{"paper_id":"0613031b-a71a-4785-91b3-de707bbf8b46","body_text":"1 \nN-acetylglucosamine supplementation fails to bypass the critical acetylation of 1 \nglucosamine-6-phosphate required for Toxoplasma gondii replication and 2 \ninvasion 3 \nMaría Pía Alberione1, Víctor González-Ruiz2,3, Serge Rudaz2, Dominique Soldati-4 \nFavre4, Luis Izquierdo1,5#, Joachim Kloehn4# 5 \n1 Barcelona Institute for Global Health (ISGlobal), Hospital Clínic-University of 6 \nBarcelona, Barcelona, Spain. 7 \n2 School of Pharmaceutical Sciences, University of Geneva, Switzerland. 8 \n3 Current affiliation: Centro de Metabolómica y Bioanálisis (CEMBIO), Facultad de 9 \nFarmacia, Universidad San Pablo-CEU, CEU Universities, 28660 Boadilla del Monte, 10 \nMadrid, Spain 11 \n4 Department of Microbiology and Molecular Medicine, University of Geneva, 12 \nSwitzerland. 13 \n5 CIBER de Enfermedades Infecciosas (CIBERINFEC), Barcelona, Spain. 14 \n 15 \n#Corresponding authors: Joachim.kloehn@unige.ch, luis.izquierdo@isglobal.org 16 \nKeywords: Toxoplasma gondii , Apicomplexa, N-acetylglucosamine, GPI -anchors, 17 \nparasites, metabolism, nutrient salvage, glycosylation, invasion 18 \nShort title 19 \nGlucosamine-6-phosphate acetylation is essential in Toxoplasma gondii 20 \n21 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 2 \nAbstract 22 \nThe cell surface of Toxoplasma gondii is rich in glycoconjugates which hold diverse and 23 \nvital functions in the lytic cycle of this obligate intracellular parasite. Additionally, the cyst 24 \nwall of bradyzoites, that shields the persistent form responsible for chronic infection from 25 \nthe immune system, is heavily glycosylated. Formation of glycoconjugates relies on 26 \nactivated sugar nucleotides , such as uridine diphosphate N-acetylglucosamine (UDP-27 \nGlcNAc). The Glucosamine-phosphate-N-acetyltransferase (GNA1) generates N-28 \nacetylglucosamine-6-phosphate critical to produce UDP-GlcNAc. Here, we demonstrate 29 \nthat downregulation of T. gondii GNA1 results in a severe reduction of UDP-GlcNAc and 30 \na concomitant drop in glycosylphosphatidylinositol (GPI), leading to impairment of the 31 \nparasite’s ability to invade and replicate in the host cell. Surprisingly, attempts to rescue 32 \nthis defect through exogenous GlcNAc supplementation fail to completely restore these 33 \nessential functions. In depth metabolomic analyses elucidate diverse causes underlying 34 \nthe failed rescue: utilization of GlcNAc is inefficient under glucose-replete conditions and 35 \nfails to restore UDP -GlcNAc levels in GNA1 -depleted parasites. In contrast, GlcNAc -36 \nsupplementation under glucose-deplete conditions fully restores UDP-GlcNAc levels but 37 \nfails to rescue the defects associated with GNA1 depletion . Our results underscore the 38 \nessentiality of GlcN6P a cetylation in governing T. gondii replication and invasion and 39 \nhighlight the potential of the evolutionary divergent GNA1 in Apicomplexa as a target for 40 \nthe development of much-needed new therapeutic strategies. 41 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 3 \nAuthor Summary 42 \nToxoplasma gondii , Plasmodium, and Cryptosporidium spp., pose serious threats to 43 \nhuman health. T. gondii, an intracellular and opportunistic pathogen, cunningly avoids 44 \nthe host immune defences by forming long-lasting tissue cysts. Finding effective drugs 45 \nto eliminate these parasites remains a challenge. 46 \nThe glucosamine-phosphate-N-acetyltransferase (GNA1) catalyses a critical key step in 47 \nthe production of activated sugar nucleotides to build glycoconjugates essential for 48 \nvarious functions in the cell. In P. falciparu m, this enzyme has been identified as a 49 \npotential target for antimalarial drugs. 50 \nIn this study, we explored the importance of this pathway in T. gondii and discovered that 51 \nthese sugar-containing compounds play a vital role in the parasite's ability to invade and 52 \nreplicate in host cells – crucial processes for its survival and ability to cause disease . 53 \nIntriguingly, unlike some organisms that can bypass the pathway, T. gondii relies critically 54 \non glucosamine -6-phosphate acetylation. This reliance sheds light on the parasite's 55 \ndistinct metabolic properties and highlights the pathway’s potential as a target for new 56 \ntherapeutic strategies. 57 \n58 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 4 \nIntroduction 59 \nThe phylum of Apicomplexa groups a vast numb er of obligate intracellular parasites, 60 \nsome of which pose a considerable threat to human health. The most ubiquitous 61 \napicomplexan, Toxoplasma gondii, causes disease in immunocompromised individuals 62 \n[1, 2], as well as abortions, stillbirths, fetal death, retinal lesions or long -term disabling 63 \nsequelae in congenitally infected children [3, 4]. At present, there is no vaccine that 64 \nprevents toxoplasmosis, and the available treatments are associated with a range of 65 \nshortcomings including high cost , toxicity and rising resistance [5]. In the accidental 66 \nhuman host, T. gondii manifests in two distinct stages: the fast -replicating tachyzoite, 67 \nresponsible for acute disease and the slow replicating bradyzoite, which persists 68 \nencysted within muscle cells and neurons throughout the lifetime of its host [6]. These 69 \npersistent parasites constitute a reservoir, that can reactivate causing life-threatening 70 \nacute toxoplasmosis when the infected individual becomes immunocompromised. The 71 \ninability to eradicate the parasite's latent form, combined with the emergence of parasites 72 \nthat are resistant to existing drugs against acute toxoplasmosis, underscores the 73 \npressing need for novel therapeutic strategies [5]. 74 \nThe endomembrane system of T. gondii is rich in glycoconjugates which play 75 \nfundamental roles in infectivity, survival , and virulence [7]. Several glycan structures 76 \nhave been characterized in T. gondii including N-glycans [8], O-glycans [9-13], C-77 \nmannose [9, 14], GPI -anchors [15, 16], and others [7]. These glycans serve various 78 \ncritical functions from invasion to O2 sensing and nutrient storage, hence contributing to 79 \nthe overall virulence of the parasite [7]. Additionally, glycans are critical components of 80 \nthe bradyzoite cyst wall and the disruption of their formation impairs the parasite’s ability 81 \nto persist [17, 18]. 82 \nThe de novo synthesis of glycans relies on activated sugar nucleotides. Uridine 83 \ndiphosphate N-acetylglucosamine (UDP -GlcNAc) serves as a donor by GlcNAc -84 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 5 \ndependent glycosyltransferases for the synthesis of N-glycans, 85 \nglycosylphosphatidylinositol (GPI) -anchors, glycoinositolphospholipids (GIPLs), and for 86 \nthe glycosylation of other protein acceptors. Given the critical roles of these structures 87 \nfor infectivity of tachyzoites [7, 8, 16, 19], and bradyzoite survival and replication [17, 18], 88 \nthe biosynthesis route of UDP-GlcNAc is a plausible target for intervention against acute 89 \ntoxoplasmosis and for eradication the chronic infection. GNA1, the enzyme catalysing 90 \nthe acetylation of glucosamine -6-phosphate (GlcN6P) is considered a promising drug 91 \ntarget in Apicomplexa . This is attributed to its independent evolutionary origin, unique 92 \nsequence features [20], and established essentiali ty for the intraerythrocytic 93 \ndevelopment of Plasmodium falciparum [21]. 94 \nIn T. gondii, a genome -wide CRISPR fitness screen underscored the significance of 95 \nUDP-GlcNAc biosynthesis for the parasite, classifying several genes encoding for 96 \nenzymes involved in the amino sugar synthesis pathway as fitness -conferring [22, 23]. 97 \nUnexpectedly, however, this study predicted GNA1 to be dispensable for T. gondii, even 98 \nthough the upstream and downstream enzymes were highly fitness-conferring [23]. 99 \nHere we demonstrate th e essential nature of GNA1 in T. gondii , revealing that its 100 \ndownregulation leads to a reduction in GPI anchors that impairs invasion and replication 101 \nwithin its host cell. Intriguingly, defects in GNA1 cannot be overcome by GlcNAc salvage. 102 \nTargeted metabolomic analyses revealed that GlcNAc salvage is inefficient in glucose -103 \nreplete conditions . In contrast, GlcNAc is effectively salvaged in glucose -deplete 104 \nconditions, but fails to rectify the defects associated with the disruption of the pathway. 105 \nThese findings highlight the potential of GNA1 as a drug targe for combatting 106 \ntoxoplasmosis. 107 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 6 \nResults 108 \nT. gondii GNA1 is essential, contradicting the prediction from a genome wide 109 \nfitness screen 110 \nT. gondii expresses several glycan structures akin to other eukaryotic cells, with few 111 \nnoteworthy characteristics ( Fig 1A). The synthesis of GPI -anchors, N-glycans and O-112 \nglycans requires as donor substrate UDP-GlcNAc, or UDP -GalNAc. The latter can be 113 \nsynthesised from UDP -GlcNAc via G alE [7], a UDP -Glc/UDP-Gal epimerase . In the 114 \namino sugar biosynthesis pathway, the glucosamine 6 -phosphate N-acetyltransferase, 115 \nGNA1, catalyses the acetylation of GlcN6P (Fig 1B). Although T. gondii has been shown 116 \nto critically rely on several glycan structures [7-9], TgGNA1 (TGGT1_243600) was 117 \nassigned a positive fitness score (+1.41) in a genome-wide fitness screen [23], indicating 118 \nits potential dispensability ( Fig 1C). This is unexpected considering the crucial role of 119 \nGNA1 in other organisms [24], including P. falciparum [21]. Additionally, enzymes acting 120 \neither downstream or upstream of GNA1 were assigned negative fitness scores in T. 121 \ngondii, suggesting their essentiality [23]. 122 \nExamination of the nanopore sequencing data on ToxoDB [25, 26] revealed two major 123 \nGNA1 transcripts, with the shorter, more prevalent transcript only covering a portion of 124 \nthe predicted protein coding sequence. In addition to the predicted GNA1 protein coding 125 \nsequence, which encodes a putative protein of 55.5 kDa , four additional in-frame open 126 \nreading frames were identified. These could give rise to GNA1 proteins of var ious 127 \nreduced sizes (45.5, 21.0, 17.2 and 16.7 kDa ) [25] (S1 Fig ). Critically, the 128 \nacetyltransferase domain is located near the C-terminus and is present in all five putative 129 \nisoforms. Consequently, all proteins, including the shortest version, could potentially be 130 \ncatalytically active. These shorter GNA1 isoforms may explain the unexpected positive 131 \nfitness score for GNA1 [23]. Concordantly, out of the ten single guide RNAs (sgRNAs) 132 \nused to target GNA1 in the genome wide fitness screen [25, 26], four bind near the 133 \nextended N -terminus, only present in the long est isoform of GNA1 (guides 134 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 7 \nsgTGGT1_243600_5, _6, _9 and _10; average fitness score +1.02), while three bind 135 \nclose to the C -terminus of GNA1 , disrupting all potential isoforms, (guides 136 \nsgTGGT1_243600_2, _3, and _4; average fitness score: -7.50,) (S1 Fig). Notably, the 137 \npublished phenotype score is calculated by averaging the value of the top five scoring 138 \nguides to mitigate the impact of stochastic losses, resulting in the positive fitness score 139 \nfor GNA1 [23]. Given the existence of diverse isoforms, the assigned fitness score likely 140 \ndoes not reflect the importance that GNA1 may play for T. gondii, prompting further 141 \ninvestigation of the enzyme. 142 \nTo examine the localization and function of GNA1 in T. gondii (TGGT1_243600), the 143 \nendogenous locus was edited using CRISPR/Cas9 [23, 27]. Simultaneously a Ty epitope 144 \ntag and a mini auxin inducible degron (mAID) domain were fused to the C-terminus of 145 \nGNA1 in parasites stably express ing the auxin receptor transport inhibitor response 1 146 \n(TIR1) from Oryza sativa (RH-TIR1) [28, 29] . The hypoxanthine-xanthine-guanine 147 \nphosphoribosyl transferase ( hxgprt) resistance cassette was inserted, for selection of 148 \npositive transfectants (S1 Fig ) [30]. T he mAID domain enables rapid and efficient 149 \ndownregulation of the protein of interest via proteasomal degradation upon addition of 150 \nauxin (indole 3-acetic acid, IAA) [29]. Successful integration of the construct at the gna1 151 \nlocus in a clonal population was confirmed by genomic PCR (S1 Fig), using primers listed 152 \nin S2 Table. GNA1 -mAID-Ty exhibited a dotty cytosolic localization by 153 \nimmunofluorescence assay (IFA) (Fig 2A). Subsequently, downregulation of GNA1 was 154 \nassessed by Western blot (2, 4 - and 18-hours IAA treatment, Fig 2B) and by IFA (18 -155 \nhours IAA treatment, Fig 2C), confirming an efficient and complete depletion of GNA1 156 \nafter 2-4 hours of IAA treatment. Interestingly, the western blot revealed several bands 157 \nbetween approximately 30 -80 kDa for GNA1 -mAID-Ty ( Fig 2B ), all of which were 158 \nefficiently downregulated upon addition of IAA. Up to five GNA1 isoforms may exist, with 159 \nmolecular weights ranging from 29.7 – 68.5 kDa, including the tag. These results indicate 160 \nthat full length GNA1 as well as shorter isoforms (S1 Fig) are synthesised, although 161 \npartial degradation cannot be excluded. 162 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 8 \nThe significance of GNA1 for the parasite lytic cycle was assessed by plaque assay. 163 \nDownregulation of GNA1 prevented the formation of plaques of lysis (Fig 2D), indicating 164 \nthat GNA1 is needed for one or several steps of the lytic cycle. The intracellular growth 165 \nassay revealed a significant impact of GNA1 depletion on the replication rate, with the 166 \naverage number of parasites during 24 hours of growth decreasing from 6.5 in the 167 \ncontrols to 4.2 following 36 hours of IAA treatment (Fig 2E). 168 \nThese results underscore the essential role of GNA1 in intracellular growth and overall 169 \nlytic cycle of T. gondii, consistent with the importance of glycoconjugates. The data 170 \nsuggest that two major transcripts are generated for GNA1, yielding up to five protein 171 \nisoforms that can be potentially catalytically active. The extended N -terminus in the 172 \nlonger isoform may be dispensable [23]. If it holds a regulatory function in other life cycle 173 \nstages remains unknown. Crucially, depletion of the GNA1 acetyltransferase domain is 174 \ndetrimental to T. gondii. 175 \n 176 \nGNA1 is active and critical for UDP-GlcNAc synthesis in T. gondii 177 \nTo examine if the UDP-GlcNAc biosynthesis pathway is active in intra- and extracellular 178 \nT. gondii, TIR1 parasites were cultured intracellularly for 24 hours in medium containing 179 \n10 mM uniformly 13C-labelled glucose (U-13C6-Glc) or extracted and purified extracellular 180 \nparasites were incubated for 3 hours in medium containing heavy Glc. Post-incubation, 181 \nthe metabolism was quenched, parasites were harvested, and metabolites extracted. 182 \nGas chromatography-mass spectrometry (GC-MS) following derivatization of the sugars 183 \nwas employed to assess the extent of label incorporation into N-acetylglucosamine-6-184 \nphosphate (GlcNAc6P) (S3 Fig). Synthesis of GlcNAc6P from labelled Glc was observed 185 \nin both parasite stages, reaching 88.8% and 25.5% labelling in intra - and extracellular 186 \nparasites, respectively ( Fig 3A). These findings unequivocally demonstrate t he active 187 \nUDP-GlcNAc biosynthesis pathway from Glc in both intra- and extracellular T. gondii. 188 \nTo assess the critical participation of GNA1 in the pathway, TIR1 and GNA1-mAID-Ty 189 \nparasites were untreated or pre-treated with IAA for 18 hours and extracellular parasites 190 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 9 \nwere incubated in medium containing 10 mM U -13C6-Glc. TIR1 −IAA parasites that 191 \nserved as control, were incubated with regular medium containing unlabelled (natural 192 \nabundance) Glc . After 5 hours of incubation, parasites were harvested, metabolites 193 \nextracted, derivatized and analysed by GC-MS in a targeted manner (S3 Fig). Under all 194 \ntested conditions, Glc6P was detected with incorporation of heavy carbons under 13C-195 \nlabelling conditi ons ( Fig 3B ). Intriguingly, glucosamine-6-phosphate (GlcN6P), the 196 \nsubstrate of GNA1, was exclusively detected in parasites depleted in GNA1, with 197 \nincorporation of considerable labelling (45.8%). In contrast, GlcNAc6P, the product of 198 \nGNA1, was detected in all conditions, except in parasites depleted in GNA1 (Fig 3B). To 199 \ngain a comprehensive understanding of the impact of GNA1 depletion on T. gondii 200 \nmetabolism, we performed metabolite profiling by GC -MS after 36 hours of 201 \ndownregulation ( S4 F ig). At this relatively late time point, pleiotropic effects were 202 \nobserved with 28 out of 64 metabolites significantly altered (>2-fold) in their abundance 203 \ncompared to TIR1 −IAA. The majority (26 metabolites) exhibited reduced abundance 204 \nincluding amino acids, TCA cycle intermediates, sugars, fatty acids, and others. Amongst 205 \nthe most dramatically reduced metabolites were GlcNAc and GlcNAc6P, consistent with 206 \nthe function of GNA1. Conversely the two significantly increased metabolites were GlcN 207 \nand myo-inositol. While GlcN accumulation directly correlates with the absence of GNA1 208 \n(following enzymatic dephosphorylation or loss of the phosphate group during sample 209 \npreparation from GlcN6P), accumulation of myo-inositol could be part of a general stress 210 \nresponse, as previously observed [31], or a consequence of impaired GPI -anchor 211 \nsynthesis. 212 \nGiven that crucial intermediates such as GlcNAc1P and the product UDP-GlcNAc cannot 213 \nbe detected by GC -MS, we turned to liquid chromatography mass spectrometry (LC -214 \nMS/MS) for more sensitive detection of all relevant pathway intermediates. Intracellular 215 \nparasites were treated with IAA or not for 18 hours, before quenching of the metabolism, 216 \nparasite harvest, metabolite extraction and analysis. While fructose-6-phoshate (Fru6P) 217 \nlevels remained unaffected by GNA1 downregulation (GNA1 -mAID-Ty +IAA), GlcN6P 218 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 10 \naccumulated dramatically to 91.8-fold higher levels compared to the controls (Fig 3C). In 219 \nsharp contrast, as observed by GC -MS, GlcNAc6P was markedly reduced (50.9 -fold). 220 \nSimilarly, the subsequent metabolites, GlcNAc1P and the product UDP -GlcNAc 221 \nexhibited reductions of 83.3- and 803.2-fold, respectively (Fig 3C). 222 \nTogether, these findings reveal that UDP -GlcNAc synthesis is active both in intra- and 223 \nextracellular T. gondii tachyzoites, Moreover, GNA1 plays a critical role in this pathway 224 \nas its disruption results in a significant reduction in the activated sugar nucleotide UDP-225 \nGlcNAc and an accumulation of its substrate GlcN6P. 226 \n 227 \nGlcNAc supplementation fails to rescue GNA1 deficiency 228 \nEfficient bypass of defects in UDP-GlcNAc synthesis is well documented in several 229 \norganisms, including P. falciparum through GlcNAc supplementation [21, 32, 33] . 230 \nGlcNAc can be taken up and phosphorylated by hexokinase, generating GlcNAc6P, 231 \neffectively circumventing the initial steps of the pathway . To test if exogenous GlcN or 232 \nGlcNAc supplementation can bypass the function of GNA1 in T. gondii, we performed 233 \nplaque assays with GNA1 -mAID-Ty parasites in presence or absence of IAA while 234 \nsupplementing different concentrations of GlcN or GlcNAc. Remarkably, none of the 235 \nsupplementations could rescue the lytic cycle defect observed in parasites depleted in 236 \nGNA1 (Fig 4A). 237 \nWe hypothesised that the inability of GlcN or GlcNAc supplementation to rescue the lytic 238 \ncycle defect in GNA1 -depleted parasites could be attributed to various reasons I) 239 \ninsufficient uptake of GlcNAc by the host cells and/or T. gondii, II) incapacity of T. gondii 240 \nhexokinase to phosphorylate GlcNAc or III) inefficient entry of phosphorylated, salvaged 241 \nGlcNAc into the UDP -GlcNAc synthesis pathway . To explore these possibi lities, we 242 \nincubated purified extracellular parasites in medium without Glc supplemented with 13C6-243 \nGlcN or 13C6-GlcNAc for 5 hours, before harvesting parasites and extracting metabolites. 244 \nGNA1-mAID-Ty and TIR1 parasites were pretreated with IAA for 18 hour s to deplete 245 \nGNA1 levels in the GNA1-mAID-Ty strain. TIR1 −IAA were incubated in regular medium 246 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 11 \nwith unlabelled (natural abundance) Glc. Remarkably, both amino sugars were efficiently 247 \nsalvaged and utilised. 13C6-GlcN was salvaged and phosphorylated under all conditions, 248 \nbut as expected, the downregulation of GNA1 prevented the formation of GlcNAc6P from 249 \nGlcN6P ( Fig 4B). Similarly, exogenous 13C6-GlcNAc was efficiently used to generate 250 \nlabelled GlcNAc6P in the control strains (Fig 4C). Parasites deficient in GNA1 could also 251 \nsalvage exogenous 13C6-GlcNAc and utilised it to generate GlcNAc6P, albeit at 252 \nsignificantly lower levels but fully 13C-labelled, consistent with the inability of Glc to 253 \ncontribute to GlcNAc6P formation. These resul ts reveal that T. gondii can salvage and 254 \nutilise GlcNAc, potentially bypassing the need for GNA1. 255 \nIt is noteworthy that this experiment was conducted with extracellular parasites which 256 \nwere pre-depleted in GNA1 over 18 hours under regular growth conditions, without 257 \nGlcNAc supplementation. Thus, parasites were expected to be impaired in their fitness 258 \nand this measurement may not reflect what occurs during intracellular development and 259 \nduring continuous supplementation. To address this, we cultured intracellular T. gondii, 260 \nTIR1 and GNA1 -mAID-Ty parasites under standard conditions for 24 hours before 261 \nchanging the culture medium to one of the following 4 conditions for 18 hours prior to 262 \nparasite harve st: regular medium −IAA; regular medium +IAA; regular medium +IAA 263 \nsupplemented with 10 mM GlcNAc; and medium without Glc +IAA supplemented with 264 \nadditional glutamine and 10 mM GlcNAc. Parasites were harvested while intracellular, 265 \nwith the medium being removed and parasite metabolism quenched prior to the harvest 266 \nof parasites, to exclude any metabolite uptake by extracellular parasites. Metabolites 267 \nwere extracted and analysed by LC -MS/MS, as above ( Fig 4D). Remarkably, GlcNAc 268 \nsupplementation had only a marginal impact in the presence of Glc. Although 269 \nsignificantly higher than in non-supplemented parasites devoid of GNA1, GlcNAc failed 270 \nto fully restore UDP-GlcNAc levels, with levels being 13.3-fold lower than in control (TIR1 271 \n-IAA) parasites (see detail in S5 Fig ). In the absence of Glc, however, GlcNAc was 272 \nefficiently salvaged and markedly increased UDP-GlcNAc levels, both in TIR1 parasites 273 \nas well as in parasites deficient of GNA1. Crucially, UDP-GlcNAc levels in GNA1-devoid 274 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 12 \nparasites supplemented with GlcNAc in the absence of Glc were 2.2-fold higher than in 275 \ncontrol (TIR1 -IAA) parasites, suggesting a full rescue of the pathway. Unexpectedly, 276 \nGlcN6P continued to accumulate to levels >100-fold higher in parasites devoid of GNA1, 277 \nregardless of the presence or absence of Glc in the medium. Since Fru6P levels were 278 \nmarkedly down in the absence of Glc, we speculate that the detected GlcN6P is not 279 \nderived from gluconeogenesis but rather from the deacetylation of GlcNAc, either 280 \nthrough deacetylases of the host cell or by the parasite. The generated GlcN6P failed to 281 \nbe converted further in the absence of GNA1. In summary, this detailed analysis of the 282 \npathway under varying conditions reveals efficient GlcNAc salvage but only in the 283 \nabsence of Glc. Under this condition, the function of GNA1 can be bypassed, fully 284 \nrestoring UDP-GlcNAc levels. Whether a potential competition between Glc and GlcNAc 285 \nhappens at the level of uptake by the host or the parasite, or at the level of 286 \nphosphorylation in the parasite remains unclear. 287 \nWhile GlcNAc supplementation failed to restore the lytic cycle defect in GNA1-depleted 288 \nparasites in the presence of Glc, we investigated whether the defects in the parasite ’s 289 \nintracellular growth could be rescued by exogen ous GlcNAc, both in the presence or 290 \nabsence of Glc. Consistent with the inefficient utilization of GlcNAc in the presence of 291 \nGlc, GlcNAc supplementation failed to rescue the growth defect in regular medium ( Fig 292 \n4E). GlcNAc supplementation in the absence of Glc, however, facilitated a significant but 293 \nmodest and incomplete rescue of the intracellular growth rate, following 48 hours of 294 \ntreatment (Fig 4E). Lastly, we explored whether certain ratios of Glc and GlcNAc could 295 \npotentially rescue parasites depleted in GNA1, by supporting central carbon metabolism 296 \n(Glc), but facilitating GNA1 bypass (GlcNAc) and potentially alleviating excessive 297 \nGlcN6P accumulation. Plaque assays were performed with parasites in varying Glc and 298 \nGlcNAc ratios, however none of the conditions were able to rescue the lytic cycle defect 299 \nassociated with GNA1 downregulation (S6 Fig). 300 \n 301 \nGNA1 is needed for GPI-anchor synthesis critical for host cell invasion by T. gondii 302 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 13 \nThe observed impairment of UDP -GlcNAc synthesis in parasites depleted in GNA1 is 303 \nexpected to impair the synthesis of glycans, including GPI -anchors, thereby likely 304 \naffecting the parasite’s ability to invade host cells. GPI-anchored proteins are critical for 305 \nparasite invasion, contributing to the expression of a series of surface antigens, which 306 \nplay a vital role during host cell attachment [16, 34, 35] . Additionally, glycosylated 307 \nproteins have been described to traffic to the apical secretory organelles and playing an 308 \nessential role in their biogenesis and function [19, 36]. To assess if GNA1 is required for 309 \nthe appropriate localization and formation of GPI-anchored proteins, we performed IFAs, 310 \nevaluating the expression and distribution of the surface antigen 1 (SAG1) [37]. 311 \nDownregulation of GNA1 resulted in a drop in SAG1 signal intensity and an aberrant 312 \ndistribution, with the signal commonly accumulating in the residual body ( Fig 5A). This 313 \nabnormal staining was observed in 68.3 and 73.3% of vacuoles following downregulation 314 \nof GNA1 for 18 or 36 hours, respectively ( Fig 5B). To investigate if this is a relatively 315 \nspecific defect or if cells devoid of GNA1 exhibit various morphological abnormalities, we 316 \nassessed the morphology of the apicoplast and mitochondrion, upon GNA1 317 \ndownregulation for the same duration (S7 Fig). The organelles were studied by IFA using 318 \nα-CPN60 (chaperonin 60) and α-5F4 (F1 ATPase beta subunit ), two specific markers 319 \nfound in the apicoplast and at the mitochondrion, respectively. Both organelles appeared 320 \nmorphologically intact and normal after 18 hours and 36 hours of IAA-treatment (S7 Fig). 321 \nNext, to confirm if the abnormal SAG1 signal, observed in GNA1 -deficient cells, can 322 \nindeed be attributed to a defect in the synthesis of GPI-anchors, we quantified the relative 323 \namount of GPI -anchors following downregulation of GNA1 for 36 hours. To this en d, 324 \nparasite lipids were extracted and subjected to methanolysis to hydrolyse 325 \nmonosaccharides off glycan structures found in the organic phase after metabolite 326 \nextraction. The derivatised sugar and fatty acid methyl esters were analysed by GC-MS 327 \nand the sig nal intensity for mannose residues was quantified relative to the signal 328 \nintensity for palmitic acid [38]. Notably, the ratio mannose signal intensity/palmitic acid 329 \nsignal intensity decreased 8.5 -fold after 36 hours of IAA treatment, c onsistent with a 330 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 14 \nmarked drop in GIPLs and GPI -anchor formation (Fig 5C). As expected, this caused a 331 \nsevere defect in host cell invasion, with only 26.5% of parasites depleted in GNA1 over 332 \n18 hours invading successfully, compared to more than 70% of parasites in all controls 333 \n(Fig 5D). 334 \nTo assess if GlcNAc supplementation, and the consequent increase in UDP -GlcNAc 335 \nlevels under Glc-deplete conditions (Fig 4D), could restore GPI-anchor synthesis, GPI-336 \nanchor levels were quantified after GlcNAc supplementation under Glc-replete or -337 \ndeplete conditions. After 18 hours of IAA-treatment and the indicated supplementations, 338 \nGlcNAc supplementation appeared to increase relative GPI levels in TIR1 parasites and 339 \nled to a slight increase in GPI levels in G NA1-depleted parasites. However, levels 340 \nremained markedly lower (~5-fold) compared to control conditions (Fig 5E). Notably, the 341 \nmodest increase in GPI-anchor levels was observed equally under Glc replete and Glc 342 \ndeplete conditions. These relative GPI level s correlated remarkably well with parasite 343 \ninvasion following 18 hours of treatment with IAA and supplementations as indicated: 344 \nGlcNAc-supplemented GNA1 -depleted parasites demonstrated a significant but still 345 \nincomplete rescue in their ability to invade host cells (Fig 5F). 346 \nOverall, GlcNAc supplementation under Glc deplete conditions fully restored UDP -347 \nGlcNAc levels in cells lacking GNA1 (Fig 4D ), but this only facilitated a modest and 348 \nincomplete rescue of the intracellular growth rate (Fig 4E), GPI abundance (Fig 5E) and 349 \nthe parasites’ ability to invade (Fig 5F). In summary, GNA1 is essential for T. gondii 350 \nreplication and invasion and cannot be bypassed by GlcNAc salvage. 351 \n 352 \nDiscussion 353 \nThe amino sugar pathway, also known as the hexosamine biosynthetic pathway, plays 354 \na crucial role in various organisms, including the apicomplexan parasite P. falciparum 355 \n[20, 21] . A critical function of the pathway in the related apicomplexan T. gondii is 356 \nconsistent with the highly negative fitness scores for most of the enzymes in the pathway, 357 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 15 \nas reported by a genome-wide CRISPR sgRNA-based fitness screen [23]. However, the 358 \nscreen assigned a positive fitness score to GNA1, which catalyses the acetylation of 359 \nGlcN6P to GlcNAc6P, contrasting with the assumed key function of this enzyme in the 360 \npathway. Our presented data confirm that the specific activity of GNA1 in T. gondii, as 361 \npreviously illustrated by in vitro activity assays [20], is essential for parasite survival. The 362 \ndiscrepancy between our finding s and the genome -wide fitness analysis likely arises 363 \nfrom an omission in the gene annotation, which failed to highlight the existence of several 364 \nshort GNA1 isoforms. Our data suggest that full size GNA1 (consistent with the 365 \nannotated sequence [25]) is synthesised alongside several shorter isoforms. These 366 \nshorter isoforms contain the essential acetyltransferase domain [39], and remain 367 \nunaffected by several individual sgRNAs employed in the genome-wide fitness screen 368 \nto disrupt GNA1 [23]. To our knowledge, most eukaryotic organisms exhibit only a single 369 \nGNA1 isoform [40]. However, within the apicomplexan GNA1 family, T. gondii GNA1 370 \nstands out due to its distinctive and elongated N -terminus [20]. The function of this 371 \nextended N -term remains unknown. Despite the valuable information provided by 372 \ngenome wide studies, our findings highlight the limitations of such approaches and 373 \nautomatic gene annotation, reinforcing the importance of studying genes individually for 374 \na thorough comprehension of their significance. 375 \nUDP-GlcNAc and UDP-GalNAc derived from UDP-GlcNAc through the activity of GalE 376 \nepimerase [7], are the main products of the amino sugar pathway. UDP -GlcNAc is key 377 \nfor the synthesis of GPI anchors and free GIPLs, which are present on the surface of all 378 \nT. gondii life stages [41-43]. SAG1, the pri mary surface antigen o f T. gondii, is a GPI 379 \nanchored protein crucial for host cell binding and invas ion [44]. Consequently GPI 380 \nanchors and GIPLs contribute to parasite virulence [45] and are essential for T. gondii 381 \nsurvival [16]. Our results reveal that GNA1 depletion leads to a mark ed drop in GPI 382 \nanchors, altering the localization of SAG1 and severely impacting host cell invasion and 383 \nintracellular growth. Furthermore, UDP-GlcNAc, along with other glycosylations, plays a 384 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 16 \ncrucial role in the biosynthesis of N-glycans, modifying numerous proteins in the T. gondii 385 \nsecretory pathway [7]. Several studies suggest that N-glycosylation is essential for 386 \nparasite invasion, motility, and viability [8, 23, 46]. Indeed, N-glycosylation, but also GPI 387 \nanchor biosynthesis and the amino sugar metabolism are among the metabolic pathways 388 \nwith the highest proportion of essential genes in T. gondii tachyzoites, as reported by a 389 \nrecent study [22]. In summary, our data emphasize the importance of GNA1 for the 390 \namino sugar pathway and UDP -GlcNAc synthesis, highlighting the pivotal role of this 391 \nmetabolic route for parasite virulence and survival. 392 \nDepletion of GNA1 results in the accumulation of GlcN6P, and the reduction or absence 393 \nof the downstream metabolites GlcNAc6P, GlcNAc1P and UDP -GlcNAc. While growth 394 \ncan be rescued by supplementing the media with high concentrations of GlcNAc in P. 395 \nfalciparum and other organisms [21, 32, 33], GlcNAc supplementation fails to rescue the 396 \nlack of GNA1 in T. gondii. GlcN supplementation also prov ed ineffective in recovering 397 \nparasite growth. Despite the inability to rescue parasite growth, the absence of Glc in the 398 \nmedia enhances GlcNAc salvaging, replenishing UDP -GlcNAc levels. This strongly 399 \nsuggests a competition between Glc and GlcNAc at the upt ake or phosphorylation 400 \nprocesses. Nevertheless, the recovery of GPI anchors and parasite growth through 401 \nGlcNAc salvage is only partial when Glc is absent. The low levels of Fru6P indicate 402 \nincomplete gluconeogenesis via glutamine, the predominant carbon source for T. gondii 403 \nin absence of Glc [47]. In addition, these scant amounts of Fru6P in GlcNAc -404 \nsupplemented TIR1 parasites under Glc depletion, strongly suggest the lack of an amino 405 \nsugar catabolic pathway in T. gondii. Notably, the pronounced accumulation of GlcN6P 406 \nobserved in T. gondii GNA1 mutants could also contribute to the limited rescue observed 407 \nwith GlcNAc supplementation [49, 50]. Intriguingly, the accumulation of GlcN6P 408 \ncorrelated more closely with a drop in GPI levels and concomitant impairment of parasite 409 \ninvasion than UDP -GlcNAc levels, indicating a potentially toxic impact of this surge in 410 \nGlcN6P. Indeed, glucosamine has been shown to interfere with P. falciparum asexual 411 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 17 \nintraerythrocytic growth at high doses [47-49], and a comparable eff ect could be 412 \noccurring in T. gondii. Regardless, the high concentration of GlcNAc needed for partial 413 \nT. gondii growth recovery remains far from physiological levels [50], suggesting that the 414 \nlikelihood of rescuing GNA1 -depleted parasites under physiological conditions is very 415 \nremote. In summary, the inco mplete rescue in GlcNAc supplemented media strongly 416 \nsuggests the inability of a metabolic bypass to overcome GNA1 deficiency. This 417 \nspotlights T. gondii GNA1 as a potential drug target to tackle toxoplasmosis. 418 \n 419 \nT. gondii GNA1 belongs to a specific gene family, with an independent evolutionary origin 420 \nwithin the phylum Apicomplexa [20]. Apicomplexan GNA1s exhibit distinct features and 421 \nconserved motifs, and a recent structural study highlighted the divergent binding sites 422 \nfor GlcN6P and acetyl -CoA in Cryptosporidium parvum GNA1 compared to human 423 \nGNA1, including important variations in key residues [21]. The key role of the amino 424 \nsugar pathway for T. gondii viability, and the predicted significance of GPI anchors and 425 \nGlcNAc-containing glycoconjugates across T. gondii’s life cycle [7, 22], underscore the 426 \npotential of GNA1 as a versatile multistage therapeutic target in toxoplasmosis that could 427 \nbe exploited for selective parasite inhibition. 428 \nCurrent drug therapies for human toxoplasmosis lack specificity, often leading to adverse 429 \neffects and inconsistent efficacy [5, 51]. Novel treatments against T. gondii must target 430 \nthe slow growing bradyzoites [52] to eradicate the chronic stage, which poses a threat to 431 \ninfected individuals if the immune system is compromised [53]. Targeting bradyzoites 432 \nefficiently is hindered by several hurdles: drugs must cross the blood -brain barrier and 433 \ntraverse the cyst wall and must act on enzymes/pathways that are critical for the poorly 434 \ncharacterized metabolism of bradyzoites [54]. Intriguingly, the cyst in which bradyzoites 435 \nreside and persist is heavily glycosylated, containing high levels of GlcNAc and GalNac 436 \nresidues [55]. A previous study highlighted that glycosylation of the cyst wall is critical for 437 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 18 \nT. gondii persistence [17]. Specifically, Caffaro et al., demonstrated that the nucleotide 438 \nsugar transporter TgNST1 is required for cyst wall glycosylation and its disruption impairs 439 \nthe ability of T. gondii to persist but is dispensable for tachyzoites in vitro and during 440 \nacute infection in vivo [17]. We demonstrate here that GNA1 is essential for tachyzoites 441 \nand can be expected to be essential for bradyzoites given the high need for UDP-GlcNAc 442 \nand UDP-GalNAc during persistence [17], making it a promising candidate for a drug 443 \ntarget. 444 \n 445 \nMaterial and Methods 446 \nParasite lines, culture and treatments 447 \nParasites stably expressing TIR1 were a generous gift from the laboratory of David 448 \nSibley [29]. These were maintained by regular passages in human foreskin fibroblasts 449 \n(HFF-1, ATCC SCRC -1041), in Dulbecco Modified Eagle Medium (DMEM, Gibco, 450 \n41966-029) supplemented with foetal bovine serum (FBS, Gibco, 10270-106, 5% v/v), 451 \nL-glutamine (Gibco, 20530-024, additional 2 mM) and Gentamycin (Gibco, 15750-045, 452 \n25 μg/ml), incubated in humidified incubators at 37 °C and 5% CO2. 453 \nAuxin (IAA, Sigma-Aldrich, I-2886) was added to cultures at 500 μM final in ethanol as 454 \nindicated for each experiment. Supplementations with sugars (Glc – Agilent, 103577, 455 \nGlcN – Sigma-Aldrich, G1514 or GlcNAc – Sigma-Aldrich, A3286) were performed as 456 \ndescribed for ea ch experiment in regular medium, as above or in DMEM without Glc 457 \n(Gibco, 11966-025) supplemented with 5% (v/v) dialysed FBS (Pan Biotech P30-2102; 458 \n10,000 Da exclusion size membrane) and additional 10 mM glutamine (Agilent, 103579). 459 \nGeneration of Transgenic Parasites 460 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 19 \nThe GNA1-mAID-Ty parasite line was generated through co-transfection of a CRISPR-461 \nCas9 expression plasmid [27] with a guide RNA (P1, S2 Table) targeting the 3’-UTR of 462 \nGNA1 (TTGT1_243600) and a homology repair template encoding the mAID domain, 463 \nthe 3-Ty domain and the hxgprt resistance cassette, amplified with the primers P2 and 464 \nP3 (S2 Table) by KOD PCR (Sigma -Aldrich). Transfected parasites were selected in 465 \nmedium containing mycophenolic acid (25 μg/ml) and xanthine (50 μg/ml) over one week 466 \nand cloned by serial dilution followed by a second round of cloning. Su bclones were 467 \nfrozen and a single clone was used in the following experiments. 468 \nCorrect integration of the homology template at the desired location was assessed by 469 \nPCR (GoTaq DNA Polymerase, Promega) on extracted genomic DNA (Promega Wizard 470 \nDNA Extraction) testing 3 amplifications using primers P4 and P5, P4 and P6 and P4 471 \nand P7 amplifying under the following conditions: 95 °C, 2 min; (95 °C, 15 s; 57 °C 15 s; 472 \n72 °C 1.5 min) × 35; 72 °C, 5 min on a SimpliAmp Thermal Cycler (Applied Biosystems). 473 \nImmunofluorescence Assays 474 \nConfluent monolayer of HFF cells grown on coverslips were inoculated with 10 μl of 475 \nfreshly egressed parasite cultures and treated with IAA as or other supplementations as 476 \nindicated for each experiment. Twenty-four hours after inoculation, parasites were fixed 477 \nwith 4% PFA and 0.05% glutaraldehyde for 10 min, before quenching with 0.1 M glycine 478 \nin PBS for 20 min. Infected host cells were permeabilized using 0.2% Triton X-100/PBS 479 \nfor 20 min, followed by 20 min incubation in (2% BSA/0.2% Triton X -100/PBS to block 480 \nunspecific binding and subsequently probed with different primary antibodies diluted in 481 \n2% BSA/0.2% Triton X-100/PBS for 1 hour. The following primary antibodies were used 482 \nas indicated for each experiment: α-Ty (1:10, mouse monoclonal, BB2), α-SAG1 (1:10, 483 \nmouse, T4 -1E5), polyclonal rabbit α -GAP45 (1:10,000, used for growth assay) [56], 484 \nmonoclonal mouse α -actin (1:20) [57], polyclonal rabbit α -CPN60 [58] and mouse 485 \nmonoclonal α-5F4 (F1 ATPase beta subunit, P. Bradle y). The probed monolayer was 486 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 20 \nwashed (3 × 5 min, 0.2% Triton X-100/PBS) and probed with a secondary antibody: anti 487 \nmouse Alexa fluor 488 (Invitrogen, A11001), anti -rabbit Alexa fluor 594 (Invitrogen, 488 \nA11012). Following 3 washing steps as above, the coversli ps were mounted on 489 \nmicroscopy slides using DAPI-containing FluoromountG (SouthernBiotech). Slides were 490 \nviewed on an Eclipse Ti inverted microscope (Nikon). For growth assays, the number of 491 \nparasites was counted in >100 vacuoles per condition for 3 independ ent biological 492 \nreplicates. Images were acquired using an LSM 700 confocal scanning microscope 493 \n(Zeiss) and images were processed using Fiji Image J software. 494 \nWestern Blots 495 \nParasites were harvested from a freshly lysed dish, washed with PBS, and resuspended 496 \nin SDS –PAGE buffer (50 mM Tris -HCl, pH 6.8, 10% glycerol, 2 mM EDTA, 2% SDS, 497 \n0.05% bromophenol blue, and 100 mM dithiothreitol (DTT)). Following boiling for 10 min, 498 \nsamples were subjected to SDS –PAGE under reducing conditions. Proteins were 499 \ntransferred to a hybond ECL nitrocellulose membrane using a wet transfer system (Bio-500 \nRad Laboratories, Hercules, CA, USA). The membrane was incubated in α-Ty antibody 501 \n(1:10, mouse monoclonal, BB2) and rabbit α-catalase as a loading control [59], diluted 502 \nin PBS, 0.05% Tween20, 5% skimmed milk. Following 3 washing steps, the membrane 503 \nwas incubated with the secondary antibodies (goat α -mouse, horse radish peroxidase 504 \nconjugated, Sigma Aldrich, A5278). Signal was visualized using the SuperSignal West 505 \nPico PLUS Chemiluminescent Substrate (ThermoFisher Scientific, 34580). Images were 506 \ntaken using the Bio -Rad ChemiDoc MP Imaging System and images were processed 507 \nusing Bio-Rad Image Lab software. 508 \nPlaque Assays 509 \nSerial dilutions of parasite cultures were incu bated on a confluent host cells monolayer 510 \nin 12- or 24-well plates for 7 days. Afterwards, the infected monolayer was washed with 511 \nPBS and fixed with 4% paraformaldehyde (PFA) for 10 min. Host cells were stained with 512 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 21 \na crystal violet solution (12.5 g crysta l violet, 125 ml ethanol mixed with 500 ml water 513 \ncontaining 1% (w/v) ammonium oxalate) over 3 hours. Wells were washed 3 times with 514 \ndeionized water to remove excess crystal violet and images of dried wells recorded. 515 \nInvasion (Red/Green) Assays 516 \nParasites from a freshly egressed culture treated as described for each experiment were 517 \ndiluted 1:10 and 150 μl of parasite solution used to infect a coverslip with confluent HFFs 518 \nin a 24 -well plate. The plate was gently spun for 1 min at 1,100 g and subsequently 519 \nincubated in a water bath at 37 °C. Cells were fixed with 4% PFA and 0.05% 520 \nglutaraldehyde for 7 min, before quenching with 0.1 M glycine in PBS for 10 min. 521 \nUnspecific binding was blocked with (2% BSA in PBS – without triton), followed by 522 \nincubation with α-SAG1 (1:10, mouse, T4-1E5) as above but without triton. Wells were 523 \nwashed 3 times with PBS before fixing cells with 4% PFA for 7 min. The next steps, 524 \npermeabilization, blocking, primary antibody incubation, washing, secondary antibody 525 \nincubation, washing and m ounting were carried out as described above for the IFA. 526 \nPolyclonal rabbit α-GAP45 (1:10,000) [56] was used as primary antibody and SAG1 and 527 \nGAP45 were revealed in green and red, respectively, using the secondary antibodies as 528 \nabove for the IFA. Slides were viewed on an Eclipse Ti inv erted microscope (Nikon). 529 \nMore than 100 parasites were counted per biological triplicate and categorised as 530 \ninvaded (red staining only) or non-invaded (red and green staining). 531 \nHarvest of parasites for mass spectrometry analyses 532 \nFreshly egressing or intra cellular parasites were harvested as follows: medium was 533 \naspirated, and the metabolism quenched through addition of ice -cold PBS. The 534 \nmonolayer was scraped, and parasites released via multiple passages through a 26G 535 \nneedle. The parasite solution was passed through a filter of 3 μm exclusion size (Merck-536 \nMillipore, TSTP04700) to remove host cell debris and collected in 15 ml conical tubes. 537 \nParasites were pelleted (2000 g, 4 °C, 25 min) and washed two more times with ice-cold 538 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 22 \nPBS. Residual PBS was removed, and pellets of 108 parasites resuspended in medium 539 \nas indicated below for labelling of extracellular parasites or stored at -80 °C until 540 \nmetabolite extraction. 541 \nStable Isotope Labelling of Extracellular Parasites 542 \nParasites pellets were resuspended in 2 ml of DMEM without Glc (Gibco, 11966-025) 543 \nsupplemented with 5% (v/v) dialysed FBS (Pan Biotech P30-2102; 10,000 Da exclusion 544 \nsize membrane) and 10 mM U-13C6-Glc (Cambridge Isotope Laboratories, CLM-1396) or 545 \nU-13C6-glucosamine (Cambridge Isotope Laboratories, CLM -9883) or U -13C6-N-546 \nacetylglucosamine (Cambridge Isotope Laboratories, CLM -1827) and incubated in a 547 \nconical tube for 5 hours in humidified incubators at 37 °C and 5% CO 2 prior to addition 548 \nof excess ice-cold PBS, centrifugation and PBS washes as described above. 549 \nSample Preparation for GC-MS Analyses 550 \nMetabolite extraction and derivatization was performed as previously described but 551 \nwithout heating step [60]. In brief, parasite pellets were placed on ice for 5 min before 552 \naddition of 50 μl chloroform followed by 200 μl methanol:ultrapure water (3:1, including 553 \nscyllo inositol as an internal standard, 1 nmol, Sigma -Aldrich, I8132). Extraction was 554 \nfacilitated through vigorous vortexing. Samples were spun (20,000 g, 4 °C, 10 min) and 555 \nthe supernatant transferred to a new vial containing 100 μl ice -cold ultrapure water. 556 \nSamples were vortexed and spun (20,000 g, 4 °C, 10 min). The lower, organic phase 557 \n(apolar, 50 μl) and the upper, polar phase (300 μl) were processed further as outlined 558 \nbelow. 559 \nSample Preparation for LC-MS Analyses 560 \nCells were harvested and washed as described above. Pellets were reconstituted in 60 561 \nμl acetonitrile:ultrapure water (4:1, containing 13C6/15N-isoleucine as internal standard, 562 \n40 μM, Cambridge Isotope Laboratories, CNLM -561-H) and vortexed vigorously. 563 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 23 \nExtracts were spun (20,000 g, 4 °C, 10 min) and the clear supernatant transferred to a 564 \nmass spectrometry vial with insert. The metabolite extraction is based on that described 565 \nin previous studies [61]. 566 \nGPI Quantification via GC-MS 567 \nGPI quantification analysis and quantification was performed via methanolysis as 568 \npreviously described [38, 62]. The apolar phase was transferred to a flame-sealed glass 569 \ntube (Sigma -Aldrich, Z328510) and dried in a centrifugal evaporator. Next, 50 μl 570 \nmethanolic hydrochloric acid (HCl, Supelco, 33354) were added, the tube flame-sealed 571 \nunder vacuum and incubated in an oven at 80 °C over night. The next day, the glass 572 \ntube was opened, and the content transferred to a mass spectrometry vial insert 573 \ncontaining 10 μl pyridine to neutralise the pH. The solution was dri ed in a centrifugal 574 \nevaporator and further derivatised through addition of 20 μl pyridine and 20 μl N, O -575 \nBis(trimethylsilyl) trifluoracetamid 99% (Supelco, B-023). Samples were analysed on an 576 \n8890 GC System (Agilent) equipped with a DB5 capillary column (J&W Scientific, 30 m, 577 \n250 μm inner diameter, 0.25-μm film thickness), with a 10-m inert duraguard, connected 578 \nto a 5977B GC/MSD in electron impact (EI) mode equipped with 7693A autosampler 579 \n(Agilent). The GC-MS settings were as follows: Inlet temperature: 270 °C, MS transfer 580 \nline temperature: 280 °C, MS source temperature: 230 °C and MS quadrupole 581 \ntemperature: 150 °C. The oven gradient during the sample run was as follows: 80 °C (2 582 \nmin); 80 °C to 140 °C at 30 °C/min; 140 °C to 250 °C at 5 °C/min; 250 °C to 310 °C at 583 \n15 °C/min; 310 °C for 2 min. Mannose and palmitic acid derivatives were identified based 584 \non the ion spectrum and retention time of authentic standards. Final analyses were 585 \nperformed in selected ion monitoring (SIM) mode, detecting the ions m/z 204 (mannose 586 \nderivative) and m/z 270 (palmitate derivative), following injection of 1 μl in split mode 587 \n(1:50). Data was analysed using MassHunter ( Quantitative Analysis and Qualitative 588 \nAnalysis 10.0, Agilent) and Excel (Microsoft). 589 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 24 \nUntargeted Polar Metabolite Profiling via GC-MS 590 \nPolar metabolites were derivatised and analysed as previously described [60]. In brief, 591 \nthe polar phase was sequentially dried within a mass spectrometry insert in a centrifugal 592 \nevaporator (50 μl at a time) and further dried and concentrated through addition of 593 \nmethanol. The dried metabolite extract was derivatised through addition of 20 μl pyridine 594 \ncontaining methoxyamine hydrochloride at 20 mg/ml and incubation at room temperature 595 \novernight. The following day, 20 μl N, O-Bis(trimethylsilyl) trifluoracetamid 99% (Supelco, 596 \nB-023) were added and samples vortexed and analysed by GC -MS. The analysis was 597 \nperformed as described above but using the following oven gradient: 70 °C (1 min); 70 598 \n°C to 295 °C at 12.5 °C/min; 295 °C to 320 °C at 25 °C/min; 320 °C for 2 min and 599 \noperating in scan mode ( m/z 70-700) with a 5.5 min solvent delay. Metabolites were 600 \nidentified based on the analysis of authentic standards or reliable predictions (NIST 601 \nlibrary, NIST MS Search 2.4, >60% confidence and manual curation). Data was analysed 602 \nusing MassHunter (Quantitative Analysis and Qualitative Analysis 10.0, Agilent) and 603 \nExcel (Microsoft). Metabolite intensities were normalised to the internal standard (scyllo 604 \ninositol) and expressed as relative abundances relative to th e control (TIR1 −IAA, 605 \nabundance = 1). 606 \nTargeted Aminosugar Profiling via GC-MS 607 \nSamples were prepped and analysed as described above for the untargeted profiling. 608 \nHowever, the MS was operated in SIM mode, detecting the ions m/z 356, 357, 358 and 609 \n359 to determine labelling in the desired sugars (see S3 Fig), as well as m/z 318 (internal 610 \nstandard, scyllo inositol). The 13C-fractional labelling was determined by measuring the 611 \nisotopologue abundance for m/z 357, 358 and 359 and correcting for occurrence of 612 \nnatural isotopes [63]. 613 \nTargeted Aminosugar Profiling via LC-MS/MS 614 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 25 \nSample analyses were performed on an Agilent LC -MS (Santa Clara, CA, USA) using 615 \nMassHunter B.08.00 software for system control and data acquisition. 1290 Infinity LC 616 \ncomprised a binary pump, HiP autosampler, column oven, and Flexible Cube module. 617 \nThe LC was hyphenated to a 6490 triple -quadrupole detector through an Agilent Jet 618 \nStream ion source. HILIC chromatographic separation was conducted on a Waters 619 \nAcquity Premier BEH Amide column (2.1 × 150 mm, 1.7 µm) kept at 35 ºC. Elution was 620 \nperformed at a flow rate of 0.4 ml min –1, using the following gradient of mobile phases 621 \n[64]: A (10 mM AF + 0.15% FA in MeCN:H 2O 85:15 v/v) and B (10 mM AF + 0.15% FA 622 \nin H2O): 0-6 min 0% B, 6.1 min 5.9% B, 10 min 17.6% B, 12 min 29.4% B and back to 0 623 \n% B from 12 to 18 minutes for column re-equilibration. Samples were kept at 6 ºC and 624 \ninjection volume was 7 µl. Ion source parameters were as follow: Jet Stream gas 625 \ntemperature and flow rate were 250 ºC and 15 l min–1 respectively, while for sheath gas 626 \nthey were set to 400 ºC and 11 l min –1. Nebulizer pressure was 40 psi, and a 3000 V 627 \ncapillary voltage was used. Ion funnel high/low pressure radiofrequencies were set to 628 \n150/60 for positive ionization transitions and 90/60 for negative ones. 629 \nMultiple reaction monitoring transitions were optimiz ed using Agilent MassHunter 630 \nOptimizer B.08.00 using individual solutions of the compounds dissolved at 100 μM in 631 \n80% ACN. Collision energy and cone voltage were optimized and at least three 632 \nfragments derived from the [M+H] +, [M+Na] + or [M -H]– precursor ion s were used to 633 \nmonitor each molecule. The specific transitions, MS source and ion funnel conditions 634 \ncan be found in S8 Table. Data was processed using Skyline 22.2. Peak identity was 635 \nconfirmed based on its qualifier transitions and retention time compared to those of 636 \nstandard compounds (RSD < 3%). The relative abundance of each molecule was 637 \nexpressed as the sum of all the areas of the corresponding transitions, normalized to the 638 \ninternal standard and expressed as relative abundance in relation to TIR1 −IAA 639 \nabundance = 1, using Excel (Microsoft). 640 \n 641 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 26 \nFunding 642 \nThis work is supported by the Indo-Swiss Joint Research Programme (ISJRP) 643 \nIZLIZ3_200277 to DSF. JK is supported by Carigest, SA. Barcelona Institute for Global 644 \nHealth (ISGlobal) is supported by the Spanish Ministry of Science and Innovation through 645 \nthe Centro de Excelencia Severo Ochoa 2019-2023 Program (grant number CEX2018-646 \n000806-S), and the Generalitat de Catalunya through the CERCA Program. This work is 647 \npart of the ISGlobal ’s Program on the Molecular Mechanisms of Malaria, partially 648 \nsupported by the Fundación Ramón Areces. LI received support by PID2019-110810RB-649 \nI00 and PID2022 -137031OB-I00 grants from the Spanish Ministry of Science & 650 \nInnovation. MPA is supported by a FI Fel lowship from the Generalitat de Catalunya 651 \nsupported by Secretaria d’Universitats i Recerca de la Generalitat de Catalunya and 652 \nFons Social Europeu (2021 FI_B 00470). MPA also received support from an EMBO 653 \nScientific Exchange Grant (9474). 654 \nAuthor contributions 655 \nJ.K., L.I., D.S.F. and M.P.A. conceived the study; M.P.A. and J.K. designed, performed 656 \nand interpreted the experimental work, with the support of L.I. and D.S.F; J.K. conducted 657 \nthe formal analysis; V.G.R. and S.R. were responsible of LC -MS experiments; J.K., L.I. 658 \nand D.S.F. supervised the research; D.S.F contributed to resources; M.P.A. and J.K. 659 \noutlined the draft. All authors contributed to the writing, review and editing of this 660 \nmanuscript. 661 \n 662 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 27 \nFigure Captions 663 \nFig 1. Glycans and UDP-GlcNAc synthesis in T. gondii. A) Proteins of the secretory 664 \npathway of Toxoplasma gondii are commonly modified as N-glycans, O-glycans or GPI-665 \nanchored. The typical glycan structures in T. gondii are shown, as well as the 666 \nmodification of Skp1, which harbours a specific O-glycosylation [65]. B) The activated 667 \nsugar nucleotide UDP-GlcNAc is synthesised from glucose in six conserved enzymatic 668 \nreactions, with the glucosamine -phosphate N-acetyltransferase (GNA1) converting 669 \nglucosamine-6-phosphate (GlcN6P) to N-acetylglucosamine-6-phosphate (GlcN6P) C) 670 \nOverview of T. gondii enzymes in UDP-GlcNAc synthesis, listing their names, accession 671 \nnumber (ID) [25], fitness score (FS) [23] and putative localisation (hLOPIT) [66], as well 672 \nas the accession number [67] and mutagenesis fitness score (MFS) [68] in the related 673 \nPlasmodium falciparum parasite. Abbreviations: PI: phosphatidylinositol; EtN : 674 \nethanolamine; GT1: glucose transporter 1; PM: plasma membrane; Fru, fructose; PI, 675 \nphosphatidylinositol; GPI, glycosylphosphatidyl inositol; UDPGlcNAc, uridine 676 \ndiphosphate N-acetylglucosamine. Other abbreviations, see panel A and C. 677 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 28 \nFig 2. GNA1 is essent ial for T. gondii. A) Immunofluorescence assay (IFA) showing 678 \nthe pellicle marker GAP45 and Ty signal in GNA1-mAID-Ty parasite line and its parental 679 \nline (TIR1). B) Western blot revealing the signal of Ty-tagged GNA1 and TIR1 at different 680 \ntime points of aux in (IAA) treatment. C) IFA showing Ty signal in GNA1 -mAID-Ty 681 \nparasites in the absence of IAA and 18 hours after IAA treatment. D) Lysis plaques 682 \nformed over one week of TIR1 and GNA1-mAID-Ty parasite cultivation in the presence 683 \nor absence of IAA. E) Growth assay of TIR1 and GNA1-mAID-Ty parasites showing the 684 \nnumber of parasites per vacuole after 24 hours of growth and varying durations of IAA 685 \ntreatment. A -D show representative data of 3 independent experiments. E shows 686 \nrepresentative data from one of three i ndependent biological replicates averaging 687 \ntechnical triplicates. p-values are given comparing the average number of parasites by 688 \ntwo-sided student’s t -test, between the indicated conditions. Abbreviations: MW, 689 \nmolecular weight; CAT, catalase. 690 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 29 \nFig 3. UDP-GlcNAc synthesis is disrupted in T. gondii that lack GNA1. 691 \nA) Percent 13C-labelling in T. gondii (TIR1) derived N-acetylglucosamine-6-phosphate 692 \n(GlcNAc6P) in unlabelled parasites (natural abundance) or after incubation of 693 \nintracellular or extracellular parasites in medium containing U -13C6-glucose for 24 or 3 694 \nhours, respectively. B) Relative abundance and fractional 13C-labelling in TIR1 and 695 \nGNA1-mAID-Ty parasite metabolite extracts, following incubation of extracellular 696 \nparasites in medium containing U -13C6-glucose in the absence of auxin (−IAA) or 697 \nfollowing 18 hours pre-treatment (+IAA). TIR1 −IAA parasites were incubated in medium 698 \nwith natural abundance glucose as an unlabelled control. Note that metabolites for which 699 \nthe abundance of labelled and unlabelled ions was too low to obtain reliable labelling 700 \ndata were deemed below limit of detection (100 vacuoles were counted and categorised, per replicate. 747 \np-values are given in B-F following two-sided student’s t-tests, comparing the indicated 748 \nconditions. Abbreviations: see Fig 3 and 4. 749 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 32 \nReferences 750 \n1. Nissapatorn V. Toxoplasma gondii and HIV: a never -ending story. Lancet HIV. 751 \n2017;4(4):e146-e7. 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Uncov ering 1040 \nthe essential genes of the human malaria parasite Plasmodium falciparum by saturation 1041 \nmutagenesis. Science. 2018;360(6388). Epub 2018/05/05. doi: 1042 \n10.1126/science.aap7847. PubMed PMID: 29724925; PubMed Central PMCID: 1043 \nPMCPMC6360947. 1044 \n 1045 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 44 \nCaptions of Supporting Material 1046 \nS1 Fig. GNA1 sequence, genome-wide fitness screen guides, locus modification 1047 \nand genomic PCR. A) GNA1 coding sequence as found on ToxoDB. The initial start 1048 \ncodon is highlighted in green, the stop codon in red. Four additional ‘in -frame’ start 1049 \ncodons were found and are also highlighted by green shading. Blue (guide with positive 1050 \nscore) and red shading (guide with negative score; intensity of shading indicating score) 1051 \nhighlights the sequence of single guide RNAs (sgRNA) used in the genome-wide fitness 1052 \nscreen. For overlapping guides, the first guide is shown as underlined, the second guide 1053 \nis shown in bold. The name/number of the guides and their respective fitness score is 1054 \nprovided. The listing is from left to right and from top to bottom i n the order of their 1055 \nappearance in the coding sequence. Guides with a positive fitness score are shown in 1056 \nblue, guides with a negative fitness score in red. Note that the first 4 guides only affect 1057 \nthe longest putative GNA1 product, while the last 4 guides affect all GNA1 products, 1058 \nincluding the shortest potential GNA1 protein (highlighted in italic). The GNAT domain, 1059 \nneeded for the catalytic activity is highlighted in purple and bold. B) Schematic depiction 1060 \nof the GNA1 locus and its modification through insertion of a mini auxin inducible degron 1061 \n(mAID) domain, a 3 -Ty tag and a hxgprt cassette for selection. C) Schematic showing 1062 \nthe binding sites of primers used to validate the successful modification of the GNA1 1063 \nlocus and integration PCR, showing the expected bands following amplification with the 1064 \nindicated primers. Sequences of these primers are listed in Supplementary Table 1. 1065 \n 1066 \nS2 Table. Description and sequence of primers used in this study. 1067 \n 1068 \nS3 Fig . Chromatogram, ion spectrum and structure of relevant metabolites. A) 1069 \nAuthentic standards of glucose -6-phosphate (Glc6P), glucosamine -6-phosphate 1070 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 45 \n(GlcN6P) and N-acetylglucosamine-6-phosphate (GlcNAc6P) were derivatised via 1071 \nmethoximation and silylated. Their gas chromatography -mass spectrometry (GC -MS) 1072 \nchromatograms were overlayed (top panel) and the corresponding ion spectra at the 1073 \nindicated retention times (RT) are shown below. Note that all 3 derivatives share a 1074 \ncommon fragment of m/z 357 (highlighted in red), which contains 2 carbons of the 1075 \nsugars. B) The structure of the methoxyamine (MeOx) and trimethylsilyl (TMS) derivative 1076 \nof 13C6-GlcNAc6P is shown. Labelled carbons are highlighted through red asterisks. The 1077 \nfragment m/z 357 (natural abundance) becomes m/z 359 in 13C-labelled sugars. 1078 \n 1079 \nS4 Fig. Untargeted metabolite profiling by gas chromatography -mass 1080 \nspectrometry (GC-MS). Relative levels of 64 metabolites were determined in TIR1 and 1081 \nGNA1-mAID-Ty parasites in presence (+IAA, 36 hours) or absence of auxin (−IAA). 1082 \nMetabolites were quantified in equal cell nu mbers, normalised to an internal standard, 1083 \nand are shown relative to levels in TIR1 −IAA (level of metabolites TIR1 −IAA = 1). 1084 \nMetabolites were grouped into different categories. The fold-change is indicated through 1085 \nred and blue shading as shown in the legend. Significantly altered levels are highlighted 1086 \nthrough bold lettering. Data represent average of 4 independent biological replicates 1087 \nfrom a single experiment. Significance indicates a p-value <0.05 in a two-sided student 1088 \nt-tests in comparison to TIR1 −IAA. 1089 \n 1090 \nS5 Fig. Detail of amino sugar abundance graph Detail of graph shown in main Fig 4D. 1091 \n 1092 \nS6 Fig P laque assay following supplementation with varying glucose/GlcNAc 1093 \nratios. Plaque assay of TIR1 and GNA1 -mAID-Ty parasites in presence or absence of 1094 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n 46 \nauxin (IAA) and with glucose (Glc) or N-acetylglucosamine (GlcNAC) supplemented in 1095 \nmedium without Glc as indicated. Image of stained plaques of a single experiment. 1096 \n 1097 \nS7 Fig. Morphology of the apicoplast and mitochondrion following downregulation 1098 \nof GNA1. Immunofluorescence assays (IFAs) were performed after auxin (IAA) 1099 \ntreatment for the indicated duration and after 24 hours of intracellular growth. Cells were 1100 \nstained with antibodies marking actin (Act) and the pellicle (GAP45), while organelles 1101 \nwere stained with CPN 60 (apicoplast) and 5F4 (mitochondrion). Intactness of the 1102 \napicoplast and mitochondrion was determined in 3 technical replicates of a single 1103 \nexperiment. Images show representatively the morphology of the apicoplast and 1104 \nmitochondrion. 1105 \n 1106 \nS8 Table. T ransitions, MS source and ion funnel conditions. Table with details 1107 \npertaining to LC-MS analyses. 1108 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}