NAD (P) transhydrogenase has vital non‐mitochondrial functions in malaria parasite transmission
2020; Springer Nature; Volume: 21; Issue: 3 Linguagem: Inglês
10.15252/embr.201947832
ISSN1469-3178
AutoresSadia Saeed, Annie Z. Tremp, Vikram Sharma, Edwin Lasonder, Johannes T. Dessens,
Tópico(s)Hormonal Regulation and Hypertension
ResumoReport17 January 2020Open Access Transparent process NAD(P) transhydrogenase has vital non-mitochondrial functions in malaria parasite transmission Sadia Saeed Sadia Saeed Department of Infection Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, UK Search for more papers by this author Annie Z Tremp Annie Z Tremp Department of Infection Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, UK Search for more papers by this author Vikram Sharma Vikram Sharma School of Biomedical Sciences, University of Plymouth, Plymouth, UK Search for more papers by this author Edwin Lasonder Edwin Lasonder School of Biomedical Sciences, University of Plymouth, Plymouth, UK Search for more papers by this author Johannes T Dessens Corresponding Author Johannes T Dessens [email protected] orcid.org/0000-0002-2070-6073 Department of Infection Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, UK Search for more papers by this author Sadia Saeed Sadia Saeed Department of Infection Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, UK Search for more papers by this author Annie Z Tremp Annie Z Tremp Department of Infection Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, UK Search for more papers by this author Vikram Sharma Vikram Sharma School of Biomedical Sciences, University of Plymouth, Plymouth, UK Search for more papers by this author Edwin Lasonder Edwin Lasonder School of Biomedical Sciences, University of Plymouth, Plymouth, UK Search for more papers by this author Johannes T Dessens Corresponding Author Johannes T Dessens [email protected] orcid.org/0000-0002-2070-6073 Department of Infection Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, UK Search for more papers by this author Author Information Sadia Saeed1, Annie Z Tremp1, Vikram Sharma2, Edwin Lasonder2 and Johannes T Dessens *,1 1Department of Infection Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, UK 2School of Biomedical Sciences, University of Plymouth, Plymouth, UK *Corresponding author. Tel: +442079272865; Fax: +442076374314; E-mail: [email protected] EMBO Reports (2020)21:e47832https://doi.org/10.15252/embr.201947832 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form (NADP) are vital for cell function in all organisms and form cofactors to a host of enzymes in catabolic and anabolic processes. NAD(P) transhydrogenases (NTHs) catalyse hydride ion transfer between NAD(H) and NADP(H). Membrane-bound NTH isoforms reside in the cytoplasmic membrane of bacteria, and the inner membrane of mitochondria in metazoans, where they generate NADPH. Here, we show that malaria parasites encode a single membrane-bound NTH that localises to the crystalloid, an organelle required for sporozoite transmission from mosquitos to vertebrates. We demonstrate that NTH has an essential structural role in crystalloid biogenesis, whilst its enzymatic activity is required for sporozoite development. This pinpoints an essential function in sporogony to the activity of a single crystalloid protein. Its additional presence in the apicoplast of sporozoites identifies NTH as a likely supplier of NADPH for this organelle during liver infection. Our findings reveal that Plasmodium species have co-opted NTH to a variety of non-mitochondrial organelles to provide a critical source of NADPH reducing power. Synopsis Membrane-bound NTH is a mitochondrial NADPH-generating enzyme. In malaria parasites it is found in distinct organelles, the crystalloid in ookinetes and the apicoplast in sporozoites, and has specific roles in sporozoite formation and infectivity. Plasmodium NAD(P) transhydrogenase (NTH) has an unusual βα domain architecture. NTH has an essential structural role in crystalloid biogenesis in malaria parasites. Crystalloid-resident NTH has an essential enzymatic role in sporozoite formation. Apicoplast-resident NTH activity contributes to the transition from sporozoite to intraerythrocytic parasites. Introduction Curbing malaria parasite transmission by mosquitoes is considered an essential part of successful malaria control and eradication programmes. In the parasite life cycle (Fig EV1), transmission starts with the uptake of haploid sexual stage precursor cells (gametocytes) from the vertebrate host with the blood meal of a feeding mosquito, setting off a rapid process of gametogenesis and fertilisation in the mosquito midgut. The resulting diploid zygotes undergo meiosis and transform into motile elongated forms called ookinetes, which traverse the midgut epithelium and then round up to form oocysts (Fig EV1). In the ensuing weeks, these young oocysts grow and divide by a process called sporogony generating thousands of haploid progeny cells named sporozoites. After egress from the oocysts, sporozoites colonise the insect's salivary glands, after which they are transmissible to new hosts by mosquito bite to infect liver cells and initiate new malaria blood-stage infections (Fig EV1). Click here to expand this figure. Figure EV1. Life cycle of malaria parasites in mouse and mosquitoParasites are depicted blue, mosquito tissues grey, liver dark red, and red blood cells red. Download figure Download PowerPoint NAD(P) transhydrogenases (NTHs) are enzymes that catalyse the reversible hydride ion transfer between NAD(H) and NADP(H). They exist as soluble (EC 1.6.1.1) and membrane-bound (EC 1.6.1.2) isoforms. The latter are integral multi-pass membrane proteins that in bacteria reside in the cytoplasmic membrane, whilst in metazoans they are situated in the inner membrane of mitochondria 1, 2 likely reflecting the evolutionary origin of the mitochondrion from a bacterial primary endosymbiont 3, 4. In membrane-bound NTH proteins, the electron transfer reaction between NAD(H) and NADP(H) is coupled to the simultaneous translocation of a proton across the membrane in which it is embedded, following the reaction: where 'in' and 'out' denote the cytosol and periplasmic space of bacteria, or the matrix and cytosol of the mitochondria, respectively 1, 2, 5. The widely accepted view of the physiological role of mitochondrial NTH is to generate NADPH (the reduced form of NADP), either required for mitochondrion-specific biosynthetic purposes, or to protect the organelle from oxidative damage caused by free radicals generated in the respiratory chain 6, 7. In support of the latter, NTH-deficient Caenorhabditis elegans and NTH-deficient C57BL/6J mice show increased sensitivity to mitochondrial oxidative stress 8, 9. In this study, we characterise a membrane-bound NTH in malaria parasites that is not present in mitochondria, but instead localises in the crystalloid, an enigmatic organelle found in ookinetes and young oocysts that is critically involved in sporogony 10-13. We show that NTH has an essential structural role in crystalloid formation, as well as a vital enzymatic role in sporogony, indicating that the organelle requires NADPH to function. NTH is also found in the sporozoite apicoplast, addressing a longstanding question about the potential source of NADPH required for some of the anabolic activities that take place in this plastid of likely red algal origin 14. Results and Discussion Plasmodium encodes a single, membrane-bound NTH Genome analysis shows that Plasmodium species encode a single, conserved membrane-bound NTH (e.g. PlasmodDB identifiers PF3D7_1453500; PVX_117805; PBANKA_1317200). Plasmodium berghei NTH is encoded by a three-exon gene and is composed of 1,201 amino acids with a calculated Mr of 135,198 and has a predicted amino-terminal ER signal peptide sequence that forms part of a bipartite apicoplast targeting sequence (PATS prediction: score 0.947 out of 1.000 15; PlasmoAP prediction: score 4 out of 5 tests positive 16; Fig 1A). The gene product forms 11 predicted transmembrane helices and has additional domains for binding NAD(H) and NADP(H) (Fig 1A). This structure conforms with the general architecture of proton-translocating NTH proteins, consisting of three functional domains: domain I, which binds NAD(H); domain II, which contains the membrane-spanning helices, and domain III, which binds NADP(H) 2 (Fig 1A and B). Functional domains I and III together facilitate hydride transfer between NAD(H) and NADP(H), whereas domain II facilitates proton translocation across the lipid bilayer in which the NTH protein is embedded (Fig 1B). Whilst prokaryotes have segmented nth genes, eukaryotic nth genes are unsegmented and encode single polypeptide NTH proteins, either linking the α subunit C-terminus to the β subunit N-terminus (αβ) as illustrated by mammalian NTH or linking the β subunit C-terminus to the α subunit N-terminus (βα) as illustrated by Plasmodium NTH (Fig 1C). Accordingly, the order of domains in Plasmodium NTH is dIIa-dIII-dI-dIIb (Fig 1A). Figure 1. Structure of the Plasmodium nth gene and gene product Schematic domain composition of PBANKA_1317200, showing predicted ER signal peptide (red), apicoplast transit peptide (green), transmembrane helices (dark grey), NADP(H) binding (pink), and NAD(H) binding (blue) modules. Functional NTH domains I, II, and III are indicated. Predicted functions of the Plasmodium berghei NTH protomer in the lipid bilayer showing functional domains I–III and corresponding functional activities. Organisation of functional NTH domains I–III (coloured bars) in Thermus thermophilus, Escherichia coli, mammals, and Plasmodium. Linker connecting functional domains is indicated by dotted line. Prokaryotic nth genes are segmented into three (T. thermophilus) or two (E. coli) segments, encoding NTH subunits α1, α2, and β (T. thermophilus) or α and β (E. coli). Eukaryotic nth genes are unsegmented and encode single polypeptide NTH of different orientations, either corresponding to an αβ linkage (mammal) or to a βα linkage (Plasmodium). The amino (N) and carboxy (C) termini of the eukaryotic NTH polypeptides are indicated. Download figure Download PowerPoint Plasmodium NTH is present in the crystalloid organelle Various Plasmodium transcriptome studies identified transcripts of the nth gene predominantly in female gametocytes and to be translationally repressed 17-19. This was a strong indication that the NTH protein is expressed in zygotes/ookinetes. To determine NTH protein expression and subcellular localisation in live P. berghei parasites, a genetically modified parasite was generated by double homologous crossover recombination that stably expresses, from its native promoter, the full length NTH fused at its carboxy-terminus to GFP (Fig EV2). The resulting parasites (termed NTH/GFP) developed normally in mouse and mosquito and were transmitted by mosquito bite, indicating that the GFP tag had not interfered with NTH function (assuming nth disruption gives a clear phenotype). In the mouse, neither asexual nor sexual blood-stage parasites displayed discernible GFP-based fluorescence consistent with the nth transcriptome data 17-19. Dispersed extranuclear GFP fluorescence was first detected in zygotes ~ 4 h after gametocyte activation (Fig 2A) consistent with a post-fertilisation lifting of translational repression and a localisation of the protein in the ER. By 24 h, mature ookinetes showed GFP fluorescence that was concentrated in typically two spots associated with pigment clusters (Fig 2B). This distribution pattern is characteristic of proteins that are trafficked to the crystalloids 11-13, 20. Click here to expand this figure. Figure EV2. Generation of genetically modified parasite lines Schematic diagram of the unmodified (parental) and modified nth alleles in parasite lines NTH/GFP, NTH-KO, NTHΔPP, and NTH/ND500LK. The nth gene is indicated with coding sequence (wide grey bars, introns not shown) and 5′ and 3′ untranslated regions (UTRs; narrow grey bars). Also indicated are the relative positions of the putative apicoplast targeting signal (black box), GFP module (gfp); the human DHFH selectable marker gene cassette (hdhfr); the XhoI and AfeI diagnostic restriction sites; and primers used for diagnostic PCR amplification (P1–P4). Sizes of PCR products are also indicated. Diagnostic PCR of parasite lines NTH/GFP and NTH-KO. Integration of the modified GFP-tagged alleles into the target locus across the 5′ integration site was carried out with primers P1 (ATATTTCCCCCTAATTTTCCCTT) and P2 (GTGCCCATTAACATCACC), amplifying expected products of ˜ 4.6 kb in NTH/GFP and 0.7 kb in NTH-KO. Absence of the unmodified nth allele was carried out with primers P1 (ATATTTCCCCCTAATTTTCCCTT) and P3 (ATTTCAATACTCGAATTTATGTTATCG), amplifying expected products of ˜ 5.2 kb from the parental parasite and 4.1 kb in NTH-KO parasites. Diagnostic PCR and restriction enzyme digests of parasite lines NTHΔPP and NTH/ND500LK. Primers P1 and P2 amplify a 4.4 kb product in NTHΔPP that is digested by XhoI into the expected two fragments of ˜ 3.7 kb and 0.7 kb, confirming presence of the mutation. Primers P1 and P2 amplify a 4.6 kb product in NTH/ND500LK that is digested by AfeI into the expected two fragments of ˜ 2.5 kb and 2.1 kb, confirming presence of the mutation. Schematic diagram of the modified acp allele (PBANKA_0305600) in parasite line ACP/mCherry. The acp gene is indicated with coding sequence (wide grey bars, introns not shown) and imc1a 5′ and 3′ untranslated regions (UTRs; narrow grey bars). Also indicated are the relative positions of the mCherry module (mCh); the human DHFR::yFCU selectable marker gene cassette (hdhfr yfcu); and primers used for diagnostic PCR amplification (P1–P3). Sizes of PCR products are also indicated. Diagnostic PCR for integration into the target imc1a locus with primers P2 and P3, giving rise to a 3.0 kb product (top panel). Diagnostic PCR with primer pair P1 and P3 amplified an ˜ 4.4 kb fragment from the parental (wild-type) parasites, and no product in the ACP/mCherry parasites, confirming absence of the unmodified imc1a allele in the clonal transgenic parasite line (bottom panel). Download figure Download PowerPoint Figure 2. Expression and subcellular localisation of NTH in Plasmodium berghei ookinetes Brightfield and fluorescence images of a NTH/GFP zygote at 4 h post-fertilisation, showing dispersed extranuclear fluorescence. Hoechst DNA staining (blue) marks nucleus. Scale bar = 5 μm. Image of an NTH/GFP ookinete, showing fluorescent spots co-localising with pigment clusters (arrows). Hoechst DNA staining (blue) marks nucleus. Scale bar = 5 μm. Schematic diagram of a genetic cross between parasite lines NTH/GFP and LAP3/mCherry and the resultant ookinete population after mosquito transmission and drug selection (oocysts, liver, and blood-stage parasites not shown). Colours represent NTH::GFP (green), LAP3::mCherry (red), and their co-localisation (orange). Spots in ookinetes depict crystalloids. Self-fertilisation events are omitted. Brightfield and fluorescence images of a zygote at 4 h post-fertilisation simultaneously expressing NTH::GFP (green) and LAP3::mCherry (red). Scale bar = 5 μm. An ookinete simultaneously expressing NTH::GFP (green) and LAP3::mCherry (red), showing co-localisation in the crystalloid. Scale bar = 5 μm. Western blot of purified NTH/GFP ookinete samples with (+) and without (−) prior in vivo crosslinking. Mr markers (kDa) are indicated on the right-hand side. Download figure Download PowerPoint Previous studies have shown that a protein complex of six modular proteins rich in putative carbohydrate binding domains, named LCCL lectin adhesive-like proteins (LAPs), resides in the crystalloid 11, 20, 21. To confirm the localisation of NTH in the crystalloid, we co-localised NTH with LAP3. To achieve this, LAP3 was fused to the red fluorescent protein mCherry 13 and the resulting parasite line (named LAP3/mCherry) was genetically crossed in vitro with parasite line NTH/GFP. Subsequent infection of mosquitoes with the resultant ookinete population gives rise to heterokaryotic polyploid oocysts containing mixtures of parental, wild-type, and double gene-tagged (i.e. possessing modified alleles for both nth and lap3) sporozoites (Fig 2C). The ensuing sporozoite population was transmitted to naive mice by mosquito bite and drug selected. Ookinete cultures derived from the transmitted parasite population contained zygotes and ookinetes with dual expression of NTH::GFP and LAP3::mCherry that co-localised both before and after crystalloid formation, respectively (n = 50; Fig 2D and E). Western blot analysis of purified ookinetes of parasite line NTH/GFP with anti-GFP antibodies produced a single band of a size corresponding to the NTH::GFP fusion protein (~ 160 kDa; Fig 2F), confirming that the nth allele had been successfully tagged. Ookinetes that were in vivo crosslinked prior to Western blot analysis showed an additional high molecular weight band migrating at approximately double the molecular size, which possibly corresponds to the NTH homodimer. This is consistent with reports that NTH proteins in other organisms operate as dimers 5, 22-25. Plasmodium NTH is required for sporogony To study the function of NTH and its contribution to parasite development and infectivity, we generated a null mutant parasite line by double homologous crossover recombination in which the coding sequence of nth was removed (Fig EV2). The resultant parasites, named NTH-KO, developed normally in the mouse consistent with the absence of NTH expression in these life stages and with a recent report that this gene is dispensable for asexual blood-stage development in P. berghei 26. NTH-KO parasites displayed normal gametocyte development, gametogenesis, and formed ookinetes of normal size and shape (Fig 3A). However, pigment in these ookinetes was more dispersed (n = 50) and not found in clusters that typically surround and highlight the crystalloids 11, 27 (Fig 3A). Absence of these pigment clusters coincides with, and is caused by, loss of crystalloid formation as demonstrated recently for null mutants of the crystalloid protein DHHC10 13. In Anopheles stephensi mosquitoes, NTH-KO parasites developed oocyst numbers comparable to their wild-type counterparts (Figs 3B and EV3A). However, despite undergoing substantial nuclear expansion these oocysts failed to produce sporozoites (1,000 oocysts examined across at least 10 midguts from two independent infections; Fig 3C) and displayed increased oocyst size (Fig 3D and EV3B). Similar phenotypes have been reported for several LAP null mutants 11, 12, 28-30. These collective findings demonstrate that NTH is required for crystalloid biogenesis and sporozoite development. Figure 3. NTH loss of function and contribution to Plasmodium berghei parasite development Brightfield images of a NTH-KO and NTH/GFP ookinete, showing normal ookinete morphology, but lack of pigment clusters associated with crystalloids (arrows) in the NTH null mutant. Scale bar = 5 μm. Aligned dot plot of oocyst numbers in Anopheles stephensi infected with parasite lines NTH/GFP, NTH-KO, and NTHΔPP. Horizontal lines mark mean values (n = 20); ns, not significantly different (Mann–Whitney). Brightfield and fluorescence images of oocysts of parasite lines NTH/GFP and NTH-KO at 15 days post-infection, showing lack of sporulation in the NTH null mutant. Hoechst DNA staining (artificially depicted red) labels parasite nuclei. The large nuclei of the epithelial midgut cells also show. Scale bar = 20 μm. Aligned dot plot of oocyst diameters in A. stephensi at 15 days post-infection with parasite lines NTH/GFP, NTH-KO, and NTHΔPP. Horizontal lines mark mean values (n = 20). ** mark statistically significant differences (P < 0.0001, Mann–Whitney); ns, not significant. Images of ookinetes of parasite lines NTHΔPP showing dispersed NTHΔPP::GFP fluorescence; LAP3/mCherry showing focal LAP3::mCherry fluorescence in crystalloids; and cross (carrying modified alleles encoding NTHΔPP::GFP and LAP3::mCherry), showing dispersed LAP3::mCherry fluorescence. Arrow marks pigment cluster associated with crystalloids. Scale bar = 5 μm. Schematic diagram of a genetic cross between parasite lines NTHΔPP and LAP3/mCherry and the resultant ookinete population after mosquito transmission and drug selection (oocysts, liver, and blood-stage parasites not shown). Colours represent NTH::GFP (green), LAP3::mCherry (red), and their co-localisation (orange). Spots in ookinetes depict crystalloids. The red cross indicates that sporozoite formation is blocked. Self-fertilisation events are omitted. PCR diagnostic for the presence of modified nth alleles in blood-stage infections obtained after sporozoite transmission of a NTHΔPP x LAP3/mCherry genetic cross (lane NTHΔPP), amplifying an ˜ 4.4 kb fragment. NTH/GFP parasites are used as a PCR control, amplifying ˜ 4.6 kb. Parental parasites provide a negative control. Image of a NTH/ND500LK ookinete, showing fluorescent spots co-localising with pigment clusters (arrows) indicative of normal crystalloid formation. Scale bar = 5 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Oocyst development in Anopheles stephensi infected with parasite lines NTH/GFP, NTH-KO, NTHΔPP, and NTH/ND500LK Aligned dot plot of oocyst numbers at 10 days post-infection. Horizontal lines mark mean values (n = 20); ns, not significantly different (Mann–Whitney). Bar chart showing percentage of oocyst with a diameter > 50 μm at 15 days post-infection. Diameters were scored at 400× magnification using an eyepiece micrometre. Five infected mosquitoes and 100 oocysts per mosquito were sampled for each parasite line. Error bars show standard deviations (n = 5). ** mark statistically significant differences (P < 0.001, Mann–Whitney); ns, not significant. Brightfield and fluorescence images of oocysts pre-sporulation 11 days post-infection, stained with Hoechst DNA stain (blue), showing similar developmental progression between NTH/GFP and mutants. Scale bar = 20 μm. Download figure Download PowerPoint A different parasite line named NTHΔPP was generated that expresses an N-terminally truncated version of NTH::GFP in which 60 amino acids were removed downstream of the ER signal peptide, including the predicted apicoplast transit peptide (Fig EV2). NTHΔPP ookinetes displayed weak dispersed GFP fluorescence that did not localise in crystalloids, and typical crystalloid-associated pigment clusters were absent (n = 50; Fig 3E), as was observed in NTH-KO ookinetes. NTHΔPP parasites formed normal numbers of oocysts in mosquitoes (Figs 3B and EV3A), but like NTH-KO parasites these failed to produce sporozoites (1,000 oocysts examined across at least 10 midguts from two independent infections) and had increased size (Figs 3D and EV3B). These collective findings indicate that the truncated NTH protein expressed in NTHΔPP parasites is structurally compromised and dysfunctional, resulting in a loss-of-function phenotype. To assess the localisation of the crystalloid protein LAP3 in parasite line NTHΔPP, the latter was genetically crossed with parasite line LAP3/mCherry (Fig 3F). The expression of the functional nth allele in heterokaryotic zygotes allows their normal developmental progression through sporogony, as shown previously for LAP null mutants 29, 31. Accordingly, LAP3/mCherry female gametes that are fertilised by NTHΔPP male gametes give rise to heterokaryotic polyploid oocysts that sporulate normally (Fig 3F). The resultant sporozoite populations were transmitted to naïve mice by mosquito bite and drug selected. Diagnostic PCR carried out on the ensuing blood-stage parasite infections showed the presence of the modified nth allele (Fig 3G). Resultant ookinete cultures contained a mixture of the two parental lines (NTHΔPP and LAP3/mCherry) as well as double mutants possessing modified alleles for both nth and lap3 (Fig 3E). Subcellular distribution of LAP3::mcherry in double mutant ookinetes was dispersed rather than concentrated in spots (Fig 3E), demonstrating that the absence of structurally intact NTH prevents LAP3 from reaching the crystalloid, consistent with the observed block in crystalloid formation. NTH has an essential structural role in crystalloid biogenesis Besides NTH null mutants, ookinetes devoid of LAP1, LAP3, or DHHC10 also lack crystalloids 11-13, 21. The effect of NTH knockout on sporozoite formation could thus be caused by the failure to form crystalloids and not reflect a direct role of NTH in sporogony. To test this hypothesis, we created a parasite line expressing a structurally intact, but enzymatically inactive version of NTH. To do so, the highly conserved aspartic acid residue at position 500 was mutated to a lysine (Fig EV2). The equivalent point mutation in bacterial NTH abolishes both hydride transfer and proton-translocating activities 32. Ookinetes of the resulting NTH functional knockout (named NTH/ND500LK; Fig EV2) displayed GFP fluorescence in discrete spots that co-localised with pigment clusters (n = 50; Fig 3H), indicative of normal crystalloid formation. This shows that NTH needs to be physically present and structurally intact, but not enzymatically active, to facilitate crystalloid biogenesis. Crystalloid proteins are trafficked via the ER, but specific sorting signals for the organelle have not been identified 10. The crystalloid is a short-lived organelle that forms in ookinetes by coordinated congregation of small ER-derived vesicles, a process that is dependent on the synthesis of some of its protein constituents 11-13. This indicates that crystalloid proteins are delivered to the organelle concurrent with its formation. One plausible explanation for the structural role of NTH in crystalloid biogenesis is that it interacts in the ER with other proteins destined for the organelle (e.g. LAPs), thereby ensuring that they are trafficked together and eliminating the need for individual proteins to possess specific crystalloid targeting signals. Indeed, interactions between LAPs have been shown to occur before crystalloid formation 21, 33. The formation of such a "crystalloid protein complex" also offers an explanation how several structurally and functionally unrelated crystalloid proteins including LAPs, DHHC10, and NTH produce very similar loss-of-function phenotypes 11-13: Disruption of highly constrained interactions between crystalloid proteins, through their removal or structural alteration, could compromise formation of the crystalloids and consequently the downstream process of sporogony. Indeed, structural modifications of LAP family members have been shown to affect their ability to interact with each other 21 and impact on crystalloid biogenesis 11, 12, 21, 28. NTH has an essential enzymatic role in sporogony The normal crystalloid formation in parasite line NTH/ND500LK allowed us to assess the impact of crystalloids with enzymatically inactive NTH on sporogony. Like NTH structural knockout parasites, NTH/ND500LK parasites developed oocysts (Fig EV3A), confirming that NTH activity is not required for oocyst development per se. Like the previous NTH mutants, NTH/ND500LK oocysts reached a larger size (Fig EV3B) and failed to sporulate (1,000 oocysts examined across at least 10 midguts from two independent infections), showing that NTH activity is essential for sporogony. The NTH functional knockout parasite is likely to have a normal protein repertoire in the organelle. The phenotype of this mutant parasite line can thus for the first time be pinpointed to the activity of a single crystalloid protein, identifying NTH as an essential molecule for crystalloid function, sporogony, and sporozoite transmission. The predicted membrane topology of NTH projects its catalytic domains I and III inside the lumen of the crystalloid subunit vesicles, providing a source of luminal NADPH for the organelle. Indeed, the opposite topology would make little sense, because cytosolic NADPH/NADP+ ratios are already high to maintain the reducing redox state of this environment. Our observation that NTH-deficient parasites are not impaired in oocyst formation implies that the ookinetes and young oocysts have normal fitness and do not suffer extra from oxidative stress encountered in the insect. It is therefore plausible that crystalloid-resident NTH is not involved in maintaining the redox state, but instead facilitates specific NADPH-dependent anabolic reactions. In this context, the unusual structure of the crystalloid being a large cluster of small vesicles rather than a single large membrane-limited compartment could have biological relevance as it greatly increases the membranous area of the organelle and thus the amount of NTH activity, and that of other membrane-bound enzymes such as DHHC10, that can be accommodated. NTH is targeted to the sporozoite apicoplast Apart from very young oocysts that still possess crystalloids, no discernible GFP fluorescence was observed in oocysts before sporulation (Fig 4A; n = 1,000). However, GFP fluorescence was observed in sporulated oocysts (Fig 4B) and in individual sporozoites (Fig 4C), indic
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