N-acetylglucosamine supplementation fails to bypass the critical acetylation of glucosamine-6-phosphate required forToxoplasma gondiireplication and invasion

Toxoplasma gondii , Apicomplexa, N-acetylglucosamine, GPI -anchors, 17 parasites, metabolism, nutrient salvage, glycosylation, invasion 18 Short title 19 Glucosamine-6-phosphate acetylation is essential in Toxoplasma gondii 20 21 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 2

22 The cell surface of Toxoplasma gondii is rich in glycoconjugates which hold diverse and 23 vital functions in the lytic cycle of this obligate intracellular parasite. Additionally, the cyst 24 wall of bradyzoites, that shields the persistent form responsible for chronic infection from 25 the immune system, is heavily glycosylated. Formation of glycoconjugates relies on 26 activated sugar nucleotides , such as uridine diphosphate N-acetylglucosamine (UDP-27 GlcNAc). The Glucosamine-phosphate-N-acetyltransferase (GNA1) generates N-28 acetylglucosamine-6-phosphate critical to produce UDP-GlcNAc. Here, we demonstrate 29 that downregulation of T. gondii GNA1 results in a severe reduction of UDP-GlcNAc and 30 a concomitant drop in glycosylphosphatidylinositol (GPI), leading to impairment of the 31 parasite’s ability to invade and replicate in the host cell. Surprisingly, attempts to rescue 32 this defect through exogenous GlcNAc supplementation fail to completely restore these 33 essential functions. In depth metabolomic analyses elucidate diverse causes underlying 34 the failed rescue: utilization of GlcNAc is inefficient under glucose-replete conditions and 35 fails to restore UDP -GlcNAc levels in GNA1 -depleted parasites. In contrast, GlcNAc -36 supplementation under glucose-deplete conditions fully restores UDP-GlcNAc levels but 37 fails to rescue the defects associated with GNA1 depletion . Our results underscore the 38 essentiality of GlcN6P a cetylation in governing T. gondii replication and invasion and 39 highlight the potential of the evolutionary divergent GNA1 in Apicomplexa as a target for 40 the development of much-needed new therapeutic strategies. 41 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 3 Author Summary 42 Toxoplasma gondii , Plasmodium, and Cryptosporidium spp., pose serious threats to 43 human health. T. gondii, an intracellular and opportunistic pathogen, cunningly avoids 44 the host immune defences by forming long-lasting tissue cysts. Finding effective drugs 45 to eliminate these parasites remains a challenge. 46 The glucosamine-phosphate-N-acetyltransferase (GNA1) catalyses a critical key step in 47 the production of activated sugar nucleotides to build glycoconjugates essential for 48 various functions in the cell. In P. falciparu m, this enzyme has been identified as a 49 potential target for antimalarial drugs. 50 In this study, we explored the importance of this pathway in T. gondii and discovered that 51 these sugar-containing compounds play a vital role in the parasite's ability to invade and 52 replicate in host cells – crucial processes for its survival and ability to cause disease . 53 Intriguingly, unlike some organisms that can bypass the pathway, T. gondii relies critically 54 on glucosamine -6-phosphate acetylation. This reliance sheds light on the parasite's 55 distinct metabolic properties and highlights the pathway’s potential as a target for new 56 therapeutic strategies. 57 58 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 4

59 The phylum of Apicomplexa groups a vast numb er of obligate intracellular parasites, 60 some of which pose a considerable threat to human health. The most ubiquitous 61 apicomplexan, Toxoplasma gondii, causes disease in immunocompromised individuals 62 [1, 2], as well as abortions, stillbirths, fetal death, retinal lesions or long -term disabling 63 sequelae in congenitally infected children [3, 4]. At present, there is no vaccine that 64 prevents toxoplasmosis, and the available treatments are associated with a range of 65 shortcomings including high cost , toxicity and rising resistance [5]. In the accidental 66 human host, T. gondii manifests in two distinct stages: the fast -replicating tachyzoite, 67 responsible for acute disease and the slow replicating bradyzoite, which persists 68 encysted within muscle cells and neurons throughout the lifetime of its host [6]. These 69 persistent parasites constitute a reservoir, that can reactivate causing life-threatening 70 acute toxoplasmosis when the infected individual becomes immunocompromised. The 71 inability to eradicate the parasite's latent form, combined with the emergence of parasites 72 that are resistant to existing drugs against acute toxoplasmosis, underscores the 73 pressing need for novel therapeutic strategies [5]. 74 The endomembrane system of T. gondii is rich in glycoconjugates which play 75 fundamental roles in infectivity, survival , and virulence [7]. Several glycan structures 76 have been characterized in T. gondii including N-glycans [8], O-glycans [9-13], C-77 mannose [9, 14], GPI -anchors [15, 16], and others [7]. These glycans serve various 78 critical functions from invasion to O2 sensing and nutrient storage, hence contributing to 79 the overall virulence of the parasite [7]. Additionally, glycans are critical components of 80 the bradyzoite cyst wall and the disruption of their formation impairs the parasite’s ability 81 to persist [17, 18]. 82 The de novo synthesis of glycans relies on activated sugar nucleotides. Uridine 83 diphosphate N-acetylglucosamine (UDP -GlcNAc) serves as a donor by GlcNAc -84 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 5 dependent glycosyltransferases for the synthesis of N-glycans, 85 glycosylphosphatidylinositol (GPI) -anchors, glycoinositolphospholipids (GIPLs), and for 86 the glycosylation of other protein acceptors. Given the critical roles of these structures 87 for infectivity of tachyzoites [7, 8, 16, 19], and bradyzoite survival and replication [17, 18], 88 the biosynthesis route of UDP-GlcNAc is a plausible target for intervention against acute 89 toxoplasmosis and for eradication the chronic infection. GNA1, the enzyme catalysing 90 the acetylation of glucosamine -6-phosphate (GlcN6P) is considered a promising drug 91 target in Apicomplexa . This is attributed to its independent evolutionary origin, unique 92 sequence features [20], and established essentiali ty for the intraerythrocytic 93 development of Plasmodium falciparum [21]. 94 In T. gondii, a genome -wide CRISPR fitness screen underscored the significance of 95 UDP-GlcNAc biosynthesis for the parasite, classifying several genes encoding for 96 enzymes involved in the amino sugar synthesis pathway as fitness -conferring [22, 23]. 97 Unexpectedly, however, this study predicted GNA1 to be dispensable for T. gondii, even 98 though the upstream and downstream enzymes were highly fitness-conferring [23]. 99 Here we demonstrate th e essential nature of GNA1 in T. gondii , revealing that its 100 downregulation leads to a reduction in GPI anchors that impairs invasion and replication 101 within its host cell. Intriguingly, defects in GNA1 cannot be overcome by GlcNAc salvage. 102 Targeted metabolomic analyses revealed that GlcNAc salvage is inefficient in glucose -103 replete conditions . In contrast, GlcNAc is effectively salvaged in glucose -deplete 104 conditions, but fails to rectify the defects associated with the disruption of the pathway. 105 These findings highlight the potential of GNA1 as a drug targe for combatting 106 toxoplasmosis. 107 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 6

108 T. gondii GNA1 is essential, contradicting the prediction from a genome wide 109 fitness screen 110 T. gondii expresses several glycan structures akin to other eukaryotic cells, with few 111 noteworthy characteristics ( Fig 1A). The synthesis of GPI -anchors, N-glycans and O-112 glycans requires as donor substrate UDP-GlcNAc, or UDP -GalNAc. The latter can be 113 synthesised from UDP -GlcNAc via G alE [7], a UDP -Glc/UDP-Gal epimerase . In the 114 amino sugar biosynthesis pathway, the glucosamine 6 -phosphate N-acetyltransferase, 115 GNA1, catalyses the acetylation of GlcN6P (Fig 1B). Although T. gondii has been shown 116 to critically rely on several glycan structures [7-9], TgGNA1 (TGGT1_243600) was 117 assigned a positive fitness score (+1.41) in a genome-wide fitness screen [23], indicating 118 its potential dispensability ( Fig 1C). This is unexpected considering the crucial role of 119 GNA1 in other organisms [24], including P. falciparum [21]. Additionally, enzymes acting 120 either downstream or upstream of GNA1 were assigned negative fitness scores in T. 121 gondii, suggesting their essentiality [23]. 122 Examination of the nanopore sequencing data on ToxoDB [25, 26] revealed two major 123 GNA1 transcripts, with the shorter, more prevalent transcript only covering a portion of 124 the predicted protein coding sequence. In addition to the predicted GNA1 protein coding 125 sequence, which encodes a putative protein of 55.5 kDa , four additional in-frame open 126 reading frames were identified. These could give rise to GNA1 proteins of var ious 127 reduced sizes (45.5, 21.0, 17.2 and 16.7 kDa ) [25] (S1 Fig ). Critically, the 128 acetyltransferase domain is located near the C-terminus and is present in all five putative 129 isoforms. Consequently, all proteins, including the shortest version, could potentially be 130 catalytically active. These shorter GNA1 isoforms may explain the unexpected positive 131 fitness score for GNA1 [23]. Concordantly, out of the ten single guide RNAs (sgRNAs) 132 used to target GNA1 in the genome wide fitness screen [25, 26], four bind near the 133 extended N -terminus, only present in the long est isoform of GNA1 (guides 134 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 7 sgTGGT1_243600_5, _6, _9 and _10; average fitness score +1.02), while three bind 135 close to the C -terminus of GNA1 , disrupting all potential isoforms, (guides 136 sgTGGT1_243600_2, _3, and _4; average fitness score: -7.50,) (S1 Fig). Notably, the 137 published phenotype score is calculated by averaging the value of the top five scoring 138 guides to mitigate the impact of stochastic losses, resulting in the positive fitness score 139 for GNA1 [23]. Given the existence of diverse isoforms, the assigned fitness score likely 140 does not reflect the importance that GNA1 may play for T. gondii, prompting further 141 investigation of the enzyme. 142 To examine the localization and function of GNA1 in T. gondii (TGGT1_243600), the 143 endogenous locus was edited using CRISPR/Cas9 [23, 27]. Simultaneously a Ty epitope 144 tag and a mini auxin inducible degron (mAID) domain were fused to the C-terminus of 145 GNA1 in parasites stably express ing the auxin receptor transport inhibitor response 1 146 (TIR1) from Oryza sativa (RH-TIR1) [28, 29] . The hypoxanthine-xanthine-guanine 147 phosphoribosyl transferase ( hxgprt) resistance cassette was inserted, for selection of 148 positive transfectants (S1 Fig ) [30]. T he mAID domain enables rapid and efficient 149 downregulation of the protein of interest via proteasomal degradation upon addition of 150 auxin (indole 3-acetic acid, IAA) [29]. Successful integration of the construct at the gna1 151 locus in a clonal population was confirmed by genomic PCR (S1 Fig), using primers listed 152 in S2 Table. GNA1 -mAID-Ty exhibited a dotty cytosolic localization by 153 immunofluorescence assay (IFA) (Fig 2A). Subsequently, downregulation of GNA1 was 154 assessed by Western blot (2, 4 - and 18-hours IAA treatment, Fig 2B) and by IFA (18 -155 hours IAA treatment, Fig 2C), confirming an efficient and complete depletion of GNA1 156 after 2-4 hours of IAA treatment. Interestingly, the western blot revealed several bands 157 between approximately 30 -80 kDa for GNA1 -mAID-Ty ( Fig 2B ), all of which were 158 efficiently downregulated upon addition of IAA. Up to five GNA1 isoforms may exist, with 159 molecular weights ranging from 29.7 – 68.5 kDa, including the tag. These results indicate 160 that full length GNA1 as well as shorter isoforms (S1 Fig) are synthesised, although 161 partial degradation cannot be excluded. 162 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 8 The significance of GNA1 for the parasite lytic cycle was assessed by plaque assay. 163 Downregulation of GNA1 prevented the formation of plaques of lysis (Fig 2D), indicating 164 that GNA1 is needed for one or several steps of the lytic cycle. The intracellular growth 165 assay revealed a significant impact of GNA1 depletion on the replication rate, with the 166 average number of parasites during 24 hours of growth decreasing from 6.5 in the 167 controls to 4.2 following 36 hours of IAA treatment (Fig 2E). 168 These results underscore the essential role of GNA1 in intracellular growth and overall 169 lytic cycle of T. gondii, consistent with the importance of glycoconjugates. The data 170 suggest that two major transcripts are generated for GNA1, yielding up to five protein 171 isoforms that can be potentially catalytically active. The extended N -terminus in the 172 longer isoform may be dispensable [23]. If it holds a regulatory function in other life cycle 173 stages remains unknown. Crucially, depletion of the GNA1 acetyltransferase domain is 174 detrimental to T. gondii. 175 176 GNA1 is active and critical for UDP-GlcNAc synthesis in T. gondii 177 To examine if the UDP-GlcNAc biosynthesis pathway is active in intra- and extracellular 178 T. gondii, TIR1 parasites were cultured intracellularly for 24 hours in medium containing 179 10 mM uniformly 13C-labelled glucose (U-13C6-Glc) or extracted and purified extracellular 180 parasites were incubated for 3 hours in medium containing heavy Glc. Post-incubation, 181 the metabolism was quenched, parasites were harvested, and metabolites extracted. 182 Gas chromatography-mass spectrometry (GC-MS) following derivatization of the sugars 183 was employed to assess the extent of label incorporation into N-acetylglucosamine-6-184 phosphate (GlcNAc6P) (S3 Fig). Synthesis of GlcNAc6P from labelled Glc was observed 185 in both parasite stages, reaching 88.8% and 25.5% labelling in intra - and extracellular 186 parasites, respectively ( Fig 3A). These findings unequivocally demonstrate t he active 187 UDP-GlcNAc biosynthesis pathway from Glc in both intra- and extracellular T. gondii. 188 To assess the critical participation of GNA1 in the pathway, TIR1 and GNA1-mAID-Ty 189 parasites were untreated or pre-treated with IAA for 18 hours and extracellular parasites 190 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 9 were incubated in medium containing 10 mM U -13C6-Glc. TIR1 −IAA parasites that 191 served as control, were incubated with regular medium containing unlabelled (natural 192 abundance) Glc . After 5 hours of incubation, parasites were harvested, metabolites 193 extracted, derivatized and analysed by GC-MS in a targeted manner (S3 Fig). Under all 194 tested conditions, Glc6P was detected with incorporation of heavy carbons under 13C-195 labelling conditi ons ( Fig 3B ). Intriguingly, glucosamine-6-phosphate (GlcN6P), the 196 substrate of GNA1, was exclusively detected in parasites depleted in GNA1, with 197 incorporation of considerable labelling (45.8%). In contrast, GlcNAc6P, the product of 198 GNA1, was detected in all conditions, except in parasites depleted in GNA1 (Fig 3B). To 199 gain a comprehensive understanding of the impact of GNA1 depletion on T. gondii 200 metabolism, we performed metabolite profiling by GC -MS after 36 hours of 201 downregulation ( S4 F ig). At this relatively late time point, pleiotropic effects were 202 observed with 28 out of 64 metabolites significantly altered (>2-fold) in their abundance 203 compared to TIR1 −IAA. The majority (26 metabolites) exhibited reduced abundance 204 including amino acids, TCA cycle intermediates, sugars, fatty acids, and others. Amongst 205 the most dramatically reduced metabolites were GlcNAc and GlcNAc6P, consistent with 206 the function of GNA1. Conversely the two significantly increased metabolites were GlcN 207 and myo-inositol. While GlcN accumulation directly correlates with the absence of GNA1 208 (following enzymatic dephosphorylation or loss of the phosphate group during sample 209 preparation from GlcN6P), accumulation of myo-inositol could be part of a general stress 210 response, as previously observed [31], or a consequence of impaired GPI -anchor 211 synthesis. 212 Given that crucial intermediates such as GlcNAc1P and the product UDP-GlcNAc cannot 213 be detected by GC -MS, we turned to liquid chromatography mass spectrometry (LC -214 MS/MS) for more sensitive detection of all relevant pathway intermediates. Intracellular 215 parasites were treated with IAA or not for 18 hours, before quenching of the metabolism, 216 parasite harvest, metabolite extraction and analysis. While fructose-6-phoshate (Fru6P) 217 levels remained unaffected by GNA1 downregulation (GNA1 -mAID-Ty +IAA), GlcN6P 218 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 10 accumulated dramatically to 91.8-fold higher levels compared to the controls (Fig 3C). In 219 sharp contrast, as observed by GC -MS, GlcNAc6P was markedly reduced (50.9 -fold). 220 Similarly, the subsequent metabolites, GlcNAc1P and the product UDP -GlcNAc 221 exhibited reductions of 83.3- and 803.2-fold, respectively (Fig 3C). 222 Together, these findings reveal that UDP -GlcNAc synthesis is active both in intra- and 223 extracellular T. gondii tachyzoites, Moreover, GNA1 plays a critical role in this pathway 224 as its disruption results in a significant reduction in the activated sugar nucleotide UDP-225 GlcNAc and an accumulation of its substrate GlcN6P. 226 227 GlcNAc supplementation fails to rescue GNA1 deficiency 228 Efficient bypass of defects in UDP-GlcNAc synthesis is well documented in several 229 organisms, including P. falciparum through GlcNAc supplementation [21, 32, 33] . 230 GlcNAc can be taken up and phosphorylated by hexokinase, generating GlcNAc6P, 231 effectively circumventing the initial steps of the pathway . To test if exogenous GlcN or 232 GlcNAc supplementation can bypass the function of GNA1 in T. gondii, we performed 233 plaque assays with GNA1 -mAID-Ty parasites in presence or absence of IAA while 234 supplementing different concentrations of GlcN or GlcNAc. Remarkably, none of the 235 supplementations could rescue the lytic cycle defect observed in parasites depleted in 236 GNA1 (Fig 4A). 237 We hypothesised that the inability of GlcN or GlcNAc supplementation to rescue the lytic 238 cycle defect in GNA1 -depleted parasites could be attributed to various reasons I) 239 insufficient uptake of GlcNAc by the host cells and/or T. gondii, II) incapacity of T. gondii 240 hexokinase to phosphorylate GlcNAc or III) inefficient entry of phosphorylated, salvaged 241 GlcNAc into the UDP -GlcNAc synthesis pathway . To explore these possibi lities, we 242 incubated purified extracellular parasites in medium without Glc supplemented with 13C6-243 GlcN or 13C6-GlcNAc for 5 hours, before harvesting parasites and extracting metabolites. 244 GNA1-mAID-Ty and TIR1 parasites were pretreated with IAA for 18 hour s to deplete 245 GNA1 levels in the GNA1-mAID-Ty strain. TIR1 −IAA were incubated in regular medium 246 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 11 with unlabelled (natural abundance) Glc. Remarkably, both amino sugars were efficiently 247 salvaged and utilised. 13C6-GlcN was salvaged and phosphorylated under all conditions, 248 but as expected, the downregulation of GNA1 prevented the formation of GlcNAc6P from 249 GlcN6P ( Fig 4B). Similarly, exogenous 13C6-GlcNAc was efficiently used to generate 250 labelled GlcNAc6P in the control strains (Fig 4C). Parasites deficient in GNA1 could also 251 salvage exogenous 13C6-GlcNAc and utilised it to generate GlcNAc6P, albeit at 252 significantly lower levels but fully 13C-labelled, consistent with the inability of Glc to 253 contribute to GlcNAc6P formation. These resul ts reveal that T. gondii can salvage and 254 utilise GlcNAc, potentially bypassing the need for GNA1. 255 It is noteworthy that this experiment was conducted with extracellular parasites which 256 were pre-depleted in GNA1 over 18 hours under regular growth conditions, without 257 GlcNAc supplementation. Thus, parasites were expected to be impaired in their fitness 258 and this measurement may not reflect what occurs during intracellular development and 259 during continuous supplementation. To address this, we cultured intracellular T. gondii, 260 TIR1 and GNA1 -mAID-Ty parasites under standard conditions for 24 hours before 261 changing the culture medium to one of the following 4 conditions for 18 hours prior to 262 parasite harve st: regular medium −IAA; regular medium +IAA; regular medium +IAA 263 supplemented with 10 mM GlcNAc; and medium without Glc +IAA supplemented with 264 additional glutamine and 10 mM GlcNAc. Parasites were harvested while intracellular, 265 with the medium being removed and parasite metabolism quenched prior to the harvest 266 of parasites, to exclude any metabolite uptake by extracellular parasites. Metabolites 267 were extracted and analysed by LC -MS/MS, as above ( Fig 4D). Remarkably, GlcNAc 268 supplementation had only a marginal impact in the presence of Glc. Although 269 significantly higher than in non-supplemented parasites devoid of GNA1, GlcNAc failed 270 to fully restore UDP-GlcNAc levels, with levels being 13.3-fold lower than in control (TIR1 271 -IAA) parasites (see detail in S5 Fig ). In the absence of Glc, however, GlcNAc was 272 efficiently salvaged and markedly increased UDP-GlcNAc levels, both in TIR1 parasites 273 as well as in parasites deficient of GNA1. Crucially, UDP-GlcNAc levels in GNA1-devoid 274 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 12 parasites supplemented with GlcNAc in the absence of Glc were 2.2-fold higher than in 275 control (TIR1 -IAA) parasites, suggesting a full rescue of the pathway. Unexpectedly, 276 GlcN6P continued to accumulate to levels >100-fold higher in parasites devoid of GNA1, 277 regardless of the presence or absence of Glc in the medium. Since Fru6P levels were 278 markedly down in the absence of Glc, we speculate that the detected GlcN6P is not 279 derived from gluconeogenesis but rather from the deacetylation of GlcNAc, either 280 through deacetylases of the host cell or by the parasite. The generated GlcN6P failed to 281 be converted further in the absence of GNA1. In summary, this detailed analysis of the 282 pathway under varying conditions reveals efficient GlcNAc salvage but only in the 283 absence of Glc. Under this condition, the function of GNA1 can be bypassed, fully 284 restoring UDP-GlcNAc levels. Whether a potential competition between Glc and GlcNAc 285 happens at the level of uptake by the host or the parasite, or at the level of 286 phosphorylation in the parasite remains unclear. 287 While GlcNAc supplementation failed to restore the lytic cycle defect in GNA1-depleted 288 parasites in the presence of Glc, we investigated whether the defects in the parasite ’s 289 intracellular growth could be rescued by exogen ous GlcNAc, both in the presence or 290 absence of Glc. Consistent with the inefficient utilization of GlcNAc in the presence of 291 Glc, GlcNAc supplementation failed to rescue the growth defect in regular medium ( Fig 292 4E). GlcNAc supplementation in the absence of Glc, however, facilitated a significant but 293 modest and incomplete rescue of the intracellular growth rate, following 48 hours of 294 treatment (Fig 4E). Lastly, we explored whether certain ratios of Glc and GlcNAc could 295 potentially rescue parasites depleted in GNA1, by supporting central carbon metabolism 296 (Glc), but facilitating GNA1 bypass (GlcNAc) and potentially alleviating excessive 297 GlcN6P accumulation. Plaque assays were performed with parasites in varying Glc and 298 GlcNAc ratios, however none of the conditions were able to rescue the lytic cycle defect 299 associated with GNA1 downregulation (S6 Fig). 300 301 GNA1 is needed for GPI-anchor synthesis critical for host cell invasion by T. gondii 302 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 13 The observed impairment of UDP -GlcNAc synthesis in parasites depleted in GNA1 is 303 expected to impair the synthesis of glycans, including GPI -anchors, thereby likely 304 affecting the parasite’s ability to invade host cells. GPI-anchored proteins are critical for 305 parasite invasion, contributing to the expression of a series of surface antigens, which 306 play a vital role during host cell attachment [16, 34, 35] . Additionally, glycosylated 307 proteins have been described to traffic to the apical secretory organelles and playing an 308 essential role in their biogenesis and function [19, 36]. To assess if GNA1 is required for 309 the appropriate localization and formation of GPI-anchored proteins, we performed IFAs, 310 evaluating the expression and distribution of the surface antigen 1 (SAG1) [37]. 311 Downregulation of GNA1 resulted in a drop in SAG1 signal intensity and an aberrant 312 distribution, with the signal commonly accumulating in the residual body ( Fig 5A). This 313 abnormal staining was observed in 68.3 and 73.3% of vacuoles following downregulation 314 of GNA1 for 18 or 36 hours, respectively ( Fig 5B). To investigate if this is a relatively 315 specific defect or if cells devoid of GNA1 exhibit various morphological abnormalities, we 316 assessed the morphology of the apicoplast and mitochondrion, upon GNA1 317 downregulation for the same duration (S7 Fig). The organelles were studied by IFA using 318 α-CPN60 (chaperonin 60) and α-5F4 (F1 ATPase beta subunit ), two specific markers 319 found in the apicoplast and at the mitochondrion, respectively. Both organelles appeared 320 morphologically intact and normal after 18 hours and 36 hours of IAA-treatment (S7 Fig). 321 Next, to confirm if the abnormal SAG1 signal, observed in GNA1 -deficient cells, can 322 indeed be attributed to a defect in the synthesis of GPI-anchors, we quantified the relative 323 amount of GPI -anchors following downregulation of GNA1 for 36 hours. To this en d, 324 parasite lipids were extracted and subjected to methanolysis to hydrolyse 325 monosaccharides off glycan structures found in the organic phase after metabolite 326 extraction. The derivatised sugar and fatty acid methyl esters were analysed by GC-MS 327 and the sig nal intensity for mannose residues was quantified relative to the signal 328 intensity for palmitic acid [38]. Notably, the ratio mannose signal intensity/palmitic acid 329 signal intensity decreased 8.5 -fold after 36 hours of IAA treatment, c onsistent with a 330 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 14 marked drop in GIPLs and GPI -anchor formation (Fig 5C). As expected, this caused a 331 severe defect in host cell invasion, with only 26.5% of parasites depleted in GNA1 over 332 18 hours invading successfully, compared to more than 70% of parasites in all controls 333 (Fig 5D). 334 To assess if GlcNAc supplementation, and the consequent increase in UDP -GlcNAc 335 levels under Glc-deplete conditions (Fig 4D), could restore GPI-anchor synthesis, GPI-336 anchor levels were quantified after GlcNAc supplementation under Glc-replete or -337 deplete conditions. After 18 hours of IAA-treatment and the indicated supplementations, 338 GlcNAc supplementation appeared to increase relative GPI levels in TIR1 parasites and 339 led to a slight increase in GPI levels in G NA1-depleted parasites. However, levels 340 remained markedly lower (~5-fold) compared to control conditions (Fig 5E). Notably, the 341 modest increase in GPI-anchor levels was observed equally under Glc replete and Glc 342 deplete conditions. These relative GPI level s correlated remarkably well with parasite 343 invasion following 18 hours of treatment with IAA and supplementations as indicated: 344 GlcNAc-supplemented GNA1 -depleted parasites demonstrated a significant but still 345 incomplete rescue in their ability to invade host cells (Fig 5F). 346 Overall, GlcNAc supplementation under Glc deplete conditions fully restored UDP -347 GlcNAc levels in cells lacking GNA1 (Fig 4D ), but this only facilitated a modest and 348 incomplete rescue of the intracellular growth rate (Fig 4E), GPI abundance (Fig 5E) and 349 the parasites’ ability to invade (Fig 5F). In summary, GNA1 is essential for T. gondii 350 replication and invasion and cannot be bypassed by GlcNAc salvage. 351 352

353 The amino sugar pathway, also known as the hexosamine biosynthetic pathway, plays 354 a crucial role in various organisms, including the apicomplexan parasite P. falciparum 355 [20, 21] . A critical function of the pathway in the related apicomplexan T. gondii is 356 consistent with the highly negative fitness scores for most of the enzymes in the pathway, 357 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 15 as reported by a genome-wide CRISPR sgRNA-based fitness screen [23]. However, the 358 screen assigned a positive fitness score to GNA1, which catalyses the acetylation of 359 GlcN6P to GlcNAc6P, contrasting with the assumed key function of this enzyme in the 360 pathway. Our presented data confirm that the specific activity of GNA1 in T. gondii, as 361 previously illustrated by in vitro activity assays [20], is essential for parasite survival. The 362 discrepancy between our finding s and the genome -wide fitness analysis likely arises 363 from an omission in the gene annotation, which failed to highlight the existence of several 364 short GNA1 isoforms. Our data suggest that full size GNA1 (consistent with the 365 annotated sequence [25]) is synthesised alongside several shorter isoforms. These 366 shorter isoforms contain the essential acetyltransferase domain [39], and remain 367 unaffected by several individual sgRNAs employed in the genome-wide fitness screen 368 to disrupt GNA1 [23]. To our knowledge, most eukaryotic organisms exhibit only a single 369 GNA1 isoform [40]. However, within the apicomplexan GNA1 family, T. gondii GNA1 370 stands out due to its distinctive and elongated N -terminus [20]. The function of this 371 extended N -term remains unknown. Despite the valuable information provided by 372 genome wide studies, our findings highlight the limitations of such approaches and 373 automatic gene annotation, reinforcing the importance of studying genes individually for 374 a thorough comprehension of their significance. 375 UDP-GlcNAc and UDP-GalNAc derived from UDP-GlcNAc through the activity of GalE 376 epimerase [7], are the main products of the amino sugar pathway. UDP -GlcNAc is key 377 for the synthesis of GPI anchors and free GIPLs, which are present on the surface of all 378 T. gondii life stages [41-43]. SAG1, the pri mary surface antigen o f T. gondii, is a GPI 379 anchored protein crucial for host cell binding and invas ion [44]. Consequently GPI 380 anchors and GIPLs contribute to parasite virulence [45] and are essential for T. gondii 381 survival [16]. Our results reveal that GNA1 depletion leads to a mark ed drop in GPI 382 anchors, altering the localization of SAG1 and severely impacting host cell invasion and 383 intracellular growth. Furthermore, UDP-GlcNAc, along with other glycosylations, plays a 384 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 16 crucial role in the biosynthesis of N-glycans, modifying numerous proteins in the T. gondii 385 secretory pathway [7]. Several studies suggest that N-glycosylation is essential for 386 parasite invasion, motility, and viability [8, 23, 46]. Indeed, N-glycosylation, but also GPI 387 anchor biosynthesis and the amino sugar metabolism are among the metabolic pathways 388 with the highest proportion of essential genes in T. gondii tachyzoites, as reported by a 389 recent study [22]. In summary, our data emphasize the importance of GNA1 for the 390 amino sugar pathway and UDP -GlcNAc synthesis, highlighting the pivotal role of this 391 metabolic route for parasite virulence and survival. 392 Depletion of GNA1 results in the accumulation of GlcN6P, and the reduction or absence 393 of the downstream metabolites GlcNAc6P, GlcNAc1P and UDP -GlcNAc. While growth 394 can be rescued by supplementing the media with high concentrations of GlcNAc in P. 395 falciparum and other organisms [21, 32, 33], GlcNAc supplementation fails to rescue the 396 lack of GNA1 in T. gondii. GlcN supplementation also prov ed ineffective in recovering 397 parasite growth. Despite the inability to rescue parasite growth, the absence of Glc in the 398 media enhances GlcNAc salvaging, replenishing UDP -GlcNAc levels. This strongly 399 suggests a competition between Glc and GlcNAc at the upt ake or phosphorylation 400 processes. Nevertheless, the recovery of GPI anchors and parasite growth through 401 GlcNAc salvage is only partial when Glc is absent. The low levels of Fru6P indicate 402 incomplete gluconeogenesis via glutamine, the predominant carbon source for T. gondii 403 in absence of Glc [47]. In addition, these scant amounts of Fru6P in GlcNAc -404 supplemented TIR1 parasites under Glc depletion, strongly suggest the lack of an amino 405 sugar catabolic pathway in T. gondii. Notably, the pronounced accumulation of GlcN6P 406 observed in T. gondii GNA1 mutants could also contribute to the limited rescue observed 407 with GlcNAc supplementation [49, 50]. Intriguingly, the accumulation of GlcN6P 408 correlated more closely with a drop in GPI levels and concomitant impairment of parasite 409 invasion than UDP -GlcNAc levels, indicating a potentially toxic impact of this surge in 410 GlcN6P. Indeed, glucosamine has been shown to interfere with P. falciparum asexual 411 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 17 intraerythrocytic growth at high doses [47-49], and a comparable eff ect could be 412 occurring in T. gondii. Regardless, the high concentration of GlcNAc needed for partial 413 T. gondii growth recovery remains far from physiological levels [50], suggesting that the 414 likelihood of rescuing GNA1 -depleted parasites under physiological conditions is very 415 remote. In summary, the inco mplete rescue in GlcNAc supplemented media strongly 416 suggests the inability of a metabolic bypass to overcome GNA1 deficiency. This 417 spotlights T. gondii GNA1 as a potential drug target to tackle toxoplasmosis. 418 419 T. gondii GNA1 belongs to a specific gene family, with an independent evolutionary origin 420 within the phylum Apicomplexa [20]. Apicomplexan GNA1s exhibit distinct features and 421 conserved motifs, and a recent structural study highlighted the divergent binding sites 422 for GlcN6P and acetyl -CoA in Cryptosporidium parvum GNA1 compared to human 423 GNA1, including important variations in key residues [21]. The key role of the amino 424 sugar pathway for T. gondii viability, and the predicted significance of GPI anchors and 425 GlcNAc-containing glycoconjugates across T. gondii’s life cycle [7, 22], underscore the 426 potential of GNA1 as a versatile multistage therapeutic target in toxoplasmosis that could 427 be exploited for selective parasite inhibition. 428 Current drug therapies for human toxoplasmosis lack specificity, often leading to adverse 429 effects and inconsistent efficacy [5, 51]. Novel treatments against T. gondii must target 430 the slow growing bradyzoites [52] to eradicate the chronic stage, which poses a threat to 431 infected individuals if the immune system is compromised [53]. Targeting bradyzoites 432 efficiently is hindered by several hurdles: drugs must cross the blood -brain barrier and 433 traverse the cyst wall and must act on enzymes/pathways that are critical for the poorly 434 characterized metabolism of bradyzoites [54]. Intriguingly, the cyst in which bradyzoites 435 reside and persist is heavily glycosylated, containing high levels of GlcNAc and GalNac 436 residues [55]. A previous study highlighted that glycosylation of the cyst wall is critical for 437 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 18 T. gondii persistence [17]. Specifically, Caffaro et al., demonstrated that the nucleotide 438 sugar transporter TgNST1 is required for cyst wall glycosylation and its disruption impairs 439 the ability of T. gondii to persist but is dispensable for tachyzoites in vitro and during 440 acute infection in vivo [17]. We demonstrate here that GNA1 is essential for tachyzoites 441 and can be expected to be essential for bradyzoites given the high need for UDP-GlcNAc 442 and UDP-GalNAc during persistence [17], making it a promising candidate for a drug 443 target. 444 445

446 Parasite lines, culture and treatments 447 Parasites stably expressing TIR1 were a generous gift from the laboratory of David 448 Sibley [29]. These were maintained by regular passages in human foreskin fibroblasts 449 (HFF-1, ATCC SCRC -1041), in Dulbecco Modified Eagle Medium (DMEM, Gibco, 450 41966-029) supplemented with foetal bovine serum (FBS, Gibco, 10270-106, 5% v/v), 451 L-glutamine (Gibco, 20530-024, additional 2 mM) and Gentamycin (Gibco, 15750-045, 452 25 μg/ml), incubated in humidified incubators at 37 °C and 5% CO2. 453 Auxin (IAA, Sigma-Aldrich, I-2886) was added to cultures at 500 μM final in ethanol as 454 indicated for each experiment. Supplementations with sugars (Glc – Agilent, 103577, 455 GlcN – Sigma-Aldrich, G1514 or GlcNAc – Sigma-Aldrich, A3286) were performed as 456 described for ea ch experiment in regular medium, as above or in DMEM without Glc 457 (Gibco, 11966-025) supplemented with 5% (v/v) dialysed FBS (Pan Biotech P30-2102; 458 10,000 Da exclusion size membrane) and additional 10 mM glutamine (Agilent, 103579). 459 Generation of Transgenic Parasites 460 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 19 The GNA1-mAID-Ty parasite line was generated through co-transfection of a CRISPR-461 Cas9 expression plasmid [27] with a guide RNA (P1, S2 Table) targeting the 3’-UTR of 462 GNA1 (TTGT1_243600) and a homology repair template encoding the mAID domain, 463 the 3-Ty domain and the hxgprt resistance cassette, amplified with the primers P2 and 464 P3 (S2 Table) by KOD PCR (Sigma -Aldrich). Transfected parasites were selected in 465 medium containing mycophenolic acid (25 μg/ml) and xanthine (50 μg/ml) over one week 466 and cloned by serial dilution followed by a second round of cloning. Su bclones were 467 frozen and a single clone was used in the following experiments. 468 Correct integration of the homology template at the desired location was assessed by 469 PCR (GoTaq DNA Polymerase, Promega) on extracted genomic DNA (Promega Wizard 470 DNA Extraction) testing 3 amplifications using primers P4 and P5, P4 and P6 and P4 471 and P7 amplifying under the following conditions: 95 °C, 2 min; (95 °C, 15 s; 57 °C 15 s; 472 72 °C 1.5 min) × 35; 72 °C, 5 min on a SimpliAmp Thermal Cycler (Applied Biosystems). 473 Immunofluorescence Assays 474 Confluent monolayer of HFF cells grown on coverslips were inoculated with 10 μl of 475 freshly egressed parasite cultures and treated with IAA as or other supplementations as 476 indicated for each experiment. Twenty-four hours after inoculation, parasites were fixed 477 with 4% PFA and 0.05% glutaraldehyde for 10 min, before quenching with 0.1 M glycine 478 in PBS for 20 min. Infected host cells were permeabilized using 0.2% Triton X-100/PBS 479 for 20 min, followed by 20 min incubation in (2% BSA/0.2% Triton X -100/PBS to block 480 unspecific binding and subsequently probed with different primary antibodies diluted in 481 2% BSA/0.2% Triton X-100/PBS for 1 hour. The following primary antibodies were used 482 as indicated for each experiment: α-Ty (1:10, mouse monoclonal, BB2), α-SAG1 (1:10, 483 mouse, T4 -1E5), polyclonal rabbit α -GAP45 (1:10,000, used for growth assay) [56], 484 monoclonal mouse α -actin (1:20) [57], polyclonal rabbit α -CPN60 [58] and mouse 485 monoclonal α-5F4 (F1 ATPase beta subunit, P. Bradle y). The probed monolayer was 486 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 20 washed (3 × 5 min, 0.2% Triton X-100/PBS) and probed with a secondary antibody: anti 487 mouse Alexa fluor 488 (Invitrogen, A11001), anti -rabbit Alexa fluor 594 (Invitrogen, 488 A11012). Following 3 washing steps as above, the coversli ps were mounted on 489 microscopy slides using DAPI-containing FluoromountG (SouthernBiotech). Slides were 490 viewed on an Eclipse Ti inverted microscope (Nikon). For growth assays, the number of 491 parasites was counted in >100 vacuoles per condition for 3 independ ent biological 492 replicates. Images were acquired using an LSM 700 confocal scanning microscope 493 (Zeiss) and images were processed using Fiji Image J software. 494 Western Blots 495 Parasites were harvested from a freshly lysed dish, washed with PBS, and resuspended 496 in SDS –PAGE buffer (50 mM Tris -HCl, pH 6.8, 10% glycerol, 2 mM EDTA, 2% SDS, 497 0.05% bromophenol blue, and 100 mM dithiothreitol (DTT)). Following boiling for 10 min, 498 samples were subjected to SDS –PAGE under reducing conditions. Proteins were 499 transferred to a hybond ECL nitrocellulose membrane using a wet transfer system (Bio-500 Rad Laboratories, Hercules, CA, USA). The membrane was incubated in α-Ty antibody 501 (1:10, mouse monoclonal, BB2) and rabbit α-catalase as a loading control [59], diluted 502 in PBS, 0.05% Tween20, 5% skimmed milk. Following 3 washing steps, the membrane 503 was incubated with the secondary antibodies (goat α -mouse, horse radish peroxidase 504 conjugated, Sigma Aldrich, A5278). Signal was visualized using the SuperSignal West 505 Pico PLUS Chemiluminescent Substrate (ThermoFisher Scientific, 34580). Images were 506 taken using the Bio -Rad ChemiDoc MP Imaging System and images were processed 507 using Bio-Rad Image Lab software. 508 Plaque Assays 509 Serial dilutions of parasite cultures were incu bated on a confluent host cells monolayer 510 in 12- or 24-well plates for 7 days. Afterwards, the infected monolayer was washed with 511 PBS and fixed with 4% paraformaldehyde (PFA) for 10 min. Host cells were stained with 512 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 21 a crystal violet solution (12.5 g crysta l violet, 125 ml ethanol mixed with 500 ml water 513 containing 1% (w/v) ammonium oxalate) over 3 hours. Wells were washed 3 times with 514 deionized water to remove excess crystal violet and images of dried wells recorded. 515 Invasion (Red/Green) Assays 516 Parasites from a freshly egressed culture treated as described for each experiment were 517 diluted 1:10 and 150 μl of parasite solution used to infect a coverslip with confluent HFFs 518 in a 24 -well plate. The plate was gently spun for 1 min at 1,100 g and subsequently 519 incubated in a water bath at 37 °C. Cells were fixed with 4% PFA and 0.05% 520 glutaraldehyde for 7 min, before quenching with 0.1 M glycine in PBS for 10 min. 521 Unspecific binding was blocked with (2% BSA in PBS – without triton), followed by 522 incubation with α-SAG1 (1:10, mouse, T4-1E5) as above but without triton. Wells were 523 washed 3 times with PBS before fixing cells with 4% PFA for 7 min. The next steps, 524 permeabilization, blocking, primary antibody incubation, washing, secondary antibody 525 incubation, washing and m ounting were carried out as described above for the IFA. 526 Polyclonal rabbit α-GAP45 (1:10,000) [56] was used as primary antibody and SAG1 and 527 GAP45 were revealed in green and red, respectively, using the secondary antibodies as 528 above for the IFA. Slides were viewed on an Eclipse Ti inv erted microscope (Nikon). 529 More than 100 parasites were counted per biological triplicate and categorised as 530 invaded (red staining only) or non-invaded (red and green staining). 531 Harvest of parasites for mass spectrometry analyses 532 Freshly egressing or intra cellular parasites were harvested as follows: medium was 533 aspirated, and the metabolism quenched through addition of ice -cold PBS. The 534 monolayer was scraped, and parasites released via multiple passages through a 26G 535 needle. The parasite solution was passed through a filter of 3 μm exclusion size (Merck-536 Millipore, TSTP04700) to remove host cell debris and collected in 15 ml conical tubes. 537 Parasites were pelleted (2000 g, 4 °C, 25 min) and washed two more times with ice-cold 538 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 22 PBS. Residual PBS was removed, and pellets of 108 parasites resuspended in medium 539 as indicated below for labelling of extracellular parasites or stored at -80 °C until 540 metabolite extraction. 541 Stable Isotope Labelling of Extracellular Parasites 542 Parasites pellets were resuspended in 2 ml of DMEM without Glc (Gibco, 11966-025) 543 supplemented with 5% (v/v) dialysed FBS (Pan Biotech P30-2102; 10,000 Da exclusion 544 size membrane) and 10 mM U-13C6-Glc (Cambridge Isotope Laboratories, CLM-1396) or 545 U-13C6-glucosamine (Cambridge Isotope Laboratories, CLM -9883) or U -13C6-N-546 acetylglucosamine (Cambridge Isotope Laboratories, CLM -1827) and incubated in a 547 conical tube for 5 hours in humidified incubators at 37 °C and 5% CO 2 prior to addition 548 of excess ice-cold PBS, centrifugation and PBS washes as described above. 549 Sample Preparation for GC-MS Analyses 550 Metabolite extraction and derivatization was performed as previously described but 551 without heating step [60]. In brief, parasite pellets were placed on ice for 5 min before 552 addition of 50 μl chloroform followed by 200 μl methanol:ultrapure water (3:1, including 553 scyllo inositol as an internal standard, 1 nmol, Sigma -Aldrich, I8132). Extraction was 554 facilitated through vigorous vortexing. Samples were spun (20,000 g, 4 °C, 10 min) and 555 the supernatant transferred to a new vial containing 100 μl ice -cold ultrapure water. 556 Samples were vortexed and spun (20,000 g, 4 °C, 10 min). The lower, organic phase 557 (apolar, 50 μl) and the upper, polar phase (300 μl) were processed further as outlined 558 below. 559 Sample Preparation for LC-MS Analyses 560 Cells were harvested and washed as described above. Pellets were reconstituted in 60 561 μl acetonitrile:ultrapure water (4:1, containing 13C6/15N-isoleucine as internal standard, 562 40 μM, Cambridge Isotope Laboratories, CNLM -561-H) and vortexed vigorously. 563 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 23 Extracts were spun (20,000 g, 4 °C, 10 min) and the clear supernatant transferred to a 564 mass spectrometry vial with insert. The metabolite extraction is based on that described 565 in previous studies [61]. 566 GPI Quantification via GC-MS 567 GPI quantification analysis and quantification was performed via methanolysis as 568 previously described [38, 62]. The apolar phase was transferred to a flame-sealed glass 569 tube (Sigma -Aldrich, Z328510) and dried in a centrifugal evaporator. Next, 50 μl 570 methanolic hydrochloric acid (HCl, Supelco, 33354) were added, the tube flame-sealed 571 under vacuum and incubated in an oven at 80 °C over night. The next day, the glass 572 tube was opened, and the content transferred to a mass spectrometry vial insert 573 containing 10 μl pyridine to neutralise the pH. The solution was dri ed in a centrifugal 574 evaporator and further derivatised through addition of 20 μl pyridine and 20 μl N, O -575 Bis(trimethylsilyl) trifluoracetamid 99% (Supelco, B-023). Samples were analysed on an 576 8890 GC System (Agilent) equipped with a DB5 capillary column (J&W Scientific, 30 m, 577 250 μm inner diameter, 0.25-μm film thickness), with a 10-m inert duraguard, connected 578 to a 5977B GC/MSD in electron impact (EI) mode equipped with 7693A autosampler 579 (Agilent). The GC-MS settings were as follows: Inlet temperature: 270 °C, MS transfer 580 line temperature: 280 °C, MS source temperature: 230 °C and MS quadrupole 581 temperature: 150 °C. The oven gradient during the sample run was as follows: 80 °C (2 582 min); 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 15 °C/min; 310 °C for 2 min. Mannose and palmitic acid derivatives were identified based 584 on the ion spectrum and retention time of authentic standards. Final analyses were 585 performed in selected ion monitoring (SIM) mode, detecting the ions m/z 204 (mannose 586 derivative) and m/z 270 (palmitate derivative), following injection of 1 μl in split mode 587 (1:50). Data was analysed using MassHunter ( Quantitative Analysis and Qualitative 588 Analysis 10.0, Agilent) and Excel (Microsoft). 589 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 24 Untargeted Polar Metabolite Profiling via GC-MS 590 Polar metabolites were derivatised and analysed as previously described [60]. In brief, 591 the polar phase was sequentially dried within a mass spectrometry insert in a centrifugal 592 evaporator (50 μl at a time) and further dried and concentrated through addition of 593 methanol. The dried metabolite extract was derivatised through addition of 20 μl pyridine 594 containing methoxyamine hydrochloride at 20 mg/ml and incubation at room temperature 595 overnight. The following day, 20 μl N, O-Bis(trimethylsilyl) trifluoracetamid 99% (Supelco, 596 B-023) were added and samples vortexed and analysed by GC -MS. The analysis was 597 performed as described above but using the following oven gradient: 70 °C (1 min); 70 598 °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 operating in scan mode ( m/z 70-700) with a 5.5 min solvent delay. Metabolites were 600 identified based on the analysis of authentic standards or reliable predictions (NIST 601 library, NIST MS Search 2.4, >60% confidence and manual curation). Data was analysed 602 using MassHunter (Quantitative Analysis and Qualitative Analysis 10.0, Agilent) and 603 Excel (Microsoft). Metabolite intensities were normalised to the internal standard (scyllo 604 inositol) and expressed as relative abundances relative to th e control (TIR1 −IAA, 605 abundance = 1). 606 Targeted Aminosugar Profiling via GC-MS 607 Samples were prepped and analysed as described above for the untargeted profiling. 608 However, the MS was operated in SIM mode, detecting the ions m/z 356, 357, 358 and 609 359 to determine labelling in the desired sugars (see S3 Fig), as well as m/z 318 (internal 610 standard, scyllo inositol). The 13C-fractional labelling was determined by measuring the 611 isotopologue abundance for m/z 357, 358 and 359 and correcting for occurrence of 612 natural isotopes [63]. 613 Targeted Aminosugar Profiling via LC-MS/MS 614 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 25 Sample analyses were performed on an Agilent LC -MS (Santa Clara, CA, USA) using 615 MassHunter B.08.00 software for system control and data acquisition. 1290 Infinity LC 616 comprised a binary pump, HiP autosampler, column oven, and Flexible Cube module. 617 The LC was hyphenated to a 6490 triple -quadrupole detector through an Agilent Jet 618 Stream ion source. HILIC chromatographic separation was conducted on a Waters 619 Acquity Premier BEH Amide column (2.1 × 150 mm, 1.7 µm) kept at 35 ºC. Elution was 620 performed at a flow rate of 0.4 ml min –1, using the following gradient of mobile phases 621 [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 in 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 % B from 12 to 18 minutes for column re-equilibration. Samples were kept at 6 ºC and 624 injection volume was 7 µl. Ion source parameters were as follow: Jet Stream gas 625 temperature and flow rate were 250 ºC and 15 l min–1 respectively, while for sheath gas 626 they were set to 400 ºC and 11 l min –1. Nebulizer pressure was 40 psi, and a 3000 V 627 capillary voltage was used. Ion funnel high/low pressure radiofrequencies were set to 628 150/60 for positive ionization transitions and 90/60 for negative ones. 629 Multiple reaction monitoring transitions were optimiz ed using Agilent MassHunter 630 Optimizer B.08.00 using individual solutions of the compounds dissolved at 100 μM in 631 80% ACN. Collision energy and cone voltage were optimized and at least three 632 fragments derived from the [M+H] +, [M+Na] + or [M -H]– precursor ion s were used to 633 monitor each molecule. The specific transitions, MS source and ion funnel conditions 634 can be found in S8 Table. Data was processed using Skyline 22.2. Peak identity was 635 confirmed based on its qualifier transitions and retention time compared to those of 636 standard compounds (RSD < 3%). The relative abundance of each molecule was 637 expressed as the sum of all the areas of the corresponding transitions, normalized to the 638 internal standard and expressed as relative abundance in relation to TIR1 −IAA 639 abundance = 1, using Excel (Microsoft). 640 641 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 26 Funding 642 This work is supported by the Indo-Swiss Joint Research Programme (ISJRP) 643 IZLIZ3_200277 to DSF. JK is supported by Carigest, SA. Barcelona Institute for Global 644 Health (ISGlobal) is supported by the Spanish Ministry of Science and Innovation through 645 the Centro de Excelencia Severo Ochoa 2019-2023 Program (grant number CEX2018-646 000806-S), and the Generalitat de Catalunya through the CERCA Program. This work is 647 part of the ISGlobal ’s Program on the Molecular Mechanisms of Malaria, partially 648 supported by the Fundación Ramón Areces. LI received support by PID2019-110810RB-649 I00 and PID2022 -137031OB-I00 grants from the Spanish Ministry of Science & 650 Innovation. MPA is supported by a FI Fel lowship from the Generalitat de Catalunya 651 supported by Secretaria d’Universitats i Recerca de la Generalitat de Catalunya and 652 Fons Social Europeu (2021 FI_B 00470). MPA also received support from an EMBO 653 Scientific Exchange Grant (9474). 654 Author contributions 655 J.K., L.I., D.S.F. and M.P.A. conceived the study; M.P.A. and J.K. designed, performed 656 and interpreted the experimental work, with the support of L.I. and D.S.F; J.K. conducted 657 the formal analysis; V.G.R. and S.R. were responsible of LC -MS experiments; J.K., L.I. 658 and D.S.F. supervised the research; D.S.F contributed to resources; M.P.A. and J.K. 659 outlined the draft. All authors contributed to the writing, review and editing of this 660 manuscript. 661 662 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 27 Figure Captions 663 Fig 1. Glycans and UDP-GlcNAc synthesis in T. gondii. A) Proteins of the secretory 664 pathway of Toxoplasma gondii are commonly modified as N-glycans, O-glycans or GPI-665 anchored. The typical glycan structures in T. gondii are shown, as well as the 666 modification of Skp1, which harbours a specific O-glycosylation [65]. B) The activated 667 sugar nucleotide UDP-GlcNAc is synthesised from glucose in six conserved enzymatic 668 reactions, with the glucosamine -phosphate N-acetyltransferase (GNA1) converting 669 glucosamine-6-phosphate (GlcN6P) to N-acetylglucosamine-6-phosphate (GlcN6P) C) 670 Overview of T. gondii enzymes in UDP-GlcNAc synthesis, listing their names, accession 671 number (ID) [25], fitness score (FS) [23] and putative localisation (hLOPIT) [66], as well 672 as the accession number [67] and mutagenesis fitness score (MFS) [68] in the related 673 Plasmodium falciparum parasite. Abbreviations: PI: phosphatidylinositol; EtN : 674 ethanolamine; GT1: glucose transporter 1; PM: plasma membrane; Fru, fructose; PI, 675 phosphatidylinositol; GPI, glycosylphosphatidyl inositol; UDPGlcNAc, uridine 676 diphosphate N-acetylglucosamine. Other abbreviations, see panel A and C. 677 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 28 Fig 2. GNA1 is essent ial for T. gondii. A) Immunofluorescence assay (IFA) showing 678 the pellicle marker GAP45 and Ty signal in GNA1-mAID-Ty parasite line and its parental 679 line (TIR1). B) Western blot revealing the signal of Ty-tagged GNA1 and TIR1 at different 680 time points of aux in (IAA) treatment. C) IFA showing Ty signal in GNA1 -mAID-Ty 681 parasites in the absence of IAA and 18 hours after IAA treatment. D) Lysis plaques 682 formed over one week of TIR1 and GNA1-mAID-Ty parasite cultivation in the presence 683 or absence of IAA. E) Growth assay of TIR1 and GNA1-mAID-Ty parasites showing the 684 number of parasites per vacuole after 24 hours of growth and varying durations of IAA 685 treatment. A -D show representative data of 3 independent experiments. E shows 686 representative data from one of three i ndependent biological replicates averaging 687 technical triplicates. p-values are given comparing the average number of parasites by 688 two-sided student’s t -test, between the indicated conditions. Abbreviations: MW, 689 molecular weight; CAT, catalase. 690 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 29 Fig 3. UDP-GlcNAc synthesis is disrupted in T. gondii that lack GNA1. 691 A) Percent 13C-labelling in T. gondii (TIR1) derived N-acetylglucosamine-6-phosphate 692 (GlcNAc6P) in unlabelled parasites (natural abundance) or after incubation of 693 intracellular or extracellular parasites in medium containing U -13C6-glucose for 24 or 3 694 hours, respectively. B) Relative abundance and fractional 13C-labelling in TIR1 and 695 GNA1-mAID-Ty parasite metabolite extracts, following incubation of extracellular 696 parasites in medium containing U -13C6-glucose in the absence of auxin (−IAA) or 697 following 18 hours pre-treatment (+IAA). TIR1 −IAA parasites were incubated in medium 698 with natural abundance glucose as an unlabelled control. Note that metabolites for which 699 the abundance of labelled and unlabelled ions was too low to obtain reliable labelling 700 data were deemed below limit of detection (<LOD, sum of ion intensity <1000 arbitrary 701 units). C) Relative metabolite levels in TIR1 and GNA1-mAID-Ty, following no treatment 702 (−IAA) or treatment with IAA for 18 hours during intracellular growth (+IAA). Data plotted 703 show the average and standard deviation of 3 (A, B) or 4 (C) independent biological 704 replicates. p-values from two-sided student t-tests are given in A and C, comparing the 705 indicated conditions. Abbreviations: Glc6P, glucose -6-phosphate; GlcN6P, 706 glucosamine-6-phosphate; GlcNAc6P, N-acetylglucosamine-6-phosphate; Fru6P, 707 fructose-6-phosphate; GlcNAc1P, N-acetylglucosamine-1-phosphate; UDPGlcNAc, 708 uridine diphosphate N-acetylglucosamine. 709 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 30 Fig 4. Disruption of TgGNA1 cannot be rescued by GlcNAc supplementation. 710 A) Lysis plaques formed by TIR1 and GNA1-mAID-Ty parasites over one week of growth 711 when treated with auxin (+IAA) or not (−IAA) and supplemented with varying 712 concentrations of glucosamine (GlcN) and/or N-acetylglucosamine (GlcNAc) as 713 indicated. B-C) Relative metabolite abundance and fractional 13C-labelling in TIR1 and 714 GNA1-mAID-Ty parasite extracts, incubated for 5 hours extracellularly in medium without 715 glucose and containing U-13C6-glucosamine (B) or U-13C6-N-acetylglucosamine (C) in the 716 absence of IAA or following IAA pre -treatment (+IAA, 18 h). TIR1 −IAA parasites were 717 incubated in medium with natural abundance glucose as an unlabelled control. Note that 718 metabolites for which the abundance of labelled and unlabelled ions was too low to 719 obtain reliable labelling data were deemed below limit of detection (<LOD, sum of ion 720 intensity <1000 arbitrary units). D) Relative metabolite levels in TIR1 and GNA1 -mAID-721 Ty, following no treatment (−IAA) or treatment with IAA (+IAA, 18 h) during intracellular 722 growth in the presence or absence of glucose and supplemented with GlcNAc as 723 indicated for the same duration. E) Intracellular growth assay showing the number of 724 parasites per vacuole after 24 hours of growth, treated for 48 hours with IAA, G lc or 725 GlcNAc as indicated. A) shows representative images of 3 independent experiments. 726 Data plotted in B -E show the average and standard deviation of 3 (B, C , E) and 4 (D) 727 independent biological replicates, respectively. p -values from two-sided student t-tests 728 are given in D and E, comparing the indicated conditions. p -values in E compare the 729 average number of parasites per vacuole. Abbreviations: Glc, glucose. Other 730 abbreviations, see Fig 3. 731 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 31 Fig 5. Lack of TgGNA1 disrupts the localization of GPI-anchored proteins 732 causing an invasion defect. 733 A) Immunofluorescence assay (IFA), showing the staining of the pellicle marker GAP45 734 and the GPI -anchored protein surface antigen 1 (SAG1) in TIR1 and GNA -mAID-Ty 735 parasites after varying durations of auxin (IAA) treatment. B) Quantification of vacuoles 736 displaying normal (even distribution) and abnormal SAG1 signal (uneven, patchy 737 distribution with predominant accumulation inside the residual body), based on IFA 738 images as shown in panel A. C) relative GPI abundance of u ntreated (−IAA) or IAA -739 treated (+IAA, 36 h) TIR1 and GNA1-mAID-Ty parasite. D) Percent of invaded TIR1 and 740 GNA1-mAID-Ty parasite in the absence ( -IAA) or after IAA treatment (18 h) as 741 determined by a red/green invasion assay. E) Quantification of GPI -anchors in cells 742 grown with the indicated treatments/supplementations for 18 hours. F) Percentage of 743 invaded TIR1 and GNA1 -mAID-Ty parasites, following the indicated treatment over 18 744 hours. A) shows representative images of 3 independent experiments. B -F show 745 representative data from one of three independent biological replicates, averaging 746 technical triplicates. For B, >100 vacuoles were counted and categorised, per replicate. 747 p-values are given in B-F following two-sided student’s t-tests, comparing the indicated 748 conditions. Abbreviations: see Fig 3 and 4. 749 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 32

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It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 44 Captions of Supporting Material 1046 S1 Fig. GNA1 sequence, genome-wide fitness screen guides, locus modification 1047 and genomic PCR. A) GNA1 coding sequence as found on ToxoDB. The initial start 1048 codon is highlighted in green, the stop codon in red. Four additional ‘in -frame’ start 1049 codons were found and are also highlighted by green shading. Blue (guide with positive 1050 score) and red shading (guide with negative score; intensity of shading indicating score) 1051 highlights the sequence of single guide RNAs (sgRNA) used in the genome-wide fitness 1052 screen. For overlapping guides, the first guide is shown as underlined, the second guide 1053 is shown in bold. The name/number of the guides and their respective fitness score is 1054 provided. The listing is from left to right and from top to bottom i n the order of their 1055 appearance in the coding sequence. Guides with a positive fitness score are shown in 1056 blue, guides with a negative fitness score in red. Note that the first 4 guides only affect 1057 the longest putative GNA1 product, while the last 4 guides affect all GNA1 products, 1058 including the shortest potential GNA1 protein (highlighted in italic). The GNAT domain, 1059 needed for the catalytic activity is highlighted in purple and bold. B) Schematic depiction 1060 of the GNA1 locus and its modification through insertion of a mini auxin inducible degron 1061 (mAID) domain, a 3 -Ty tag and a hxgprt cassette for selection. C) Schematic showing 1062 the binding sites of primers used to validate the successful modification of the GNA1 1063 locus and integration PCR, showing the expected bands following amplification with the 1064 indicated primers. Sequences of these primers are listed in Supplementary Table 1. 1065 1066 S2 Table. Description and sequence of primers used in this study. 1067 1068 S3 Fig . Chromatogram, ion spectrum and structure of relevant metabolites. A) 1069 Authentic standards of glucose -6-phosphate (Glc6P), glucosamine -6-phosphate 1070 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 45 (GlcN6P) and N-acetylglucosamine-6-phosphate (GlcNAc6P) were derivatised via 1071 methoximation and silylated. Their gas chromatography -mass spectrometry (GC -MS) 1072 chromatograms were overlayed (top panel) and the corresponding ion spectra at the 1073 indicated retention times (RT) are shown below. Note that all 3 derivatives share a 1074 common fragment of m/z 357 (highlighted in red), which contains 2 carbons of the 1075 sugars. B) The structure of the methoxyamine (MeOx) and trimethylsilyl (TMS) derivative 1076 of 13C6-GlcNAc6P is shown. Labelled carbons are highlighted through red asterisks. The 1077 fragment m/z 357 (natural abundance) becomes m/z 359 in 13C-labelled sugars. 1078 1079 S4 Fig. Untargeted metabolite profiling by gas chromatography -mass 1080 spectrometry (GC-MS). Relative levels of 64 metabolites were determined in TIR1 and 1081 GNA1-mAID-Ty parasites in presence (+IAA, 36 hours) or absence of auxin (−IAA). 1082 Metabolites were quantified in equal cell nu mbers, normalised to an internal standard, 1083 and are shown relative to levels in TIR1 −IAA (level of metabolites TIR1 −IAA = 1). 1084 Metabolites were grouped into different categories. The fold-change is indicated through 1085 red and blue shading as shown in the legend. Significantly altered levels are highlighted 1086 through bold lettering. Data represent average of 4 independent biological replicates 1087 from a single experiment. Significance indicates a p-value <0.05 in a two-sided student 1088 t-tests in comparison to TIR1 −IAA. 1089 1090 S5 Fig. Detail of amino sugar abundance graph Detail of graph shown in main Fig 4D. 1091 1092 S6 Fig P laque assay following supplementation with varying glucose/GlcNAc 1093 ratios. Plaque assay of TIR1 and GNA1 -mAID-Ty parasites in presence or absence of 1094 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint 46 auxin (IAA) and with glucose (Glc) or N-acetylglucosamine (GlcNAC) supplemented in 1095 medium without Glc as indicated. Image of stained plaques of a single experiment. 1096 1097 S7 Fig. Morphology of the apicoplast and mitochondrion following downregulation 1098 of GNA1. Immunofluorescence assays (IFAs) were performed after auxin (IAA) 1099 treatment for the indicated duration and after 24 hours of intracellular growth. Cells were 1100 stained with antibodies marking actin (Act) and the pellicle (GAP45), while organelles 1101 were stained with CPN 60 (apicoplast) and 5F4 (mitochondrion). Intactness of the 1102 apicoplast and mitochondrion was determined in 3 technical replicates of a single 1103 experiment. Images show representatively the morphology of the apicoplast and 1104 mitochondrion. 1105 1106 S8 Table. T ransitions, MS source and ion funnel conditions. Table with details 1107 pertaining to LC-MS analyses. 1108 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 18, 2024. ; https://doi.org/10.1101/2024.01.18.576165doi: bioRxiv preprint .CC-BY 4.0 International licenseperpetuity. 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