(C) PLOS One This story was originally published by PLOS One and is unaltered. . . . . . . . . . . N-acetylglucosamine supplementation fails to bypass the critical acetylation of glucosamine-6-phosphate required for Toxoplasma gondii replication and invasion [1] ['María Pía Alberione', 'Barcelona Institute For Global Health', 'Isglobal', 'Hospital Clínic-University Of Barcelona', 'Barcelona', 'Víctor González-Ruiz', 'School Of Pharmaceutical Sciences', 'University Of Geneva', 'Geneva', 'Olivier Von Rohr'] Date: 2024-07 The cell surface of Toxoplasma gondii is rich in glycoconjugates which hold diverse and vital functions in the lytic cycle of this obligate intracellular parasite. Additionally, the cyst wall of bradyzoites, that shields the persistent form responsible for chronic infection from the immune system, is heavily glycosylated. Formation of glycoconjugates relies on activated sugar nucleotides, such as uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). The glucosamine-phosphate-N-acetyltransferase (GNA1) generates N-acetylglucosamine-6-phosphate critical to produce UDP-GlcNAc. Here, we demonstrate that downregulation of T. gondii GNA1 results in a severe reduction of UDP-GlcNAc and a concomitant drop in glycosylphosphatidylinositols (GPIs), leading to impairment of the parasite’s ability to invade and replicate in the host cell. Surprisingly, attempts to rescue this defect through exogenous GlcNAc supplementation fail to completely restore these vital functions. In depth metabolomic analyses elucidate diverse causes underlying the failed rescue: utilization of GlcNAc is inefficient under glucose-replete conditions and fails to restore UDP-GlcNAc levels in GNA1-depleted parasites. In contrast, GlcNAc-supplementation under glucose-deplete conditions fully restores UDP-GlcNAc levels but fails to rescue the defects associated with GNA1 depletion. Our results underscore the importance of glucosamine-6-phosphate acetylation in governing T. gondii replication and invasion and highlight the potential of the evolutionary divergent GNA1 in Apicomplexa as a target for the development of much-needed new therapeutic strategies. In this study, we explored the importance of this pathway in T. gondii and discovered that these sugar-containing compounds play a vital role in the parasite’s ability to invade and replicate in host cells–crucial processes for its survival and ability to cause disease. Intriguingly, unlike some organisms that can bypass the pathway, T. gondii relies critically on glucosamine-6-phosphate acetylation. This reliance sheds light on the parasite’s distinct metabolic properties and highlights the pathway’s potential as a target for new therapeutic strategies. Funding: This work is supported by the Indo-Swiss Joint Research Programme (ISJRP) IZLIZ3_200277 awarded to DSF; by the Carigest SA ( https://carigest.ch/ ) to DSF; by the Novartis Foundation for Medical Biological Research (22C164 awarded to JK). The Barcelona Institute for Global Health (ISGlobal) is supported by the Spanish Ministry of Science and Innovation through the Centro de Excelencia Severo Ochoa 2019-2023 Program (CEX2018-000806-S). The work is also supported by the Generalitat de Catalunya through the CERCA Program ( https://cerca.cat ). This work is part of the ISGlobal’s Program on the Molecular Mechanisms of Malaria, partially supported by the Fundación Ramón Areces ( https://www.fundacionareces.es ). LI received support by PID2019-110810RB-I00 and PID2022-137031OB-I00 grants from the Spanish Ministry of Science & Innovation. The work is supported by a FI Fellowship from the Generalitat de Catalunya supported by Secretaria d’Universitats i Recerca de la Generalitat de Catalunya and Fons Social Europeu (2021 FI_B 00470 to MPA). MPA also received support from an EMBO Scientific Exchange Grant (9474). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Here we demonstrate the critical role of GNA1 for T. gondii, revealing that its downregulation leads to a reduction in GPI anchors that impairs invasion and replication within its host cell. Intriguingly, defects in GNA1 cannot be overcome by GlcNAc salvage. Targeted metabolomic analyses revealed that GlcNAc salvage is inefficient in glucose-replete conditions. In contrast, GlcNAc is effectively salvaged in glucose-deplete conditions, but fails to rectify the defects associated with the disruption of the pathway. These findings highlight the potential of GNA1 as a drug target to combat toxoplasmosis. In T. gondii, a genome-wide CRISPR fitness screen underscored the significance of UDP-GlcNAc biosynthesis for the parasite, classifying several genes encoding for enzymes involved in the amino sugar synthesis pathway as fitness-conferring [ 22 , 23 ]. Unexpectedly, however, this study predicted GNA1 to be dispensable for T. gondii, even though the upstream and downstream enzymes were classified as highly fitness-conferring [ 23 ]. The de novo synthesis of glycans relies on activated sugar nucleotides. Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) serves as a donor by GlcNAc-dependent glycosyltransferases for the synthesis of N-glycans, glycosylphosphatidylinositol (GPI) -anchors, glycoinositolphospholipids (GIPLs), and for the glycosylation of other protein acceptors. Given the critical roles of these structures for infectivity of tachyzoites [ 7 , 8 , 16 , 19 ], and bradyzoite survival and replication [ 17 , 18 ], the biosynthesis route of UDP-GlcNAc is a plausible target for intervention against acute toxoplasmosis and for eradication the chronic infection. GNA1, the enzyme catalysing the acetylation of glucosamine-6-phosphate (GlcN6P) is considered a promising drug target in Apicomplexa. This is attributed to its independent evolutionary origin, unique sequence features [ 20 ], and established essentiality for the intraerythrocytic development of Plasmodium falciparum [ 21 ]. The endomembrane system of T. gondii is rich in glycoconjugates which play fundamental roles in infectivity, survival, and virulence [ 7 ]. Several glycan structures have been characterized in T. gondii including N-glycans [ 8 ], O-glycans [ 9 – 13 ], C-mannose [ 9 , 14 ], GPI-anchors [ 15 , 16 ], and others [ 7 ]. These glycans serve various critical functions from invasion to O 2 sensing and nutrient storage, hence contributing to the overall virulence of the parasite [ 7 ]. Additionally, glycans are critical components of the bradyzoite cyst wall and the disruption of their formation impairs the parasite’s ability to persist [ 17 , 18 ]. The phylum of Apicomplexa groups a vast number of obligate intracellular parasites, some of which pose a considerable threat to human health. The most ubiquitous apicomplexan, Toxoplasma gondii, causes disease in immunocompromised individuals [ 1 , 2 ], as well as abortions, stillbirths, fetal death, retinal lesions or long-term disabling sequelae in congenitally infected children [ 3 , 4 ]. At present, there is no vaccine that prevents toxoplasmosis, and the available treatments are associated with a range of shortcomings including high cost, toxicity and rising resistance [ 5 ]. In the accidental human host, T. gondii manifests in two distinct stages: the fast-replicating tachyzoite, responsible for acute disease and the slow replicating bradyzoite, which persists encysted within muscle cells and neurons throughout the lifetime of its host [ 6 ]. These persistent parasites constitute a reservoir, that can reactivate causing life-threatening acute toxoplasmosis when the infected individual becomes immunocompromised. The inability to eradicate the parasite’s latent form, combined with the emergence of parasites that are resistant to existing drugs against acute toxoplasmosis, underscores the pressing need for novel therapeutic strategies [ 5 ]. Results GNA1 is active and critical for UDP-GlcNAc synthesis in T. gondii To examine if the UDP-GlcNAc biosynthesis pathway is active in intra- and extracellular T. gondii, TIR1 parasites were cultured intracellularly for 24 hours in medium containing 10 mM uniformly 13C-labelled glucose (U-13C 6 -Glc) or extracted and purified extracellular parasites were incubated for three hours in medium containing heavy Glc. Post-incubation, the metabolism was quenched, parasites were harvested, and metabolites extracted. Gas chromatography-mass spectrometry (GC-MS) was employed following derivatization of the sugars to assess the extent of label incorporation into N-acetylglucosamine-6-phosphate (GlcNAc6P) (Figs 3A and S2). Labelling was quantified in a fragment (m/z 357), which contains the two carbons proximal to the phosphate group and can be found in most sugar-phosphates, including glucose-6-phosphate (Glc6P) and GlcNAc6P (S2A–S2C Fig). The fragment shifts to m/z 359 upon incorporation of heavy carbons from U-13C 6 -Glc (S2D–S2J Fig). Synthesis of GlcNAc6P from labelled Glc was observed in both parasite stages, reaching 88.8% and 25.5% labelling in intra- and extracellular parasites, respectively (Fig 3A). These findings suggest an active UDP-GlcNAc biosynthesis pathway from Glc in both intra- and extracellular T. gondii. PPT PowerPoint slide PNG larger image TIFF original image Download: Fig 3. UDP-GlcNAc synthesis is disrupted in T. gondii that lack GNA1. A) Percent 13C-labelling in T. gondii (TIR1) derived N-acetylglucosamine-6-phosphate (GlcNAc6P) in unlabelled parasites (natural abundance) or after incubation of intracellular or extracellular parasites in medium containing U-13C 6 -glucose for 24 or three hours, respectively. B) Relative abundance and fractional 13C-labelling in TIR1 and GNA1-mAID-Ty parasite metabolite extracts, following incubation of extracellular parasites in medium containing U-13C 6 -glucose for five hours in the absence of auxin (−IAA) or following 18 hours pre-treatment (+IAA). TIR1 −IAA parasites were incubated in medium with natural abundance glucose as an unlabelled control. Note that metabolites for which the abundance of labelled and unlabelled ions was too low to obtain reliable labelling data were deemed below limit of detection (