Plasmodium sporozoite excystation involves local breakdown of the oocyst capsule.

Plasmodium oocysts develop on the abluminal side of the mosquito midgut in relatively small numbers. Oocysts possess an extracellular cell wall-the capsule-to protect them from the insect's haemolymph environment. To further maximise transmission, each oocyst generates hundreds of sporozoites through an asexual multiplication step called sporogony. Completion of transmission requires sporozoite egress from the capsule (excystation), but this process remains poorly understood. In this study, we fused the parasite-encoded capsule protein Cap380 with green fluorescent protein in a transgenic P. berghei line, allowing live fluorescence imaging of capsules throughout sporogony and sporozoite excystation. The results show that capsules progressively weaken during sporulation ultimately resulting in sporozoite exit through small holes. Prior to formation of the holes, local thinning of the capsule was observed. Our findings support an excystation model based on local, rather than global, weakening of the capsule likely facilitated by local re-orientation of sporozoites and apical secretion.

Plasmodium berghei oocysts possess fatty acid synthesis and scavenging routes.

Malaria parasites carry out fatty acid synthesis (FAS) in their apicoplast organelle via a bacterially related (type II) enzymatic pathway. In the vertebrate host, exoerythrocytic Plasmodium stages rely on FAS, whereas intraerythrocytic stages depend on scavenging FA from their environment. In the mosquito, P. falciparum oocysts express and rely on FAS enzymes for sporozoite formation, but P. yoelii oocysts do not express, nor depend on, FAS enzymes and thus rely on FA scavenging to support sporogony. In P. berghei, FAS enzymes are similarly expendable for sporogony, indicating it conforms to the P. yoelii scenario. We show here that P. berghei, unexpectedly, expresses FAS enzymes throughout oocyst development. These findings indicate that P. berghei can employ FAS alongside FA scavenging to maximise sporogony and transmission, and is more similar to P. falciparum than previously assumed with respect to FA acquisition by the oocyst. The ability of oocysts to switch between FAS and scavenging could be an important factor in the non-competitive relationship of resource exploitation between Plasmodium parasites and their mosquito vectors, which shapes parasite virulence both in the insect and vertebrate.

NAD(P) transhydrogenase isoform distribution provides insight into apicomplexan evolution.

Membrane-located NAD(P) transhydrogenase (NTH) catalyses reversible hydride ion transfer between NAD(H) and NADP(H), simultaneously translocating a proton across the membrane. The enzyme is structurally conserved across prokaryotes and eukaryotes. In heterotrophic bacteria NTH proteins reside in the cytoplasmic membrane, whereas in animals they localise in the mitochondrial inner membrane. Eukaryotic NTH proteins exists in two distinct configurations (isoforms) and have non-mitochondrial functions in unicellular eukaryotes like Plasmodium, the causative agent of malaria. In this study, we carried out a systematic analysis of nth genes across eukaryotic life to determine its prevalence and distribution of isoforms. The results reveal that NTH is found across all major lineages, but that some organisms, notably plants, lack nth genes altogether. Isoform distribution and phylogenetic analysis reveals different nth gene loss scenarios in apicomplexan lineages, which sheds new light on the evolution of the Piroplasmida and Eimeriidae.

Plasmodium berghei leucine-rich repeat protein 1 downregulates protein phosphatase 1 activity and is required for efficient oocyst development.

Protein phosphatase 1 (PP1) is a key enzyme for Plasmodium development. However, the detailed mechanisms underlying its regulation remain to be deciphered. Here, we report the functional characterization of the Plasmodium berghei leucine-rich repeat protein 1 (PbLRR1), an orthologue of SDS22, one of the most ancient and conserved PP1 interactors. Our study shows that PbLRR1 is expressed during intra-erythrocytic development of the parasite, and up to the zygote stage in mosquitoes. PbLRR1 can be found in complex with PbPP1 in both asexual and sexual stages and inhibits its phosphatase activity. Genetic analysis demonstrates that PbLRR1 depletion adversely affects the development of oocysts. PbLRR1 interactome analysis associated with phospho-proteomics studies identifies several novel putative PbLRR1/PbPP1 partners. Some of these partners have previously been characterized as essential for the parasite sexual development. Interestingly, and for the first time, Inhibitor 3 (I3), a well-known and direct interactant of Plasmodium PP1, was found to be drastically hypophosphorylated in PbLRR1-depleted parasites. These data, along with the detection of I3 with PP1 in the LRR1 interactome, strongly suggest that the phosphorylation status of PbI3 is under the control of the PP1-LRR1 complex and could contribute (in)directly to oocyst development. This study provides new insights into previously unrecognized PbPP1 fine regulation of Plasmodium oocyst development through its interaction with PbLRR1.

Crystalloids: Fascinating Parasite Organelles Essential for Malaria Transmission.

Crystalloids are malaria parasite organelles exclusive to the ookinete and young oocyst life stages that infect the mosquito. The organelles have key roles in sporozoite development and infectivity but the way this is facilitated on a molecular level remains poorly understood. Recent discoveries have shed new light on these processes.

Plasmodium berghei LAPs form an extended protein complex that facilitates crystalloid targeting and biogenesis.

Passage of malaria parasites through mosquitoes involves multiple developmental transitions, from gametocytes that are ingested with the blood meal, through to sporozoites that are transmitted by insect bite to the host. During the transformation from gametocyte to oocyst, the parasite forms a unique transient organelle named the crystalloid, which is involved in sporozoite formation. In Plasmodium berghei, a complex of six LCCL domain-containing proteins (LAPs) reside in the crystalloid and are required for its biogenesis. However, little else is known about the molecular mechanisms that underlie the crystalloid's role in sporogony. In this study, we have used transgenic parasites stably expressing LAP3 fused to GFP, combined with GFP affinity pulldown and high accuracy mass spectrometry, to identify an extended LAP interactome of some fifty proteins. We show that many of these are targeted to the crystalloid, including members of two protein families with CPW-WPC and pleckstrin homology-like domains, respectively. Our findings indicate that the LAPs are part of an intricate protein complex, the formation of which facilitates both crystalloid targeting and biogenesis. SIGNIFICANCE: Reducing malaria parasite transmission by mosquitoes is a key component of malaria eradication and control strategies. This study sheds important new light on the molecular composition of the crystalloid, an enigmatic parasite organelle that is essential for sporozoite formation and transmission from the insect to the vertebrate host. Our findings provide new mechanistic insight into how proteins are delivered to the crystalloid, and indicate that the molecular mechanisms that underlie crystalloid function are complex, involving several protein families unique to Plasmodium and closely related organisms. The new crystalloid proteins identified will form a useful starting point for studies aimed at unravelling how the crystalloid organelle facilitates sporogony and transmission.

NAD(P) transhydrogenase has vital non-mitochondrial functions in malaria parasite transmission.

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.

Distinct Functional Contributions by the Conserved Domains of the Malaria Parasite Alveolin IMC1h.

Invasive, motile life cycle stages (zoites) of apicomplexan parasites possess a cortical membrane skeleton composed of intermediate filaments with roles in zoite morphogenesis, tensile strength and motility. Its building blocks include a family of proteins called alveolins that are characterized by conserved "alveolin" domains composed of tandem repeat sequences. A subset of alveolins possess additional conserved domains that are structurally unrelated and the roles of which remain unclear. In this structure-function analysis we investigated the functional contributions of the "alveolin" vs. "non-alveolin" domains of IMC1h, a protein expressed in the ookinete and sporozoite life cycle stages of malaria parasites and essential for parasite transmission. Using allelic replacement in Plasmodium berghei, we show that the alveolin domain is responsible for targeting IMC1h to the membrane skeleton and, consequently, its deletion from the protein results in loss of function manifested by abnormally-shaped ookinetes and sporozoites with reduced tensile strength, motility and infectivity. Conversely, IMC1h lacking its non-alveolin conserved domain is correctly targeted and can facilitate tensile strength but not motility. Our findings support the concept that the alveolin module contains the properties for filament formation, and show for the first time that tensile strength makes an important contribution to zoite infectivity. The data furthermore provide new insight into the underlying molecular mechanisms of motility, indicating that tensile strength is mechanistically uncoupled from locomotion, and pointing to a role of the non-alveolin domain in the motility-enhancing properties of IMC1h possibly by engaging with the locomotion apparatus.

Dysregulated gene expression in oocysts of Plasmodium berghei LAP mutants.

Malaria parasite oocysts generate sporozoites by a process termed sporogony. Essential for successful sporogony of Plasmodium berghei in Anopheles stephensi mosquitoes is a complex of six LCCL lectin domain adhesive-like proteins (LAPs). LAP null mutant oocysts undergo growth and mitosis but fail to form sporozoites. At a cytological level, LAP null mutant oocyst development is indistinguishable from its wildtype counterparts for the first week, supporting the hypothesis that LAP null mutant oocysts develop normally before cytokinesis. We show here that LAP1 null mutant oocysts display highly reduced expression of sporozoite proteins and their transcription factors. Our findings indicate that events leading up to the cytokinesis defect in LAP null mutants occur early in oocyst development.

The Plasmodium LAP complex affects crystalloid biogenesis and oocyst cell division.

Malaria parasite oocysts located on the mosquito midgut generate sporozoites by a process called sporogony. Plasmodium berghei parasites express six LCCL lectin domain adhesive-like proteins (LAPs), which operate as a complex and share a localisation in the crystalloid - an organelle found in the ookinete and young oocyst. Depletion of LAPs prevents crystalloid formation, increases oocyst growth, and blocks sporogony. Here, we describe a LAP4 mutant that has abnormal crystalloid biogenesis and produces oocysts that display reduced growth and premature sporogony. These findings provide evidence for a role of the LAP complex in regulating oocyst cell division via the crystalloid.

LCCL protein complex formation in Plasmodium is critically dependent on LAP1.

Successful sporogony of Plasmodium berghei in vector mosquitoes requires expression of a family of six modular proteins named LCCL lectin domain adhesive-like proteins (LAPs). The LAPs share a subcellular localization in the crystalloid, a unique parasite organelle that forms during ookinete development. Here, LAP interactions in P. berghei were studied using a series of parasite lines stably expressing reporter-tagged LAPs combined with affinity purification and high accuracy label free quantitative mass spectrometry. Our results show that abundant complexes containing LAP1, LAP2 and LAP3 are formed in gametocytes through high avidity interactions. Following fertilization, LAP4, LAP5 and LAP6 are recruited to this complex, a process that is facilitated by LAP1 chiefly through its scavenger receptor cysteine-rich modules. These collective findings provide new insight into the temporal and molecular dynamics of protein complex formation that lead up to, and are required for, crystalloid biogenesis and downstream sporozoite transmission of malaria parasites.

Palmitoylation of Plasmodium alveolins promotes cytoskeletal function.

S-palmitoylation is a post-translational lipid modification that is widespread among Plasmodium proteins and essential for parasite development. Little is known about the contribution of palmitoylation to the function of individual parasite molecules and structures. Alveolins are major components of the subpellicular network (SPN), a cortical cytoskeleton primarily involved in providing mechanical strength to the cell. We show here that the alveolin IMC1c is palmitoylated on a conserved cysteine motif, and that non-palmitoylated IMC1c displays normal expression, stability and trafficking. However, mutant parasites exhibit reduced osmotic stress resistance and tensile strength. These findings support the hypothesis that alveolin palmitoylation enhances cytoskeletal function by strengthening the connection between the SPN and the adjoining inner membrane complex via lipid anchoring.

The Plasmodium alveolin IMC1a is stabilised by its terminal cysteine motifs and facilitates sporozoite morphogenesis and infectivity in a dose-dependent manner.

Apicomplexan parasites possess a unique cortical cytoskeleton structure composed of intermediate filaments. Its building blocks are provided by a conserved family of proteins named alveolins. The core alveolin structure is made up of tandem repeat sequences, thought to be responsible for the filamentous properties of these proteins. A subset of alveolins also possess conserved motifs composed of three closely spaced cysteine residues situated near the ends of the polypeptides. The roles of these cysteine motifs and their contribution to alveolin function remains poorly understood. The sporozoite-expressed IMC1a is unique within the Plasmodium alveolin family in having conserved cysteine motifs at both termini. Using transgenic Plasmodium berghei parasites, we show in this structure-function analysis that mutagenesis of the amino- or carboxy-terminal cysteine motif causes marked reductions in IMC1a protein levels in the parasite, which are accompanied by partial losses of sporozoite shape and infectivity. Our findings give new insight into alveolin function, identifying a dose-dependent effect of alveolin depletion on sporozoite size and infectivity, and vital roles of the terminal cysteine motifs in maintaining alveolin stability in the parasite.

Biogenesis of the crystalloid organelle in Plasmodium involves microtubule-dependent vesicle transport and assembly.

Malaria parasites possess unique subcellular structures and organelles. One of these is the crystalloid, a multivesicular organelle that forms during the parasite's development in vector mosquitoes. The formation and function of these organelles remain poorly understood. A family of six conserved and modular proteins named LCCL-lectin adhesive-like proteins (LAPs), which have essential roles in sporozoite transmission, localise to the crystalloids. In this study we analyse crystalloid formation using transgenic Plasmodium berghei parasites expressing GFP-tagged LAP3. We show that deletion of the LCCL domain from LAP3 causes retarded crystalloid development, while knockout of LAP3 prevents formation of the organelle. Our data reveal that the process of crystalloid formation involves active relocation of endoplasmic reticulum-derived vesicles to common assembly points via microtubule-dependent transport. Inhibition of microtubule-dependent cargo transport disrupts this process and replicates the LCCL domain deletion mutant phenotype in wildtype parasites. These findings provide the first clear insight into crystalloid biogenesis, demonstrating a fundamental role for the LAP family in this process, and identifying the crystalloid and its formation as potential targets for malaria transmission control.

Plasmodium alveolins possess distinct but structurally and functionally related multi-repeat domains.

The invasive and motile life stages of malaria parasites (merozoite, ookinete and sporozoite) possess a distinctive cortical structure termed the pellicle. The pellicle is characterised by a double-layered 'inner membrane complex' (IMC) located underneath the plasma membrane, which is supported by a cytoskeletal structure termed the subpellicular network (SPN). The SPN consists of intermediate filaments, whose major constituents include a family of proteins called alveolins. Here, we re-appraise the alveolins in the genus Plasmodium with respect to their repertoire, structure and interrelatedness. Amongst 13 family members identified, we distinguish two domain types that, albeit distinct at the primary structure level, are structurally related and contain tandem repeats with a consensus 12-amino acid periodicity. Analysis in Plasmodium berghei of the most divergent alveolin, PbIMC1d, reveals a zoite-specific expression in ookinetes and a subcellular localisation in the pellicle, consistent with its predicted role as a SPN component. Knockout of PbIMC1d gives rise to a wild-type phenotype with respect to ookinete morphogenesis, tensile strength, gliding motility and infectivity, presenting the first example of apparent functional redundancy amongst alveolin family members.

Distinct temporal recruitment of Plasmodium alveolins to the subpellicular network.

The zoite stages of malaria parasites (merozoite, ookinete and sporozoite) possess a distinctive cortical structure termed the pellicle, which is defined by a double membrane layer named the inner membrane complex (IMC). The IMC is supported by a cytoskeleton of intermediate filaments, termed the subpellicular network (SPN). Plasmodium IMC1 proteins, or alveolins, make up a conserved family of structurally related proteins that comprise building blocks of the SPN. Here, using green fluorescent protein (GFP) tagging in P. berghei, we show that the alveolins PbIMC1c and PbIMC1e are expressed in all three zoite stages. Our data reveal that PbIMC1e is assembled into the SPN concurrent with pellicle development, while PbIMC1c is assembled after pellicle formation. In the sexual stages, these processes are accompanied by different gene expressions from maternal and paternal alleles: PbIMC1e is expressed uniquely from the maternal allele, while PbIMC1c is expressed from the maternal allele in gametocytes, but from both parental alleles during ookinete development. These findings establish biogenesis of the cortical cytoskeleton in Plasmodium to be a complex and dynamic process, involving distinct parental gene expression and chronological recruitment of its protein constituents. While allelic replacement of the pbimc1c and pbimc1e genes with GFP-tagged versions was readily achieved using double crossover homologous recombination, attempts to disrupt these genes by this strategy only resulted in the integration of the selectable marker and GFP reporter into non-specific genomic locations. The recurrent inability to disrupt these genes provides the first genetic evidence that alveolins are necessary for asexual blood-stage parasite development in Plasmodium.

Morphogenesis of Plasmodium zoites is uncoupled from tensile strength.

A shared feature of the motile stages (zoites) of malaria parasites is a cortical cytoskeletal structure termed subpellicular network (SPN), thought to define and maintain cell shape. Plasmodium alveolins comprise structural components of the SPN, and alveolin gene knockout causes morphological abnormalities that coincide with markedly reduced tensile strength of the affected zoites, indicating the alveolins are prime cell shape determinants. Here, we characterize a novel SPN protein of Plasmodium berghei ookinetes and sporozoites named G2 (glycine at position 2), which is structurally unrelated to alveolins. G2 knockout abolishes parasite transmission and causes zoite malformations and motility defects similar to those observed in alveolin null mutants. Unlike alveolins, however, G2 contributes little to tensile strength, arguing against a cause-effect relationship between tensile strength and cell shape. We also show that G2 null mutant sporozoites display an abnormal arrangement of their subpellicular microtubules. These results provide important new understanding of the factors that determine zoite morphogenesis, as well as the potential roles of the cortical cytoskeleton in gliding motility.

Translational repression controls temporal expression of the Plasmodium berghei LCCL protein complex.

Plasmodium LCCL proteins comprise a family of six proteins that function as a protein complex and have essential roles in sporozoite transmission. In Plasmodium berghei, family members PbLAP1, PbLAP2 and PbLAP3 have been shown to be expressed in gametocytes and, following gametogenesis and fertilization, to be targeted to distinctive multivesicular organelles termed crystalloids that form in the ookinete. Here, we show by GFP-tagging that PbLAP4, PbLAP5 and PbLAP6, like their family members, are associated with the crystalloids. However, in contrast to their family members, protein expression of PbLAP4, PbLAP5 and PbLAP6 was not detected in gametocytes, even though transcription of the corresponding genes is most prominent in the sexual blood stage parasites. These results suggest that translational repression controls expression of the LCCL protein repertoire and, consequently, the temporal function of the protein complex during P. berghei development in the mosquito.

Conformational co-dependence between Plasmodium berghei LCCL proteins promotes complex formation and stability.

Malaria parasites express a conserved family of LCCL-lectin adhesive-like domain proteins (LAPs) that have essential functions in sporozoite transmission. In Plasmodium falciparum all six family members are expressed in gametocytes and form a multi-protein complex. Intriguingly, knockout of P. falciparum LCCL proteins adversely affects expression of other family members at protein, but not at mRNA level, a phenomenon termed co-dependent expression. Here, we investigate this in Plasmodium berghei by crossing a PbLAP1 null mutant parasite with a parasite line expressing GFP-tagged PbLAP3 that displays strong fluorescence in gametocytes. Selected and validated double mutants show normal synthesis and subcellular localization of PbLAP3::GFP. However, GFP-based fluorescence is dramatically reduced without PbLAP1 present, indicating that PbLAP1 and PbLAP3 interact. Moreover, absence of PbLAP1 markedly reduces the half-life of PbLAP3, consistent with a scenario of misfolding. These findings unveil a potential mechanism of conformational interdependence that facilitates assembly and stability of the functional LCCL protein complex.

Malaria crystalloids: specialized structures for parasite transmission?

Malaria parasites possess many unique subcellular structures and organelles that are essential for the successful completion of the complex life cycle of Plasmodium in the vertebrate host and mosquito vector. Among these are the crystalloids: transient structures whose presence is restricted to the mosquito-specific ookinete and young oocyst stages of the parasite. Nearly five decades after they were first described, the crystalloids are back in the spotlight, with recent discoveries pointing to an important role in protein trafficking and sporozoite transmission that could be exploited as new targets for control of malaria transmission.