Paracrine rescue of MYR1-deficient Toxoplasma gondii mutants reveals limitations of pooled in vivo CRISPR screens

Reviewer #1 (Public review):

Previous studies have highlighted some of these paracrine activities of Toxoplasma - and Rasogi et al (mBio, 2020) used a single cell sequencing approach of cells infected in vitro with the WT or MYR KO parasites - and one of their conclusions was that MYR-1 dependent paracrine activities counteract ROP-dependent processes. Similarly, Chen et al (JEM 2020) highlighted that a particular rhoptry protein (ROP16) could be injected into uninfected macrophages and move them to an anti-inflammatory state that might benefit the parasite.

There are caveats around immunity and as yet no insight into how this works. In Figure 2 there is a marked defect in the ability of the parasites to expand at day 2 and day 5. Together, these data sets suggest that this paracrine effect mediated by MYR-1 works early - well before the development of adaptive responses.

Reviewer #2 (Public review):

Summary:

In this manuscript by Torelli et al., the authors propose that the major function of MYR1 and MYR1-dependent secreted proteins is to contribute to parasite survival in a paracrine manner rather than to protect parasites from cell-autonomous immune response. The authors conclude that these paracrine effects rescue ∆MYR1 or knockouts of MYR1-dependent effectors within pooled in vivo CRISPR screens.

Strengths:

The authors raised a more general concern that pooled CRISPR screens (not only in Toxoplasma but also other microbes or cancers) would miss important genes by "paracrine masking effect". Although there is no doubt that pooled CRISPR screens (especially in vivo CRISPR screens) are powerful techniques, I think this topic could be of interest to those fields and researchers.

Weaknesses:

In this version, the reviewer is not entirely convinced of the 'paracrine masking effect' because the in vivo experiments should include appropriate controls (see major point 2).

(1) It is convincing that co-infection of WT and ∆MYR1 parasites could rescue the growth of ∆MYR1 in mice shown by in vivo luciferase imaging. Also, this is consistent with ∆MYR1 parasites showing no in vivo fitness defect in the in vivo CRISPR screens conducted by several groups. Meanwhile, it has been reported previously and shown in this manuscript that ∆MYR1 parasites have an in vitro growth defect; however, ∆MYR1 parasites show no in vitro fitness defect the in vitro pooled CRISPR screen. The authors show that the competition defect of ∆MYR1 parasites cannot be rescued by co-infection with WT parasites in Figure 1c, which might indicate that no paracrine rescue occurred in an in vitro environment. The authors seem not to mention these discrepancies between in vitro CRISPR screens and in vitro competition assays. Why do ∆MYR1 parasites possess neutral in vitro fitness scores in in vitro CRISPR screens? Could the authors describe a reasonable hypothesis?

(2) The authors developed a mixed infection assay with an inoculum containing a 20:80 ratio of ΔMYR1-Luc parasites with either WT parasites or ΔMYR1 mutants not expressing luciferase, showing that the in vivo growth defect of ∆MYR1 parasites is rescued by the presence of WT parasites. Since this experiment lacks appropriate controls, interpretation could be difficult. Is this phenomenon specific to MYR1? If a co-inoculum of ∆GRA12-Luc with either WT parasites or GRA12 parasites not expressing luciferase is included, the data could be appropriately interpreted.

(3) In the Discussion part, the authors argue that the rescue phenotype of mixed infection is not due to co-infection of host cells (lines 307-310). This data is important to support the authors' paracrine hypothesis and should be shown in the main figure.

(4) In the Discussion part, the authors assume that the rescue phenotype is the result of multiple MYR1-dependent effectors. I admit that this hypothesis could be possible since a recently published paper described the concerted action of numerous MYR1-dependent or independent effectors contributing to the hypermigration of infected cells (Ten Hoeve et al., mBio, 2024). I think this paragraph would be kind of overstated since the authors did not test any of the candidate effectors. Since the authors possess ∆IST parasites, they can test whether IST is involved in the "paracrine masking effect" or not to support their claim.