROPs and GRAs on host gene expression, we developed a robust, novel 34 protocol to enrich for ultra-pure populations of a naturally occurring and reproducible 35 population of host cells called uninfected-injected (U-I) cells, which Toxoplasma injects 36with ROPs but subsequently fails to invade. We then performed single cell 37 transcriptomic analysis at 1-3 hours post-infection on U-I cells (as well as on uninfected 38 and infected controls) arising from infection with either wild type parasites or parasites 39 lacking the MYR1 protein, which is required for soluble GRAs to cross the 40 parasitophorous vacuole membrane (PVM) and reach the host cell cytosol. Based on 41 comparisons of infected and U-I cells, the host's earliest response to infection appears 42 to be driven primarily by the injected ROPs, which appear to induce immune and 43 cellular stress pathways. These ROP-dependent pro-inflammatory signatures appear to 44 be counteracted by at least some of the MYR1-dependent GRAs and may be enhanced 45 by the MYR-independent GRAs, (which are found embedded within the PVM). Finally, 46 signatures detected in uninfected bystander cells from the infected monolayers 47 suggests that MYR1-dependent paracrine effects also counteract inflammatory ROP-48 dependent processes. 49 50 IMPORTANCE 51 This work performs the first transcriptomic analysis of U-I cells, captures the 52 earliest stage of a host cell's interaction with Toxoplasma gondii, and dissects the 53 effects of individual classes of parasite effectors on host cell biology. 54 55 MAIN TEXT 56 Introduction 57The obligate intracellular parasite Toxoplasma gondii parasitizes a wide range of 58 avian and mammalian organisms, including humans (1). During the acute phase of 59 infection, this unicellular eukaryote rapidly expands within host tissues by penetrating 60 host cells, establishing and replicating within an intracellular parasitophorous vacuole 61 (PV), and simultaneously avoiding clearance by the host immune system (reviewed in 62(2)). To orchestrate these events (Fig. 1A), Toxoplasma employs a vast repertoire of 63 effector proteins housed primarily in two secretory organelles, the rhoptries and dense 64 granules. The rhoptry organelle effectors (ROPs) are secreted by the parasite into the 65 host cytosol during or immediately prior to invasion by an as yet undefined mechanism 66 (reviewed in (3)). The handful of ROPs that have been characterized to date include 67 virulence factors that disrupt immune clearance mechanisms (4-7), remodel the host's 68 cortical actin cytoskeleton at the point of parasite penetration (8, 9), and co-opt the 69 STAT3 and STAT6 pathways (10-12). In contrast, the dense granule effectors (GRAs) 70 are thought to be secr...

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