Overexpression screen of interferon-stimulated genes identifies RARRES3 as a restrictor of Toxoplasma gondii infection
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Evaluation Summary:
Toxoplasma gondii is a widespread parasite of warm blooded animals, with estimates suggesting 2 billion people are currently and chronically infected with this pathogen. Many questions remain as to how humans control and eliminate T. gondii following infection. In this manuscript, Rinkenberger et al. reveal a previously unidentified and understudied host factor, RARRES3 that promotes cell autonomous control of T. gondii in human cells. The precise mechanism of control and its in vivo relevance remain areas for additional work.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Abstract
Toxoplasma gondii is an important human pathogen infecting an estimated one in three people worldwide. The cytokine interferon gamma (IFNγ) is induced during infection and is critical for restricting T. gondii growth in human cells. Growth restriction is presumed to be due to the induction of interferon-stimulated genes (ISGs) that are upregulated to protect the host from infection. Although there are hundreds of ISGs induced by IFNγ, their individual roles in restricting parasite growth in human cells remain somewhat elusive. To address this deficiency, we screened a library of 414 IFNγ induced ISGs to identify factors that impact T. gondii infection in human cells. In addition to IRF1, which likely acts through the induction of numerous downstream genes, we identified RARRES3 as a single factor that restricts T. gondii infection by inducing premature egress of the parasite in multiple human cell lines. Overall, while we successfully identified a novel IFNγ induced factor restricting T. gondii infection, the limited number of ISGs capable of restricting T. gondii infection when individually expressed suggests that IFNγ-mediated immunity to T. gondii infection is a complex, multifactorial process.
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Author Response:
Reviewer #1 (Public Review):
Rinkenberger et al. take a forward genetics ORF overexpression approach to identify human interferon (IFN)-inducible gene (ISG) products driving host defense to the protozoan pathogen Toxoplasma. The screen encompassing approximately 500 ISG identifies 3 ISG candidates and is able to validate 2 of these 3, namely the transcription factor IRF1 and the retinoic acid receptor responsive gene RARRES3, which becomes the focus of the study. Using gain- and loss-of-function approaches the study demonstrates that RARRES3 promotes the reduction of parasitic burden in human cell lines. Importantly, the study provides evidence linking RARRES3 functionally to the previously reported interferon-inducible defense mechanism of host-mediated parasite extrusion. Overall, the discovery of RARRES3 as an …
Author Response:
Reviewer #1 (Public Review):
Rinkenberger et al. take a forward genetics ORF overexpression approach to identify human interferon (IFN)-inducible gene (ISG) products driving host defense to the protozoan pathogen Toxoplasma. The screen encompassing approximately 500 ISG identifies 3 ISG candidates and is able to validate 2 of these 3, namely the transcription factor IRF1 and the retinoic acid receptor responsive gene RARRES3, which becomes the focus of the study. Using gain- and loss-of-function approaches the study demonstrates that RARRES3 promotes the reduction of parasitic burden in human cell lines. Importantly, the study provides evidence linking RARRES3 functionally to the previously reported interferon-inducible defense mechanism of host-mediated parasite extrusion. Overall, the discovery of RARRES3 as an anti-parasitic factor is potentially of broad interest to the field of innate immunity, parasitology and, more generally, microbial pathogenesis, although its physiologically importance or its role in host defense to pathogens other than Toxoplasma was not explored in this study.
Strengths:
The paper takes an unbiased genetics approach to identify novel human genes that execute cell-autonomous host defense against the parasite Toxoplasma
The study is well controlled and convincingly demonstrates that RARRES3 limits parasitic burden in human cell lines, using both gain- and loss-of-function approaches.
The study provides indirect evidence that RARRES3 mediates the expulsion of parasites from infected cells
The study shows that some clonal lineages of Toxoplasma are resistant to RARRES3-mediated immunity suggesting that some Toxoplasma strains may have evolved mechanisms to counteract the host defense pathway(s) regulated by RARRES3.
Weaknesses:
The physiological relevance of RARRES3-mediated parasite egress during the course of Toxoplasma infections is unclear and not discussed.
We added a section in the Discussion covering the potential physiological relevance of RARRES3.
Regarding the failure to see an IDO phenotype (Fig. 1F), the authors may consider that there standard media and serum contains relatively high concentrations of tryptophan (Materials and Methods doesn't provide any information on the exact trp concentration used) and that IDO cannot catabolize the excess amount of tryptophan present in media + serum to achieve tryptophan starvation conditions. I believe previous studies demonstrating IDO-mediated nutritional immunity in cell culture used trp-limited culture conditions. Without any careful experiments using titrated concentrations of trp, the conclusion that IDO cannot restrict Toxo in A549 cells does not seem justified
A brief discussion of this point has been added to the results discussing figure 1F and the conclusions have been toned down. We have specified in the text that our culture medium (DMEM containing 10% FBS) contains 16 ug/ml concentration of tryptophan. Although this might mask the effects of IDO, we are able to appreciate inhibition of parasite growth in response to INF-g, suggesting this pathway is not the most important in A549 cells. We agree with the importance and requirement of tryptophan for parasite growth, we just didn't observe the involvement of IDO1 in our experimental set up.
The authors state that RARRES3 deficiency was complemented with RARRES3 ectopic expression. However, it is unclear from the data presentation whether complemented KOs are statistically different from controls (KO + FLUC) under IFNgamma primed conditions (Fig. 5B) and thus whether complementation was actually achieved.
The complemented KO is not significantly different from WT demonstrating complementation. A “ns” comparison between these bars has been added to the figure for clarity.
The paper lacks any direct evidence for RARRES3-mediated parasite egress.
We conducted a live imaging experiment to directly observed parasite egress in RARRES3 and FLUC ectopically expressing A549s. The data is presented in figure 6C and videos 1-2.
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Evaluation Summary:
Toxoplasma gondii is a widespread parasite of warm blooded animals, with estimates suggesting 2 billion people are currently and chronically infected with this pathogen. Many questions remain as to how humans control and eliminate T. gondii following infection. In this manuscript, Rinkenberger et al. reveal a previously unidentified and understudied host factor, RARRES3 that promotes cell autonomous control of T. gondii in human cells. The precise mechanism of control and its in vivo relevance remain areas for additional work.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Rinkenberger et al. take a forward genetics ORF overexpression approach to identify human interferon (IFN)-inducible gene (ISG) products driving host defense to the protozoan pathogen Toxoplasma. The screen encompassing approximately 500 ISG identifies 3 ISG candidates and is able to validate 2 of these 3, namely the transcription factor IRF1 and the retinoic acid receptor responsive gene RARRES3, which becomes the focus of the study. Using gain- and loss-of-function approaches the study demonstrates that RARRES3 promotes the reduction of parasitic burden in human cell lines. Importantly, the study provides evidence linking RARRES3 functionally to the previously reported interferon-inducible defense mechanism of host-mediated parasite extrusion. Overall, the discovery of RARRES3 as an anti-parasitic factor …
Reviewer #1 (Public Review):
Rinkenberger et al. take a forward genetics ORF overexpression approach to identify human interferon (IFN)-inducible gene (ISG) products driving host defense to the protozoan pathogen Toxoplasma. The screen encompassing approximately 500 ISG identifies 3 ISG candidates and is able to validate 2 of these 3, namely the transcription factor IRF1 and the retinoic acid receptor responsive gene RARRES3, which becomes the focus of the study. Using gain- and loss-of-function approaches the study demonstrates that RARRES3 promotes the reduction of parasitic burden in human cell lines. Importantly, the study provides evidence linking RARRES3 functionally to the previously reported interferon-inducible defense mechanism of host-mediated parasite extrusion. Overall, the discovery of RARRES3 as an anti-parasitic factor is potentially of broad interest to the field of innate immunity, parasitology and, more generally, microbial pathogenesis, although its physiologically importance or its role in host defense to pathogens other than Toxoplasma was not explored in this study.
Strengths:
The paper takes an unbiased genetics approach to identify novel human genes that execute cell-autonomous host defense against the parasite Toxoplasma
The study is well controlled and convincingly demonstrates that RARRES3 limits parasitic burden in human cell lines, using both gain- and loss-of-function approaches.The study provides indirect evidence that RARRES3 mediates the expulsion of parasites from infected cells
The study shows that some clonal lineages of Toxoplasma are resistant to RARRES3-mediated immunity suggesting that some Toxoplasma strains may have evolved mechanisms to counteract the host defense pathway(s) regulated by RARRES3.
Weaknesses:
The physiological relevance of RARRES3-mediated parasite egress during the course of Toxoplasma infections is unclear and not discussed.
Regarding the failure to see an IDO phenotype (Fig. 1F), the authors may consider that there standard media and serum contains relatively high concentrations of tryptophan (Materials and Methods doesn't provide any information on the exact trp concentration used) and that IDO cannot catabolize the excess amount of tryptophan present in media + serum to achieve tryptophan starvation conditions. I believe previous studies demonstrating IDO-mediated nutritional immunity in cell culture used trp-limited culture conditions. Without any careful experiments using titrated concentrations of trp, the conclusion that IDO cannot restrict Toxo in A549 cells does not seem justified
The authors state that RARRES3 deficiency was complemented with RARRES3 ectopic expression. However, it is unclear from the data presentation whether complemented KOs are statistically different from controls (KO + FLUC) under IFNgamma primed conditions (Fig. 5B) and thus whether complementation was actually achieved.
The paper lacks any direct evidence for RARRES3-mediated parasite egress.
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Reviewer #2 (Public Review):
The manuscript by Rinkenberger et al. titled "Over-expression Screen of Interferon-Stimulated Genes Identifies RARRES3 as a Restrictor of Toxoplasma gondii Infection" describes a series of experiments to investigate the role of IFNg-induced genes, or 'ISGs', in T. gondii restriction in human cells. In humans, mechanisms of Toxoplasma gondii restriction are both cell-type specific and diverse, not relying solely on the IRG system observed in mice. Hence there are many unanswered questions as to how humans control and ultimately clear this widespread parasite of warm-blooded animals. Importantly, the authors use an unbiased over-expression ISG library to understand what additional host genes and mechanisms are employed by human cells to control parasitic infection.
The initial screen and experimental …
Reviewer #2 (Public Review):
The manuscript by Rinkenberger et al. titled "Over-expression Screen of Interferon-Stimulated Genes Identifies RARRES3 as a Restrictor of Toxoplasma gondii Infection" describes a series of experiments to investigate the role of IFNg-induced genes, or 'ISGs', in T. gondii restriction in human cells. In humans, mechanisms of Toxoplasma gondii restriction are both cell-type specific and diverse, not relying solely on the IRG system observed in mice. Hence there are many unanswered questions as to how humans control and ultimately clear this widespread parasite of warm-blooded animals. Importantly, the authors use an unbiased over-expression ISG library to understand what additional host genes and mechanisms are employed by human cells to control parasitic infection.
The initial screen and experimental validation, using ectopical expression, reveal IRF1 and RARRES3 as important host factors capable to restrict T. gondii infection in human cells. Importantly, RARRES3 induces premature parasite egress which can be blocked by Compound 1, a parasite egress inhibitor. Moreover, RARRES3 acts independently of host cell death pathways and appears to work autonomously in several contexts suggesting a new mode of parasite restriction not yet described.
The manuscript is well written. The methods employed to test the hypothesis and RARRES3 function are adequate and relevant. Little is known about RARRES3. The discussion is informative and addresses why so few ISGs were found to impact parasite restriction, whereas similar screens for viral pathogens appear to turn up many ISGs with anti-pathogen capabilities. Potential mechanisms are discussed.
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