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ROP16-mediated activation of STAT6 enhances cyst development of type III Toxoplasma gondii in neurons [1]

['Joshua A. Kochanowsky', 'Department Of Immunobiology', 'University Of Arizona', 'Tucson', 'Arizona', 'United States Of America', 'Institute', 'Sambamurthy Chandrasekaran', 'Jacqueline R. Sanchez', 'Postbaccalaureate Research Education Program']

Date: 2023-06

Toxoplasma gondii establishes a long-lived latent infection in the central nervous system (CNS) of its hosts. Reactivation in immunocompromised individuals can lead to life threatening disease. Latent infection is driven by the ability of the parasite to convert from the acute-stage tachyzoite to the latent-stage bradyzoite which resides in long-lived intracellular cysts. While much work has focused on the parasitic factors that drive cyst development, the host factors that influence encystment are not well defined. Here we show that a polymorphic secreted parasite kinase (ROP16), that phosphorylates host cell proteins, mediates efficient encystment of T. gondii in a stress-induced model of encystment and primary neuronal cell cultures (PNCs) in a strain-specific manner. Using short-hairpin RNA (shRNA) knockdowns in human foreskin fibroblasts (HFFs) and PNCs from transgenic mice, we determined that ROP16’s cyst enhancing abilities are mediated, in part, by phosphorylation—and therefore activation—of the host cell transcription factor STAT6. To test the role of STAT6 in vivo, we infected wild-type (WT) and STAT6KO mice, finding that, compared to WT mice, STAT6KO mice have a decrease in CNS cyst burden but not overall parasite burden or dissemination to the CNS. Finally, we found a similar ROP16-dependent encystment defect in human pluripotent stem cell-derived neurons. Together, these findings identify a host cell factor (STAT6) that T. gondii manipulates in a strain-specific manner to generate a favorable encystment environment.

Toxoplasma gondii is a parasite that establishes a latent infection in the brain of its host. In humans who become immunocompromised, this latent infection can reactivate to cause serious neurologic complications. The parasite establishes a latent infection by transitioning from a fast-growing state to a slow growing one. This slow growing form resides within cysts, which are commonly found in neurons. While several parasite factors that drive cyst formation have been identified, only a few host cell genes that promote encystment have been found. Using different models of encystment, including human and mouse primary neuronal cultures, we show that phosphorylation and, thus, activation of the host cell transcription factor STAT6 by the parasite kinase ROP16 is required for efficient encystment of certain T. gondii strains but not others. Collectively, this work suggests that T. gondii strain-specific pathways to encystment exist and identifies a direct link between parasite manipulation of the host cell and encystment.

Funding: Funding was provided by the National Institute of Neurologic Disorders and Stroke (NS095994 [A.A.K.], NS095994-02S1 [J.A.K.]), https://www.ninds.nih.gov/ ; the National Institute of Allergy and Infectious Diseases (F31AI147711 [J.A.K.], AI147756 [A.A.K.]), https://www.niaid.nih.gov/ ; the National Institute of General Medical Sciences (GM121228 [JRS]), https://www.nigms.nih.gov/ ; and the BIO5 Institute, University of Arizona (A.A.K.), http://bio5.org/ . The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2023 Kochanowsky et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

To address this possibility, we compared cyst formation in type II and type III strains that lacked ROP16 (IIΔrop16 and IIIΔrop16 respectively). We observed that deletion of ROP16 had no effect on type II encystment but significantly decreased type III encystment in both a stress model of encystment and in murine primary neuronal cell cultures (PNCs). Using IIIΔrop16 strains complemented with mutated ROP16s, we determined that the ROP16-dependent effect on type III encystment required a ROP16 that is capable of phosphorylating STATs. By using PNCs derived from mice deficient in STAT6 (STAT6KO) [ 39 ] and infection of STAT6KO mice with wild-type type III parasites (WT III ), we identified that efficient encystment required STAT6 in vitro and in vivo. Finally, we determined that ROP16 was also required for efficient encystment of type III parasites in human neurons. Together these results highlight a mechanism by which an allele of a parasite kinase (ROP16) enhances encystment via activation of a host cell transcription factor (STAT6). To the best of our knowledge, this study identifies the first host cell gene that influences encystment in a strain-specific manner.

Though many T. gondii strain types encyst and cause clinically relevant disease [ 20 – 24 ], most work on encystment has been done using type II strains. The ability to compare strains has highlighted that strain-specific effectors influence acute infection [ 25 – 29 ], leading us to question whether strain-specific pathways that influence encystment might also exist. We were particularly interested in the polymorphic effector protein ROP16 for several reasons [ 30 ]. ROP16s from strain types I, II, and III translocate into the host cell nucleus and have a functional kinase domain, but only the type I and III alleles (ROP16 I and ROP16 III respectively), which are 99% identical in their amino acid sequence, cause prolonged phosphorylation and activation of the host transcription factors STAT3, STAT5a, and STAT6 [ 30 – 35 ]. The importance of this STAT activation is strain-specific, as lacking ROP16 does not affect type I strain virulence in mice, but is essential for type III strain survival in vivo [ 31 , 36 , 37 ]. In addition, ROP16 III -dependent phosphorylation of STAT6 dampens host cell ROS production in human and murine cells, enabling improved type III survival in vitro [ 38 ]. Considering the profound impact of ROP16 on host cell signaling and tachyzoite survival, we wondered if ROP16 might also influence encystment.

Given T. gondii’s clinical importance, many studies have focused on understanding persistence. These studies have identified many of the parasite factors that define and drive stage conversion and encystment and determined that a range of exogenous stresses (e.g., high pH) can trigger encystment in non-permissive cells (e.g., fibroblasts, macrophages) [ 8 – 11 ]. In permissive cells (neurons and myocytes), high levels of encystment can be achieved without the addition of exogenous stress [ 12 – 16 ]. Collectively, these data suggest that specific host cell pathways promote encystment, but only a single host gene (CDA-1) that influences encystment has been identified in myocytes [ 15 , 17 ]. Glutamine starvation and reactive oxygen species (ROS) production were recently identified as triggers of encystment in murine skeletal muscle cells and IFN-γ stimulated human induced pluripotent stem cells (iPSC)-derived glutamatergic neurons consistent with the observation that encystment may be a generalized response to stress [ 18 , 19 ].

A broad range of microbes—viruses, bacteria, and parasites—establish long-term, latent infections by switching from a rapidly replicating state to a slow-growing quiescent one. While these latent infections enable microbial spread to new hosts, they can also reactivate to cause overt disease when the host becomes immunocompromised. Toxoplasma gondii, a common obligate intracellular parasite that infects most warm-blooded animals including humans, switches from a rapidly replicating state, the tachyzoite, to a slow-growing, encysted state, the bradyzoite [ 1 – 5 ]. Bradyzoite-filled cysts are the hallmark of latent infection and are primarily found in neurons in the central nervous system (CNS) and myocytes in skeletal and cardiac muscle [ 1 , 3 ]. For T. gondii, encystment allows for infection of the definitive host (felids) and passage between intermediate hosts, thereby enabling both the sexual and asexual life cycle of the parasite [ 1 ]. In humans, latent infection is generally asymptomatic, but in the setting of acquired immune deficiencies, recrudescence can lead to severe pathology and even death. During the height of the HIV/AIDs epidemic, toxoplasmic encephalitis was the most common focal neurologic finding in AIDS patients [ 6 , 7 ].

Results

ROP16 facilitates cyst development in a strain-specific manner To assess how ROP16 affected encystment, we infected human foreskin fibroblasts (HFFs) with WT III (CEP) [40], IIIΔrop16 (CEPΔrop16), or strains where we re-introduced the type III allele of rop16 (ROP16 III ) or the type II allele of rop16 (ROP16 II ) [36–38]. Of note, these strains all constitutively express an RFP (WT III expresses mCherry and the others express tdTomato). We induced encystment by placing the infected cultures under alkaline stress (pH 8.2) in combination with CO 2 deprivation (<5%) and tracked differentiation using immunofluorescent staining of T. gondii stage-specific surface antigens [41,42] and Dolichos biflorus agglutinin (DBA), which binds sugar moieties present on the proteins that form the cyst wall [43]. At 6 days post infection (dpi) the WT III strain no longer expressed the tachyzoite-specific antigen SAG1 [41], instead expressing the bradyzoite-specific surface antigen SRS9 [42] and staining positive for DBA, indicating formation of the cyst wall (S1 Fig). In contrast, IIIΔrop16 parasites displayed a defect in SRS9 expression and DBA staining, which could be rescued by complementation with ROP16 III but not by complementation with ROP16 II (S1 Fig). To further characterize and quantify this defect in cyst development, we established an automated imaging pipeline using the Operetta CLS platform and Harmony software (Fig 1A). This automated system allowed us to track all parasitophorous vacuoles (PVs) through staining with a polyclonal anti-T. gondii antibody [44] and cysts through staining with DBA. Using this system, we tracked the encystment rate of our strains over the course of eight days. Compared to WT III and ROP16 III parasites, IIIΔrop16 and ROP16 II parasites showed a significant reduction (30–50%) in encystment at 4–8 dpi (Fig 1B). To ensure that this decrease in encystment was not due to a decrease in overall parasite survival of the IIIΔrop16 and ROP16 II parasites, we used this automated system to track the accumulation of the total number of PVs over time. There were no significant differences in the accumulation of PVs between any of our strains (S2A Fig). In addition, the rate of encystment did not vary by the number of parasites within a PV (S2B Fig). Collectively, these data indicate that the defect in encystment that we observed is unlikely to be explained by a defect in parasite survival or a delay in stage conversion in the strains that lack ROP16 III . PPT PowerPoint slide

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TIFF original image Download: Fig 1. ROP16 III facilitates cyst formation of a type III parasites in multiple models of cyst development. (A) Schematic of automated cyst quantification using Operetta CLS and Harmony software. Parasitophorous vacuoles (PVs) were identified by staining with anti-T. gondii antibody. Cysts were identified by DBA staining. (B) Quantification of encystment of over time in a stress model of encystment for listed type III strains. (C) Quantification of encystment over time as in (B) except in primary neuronal cultures (PNCs). (D) Quantification of encystment over time in stress model of encystment for listed type II parasites. (E) Quantification of encystment over time as in (D) except in PNCs. (B-E) % Cyst = (# of DBA+ Vacuoles with ≥2 parasites /# of Total Vacuoles with ≥2 parasites)*100. Bars, mean ± SEM. Black dots = Average % cyst for 1 experiment. (B, D) N = 10 wells/experiment, 3 experiments total. (C, E) N = 5 wells/experiment, 4 experiments total. (B-E) *p≤0.05, **p≤0.005, and ****p≤0.0001. ns = not significant, two-way ANOVA, Dunnett’s multiple comparisons test compared to WT III or WT II . https://doi.org/10.1371/journal.ppat.1011347.g001 Although the alkaline and CO 2 stress model is commonly used to increase encystment, it is a highly artificial setting. As neurons are the major host cell type for persistent infection in the CNS [45–50], we decided to use PNCs, which prior studies suggest induce encystment without the need for exogenous stress [12,13,16]. To test how ROP16 influenced type III encystment in neurons, we infected PNCs with WT III , IIIΔrop16, or the two complemented strains and tracked cyst wall formation using DBA staining. Consistent with what we found in alkaline-stressed HFFs, we observed a defect in encystment in PNCs infected with IIIΔrop16 and ROP16 II compared to WT III and ROP16 III -infected cultures (S3 Fig). To quantify this defect more robustly, we used our automated cyst quantification system to track the encystment rate over 3 days. Compared to WT III and ROP16 III parasites, IIIΔrop16 and ROP16 II parasites showed a 50–75% reduction in encystment from 1–3 dpi in PNCs (Fig 1C). Akin to our findings in the stress model of encystment, in PNCs there were no significant differences in the accumulation of PVs between any of the strains nor did the rate of encystment vary with the number of parasites within a PV (S2C and S2D Fig). To determine if this ROP16-dependent defect in encystment was restricted to the type III strain or was also relevant to genetically distinct T. gondii strain types, we generated a ROP16 deficient type II strain (PrugniaudΔrop16 or IIΔrop16 for simplicity) using a CRISPR/CAS9 approach [36,38]. We then tested encystment of WT II (Prugniaud/PRU) [51] and IIΔrop16 parasites in the alkaline stress model and PNCs. Unlike the defect observed in the IIIΔrop16 strain, deletion of ROP16 in a type II background had no effect on cyst development in stressed HFFs or in PNCs (Figs 1D, 1E and S4). Together these results indicate that ROP16 is needed for maximal encystment of type III parasites, but not for type II parasites, suggesting that strain-specific differences in encystment exist.

ROP16 facilitates encystment through host cell manipulations While ROP16 is thought to primarily phosphorylate host cell proteins [30], we sought to ensure that our cyst defect was not due to a direct effect of ROP16 on parasite proteins. To accomplish this goal, we tested whether encystment of the IIIΔrop16 strain could be restored by co-infection of a host cell with WT III GFP-expressing parasites (WT III ::GFP) (Fig 2A). We hypothesized that if ROP16 facilitates cyst development through host cell manipulations then the IIIΔrop16 cyst defect should be rescued in host cells which also house WT III ::GFP parasites that have injected a functional copy of ROP16. However, if ROP16 is required for phosphorylation of a parasite protein involved in encystment then co-infection with WT III ::GFP parasites should be insufficient to restore encystment. Co-infection with WT III ::GFP was sufficient to restore encystment of IIIΔrop16 in an alkaline stress model of encystment, indicating that ROP16 facilitates cyst development through host cell manipulations (Fig 2B). Together these results indicate that ROP16 enhances encystment via host cell manipulations. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Co-infection of IIIΔrop16 with WT parasites restores encystment. (A) Schematic of co-infection experiment. WT III :GFP parasites are type III (CEP) parasites that express GFP. The dark circles represent non-ROP16 rhoptry protein secretion. The red circles represent ROP16 secretion by the WT III ::GFP parasites. (B) Quantification of encystment at 6 dpi in alkaline stress model of encystment. Bars, mean ± SEM. Black dots = Average % cyst for 1 experiment. N = 3 replicates/experiment, 3 experiments total. *p≤0.05, ns = not significant, one-way ANOVA, Dunnett’s multiple comparisons test compared to WT III . https://doi.org/10.1371/journal.ppat.1011347.g002

Efficient encystment requires a ROP16 with an active kinase domain and a leucine at position 503 Having determined that ROP16 III -dependent host cell manipulation is essential for efficient type III encystment, we next sought to determine what domains or functions of ROP16 were required for this phenotype. To accomplish this goal, we used a previously generated panel of IIIΔrop16 complemented strains [38]. This panel of strains includes ROP16 mutants which are catalytically inactive (ROP16 IIIKD ) or lack a nuclear localization sequence (ROP16 IIIΔNLS ) (Fig 3A) [38]. In addition, as prior work demonstrated that a single polymorphic amino acid on ROP16 determined the strain-specific activation of STAT3/5a/6 [34], this panel also includes strains which express a ROP16 III in which the leucine at position 503 was switched to a serine, rendering it “STAT-dead” (ROP16 IIISD ), and a strain that expresses a ROP16 II in which the serine at position 503 was changed to a leucine, rendering it “STAT-active” (ROP16 IISA ) [38] (Fig 3A). We tested the ability of this panel of parasites to encyst in the stress model of encystment and in PNCs. In both models, IIIΔrop16, ROP16 II , ROP16 IIISD, and ROP16 IIIKD parasites were defective in forming cysts compared to WT III , ROP16 III , or ROP16 IISA parasites (Fig 3B and 3C). Together these data indicate that efficient in vitro encystment is dependent on kinase activity and a leucine at position 503, while the nuclear localization sequence (NLS) is dispensable. Additionally, the ability of ROP16 IISA to restore these defects suggested a role for phosphorylation/activation of STATs in cyst development. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Efficient encystment requires a ROP16 with a functional kinase domain and a leucine at position 503. (A) Schematic of ROP16 and mutations in ROP16 constructs. (B) Quantification of encystment at 4 dpi in alkaline stress model of encystment. Bars, mean ± SEM. Black dots = Average % cyst for 1 experiment. N = 10 wells/experiment, 4 experiments total. (C) Quantification of encystment at 2 dpi in PNCs. Bars, mean ± SEM. Black dots = Average % cyst for 1 experiment. N = 3 wells/experiment, 4 experiments total. (B, C) **p≤0.005, ***p≤0.0005, and ****p≤0.0001, one-way ANOVA, Dunnett’s multiple comparisons test compared to WT III . https://doi.org/10.1371/journal.ppat.1011347.g003

STAT6 is required for efficient encystment in vitro To test the role of individual STATs in type III encystment, we used previously generated STAT3, 5a, and 6 Lentiviral-mediated knock-down HFFs [38] in a cyst assay. Compared to encystment in non-targeting shRNA control HFFs, knock-down of STAT6 significantly reduced encystment of WT III parasites at both 2 and 4 dpi, while knockdown of STAT5a reduced encystment only at 4 dpi (S5 Fig). Knock-down of STAT3 had no effect on encystment at 2 or 4 dpi (S5 Fig). As the STAT6 knock-down showed the strongest effect on cyst formation and STAT6KO mice are commercially available, we decided to test the role of STAT6 in cyst development using PNCs from STAT6KO mice [39]. We observed a significant decrease in encystment of WT III parasites in STAT6KO PNCs at all time points compared to parasites in PNCs from BL6 control mice (Fig 4A and 4B). To determine if the type III ROP16 encystment defect was solely mediated by STAT6, we infected STAT6KO PNCs with WT III , IIIΔrop16, or the ROP16 III complement and tracked cyst formation over time. Compared to WT III or ROP16 complemented parasites, the IIIΔrop16 mutant continued to show a significant decrease in cyst formation (Fig 4C). Finally, to determine if host cell STAT6 was required for efficient encystment in other T. gondii strains, we infected STAT6KO PNCs with two type II strains (ME49 and PRU/WT II ) and two type III strains (CEP/WT III and VEG). At 3 dpi neither of the type II strains showed a defect in encystment, while both type III strains displayed a statistically significant decrease in encystment in STAT6KO PNCs (Fig 4D). Taken together these results indicate that STAT6 enhances cyst development in HFFs and PNCs for type III strains, but not type II strains. In addition, the data suggest that other targets of ROP16 (e.g., STAT5a) also play a role in cyst formation. PPT PowerPoint slide

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TIFF original image Download: Fig 4. STAT6 facilitates encystment of type III, but not type II parasites, in vitro. (A) IFA of cyst assay. BL6 (control) or STAT6KO PNCs were infected with WT III (MOI 0.1) for 2 dpi. Images depict anti-TUJ1 (green, neurons), mCherry (red, parasites), DBA (magenta), and DAPI (blue). Scale bar = 10μm. (B) Quantification of encystment of WT III parasites overtime in identified PNCs. (C) Quantification of encystment of WT III , IIIΔrop16, and ROP16 III parasites overtime in STAT6KO PNCs. (D) Quantification of encystment in identified PNCs at 3 dpi for listed type II and type III strains. (B-D) Bars, mean ± SEM. N = 5 wells/experiment, 4 experiments total. (B, D) *p≤0.05, **p≤0.005, ***p≤0.0005, ns = not significant, two-way ANOVA, Dunnett’s multiple comparisons test compared to BL6. (C) *p ≤ 0.05, ****p≤0.0001, ns = not significant, two-way ANOVA, Dunnett’s multiple comparisons test compared to WT III . https://doi.org/10.1371/journal.ppat.1011347.g004

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