The <i>Toxoplasma gondii</i> virulence factor ROP16 acts in cis and trans, and suppresses T cell responses

Chen, Longfei; Christian, David A.; Kochanowsky, Joshua A.; Phan, Anthony T.; Clark, Joseph T.; Wang, Shuai; Berry, Corbett T.; Oh, Jung Yeol; Chen, Xiaoguang; Roos, David S.; Beiting, Daniel P.; Koshy, Anita A.; Hunter, Christopher A.

doi:10.1084/jem.20181757

Cited by 49 publications

(55 citation statements)

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“…3B, C, Table S2). Collectively, these findings are consistent with prior studies of infected non-neuronal cells 3,38,39 , indicating that injection with…”

Section: Isolation Of Neurons Using Laser Capture Microdissectionsupporting

confidence: 91%

Transcriptional profiling reveals T cells cluster around neurons injected withToxoplasma gondiiproteins

Merritt

Johnson

Wong

et al. 2020

Preprint

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Toxoplasma gondii's tropism for and persistence in the CNS underlies the symptomatic disease Toxoplasma causes in humans. Our recent work has shown that neurons are the primary CNS cell with which Toxoplasma interacts and infects in vivo.This predilection for neurons suggests that Toxoplasma's persistence in the CNS depends specifically upon parasite manipulation of the host neurons. Yet, most work on Toxoplasma-host cell interactions has been done in vitro and in non-neuronal cells. We address this gap by utilizing our Toxoplasma-Cre system that allows permanent marking and tracking of neurons injected with parasite effector proteins in vivo. Using laser capture microdissection (LCM) and RNA-seq, we isolated and transcriptionally profiled Toxoplasma-injected neurons (TINs), Bystander neurons (nearby non-Toxoplasma injected neurons), and neurons from uninfected mice (controls). These profiles show that TINs transcriptomes significantly differ from the transcriptomes of Bystander and control neurons and that much of this difference is driven by increased levels of transcripts from immune cells, especially CD8 + T cells and monocytes. These data suggest that when we used LCM to isolate neurons from infected mice, we also picked up fragments of CD8 + T cells and monocytes clustering in extreme proximity around TINs and, to a lesser extent, Bystander neurons. In addition, we found that Toxoplasma transcripts were primarily found in the TINs transcriptome, not in the Bystander transcriptome. Collectively, these data suggest that, contrary to common perception, neurons that directly interact with or harbor parasites can be recognized by CD8 + T cells.

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“…3B, C, Table S2). Collectively, these findings are consistent with prior studies of infected non-neuronal cells 3,38,39 , indicating that injection with…”

Section: Isolation Of Neurons Using Laser Capture Microdissectionsupporting

confidence: 91%

Transcriptional profiling reveals T cells cluster around neurons injected withToxoplasma gondiiproteins

Merritt

Johnson

Wong

et al. 2020

Preprint

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“…In this study, we examined host responses to infection with the parasite Toxoplasma gondii using scRNA-seq on in vitro uninfected-injected (U-I) host cells, which arise from aborted invasion events and that have until very recently (44) been characterized primarily morphologically. Key fixtures of our experimental pipeline included: 1) early time points, to limit isolating false positive U-I cells arising from mechanisms besides aborted invasion; 2) FACS sorts, which purified rare U-I cells and relatively rare infected cells at early time points; and 3) single cell resolution, which enabled bioinformatic validation of all cells’ infection status.…”

Section: Discussionmentioning

confidence: 99%

“…They may also explain the recent finding that the avirulent phenotype of !′1myr1 parasites during in vivo mouse infections is rescuable by co-infecting animals with both wild type and MYR1-deficient parasites (46); parasites expressing MYR1 may induce host cells to secrete paracrine factors that suppress transcription of inflammatory gene products that would otherwise limit !′1myr1 parasite infections. Note that while this manuscript was in preparation, a transcriptomic study of U-I macrophages was published by Hunter and colleagues (44). While they used different strains (Pru and CEP), looked at later time points (20-24 hpi), and did not look at MYR1-dependent effects, their primary conclusions that U-I cells experience a major impact of rhoptry effectors (in their case, specifically ROP16), and that paracrine effects are also in play, are similar to the conclusions reached here.…”

Section: Discussionmentioning

confidence: 99%

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

Rastogi

Xue

Quake

et al. 2020

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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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“…Together, GRA15 and GRA24 drive the classical activation of macrophages (M1) via the activation of NFκB and p38 MAPK (35, 37, 38). By contrast, the polymorphic kinase ROP16 from type I and type III strains drives the alternative activation of macrophages (M2) via the phosphorylation of the STAT6 and STAT3 transcription factors (37, 39, 40). It is likely that the deliberate activation of the immune response by Toxoplasma effectors is a strategy to limit its virulence thereby promoting the survival of its host and the formation of tissue cysts, which are the only stages in the intermediate host that are orally infectious.…”

Section: Introductionmentioning

confidence: 99%

ToxoplasmaGRA15 and GRA24 are important activators of the host innate immune response in the absence of TLR11

Mukhopadhyay

Arranz-Solís

2020

Preprint

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15The murine innate immune response against Toxoplasma gondii is predominated by the 16 interaction of TLR11/12 with Toxoplasma profilin. However, mice lacking Tlr11 or 17 humans, who do not have functional TLR11 or TLR12, still elicit a strong innate immune 18 93 and GRA24 drive the classical activation of macrophages (M1) via the activation of 94 NFκB and p38 MAPK (35, 37, 38). By contrast, the polymorphic kinase ROP16 from 95 type I and type III strains drives the alternative activation of macrophages (M2) via the 96 phosphorylation of the STAT6 and STAT3 transcription factors (37, 39, 40). It is likely 97 that the deliberate activation of the immune response by Toxoplasma effectors is a 98 strategy to limit its virulence thereby promoting the survival of its host and the formation 99 of tissue cysts, which are the only stages in the intermediate host that are orally 100 infectious. 101Given the large impact of GRA15 and GRA24 on macrophage gene expression 102 and production of IL12 and IL1β, it seems surprising that their in vivo effect on parasite 103 virulence is relatively minor (31, 35). Mice infected with type II Δgra15 or Δgra24 104 parasites had elevated parasite numbers early after infection, but as the infection 105 progressed, parasite burden and host susceptibility were no different from those 106 following wild-type type II strain infections (31, 35, 41). Increased type II Δgra15 early 107 parasite burdens were associated with decreased IL12 and IFNγ levels 2 days after 108 infection (31). Similar results were obtained after infection of mice with the Δgra24 strain 109 (35). Possibly, the effects of GRA15 and GRA24 in these studies were masked by 110 profilin: as the infection progresses and parasites lyse out of host cells, PAMPS, such 111 as profilin, are released and activate TLR11/12. At this stage, IL12/IL1β and subsequent 112 IFNγ production are probably no longer dependent on GRA15 and GRA24. However, 113 humans and many animals do not have functional TLR11/12 or IFNγ-inducible IRGs 114 (17), and therefore Toxoplasma virulence of a particular strain in mice might not 115 correlate with virulence in other species. In Tlr11 -/mice, neutrophils are the main 116 producers of IFNγ with a minor role of NK and T cells (42). The production of IFNγ by 117 neutrophils is dependent on IL1β and TNFα, but not on IL12 (42). In addition, IL18 118 secreted upon inflammasome activation plays a key role in the IFNγ response from 119 CD4 + T cells and the subsequent disease outcome in Tlr11 -/mice (43). Thus, GRA15 120 and GRA24, by inducing IL1β, IL18, TNFα and IL12, might play an important role in the 121 production of IFNγ in hosts lacking TLR11. Herein, we tested this hypothesis by 122 infecting Tlr11 -/mice with wild-type, Δgra15, Δgra24 and Δgra15/24 parasites. Our data 123 indicate that although parasites that do not express GRA15 and/or GRA24 induced 124 significantly less inflammatory cytokines, a significant increase in virulence compared to 125 wild-type was only observed after subcutaneous infecti...

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The Toxoplasma gondii virulence factor ROP16 acts in cis and trans, and suppresses T cell responses

Cited by 49 publications

References 70 publications

Transcriptional profiling reveals T cells cluster around neurons injected withToxoplasma gondiiproteins

Transcriptional profiling reveals T cells cluster around neurons injected withToxoplasma gondiiproteins

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

ToxoplasmaGRA15 and GRA24 are important activators of the host innate immune response in the absence of TLR11

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