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The Toxoplasma oxygen-sensing protein, TgPhyA, is required for resistance to interferon gamma-mediated nutritional immunity in mice [1]

['Charlotte Cordonnier', 'Department Of Microbiology', 'Immunology', 'University At Buffalo School Of Medicine', 'Buffalo', 'New York', 'United States Of America', 'Msano Mandalasi', 'Department Of Biochemistry', 'Molecular Biology']

Date: 2024-06

As Toxoplasma gondii disseminates through its host, the parasite must sense and adapt to its environment and scavenge nutrients. Oxygen (O 2 ) is one such environmental factor and cytoplasmic prolyl 4-hydroxylases (PHDs) are evolutionarily conserved O 2 cellular sensing proteins that regulate responses to changes in O 2 availability. Toxoplasma expresses 2 PHDs. One of them, TgPHYa hydroxylates SKP1, a subunit of the SCF-E3 ubiquitin ligase complex. In vitro, TgPHYa is important for growth at low O 2 levels. However, studies have yet to examine the role that TgPHYa or any other pathogen-encoded PHD plays in virulence and disease. Using a type II ME49 Toxoplasma TgPHYa knockout, we report that TgPHYa is important for Toxoplasma virulence and brain cyst formation in mice. We further find that while TgPHYa mutant parasites can establish an infection in the gut, they are unable to efficiently disseminate to peripheral tissues because the mutant parasites are unable to survive within recruited immune cells. Since this phenotype was abrogated in IFNγ knockout mice, we studied how TgPHYa mediates survival in IFNγ-treated cells. We find that TgPHYa is not required for release of parasite-encoded effectors into host cells that neutralize anti-parasitic processes induced by IFNγ. In contrast, we find that TgPHYa is required for the parasite to scavenge tryptophan, which is an amino acid whose levels are decreased after IFNγ up-regulates the tryptophan-catabolizing enzyme, indoleamine dioxygenase (IDO). We further find, relative to wild-type mice, that IDO knockout mice display increased morbidity when infected with TgPHYa knockout parasites. Together, these data identify the first parasite mechanism for evading IFNγ-induced nutritional immunity and highlight a novel role that oxygen-sensing proteins play in pathogen growth and virulence.

Funding: This work was supported by the National Institutes of Health grants AI169849 and AI169986 to IJB/CMW, AI162756 to EAW, and AI159200 to JPG. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2024 Cordonnier 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.

Here, we report that TgPHYa deletion in a cystogenic strain leads to decreased virulence in both oral and intraperitoneal infection models. Furthermore, TgPHYa is required for resistance to IFNγ-mediated killing and does so by evading IFNγ-inhibition of tryptophan utilization. Significantly, TgPHYa knockout parasites display increased virulence in IDO1 knockout mice. Taken together, these data reveal that a parasite-encoded cytoplasmically localized prolyl hydroxylase mediates Toxoplasma evasion of a protective host nutritional immune response pathway.

Interferon gamma (IFNγ) is a cytokine critically required for host resistance to Toxoplasma [ 13 , 14 ]. It acts by triggering the expression of IFNγ-responsive genes whose products are involved in oxygen radical generation and degradation of the parasitophorous vacuole membrane via the activity of immunity-related guanosine triphosphatases (IRGs) and guanylate-binding proteins (GBPs) [ 15 ]. In addition, IFNγ induces the expression of indoleamine-pyrrole 2,3-dioxygenase (IDO) that catabolizes tryptophan, which Toxoplasma must scavenge from its host [ 16 ]. The importance of IDO in nutritional immunity to Toxoplasma has remained enigmatic as it is important for resistance in some human cells but appears to be dispensable in mice [ 16 – 18 ]. The most likely reason for this is that, unlike humans, mice express 2 IDO isoforms. Thus, it remains unknown whether tryptophan scavenging/utilization is a viable anti-Toxoplasma drug target [ 18 ].

As Toxoplasma traffics through a host, it encounters diverse environments that vary in essential nutrients and other compounds. O 2 is one such compound that all aerobic cells must sense and respond to changes in its levels to optimize growth and viability [ 4 ]. Cytoplasmic prolyl 4-hydroxylases (PHDs) are dioxygenases that use α-ketoglutarate and O 2 as substrates and function as the key cellular O 2 sensors [ 5 ]. PHDs hydroxylate proline residues in substrates and in metazoans the best-recognized PHD substrate is the α subunit of the hypoxia-inducible transcription factor (HIFα). Prolyl-hydroxylated HIFα is ubiquitinated by the Von Hippel–Lindau ubiquitin ligase complex, which targets it for proteasomal degradation [ 6 ]. Under hypoxic conditions, HIFα is stabilized and able to regulate the expression of genes important for growth and survival at low O 2 . While HIFα is not conserved in protist genomes, PHDs are, indicating that protists likely respond differently to changes in O 2 availability [ 4 , 7 ]. The best characterized protozoan PHD is DdPhyA from Dictyostelium discoideum that modifies a proline in DdSkp1, which is an adaptor protein in the SCF (Skp1/Cullin/F-box protein) E3 polyubiquitin ligase complex [ 8 ]. The resulting hydroxyproline is subsequently modified by a series of glycosyltransferases that alters the binding affinity of SKP1 towards different F-Box proteins and thus differential protein ubiquitination by the SCF complex [ 8 – 10 ]. Two PHDs have been identified in Toxoplasma and one of these, TgPHYa also modifies SKP1 [ 11 , 12 ]. While TgPHYa is not essential, TgPHYa knockouts in type 1 strain parasites display reduced in vitro growth at low O 2 [ 12 ]. But TgPHYa’s function in vivo remains to be determined.

Toxoplasma gondii is an intracellular apicomplexan parasite that chronically infects up to one-third of the world’s population [ 1 ]. This opportunistic pathogen causes toxoplasmosis, a disease that can be life-threatening in immunocompromised individuals and developing fetuses. Infection in humans follows consumption of meat harboring bradyzoite-containing tissue cysts or water and produce contaminated with sporozoite-containing oocysts [ 2 ]. Gastric enzymes rupture cysts or oocysts and the released parasites go on to infect the small intestine. At this juncture, the parasites convert into replicative tachyzoites and trigger an immune response that results in recruitment of innate immune cells such as inflammatory monocytes, innate lymphoid cells, and neutrophils. These cells are in turn infected and used to disseminate to peripheral tissues where tachyzoites differentiate into bradyzoites and form tissue cysts that can persist for the host’s lifetime [ 3 ].

Results

Generation of TgPHYa knockout in the type II ME49 strain To assess a role for TgPHYa during in vivo infection, CRISPR was used to generate a TgPHYa knockout mutant in a type II Toxoplasma strain that also expresses red fluorescent protein (RFP) (TgPHYaKO II ). PCR analysis indicated that TgPHYa was properly targeted by the CRISPR construct and that the TgPHYa gene was disrupted (Fig 1A and 1B). Because TgPHYa antisera is lacking, we assessed whether the gene disruption affected TgPHYa protein by analyzing TgSKP1 mobility by SDS-PAGE. We observed that the apparent molecular weight of TgSKP1 was reduced in TgPHYaKO II parasites indicating decreased TgSKP1 prolyl hydroxylation/glycosylation (Fig 1C) [12]. Similar to the type I strain TgPHYa knockout [12], in vitro TgPHYaKO II growth was slightly decreased at 21% O 2 but significantly at 0.5% O 2 (Fig 1D and 1E). PPT PowerPoint slide

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TIFF original image Download: Fig 1. TgPHYa knockout in type II strain parasites. (A) Scheme for disrupting the TgPHYa locus by CRISPR. (B). Genomic DNA from ME49 WT and TgPHYaKO II parasites were analyzed by PCR using the primers depicted in (A). (C) Western blot analysis to detect SKP1 in lysates from the indicated strains. Note the increased mobility of the SKP1 in the TgPHYaKO II lysate indicating is loss of prolyl hydroxylation/glycosylation. (D) HFFs infected with WT or TgPHYaKO II parasites were grown for 12 days at 21% or 0.5% O 2 . The cells were then fixed, stained with crystal violet, and plaque sizes measured. Shown are averages ± SD from 3 independent experiments. (E) Representative images of plaques from (D). The data underlying the graph in this figure can be found in S2 Table and blot in S1 Raw Images. HFF, human foreskin fibroblast; WT, wild-type. https://doi.org/10.1371/journal.pbio.3002690.g001

TgPHYaKO II displays reduced virulence and decreased numbers of brain cysts C57BL/6J mice were infected by oral gavage with 50 or 100 wild-type (WT) or TgPHYaKO II tissue cysts and survival and body weight were monitored for 30 days. All mice infected with 50 TgPHYaKO II cysts survived up to 30 days post infection, while 40% of mice infected with the parental WT strain died (p = 0.06 Mantel–Cox test) (Fig 2A). Moreover, all mice infected with 100 WT cysts succumbed during the acute stage of the infection (within 8 days post infection), while <40% of the mice succumbed when infected with 100 TgPHYaKO II cysts (p = 0.02 Mantel–Cox test) (Fig 2A). Similar to decreased mortality, mice infected with the TgPHYa knockout lost less weight than those infected with WT parasites and their weight loss rebounded, whereas weight loss was sustained in the surviving WT infected mice (Fig 2B). PPT PowerPoint slide

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TIFF original image Download: Fig 2. C57BL/6 mice are less susceptible to TgPHYaKO II strain infection correlating with fewer number of brain cysts. (A) Survival of C57BL/6J mice infected orally with 50 or 100 cysts of the indicated strains. Cumulative data from 3 independent trials and 9 mice for each strain and dose. Curves were compared by log-rank survival analysis of Kaplan–Meier curves, 50 cysts p = 0.0649, 100 cysts *P = 0.02. (B) Weight loss was monitored in mice infected with 50 cysts. Shown are cumulative data from 3 independent trials (mean ± SD *P < 0.05, multiple Student’s t test with Holm–Sidak correction). Brain cyst burden (C) and diameter (D) were calculated from mice 30, 60, or 90 days after infection. Shown are cumulative data from 3 independent trials (mean ± SD *P < 0.05, multiple Student’s t test with Holm–Sidak correction). (E) In vitro cyst development was assessed by Dolichos biflorus agglutinin lectin staining of parasites grown in HFFs for 5 days in in pH 8.2 medium. Shown is percentage of lectin+ vacuoles from 3 independent assays (50 randomly selected vacuoles were counted for each sample). The data underlying the graphs in this figure can be found in S2 Table. HFF, human foreskin fibroblast; WT, wild-type. https://doi.org/10.1371/journal.pbio.3002690.g002 Next, brain cyst burdens were enumerated in mice 30-, 60-, or 90-days post infection (dpi) with 50 cysts. We found that at 30 dpi, there was a statistically significant decrease in numbers of cysts in brains of TgPHYaKO II infected mice (Fig 2C). But, at later time points differences were not noted. Brain cyst sizes were also analyzed and at each time point the TgPHYaKO II cysts were significantly smaller than the parental strain cysts (Fig 2D). To test whether TgPHYaKO II brain cyst phenotypes were a result of the prolyl hydroxylase functioning in cyst formation, cyst formation was examined in parasites exposed to alkaline medium (pH 8.2) at ambient CO 2 [19]. After 5 days of induction, cysts were examined using DBA-FITC and no differences in the ability for TgPHYaKO II to form in vitro cysts were noted (Fig 2E). Together, these data indicate that TgPHYa contributes to virulence and brain cyst development.

TgPHYa is important for parasite dissemination One possible explanation for decreased numbers of TgPHYaKO II brain cysts at 30 dpi is delayed dissemination from the gut to the brain and other tissues. To examine this, we first assessed a role for TgPHYa in establishing an infection in the gut by gavage infecting mice with 50 WT or TgPHYaKO II cysts and 7 days later enumerating parasite burdens by quantitative real time PCR (qPCR) in different segments of the small intestine. No significant differences in parasite numbers were observed throughout the intestine (Fig 3A). We also assessed intestinal inflammation by histological analysis at 7 dpi (Fig 3B) and 12 dpi (Fig 3C) and no significant differences were noted at either time point. PPT PowerPoint slide

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TIFF original image Download: Fig 3. TgPHYaKO II is not required for colonization of intestinal epithelium but is important for parasite dissemination to peripheral tissues. (A) ME49 WT or TgPHYaKO II burdens were analyzed by quantitative RT-PCR in the indicated intestinal sections 7 days following gavage infection with 50 cysts. Shown are means ± SEM of 3 independent experiments with 3 mice per experiment (ns = not significant, using one-way ANOVA). (B, C) HE-stained small intestinal sections from mice gavage infected with 50 WT or TgPHYaKO II cysts on day 7 (B) or day 12 (C) post infection. Asterisks highly inflammatory infiltrates in the villi and LP and arrowheads highlight destruction of villi. Shown are representative images and plots represent mean ± SEM of histological scores of 50 randomly selected section per mouse. (D) Parasite burdens were determined by qPCR of genomic DNA isolated from spleens, lungs, hearts, and brains of mice gavage infected with 50 of ME49 WT or TgPHYaKO II cysts. Data shown are mean ± SEM from a total of 7 to 9 mice from 3 independent experiments (ns = not significant, *P < 0.01, ***P < 0.001, using one-way ANOVA). The data underlying the graphs in this figure can be found in S2 Table. LP, lamina propria; WT, wild-type. https://doi.org/10.1371/journal.pbio.3002690.g003 Next, parasite burdens in peripheral tissues at 7 and 12 dpi were determined by qPCR. Except for the spleen, there were no remarkable differences in parasite burdens in the organs on day 7 of infection (Fig 3D). In contrast, significantly fewer TgPHYaKO II parasites were observed in the spleens, hearts, and brains of the infected mice at 12 dpi. Parasite burdens in lungs were also lower although this difference was not statistically significant (P = 0.16). These data indicate that while TgPHYaKO II parasites can establish an infection in the intestine, they are unable to disseminate efficiently to peripheral tissues.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002690

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