Glycosylation in malaria parasites: what do we know?

Abstract

In malaria parasites, although posttranslational modification of proteins with N-. O-, and C-glycosidic bond-linked glycans is limited, it confines to relatively fewer proteins in which the glycans are present at significant levels and may have important functions. Furthermore, several proteins are modified with glycosylphosphatidylinositols (GPIs) which represent the predominant glycan synthesized by parasites. Modification of proteins with GPIs is obligatory for parasite survival as GPI-anchored proteins play essential roles in all lifecycle stages of the parasites, including development, egress, gametogenesis, motility, and host cell adhesion and invasion. Here, we discuss the current knowledge on the structures and potential functions of the glycan moieties of parasite proteins. The knowledge has important implications for the development of drugs and vaccines for malaria.

Malaria, malaria parasites, and protein glycosylation

Malaria, a complex disease caused by Plasmodium species of protozoan parasites [1–3], is characterized by a wide range of mild to severe systemic clinical conditions, and organ-specific fatal pathologies, including cerebral malaria, liver, lung and kidney injuries and dysfunction, and placental malaria [1–7]. Malaria is primarily prevalent in the tropical and subtropical regions of the world [6,7] and an estimated 247 million clinical malaria infections and 625,000 deaths have occurred in 2021 [8]. Five species of Plasmodia parasites can infect human; however, two species, namely, Plasmodium falciparum and P. vivax, are responsible for most of the malaria disease burden [2,9]. These parasites are widely and differentially prevalent in endemic areas. In Africa, P. falciparum is most widespread with an undefined low P. vivax prevalence, whereas in South America, Southeast Asia, Western Pacific, and the Eastern Mediterranean regions, both P. falciparum and P. vivax co-exist in different proportions [10,11]. Compared to P. vivax, P. falciparum is highly virulent and accounts for most of the malarial deaths that occur in Africa and other regions. Malaria parasites use a vertebrate and a mosquito to complete full lifecycles and have adopted complex lifecycles in both hosts to efficiently propagate [12–14].

Glycosylation of proteins, a major post-translational modification of proteins with carbohydrates, commonly called glycans, in the endoplasmic reticulum (ER) and the Golgi, to produce glycoproteins is ubiquitous and abundantly occurs in almost all eukaryotes, including animals, many microorganisms and protozoan parasites [15–18]. Glycans are linked to proteins via an N-glycosidic bond involving the amide group of asparagine side chain in the N-X-S/T consensus sequence (X can be any amino acid except proline), via an O-glycosidic bond formed with the side chain hydroxyl groups of serine and threonine, or via a C-glycosidic bond between mannose and tryptophan (W) of WXXW/C, WXXWXXC, and WXXWXXWXXC motifs (X, any amino acids) of type 1 cytokines and ThromboSpondin Repeats (TSR) domains of proteins [19]. The glycan moieties bestow a range of intrinsic properties to proteins, including folding, sorting, intracellular targeting, localization, and maintaining functional conformation, and stability [18,20,21]. They are also involved in maintaining membrane integrity and cell shape, providing aqueous environment to biochemical interactions, and protecting proteins and cells from proteolytic attack. Additionally, the glycan moieties of glycoproteins are involved in various cell biological and physiological processes, including, cell migration, cell-cell interactions and communications, cell-matrix interactions, receptor-ligand interactions, cell adhesion, and modulation of immunological properties [22].

Many proteins and glycoproteins of eukaryotes are modified with distinct type of plasma membrane glycolipids called glycosylphosphatidylinositol (GPI) anchors via amide linkage (glypiation), involving the carboxyl group of C-terminal amino acid [23,24]. GPIs are evolutionary conserved glycolipids found in almost all eukaryotes and they play diverse functional roles essential for normal physiology of organisms [23,24]. The attachment of GPIs confers proteins an ability to tether to the cell membrane and regulate cell functions. GPI-anchored proteins (GPI-APs) and glycoproteins perform diverse biological functions, including functioning as enzymes, receptors, complement regulators, co-receptors of signaling complex, and cell adhesion molecules [20,21,25].

Malaria parasites process only an ER glycosylation machinery; they lack glycan processing enzymes in the Golgi (Figure 1). In P. falciparum, the overall abundance of N-, O-, and C-linked glycans together is ~10% of the total carbohydrate synthesized and GPIs account for the remaining ~90% [26–28]. Despite the low abundance, N-, O-, and C-glycosylation seems to occur significantly in some proteins in which the glycan moieties may perform important functions. This review provides an overview of our current understanding of the nature, structures, and biological relevance of glycan modification in parasites, and in addition, it draws attention to further studies needed to gain insight into the aspects of glycosylation that remain unclear and not understood.

Figure 1. The glycosylation machinery of malaria parasites.

Drawing shows the nucleus, ER, and Golgi of parasites and is intended to represent morphologies of all lifecycle stages. In malaria parasites, the glycosylation is exclusively confined to the ER. GPI synthesis is initiated in the cytosolic side of the ER and after two initial steps, the intermediate flips over to the luminal side to complete the production of matured GPIs (see Figure 2B). GPI anchoring of proteins by the action of transamidase complex also occur in the ER lumen, and then the GPI-anchored proteins are trafficked to the plasma membrane. N-glycosylation is also initiated in the cytoplasmic side of the ER, producing Dol-P-P-GlcNAc and Dol-P-P-GlcNAc₂, which flip over to the lumen of the ER, where the glycan moieties are transferred to proteins by OST (see Table 1 and Figure 2C). Also, O-fucosylation and tryptophan C-mannosylation occur in the ER lumen (see Figure 2E and 2F). Parasites have a rudimentary Golgi apparatus and mammalian analogs of glycan processing enzymes of the Golgi are completely absent in parasites. As such, the glycan moieties of proteins that are added in the ER are not further modified in the Golgi. Malaria parasites also appear to lack fatty acid acylases and deacylases and this could be the reason for the absence GPI fatty acid remodeling in the Golgi.

P. falciparum does not synthesize detectable levels of glycolipids other than GPIs [27]. The GPIs exist as either free glycolipids (free GPIs) in membranes or as part of GPIs-APs [26–29]. Quantitatively, the free GPIs in the parasite are 4–5 times higher than that present in GPI-APs [26,29]. The GPI-APs are mainly expressed on the surfaces of parasites at all lifecycle stages, including, trophozoites, schizonts, merozoites, gametocytes, ookinetes, oocysts, and sporozoites (see Glossary). The GPI-APs play crucial roles in processes that are critical for parasite survival and propagation, including development in hosts, egress, and motility, and adhesion to and invasion of host cells [30]. While the structures of parasite GPIs are known [26–29], their functions beyond anchoring and trafficking proteins to the plasma membrane remain unknown.

Structure, biosynthesis, and potential functions of GPIs of malaria parasites

Structures of parasite GPIs

The complete structure of blood stage P. falciparum GPIs, determined by detailed biochemical analyses [26–29,31–34], is shown in Figure 2 (key figure), panel A. Overall, the structures of parasite GPIs are less complex compared to GPIs of most organisms (Box 1). The lipid compositions of free and protein-linked GPIs of P. falciparum [29] suggest that, unlike in yeast and mammalian cells [23,24], malaria parasites do not remodel the fatty acid substituents of GPIs and retain all three original fatty acids without modification (Figure 2A).

Figure 2 (key figure). The structure of the GPIs of malaria parasites, GPI biosynthesis pathway, and glycosylation of parasite proteins.

(A) The structure of GPIs of the blood stage P. falciparum as determined by biochemical analyses and mass spectrometry. GPIs anchoring proteins via an amide bond between the terminal carboxyl group of proteins and amino group of ethanolamine phosphate (EtNP) is indicated. The glycan structure, Man₄-GlcN-myo-insoitol, is conserved across various P. falciparum isolates and rodent malaria parasite species. The fatty acid substitution of P. falciparum GPIs is highly heterogeneous and is dependent on host blood fatty acid composition during infection, as parasites predominantly use fatty acids of host blood for the synthesis of various membrane lipids. The fatty acid composition of GPIs of P. falciparum grown in pooled human sera is indicated. Note: The sn-1 and sn-2 positions are invariably substituted with saturated and unsaturated fatty acids, respectively, and inositol is substituted with shorter saturated fatty acids. (B) Upper panel shows the two matured GPIs containing EtNP substitution and the GPI biosynthetic intermediates of P. falciparum that have been biochemically identified. Lower panel shows the parasite GPI biosynthetic pathway. (C-F) Glycosylation features of parasites: N-glycosylation (C); O-GlcNAcylation (D); O-fucosylation of the TSR domains (E); C-mannosylation of TSR domains (F). (E) The TSR domains are mainly modified with single residues of Fuc and in some TSR domains, Fuc is substituted with a terminal Glc (not shown). Ino, inositol; GlcNAc, N-acetylglucosamine; GlcN, glucosamine; Man, mannose; Fuc, fucose; Glc, glucose.

Box 1. Structure of GPIs of eukaryotes.

GPIs are found in almost all eukaryotes, including all animals, protozoan parasites, plants, yeast, fungi, and slime mold [24]. GPIs of most organisms, including malaria parasites, share a common glycan core structure, Manα1–2Manα1–6Manα1–4GlcN, in which glucosamine (GlcN) is linked to myo-inositol of membrane phosphatidylinositol (PI) via α1–6 glycosidic bond (see Figure 2A). Note: Man is mannose, GlcN is glucosamine, and PI is phosphatidylinositol consisting of myo-inositol linked to diacylglycerol (DAG) via a phosphate residue. The nonreducing end mannose-3 (Man-3) is substituted with ethanolamine phosphate (EtNP) at O-6 to produce a matured GPI having a conserved glycan core, EtNP-6Manα1–2Manα1–6Manα1–4GlcNα1–6PI, which has the potential to anchor proteins to the membrane through an amide bond between the amino group of EtNP and the carboxyl group of C-terminal amino acids of proteins. Some proportions of GPIs of animals, including humans, yeast, and parasites such as T. Cruzi carry a fourth mannose (Man-4) α1–2 linked to Man-3 of the core structure [24] and thus, occur as EtNP-(Manα1–2)6Manα1–2Manα1–6Manα1–4GlcN-PI. Additionally, GPIs of animals carry EtNP substitution on Man-1 and Man-2 and sugar substituents on Man-1, Man-2, and Man-3 [24]. For example, protein-linked GPIs of mammals contain N-acetylgalactosamine (GalNAc) attached to the O-4 of Man-1, and some GPIs carry glycan branches, such as galactose (Gal) or sialic acid-Gal linked to GalNAc [23,24].

Furthermore, GPIs of various eukaryotes differ substantially in the nature and compositions of fatty acids [23,24]. While some GPIs contain 1,2-diacylglycerol moiety having variable fatty acid chain lengths or different proportions of saturated and unsaturated fatty acids, certain other GPIs contain different lipid composition on glycerol; for example, 1-lyso-2-acyl-gycerol, 1-alkyl-2-acyl-glycerol, and 1-alkenyl-2-acyl-glycerol. Furthermore, some proportions of GPIs of lower eukaryotes, such as yeast and fungi, contain ceramide moiety instead of 1,2-diarylglycerol or 1-alkyl-2-arylglycerol. Additionally, most protein-linked GPIs of mammals and yeast do not contain acyl substituent on inositol. However, in animal erythroblasts, the GPI moieties of GPI-APs contain acylated inositol [24].

P. falciparum synthesizes two matured GPIs: Ethanolamine phosphate (EtNP)-(Manα1–2)6Manα1–2Manα1–6Manα1–4GlcN-PI (major product) and EtNP-6Manα1–2Manα1–6Manα1–4GlcN-PI (minor product) [26–29,31–34] (Figure 2B). Although both GPIs can anchor proteins, structural analysis of the GPIs moieties of several GPI-APs indicates that only the former GPI is used [26] (Figure 2A). About 30 proteins of malaria parasites having size ranging from ~14 kDa-260 kDa are modified with the GPIs [26,30]. All proteins, except the ~260-kDa protein are expressed at the mid to late trophozoite of the blood stages. The ~260-kDa protein is expressed at the late ring to early trophozoite stages [26]; its amino acid sequence and function are unknown.

The glycan structures of P. falciparum GPIs are highly conserved among various parasite isolates from diverse geographical locations [32] and are identical to the corresponding GPIs of the FCR-3 and FCBR strains [26,27,35]. However, the GPIs are highly heterogenous with respect to the fatty acid substituents of both inositol and diacylglycerol (DAG) [29] (Figure 2A): This is because parasites use fatty acids of the host blood for the synthesis of membrane lipids, including GPIs [36].

The glycan moiety of GPIs is also highly conserved across various Plasmodium species. Like P. falciparum [26,27,35], the rodent malaria parasites, P. chabaudi chabaudi AS and P. yoelii, synthesize two matured GPI glycolipids [33,34], and use mainly EtN-Man₄-GlcN-PI for anchoring proteins [34]. However, the fatty acid compositions of rodent parasite GPIs are unknown.

Biosynthesis of GPIs

The available structural information on the matured free GPIs and the biosynthetic intermediates of P. falciparum and the rodent parasites, P. yoelii and P. chabaudi chabaudi AS [27,33,34], indicates that the GPI biosynthetic pathway of malaria parasites closely resemble that of mammalian cells (Box 2). P. falciparum contains mammalian homologues of phosphatidylinositol glycan (PIG) genes that are required for its GPI biosynthesis [37–40] (Figure 2B and Table 1). The PIG gene syntenic orthologs of P. falciparum are present in the genome of P. vivax, P. berghei, P. yoelii, P. knowlesii, P. cynomolgy, P. reichenowi, and P. chabaudi [40]. The structures and properties of the enzymes and their accessary proteins of the parasite PIG genes have not been investigated. However, P. falciparum PIG-B has been found to be ManT-III as a PIG-B siRNA inhibited the formation of Man₃-GlcN-PI intermediate, presumably via oligonucleotide antisense activity [41]. Further, although the mammalian homolog of PIG-Z (Man-IV) is absent in parasites [37–39], the P. falciparum PIG-B can restore the genetic defect of the yeast mutant, which lacks activity for the addition of a mannose to Man₃-GlcN-PI to generate Man₄-GlcN-PI [42], suggesting that the parasite PIG-B possesses both ManT-III and ManT-IV activities.

Box 2. Biosynthesis of GPIs in eukaryotes.

The biosynthesis of GPIs has been extensively studied in mammalian cells, yeast, and trypanosomes [23,24]. GPIs are synthesized in the ER as glycolipids by stepwise addition of sugars to the membrane PI by the action of various enzymes, some of which exist as multiprotein complex with accessary proteins. The genes of enzymes and associated accessary proteins of the GPI biosynthesis are designated as phosphatidylinositol glycan (PIG) genes. The first two reactions of GPI biosynthesis occur in the cytoplasmic side of the ER. These are: (i) the transfer of GlcNAc to the O-6 of myo-inositol of PI to yield GlcNAc-PI, catalyzed by PIG-A multiprotein complex and (ii) de-N-acetylation of GlcNAc by PIG-L to produce GlcN-PI, which flips over to the luminal side, likely by the action of a flippase. In the luminal side of ER, the myo-inositol of PI is acylated at the O-2 by PIG-W to produce GlcN-(inositolacyl)PI. Then the original fatty acids of 1,2-diacylglycerol are replaced to produce a mixture of 1-alkyl-2-acylglycerol (major) and 1,2-diacylglycerol (minor) by fatty acid remodeling [23,24]. Two mannoses are then consecutively transferred from dolichol phosphate mannose (Dol-P-Man) to GlcN-(inositolacylated)PI by two mannosyltransferases (ManTs), PIG-M (ManT-I) and PIG-V (ManT-II), respectively. Next, an EtNP is added to the O-2 position of Man-1 by PIG-N followed by the addition of a Man by PIG-B (ManT-III), and two EtNP, one to the O-6 of Man-3 and the other to O-6 of Man 2 by PIG-F to produce EtNP-6Manα1–2(EtNP-6)Manα1–6(EtNP-2)Manα-4GlcNα1-(inositolacylated)PI. This matured GPI is competent for anchoring proteins. Further, a portion of the GPI is modified by the addition of a Man at the O-2 of Man-3 by PIG-Z (ManT-IV) to generate EtNP-6(Manα1–2)Manα1–2(EtNP-6)Manα1–6(EtNP-2)Manα-4GlcNα1-(inositolacyl)PI, which is also competent for anchoring proteins [23,24].

The mature GPIs are transferred to proteins by a multicomponent transamidase (see Table 1), which cleaves the C-terminal GPI-signal sequence and attach the GPI moiety, producing GPI-APs tethered to the ER membrane [23,24]. Subsequently, in most mammalian cells, except in erythroblasts, acyl residue of inositol and EtNP substituent of Man-2 are removed by PGAP1 (a deacetylase) and PGAP5 (a phosphatase), respectively, and the GPI-APs are transported to the Golgi. In the Golgi, protein-linked GPI moiety undergoes another round of fatty acid remodeling, whereby the unsaturated 2-acyl chains of DAG are replaced by saturated 2-acyl chains. Additionally, a GalNAc is attached to Man-1 at O-3, and GalNAc may be further substituted with a Gal or sialic acid-Gal.

Table 1.

Enzymes of the glycosylation pathways of malaria parasites

Enzyme activity	Enzyme/accessory subunit name	Enzyme number	P. falciparum gene PlasmoDB #**
Enzymes of GPI biosynthesis
Phosphatidylinositol N-acetylglucosaminyl- transferase	PIGA complex	EC 2.4.1.198	PF3D7_0618900
• Subunits of PIG A complex	PIGA/GPI3*	-	PF3D7_1032400
	PIGQ/GPI1	-	PF3D7_0618900.1
	PIGC/GPI2	-	PF3D7_0911000
	PIGP/GPI19	-	PF3D7_0935300
	PIGH/GPI15	-	PF3D7_1141400
	PIGY/Eri1	-	Not identified
Dolichol-phosphate mannosyltransferase 1	DPM1	EC 2.4.1.83	PF3D7_1141600
Inositol acyltransferase	PIGW	EC 2.3.-.-	PF3D7_0615300
N-Acetylglucosaminylphosphatidylinositol de-N-acetylase	PIGL/GPI12	EC 3.5.1.89	PF3D7_0624700
GPI mannosyltransferase I	PIGM/GPI14	EC 2.4.1.-	PF3D7_1210900
GPI mannosyltransferase II	PIGV/GPI18	EC 2.4.1.-	PF3D7_1247300
GPI mannosyltransferase III	PIGB/GPI10	EC 2.4.1.-	PF3D7_1341600
GPI mannosyltransferase IV	SMP3	EC 2.4.1.-	Not identified
GPI ethanolamine phosphate transferase	PIGO/GPI13	EC 2.7.-.-	PF3D7_1214100
GPI-anchor transamidase	PIGK complex	EC 3.-.-.-
• Subunits of PIGK complex	PIGK/GPI8*		PF3D7_1128700
	PIGT/GPI16		PF3D7_1122100
	GPAA1/GAA1		PF3D7_1369000
	PIGU/Gab1		PF3D7_1330700
	PIGS/GPI17		Not identified
Enzymes of N-glycosylation
UDP-GlcNAc Dol-P N-acetylglucosamine phosphotransferase	ALG7	EC 2.7.8.15	PF3D7_0321200
Dol-P-P-GlcNAc N-acetylglucosaminyl-transferase complex	ALG13* ALG14	EC 2.4.1.141	PF3D7_0806400 PF3D7_0211600
Oligosaccharyltransferase	OST	EC 2.4.1.119
• Subunits of OST complex	STT3*		PF3D7_1116600
	WBP1		PF3D7_0919600
	OST1		PF3D7_0311600
	OST2		PF3D7_0726800
	OST3/OST6		PF3D7_0107700
	OST4		PF3D7_1233050
	OST5		PF3D7_1243200
	Swp1		Not identified
O-Fucosylation enzyme
GDP-fucose protein O-fucosyltransferase 2	POFUT2	EC 2.4.1.221	PF3D7_0909200
Tryptophan C-mannosylation enzyme
GDP-mannose protein C-mannosyltransferase	DPY19	EC 2.4.1.-	PF3D7_0806200
Cytoplasmic and nuclear O-GlcNAcylation
O-Linked N-acetylglucosamine transferase	OGT	EC 2.4.1.255	Not identified
Protein β-N-acetylhexosaminidase	OGA	EC 3.2.1.169	Not identified

^*Catalytic subunit of enzyme complex.

^**Syntenic orthologs of P. falciparum genes have been identified in P. vivax, P. knowlesii, P. cynomolgy, P. reichenowi, P. berghei, P. yoelii, and P. chabaudi [40]. Some amount of information complied in this table is taken from [39–40].

Although the biosynthesis of GPIs in malaria parasites occurs similarly to that in mammals and yeast, there are some differences. As noted above, unlike in mammals and yeast [23,24], malaria parasites neither add EtNP to Man-1 and Man-2 nor remove acyl residue from inositol, and do not remodel fatty acids. This is because malaria parasites have only remnants of the Golgi [43], and lack many processing enzymes in the Golgi (see Figure 1), including glycosyltransferases and glycosidases [40].

GPI-APs of P. falciparum

Biochemical studies and bioinformatic analysis have identified ~30 GPI-APs in P. falciparum [30,44,45]. Mass spectrometry and Western blotting analysis showed that merozoite surface protein 1 (MSP-1), MSP-2, MSP-4, MSP-5, MSP-8, MSP-10, rhoptry-associated membrane antigen (RAMA), apical sushi protein (ASP), Pf92, Pf38, Pf12, and Pf34 are GPI-linked [30]. These 11 proteins account for >90% of all GPI-APs of the blood stage parasite [30]. The apical membrane protein Pf34 is also GPI-anchored [46]. Analysis of parasite proteome using software programs that predict peptide signal sequences for GPI-anchoring, identified ~30 P. falciparum GPI-APs expressed at different developmental stages [30]. Bioinformatic analysis have predicted the presence of ~30 GPI-APs in P. vivax [44]. A recently developed software program reports that P. falciparum and P. vivax proteome contain, respectively, ~27 and ~28 GPI-APs [45].

Potential functions of malaria parasite GPIs

GPI biosynthesis is essential for the development of parasites at all lifecycle stages. As such, treatment of P. falciparum cultures with inhibitory small molecules or sugar inhibitors of GPI biosynthesis arrested the growth at the asexual trophozoite stage [35,39,47]. Disruption of P. falciparum phosphomannomutase that converts mannose 6-phosphate into mannose 1-phosphate required for the production UDP-Man prevents merozoite egress and erythrocyte invasion [48]. [Note: UDP-Man is required for synthesis of dolichol phosphate mannose (Dol-P-Man), the mannose donor for GPI biosynthesis]. Moreover, transposon-based genome-wide saturation mutagenesis of Plasmodium falciparum indicated that several genes of the GPI biosynthesis including, PIGA subunits (PF3D7_103400, PF3D7_061900.1 and PF3D7_091000), PIGL (PF3D7_0624700), PIGW (PF3D7_0615300), PIGM (PF3D7_1210900), PIGV (PF3D7_1247300), and PIGK catalytic subunit (PF3D7_1330700) are essential for the optimal growth of asexual blood-stage parasite (see Table S8 in [38]). The essentialness of GPIs for parasite growth and survival is because many proteins, including several MSPs, RAMA, ASP, circumsporozoite protein (CSP), gamete surface proteins Pfs48/45, ookinete surface proteins Pf25/28, that play critical in parasite development in different lifecycle stages or host cell invasion are GPI-APs [30,45]. Deficiency in GPI anchoring abrogates the functionally important proteins targeting to their cellular locations and their functions. For example, GPI-anchoring of CSP is essential for oocyst development, sporozoite formation and budding, and traversing endothelial barrier and hepatocyte invasion [49]. GPI anchoring of the micronemal antigen (GAMA) is crucial for ookinete invasion of mosquito midgut, sporozoite egress from oocysts, and sporozoite motility [50]. Deficiency in GPI anchoring and thus surface expression of MSP-1 show merozoite egress defect [51]. The GPI-anchored Pfs48/45 of male and female P. falciparum gametes are important for zygote development and malaria transmission, and studies in P. berghei show that Pfs48/45 play crucial role in the fertility of male gametes [52]. P. falciparum Pf25/f28 play important roles in ookinete and oocyst development and ookinetes traversing the mosquito midgut epithelium [53].

Despite these essential roles, whether and how the GPI moieties of proteins contribute to processes, other than protein trafficking, critical for the parasite biology is unknown. In animals, the GPIs play important functions, including cell signaling that are essential for their normal physiology [23–25,54–60] (Box 3). Similarly, parasite GPIs are likely to play critical roles in signaling, membrane endocytosis, and regulation of cellular processes.

Box 3. Functions of GPIs.

In most organisms, GPIs perform diverse functions, including intracellular sorting and trafficking of proteins, targeting proteins to the plasma membranes and apical membranes in polarized cells, and GPI-APs play roles in nutrient uptake, endocytosis, and signaling [23–25]. A common feature of GPI-APs is their association with detergent resistant, laterally organized cholesterol-sphingolipid/glycospingolipid enriched membrane microdomains called lipid rafts. Lipid rafts are dynamic and function as cell signaling platforms, in which non-receptor Src family of protein tyrosine kinases, such as Src, Lyn, Fyn, Yes and Lck, and heterotrimeric G protein α (Gα) subunits, are assembled in the inner leaflet of lipid bilayer in response to interaction of GPI-AP receptors on the membrane outer leaflet with ligands [54–57]. Signal generated outside by receptor-ligand interactions is transduced into cells. The interactions of GPI-APs with their ligands lead to clustering of GPI-APs and activation of inhibitory Gα2 (Gαi2) and Src family of protein tyrosine kinases in the inner leaflet. This results in the activation of PLC, which generates IP3 second messenger from PIP2, leading to IP₃-Ca²⁺ signaling. Examples of GPI-AP receptors inducing cell signaling by interacting with their cognate ligands include complement regulation by complement decay accelerating factor (DAF, C55) which binds C3bBb and C4bC2a, complement regulatory protein CD59 that binds C8 and C9, CD14 that binds LPS and present it to MD2-TLR4 complex for signaling, and CD90 (Thy-1) that binds FasL.

Studies using an anti-CD59 antibody crosslinking model have dissected important molecular events in GPI-linked CD59-induced cell signaling [58,59]. Upon anti-CD59 antibody binding, several molecules of GPI-CD59 are clustered in lipid rafts, resulting in transient recruitment of Gαi2 and Lyn to the CD59 clusters through lipid-lipid and protein-protein interactions. Gαi2 activates Lyn leading to CD59 clusters binding cytoskeletal F-actin and immobilization of CD59 cluster temporarily arresting lateral diffusion. At the same time, PLC is transiently recruited from the cytoplasm to the CD59 clusters, generating a pulse of IP₃-Ca²⁺ signal [60]. The total IP₃-Ca²⁺ signal produced in the cell is the sum of the short-lived IP₃-Ca²⁺ pulses, each produced by the transient recruitment of PLC to the immobilized CD59 cluster. It has been suggested that one or more transmembrane proteins are recruited to the stabilized lipid rafts, which then recruit acylated signaling molecules to the inner leaflet, leading to the formation of signaling assembly through protein-protein and/or protein-lipid interactions. Thus, the signal produced in the outer leaflet is transmitted outside-in [60].

Cellular activation and induction of PI-PLC-mediated inositol 1,4,5-trisphosphate(IP3)-Ca²⁺ signaling plays a central role in all key processes of malaria parasite life cycle stages, including egress of merozoites, invasion of erythrocytes, gametogenesis, sporogenesis, and sporozoite gliding and motility [61]. In all these processes, calcium-dependent protein kinase 1 (PfCDPK1) and several kinases downstream of PI-PLC-mediated signaling play crucial roles. PfCDPK1 phosphorylates merozoite glidosome motor-associated proteins, GAP40, GAP45 and MyoA [62] and thus, PfCDPK1 contributes to merozoite invasion. In eukaryotes, activated PI-PLC hydrolyses membrane phosphatidylinositol 4,5-bisphosphate (PIP2) to release second messengers, DAG and IP3; DAG activates protein kinase C (PKC), whereas IP3 induces the calcium release from the ER and other intracellular compartments [63]. Similarly, the PI-PLC-mediated signaling is likely to be at the top of hierarchical signaling pathways crucial for the progression of various lifecycle stages of malaria parasites. P. falciparum cyclic GMP-dependent protein kinase A (PfPKG) and cyclic AMP-dependent kinase A (PfPKA) also play important roles in parasite lifecycles, including merozoite invasion of red blood cells [62,64]. PfKPG activates PfCDPK, and PfPKA activates P. falciparum apical membrane antigen 1 (PfAMA1), a cAMP-dependent event crucial for successful merozoite invasion [65].

Although a significant amount of information is available on the PLC-mediated IP3-Ca²⁺ signaling and downstream protein kinases in parasite life cycle processes [62,64,65], studies are lacking to explain how the signaling generated upon GPI-APs such as MSP1 complex interacting with erythrocytes is transduced outside-in. Based on the known knowledge of signaling in animal cells, one could envisage the involvement of homologs of G-protein coupled receptors and homologs of Src family of kinases assembled in the inner membranes of lipid rafts containing GPI-AP receptor clusters in activating PI-PLC-mediated IP3-Ca²⁺ signaling and cGMP- and cAMP-dependent protein kinases. Consistent with this notion, it has been reported that a small Ras-like G-protein and a Gα subunit of heterotrimeric GTP-binding protein are present in P. falciparum [66]. The proteins are present in matured schizonts/merozoites and are suggested to be involved in signaling events in merozoites [66]. P. falciparum expresses a non-canonical Ras-like G protein with GTP binding capacity and GTPase activity at the schizont stage [67]. Another study has shown the presence of two or more heteromeric Gα-proteins belonging to both stimulatory (Ga_s) and inhibitory (Ga_i) classes and their involvement in gametogenesis and merozoite invasion [68]. Thus, parasite GPI-AP receptors, upon interacting with host cells, may activate G protein-coupled proteins and/or non-receptor kinases, and play important roles in parasite life cycle processes such as merozoite invasion, gametogenesis, and sporozoite gliding, motility, and invasion.

Functionally important proteins including several GPI-anchored and nonGPI-anchored MSPs, rhoptry-associated proteins such as RAP1/2, RAP2/3, Rhop1, Rhop2 and Rhop3, cytoskeletal proteins such as GAP45, GAP50 and myosin-A, and PfCDPK1 are present in detergent-resistant lipid raft membranes [46,69,70]. The association of these proteins in lipid rafts suggest that, like in other eukaryotes, GPIs of GPI-APs play important roles in signaling, membrane endocytosis, and regulation of various cellular processes. For example, it is known that several secreted MSPs, including MSP3, MSP6, MSP7, MSPDBL1 and MSPDBL7, bind MSP1 to form multiprotein complexes [71]. During merozoite invasion of erythrocytes, the interaction of MSP1 complex with erythrocytes may generate a signal, which is transmitted to parasites through G-protein coupled receptors and/or nonreceptor kinases, leading to the activation of downstream kinases to facilitate invasion. Another known function of eukaryote GPIs is involvement in endocytic processes [25]. As noted above P. falciparum membranes contain 4–5 times more of free GPIs compared to protein-linked GPIs [26,29]. Free GPIs interact with erythrocyte moesin [72], an erythrocyte cytoskeletol protein. It is possible that interaction GPIs with moesin facilitates the formation of parasitoporus vacuolar membrane (PVM) by regulating membrane-cortex interactions and signaling [73].

N-glycosylation

To date, the structures-functions of N-glycans of malaria parasite proteins remain unclear. Two early studies have reported that N-linked glycans of P. falciparum comprised single residues of GlcNAc, GlcNaAc₂ disaccharide, and Man_1–3GlcNAc_2, i.e., 1 to 3 Man linked to GlcNAc₂ [26,74]. However, a subsequent study has reported that P. falciparum proteins contain exclusively single residues of GlcNAc and GlcNAc₂ [75]. The genomes of P. falciparum and P. vivax contain ALG7 and ALG13/ALG14, which are involved, respectively, in the synthesis of GlcNAc-P-P-Dol and GlcNAc₂-P-P-Dol, the glycan donors for N-glycosylation of parasite proteins (Figure 2C) [75]. Proteomic and transcriptomic data in PlasmoDB also indicate the presence of ALG7, ALG13/ALG14, and oligosaccharyltransferase (OST) genes in P. falciparum (Table 1); no other ALG genes are evident in the parasite [39]. Syntenic orthologs of P. falciparum ALG7, ALG13/14, and OST are in P. vivax, P. knowlesii, P. cynomolgy, P. reichenowi, P. berghei, P. yoelii, and P. chabaudi [40]. Seven subunits of OST, including the catalytic STT3 subunit of P. falciparum have been identified (Table 1) [76]. Collectively, these results indicate that N-glycans of parasite proteins consist of mainly single residues of GlcNAc and GlcNAc₂ disaccharide. However, the reason for previously observed short chain 1–3 mannoses containing N-glycans remain unclear [26,74].

In P. falciparum, although overall abundance N-glycans is low [26], they appear to be present at significant levels in a limited number of proteins. Based on mass spectrometry of proteins bound to lectins having specificity to terminal GlcNAc, it has been suggested that P. falciparum, proteins such as MSP-1, RAP1, RAP2 and RAP3 and glyceraldehyde 3-phosphate dehydrogenase, endoplasmin, HSP70, EF1-α, enolase, fructose-bisphosphate aldolase and ornithine aminotransferase are N-glycosylated [75]. However, as described below, a subsequent proteomic study found that several of these proteins are actually O-glycosylated with single residues of GlcNAc [77]. Since lectins used in the earlier study [75] can bind proteins containing terminal GlcNAc, irrespective of whether O- or N-linked, the identity of the proteins that were suggested to be N-glycosylated remains questionable.

If proteins such as MSP-1, RAP1, and RAP2 are indeed N-glycosylated as reported [75], considering that these proteins are located in the plasma membrane or in rhoptries, it is likely that N-glycans have important functions in the parasite biology. Evidence that suggest important biological functions of N-glycans in parasite biology includes: (i) N-glycosylation-specific, ALG7, ALG13/ALG14, and OST are conserved across various Plasmodium species [40], despite lacking N-glycan-specific mannosyltransferases; (ii) the transposon-based saturation mutagenesis study by Zhang et al. showed that several genes of N-glycosylation pathway, namely ALG7 (PF3D7_0321200), ALG14 (PF3D7_0211600), and OST subunits STT3 (PF3D7–0311600) and OST2/DAD1 (PF3D7_0726800), are essential for the optimal growth of the blood-stage P. falciparum (Table 1, and also see Table S8 in [38]); (iii) tunicamycin, an inhibitor of N-glycosylation, impairs P. falciparum trophozoite differentiation into schizonts [39,74].

O-Glycosylation

Two types of protein O-glycosylation are known in P. falciparum: (i) single residues of GlcNAc β-O-linked to Ser/Thr, and (ii) either single residues of α-Fuc or Glcβ1–3Fuc disaccharide α-O-linked to Ser/Thr.

Glycosylation with O-linked GlcNAc

Several blood stage P. falciparum proteins, including MSP-1 and MSP-2 that displayed on the parasite surface as GPI-anchored proteins, have been reported to be glycosylated with single residues of GlcNAc O-linked to Ser/Thr [78–81]. A glycosyltransferase that can transfer O-GlcNAc to Ser/Thr residues of an acceptor peptide substrate is present in P. falciparum [82]. These results indicate that certain parasite proteins are glycosylated with GlcNAc β-O-linked to Ser/Thr. However, whether the glycosyltransferase that adds β-O-GlcAc to MSP1, MSP2, and other cell surface-displayed proteins is the same as the cytoplasmic and nuclear O-N-acetylglucosaminyltransferase (OGT) discussed below or a different enzyme remains unknown. As cell surface proteins are trafficked from the ER by vesicle encapsulation, the N-acetylglucosaminyltransferase that adds β-O-GlcAc to membrane proteins predicts to be ER-localized. However, alternatively, as merozoites are exposed to the host red blood cell cytosol before their egress, it is possible that β-O-GlcAcylation of parasite’s cell surface proteins is by host OGT in the cytosol of red blood cells [83].

Recently, mass spectrometry of the N-glycanase-treated parasite proteins enriched by two alternative approaches - lectin-affinity binding or avidin-affinity binding after labeling of GlcNAc by the enzymatic transfer of N-azidogalactose followed by biotin tagging with click chemistry - has identified 13 proteins having terminal O-GlcNAc [77]. These are: actin-1, actin-2, fructose-bisphosphate aldolase, chaperonin CPN60, HSP70, EF-1α, glucose regulated protein, hexokinase, ornithine aminotransferase, phosphoglycerate kinase, myosin-A, casein kinase 1, and α-tubulin. The identified O-GlcNAcylated proteins closely resemble similar classes of proteins in other eukaryotes [84] (Box 4): transcription factors (EF-1α), energy metabolism/glycolysis (hexokinase, fructose-bisphosphate, phosphoglycerate kinase, ornithine aminotransferase, and aldolase), stress response (chaperonin CPN60 and HSP70–2), chaperone and stress sensors in the ER (78-kDa glucose-regulated protein), cytoskeletal motor assembly (myosin A, actin-1, actin-2, and α-tubulin), and signaling (casein kinase 1).

Box 4. O-GlcNAcylation in eukaryotes.

In most eukaryotes, O-GlcNAcylation is found in a wide range of proteins belonging to diverse functional classes, including nuclear, mitochondrial, cytoskeletal, and cytoplasmic compartments [84]. In many proteins, O-GlcNAc is dynamically added to the Ser/Thr by the O-GlcNAc transferase (OGT) and is reversibly removed by the hydrolyzing enzyme O-GlcNAcase (OGA) (See Figure 2D). In some proteins, O-GlcNAcylation competes with Ser/Thr phosphorylation, leading to O-GlcNAcylation and phosphorylation rapidly cycling by enzyme complexes comprising OGT and a phosphatase or OGA and protein kinases [84]. O-GlcNAcylation is particularly abundant in the nucleus and the cytoplasm, regulating various cellular processes, including transcription, translation, nutrient sensing, signal transduction, stress responses, and energy metabolism [84–87]. Many proteins of the mitochondria are dynamically O-GlcNAcylated. Cellular stress signaling results in a rapid O-GlcNAcylation of many proteins.

Rapid O-GlcNAcylation also occurs in response to cellular injury, heat shock, and exposure to ethanol, UV, hypoxia, and reductive, oxidative and osmotic stress. Additionally, O-GlcNAc modulates protein localizations, stability, and interactions in response to environmental, nutritional, and developmental cues. O-GlcNAcylation is also found in cytoskeletal regulatory proteins involved in the regulation of actin and tubulin assemblies; α-tubulin is known to be dynamically modified with O-GlcNAc.

In most eukaryotes, many cytoplasmic and nuclear proteins, including transcription factors, RNA polymerase II, proteins of energy metabolism pathway, and signaling, stress response and cytoskeletal proteins are modified with single residues of O-β-linked GlcNAc (O-GlcNAcylation) [84–87] (Box 4). O-GlcNAcylation is a dynamic process wherein GlcNAc is added to the hydroxyl group of Ser/Thr by the O-N-acetylglucosaminyltransferase (OGT) and the added GlcNAc moiety is reversibly removed from proteins by the hydrolyzing enzyme O-GlcNAcase (OGA) (Figure 2D). Previously Perez-Cervera et al. have reported modification of several proteins of Taxoplasma gondii and P. falciparum with O-GlcNAc [88]. However, the expression of OGT and its enzyme activity were observed in T. gondii but not in P. falciparum. Detailed bioinformatic analyses revealed the presence of a putative OGT (TGGT1_112580) in T. gondii, but not in P. falciparum [88]. Incidentally, the identified OGT of T. gondii gene was subsequently found to be an O-fucosyltansferase having high homology to mammalian OGT [89]. In two separate studies, bioinformatic analysis showed the presence of mammalian homologs of neither OGT nor OGA [77,88]. Thus, it remains unknown whether the previously identified O-GlcNAc transferase of P. falciparum is the actual OGT having highly divergent gene sequence [82]. As O-GlcNAcylation may perform important functions in parasites, identification of O-GlcAcylation sites in proteins and site-specific gene mutational studies are needed to establish the functional relevance.

O-Fucosylation

Several TSR domain-containing proteins of malaria parasites, including CSP, thrombospondin-related anonymous protein (TRAP), and TRAP-related anonymous protein (CTRP) expressed during various lifecycle stages, including asexual, sexual, ookinete, and sporozoite stages, are functionally important for parasite egress, gliding motility, and invasion of hepatocytes [90–93]. The conserved CX_2–3S/TCX₂G motifs of TSR domains of significant portions of P. falciparum CSP and TRAP are modified mainly with O-linked fucose and to some extent with glucosylfucose disaccharide (Figure 2E) [90,94,95]. The TSR domains of CSP and TRAP of P. vivax and P. yoelii are also modified with single residues of O-linked fucose [94,95]. A homolog of mammalian protein O-fucosyltransferase 2 (POFUT2; PF3D7_0909200), an ER localized enzyme, is present in Plasmodium parasites [40], and the parasite-infected erythrocyte surface protein (PIESP1) has been suggested as the glucosyltransferase involved in the formation of glucosylfucose [96]. Both POFUT2 and PIESP1 proteins are expressed in the salivary gland P. falciparum sporozoites [97]. While O-fucosylation of TSR domains is not required for the propagation of the blood stage P. falciparum and development of gametocytes [91,92], it is essential for ookinetes infecting mosquito midgut, oocyst development, and sporozoite gliding motility, traversal and infection of cultured hepatocytes and infecting liver cells in humanized mice, and the production of exoerythrocytic merozoites [91]. In the absence of O-fucosylation, P. falciparum sporozoites were abnormal, and had reduced infectivity and reactivity with anti-TRAP antibody. The essentiality of O-fucosylation for the parasite lifecycle stages in mosquitos was found to be due to its requirement for proper folding and trafficking of sporozoite TRAPs [91]. However, another study has found that O-fucosylation is not necessary for the P. falciparum oocyst maturation and sporozoites colonizing salivary glands; whether O-fucosylation is required for gliding motility and infectivity was not investigated (89). Curiously, in a P. berghei mouse infection model, O-fucosylation was not required for sporogenesis and liver infection [92], but it is possible that rodent malaria sporozoites either lack O-fucosylation or have unique O-fucosylation pattern as compared to P. falciparum. Thus, independent studies are needed to determine whether rodent parasite sporozoites are O-fucosylated and whether O-fucosylation is required for the TSR domain stability.

C-Mannosylation

In addition to the O-fucosylation, the TSR domains of P. falciparum TRAP, and P. yoelii TRAP and CSP, are C-mannosylated at the second tryptophan of the conserved WXXWXXC motifs (Figure 2F) [95]. The TSR domains of merozoite TRAP-like protein (MTRAP) and the secreted protein with altered thrombospondin repeat domain (SPATR) are also tryptophan C-mannosylated [93]. Consistent with these findings, a mammalian homolog of ER-resident mannosyltransferase, termed DPY19 (PF3D7_0806200), possessing tryptophan C-mannosylation activity that can C-mannosylate TSR domains has been identified in P. falciparum [98]. While tryptophan C-mannosylation of TSR domains is not necessary for the propagation or gametocyte development of the blood stage P. falciparum and P. berghei [93,99,100], it is essential for the stability and maintenance of the functional structures of TSR domains and for the normal egress of gametocytes, ookinete gliding, and ookinete infection of the mosquito midgut [93,100]. Thus, tryptophan C-mannosylation plays a critical role in malarial parasite transmission from the mosquito.

α-Galactosylation

Current information indicates that α-Gal is absent in the glycoproteins and glycolipids of the blood stage P. falciparum [101], and the parasite lacks α1–3 galactosyltransferase (α1–3GalT) [101]. Nevertheless, based on lectin and anti-α-Gal antibody binding analyses, it has been reported that P. falciparum sporozoite proteins contain Galα1–3Galβ1–4GlcNAc- epitope [102]. However, the reported results show the presence of low levels of α-Gal in the salivary gland proteins of uninfected mosquitoes that correspond to the electrophoretic mobility of high levels of α-Gal-containing proteins in the salivary glands of infected mosquitos [102]. Thus, it is unclear whether the reported α-Gal-containing proteins of infected salivary glands are from sporozoites or of salivary gland itself. Hence, the results do not unequivocally support the conclusion that Galα1–3Galβ1–4GlcNAc- epitope is present on sporozoite proteins. Furthermore, although mice deficient in α1–3GalT that express anti-α-Gal antibodies and wildtype mice immunized with α-Gal-conjugated bovine serum albumin were reported to be protected from malaria infection [102], there was no direct evidence that show that anti-α-Gal antibodies protect humans from malaria. Given the potential importance of the suggested α-Gal-based vaccine for malaria [102], glycan and proteomic analyses using purified sporozoites are needed to determine whether α-Gal is present in malarial sporozoite proteins, regardless of whether parasites endogenously α-Galactosylate proteins or α-Gal is acquired from mosquito glycoproteins.. Additionally, although P. falciparum synthesizes a significant pool of UDP-Gal [103], its relevance remains unknown.

Concluding remarks

During the past decades, a significant knowledge has been gained on the nature and structures of glycan moieties of malaria parasite proteins. However, very little is known about the functions of glycans in parasites. Although GPI synthesis and GPI anchoring of proteins are essential for the normal development of parasites at various lifecycle stages, whether GPI perform functions other than trafficking GPI-APs to the cell surface is unknown (see Outstanding questions). Like in other eukaryotes, GPIs may play critical roles by facilitating GPI-APs clustering in plasma membrane lipid rafts, efficient interactions with ligands, and outside-in signal transmission during processes, such as merozoite egress, fertilization of gametes, ookinete and oocyst development, sporogenesis, and sporozoite invasion. Additionally, GPIs by interacting with moesin, may facilitate the formation of PVM by an endocytic-like processes during invasion by regulating membrane-cortex interactions and signaling. The identification of proteins bearing N-glycans and N-glycosylation sites would help understanding their functions. The occurrence of O-GlcNAcylation in functionally important classes of proteins suggest that O-GlcNAc glycan modification may have important roles in modulating protein structure and/or function such as dynamically competing with phosphorylation sites for signaling. Also, no information is available on glycosylation status, other than GPI modification, in gametes, ookinetes, and oocysts (see Outstanding questions). Furthermore, although α-Gal-based vaccine has been suggested for treating malaria, whether α-Gal modification is present in sporozoite proteins remains unclear. If indeed α-Gal is present in sporozoite proteins, which proteins contain α-Gal and whether sporozoites add α-Gal to proteins by using de novo synthesized UDP-Gal or α-Gal is acquired from the mosquito host needs investigation. The importance of suggested α-Gal-based vaccine development requires that some other group repeat the work. As GPIs, N-glycosylation, and tryptophan C-mannosylation are essential for parasite development in several life cycle stages, the enzymes of glycan biosynthesis, including phosphomannomutase, can be important targets for malarial drug development. Recent advances in mass spectrometry, CRISPR/Cas9 techniques, and other approaches should help determining the glycosylation sites, and glycan structures and functions (see Outstanding questions), providing comprehensive knowledge on parasite glycobiology, revealing targets for malaria therapy.

Outstanding questions.

• Do GPIs of malaria parasites have roles in outside-in transmission of signals generated upon GPI-AP receptors interacting with their ligands during lifecycle processes, such as egress, invasion, gamete fusion, sporogenesis, and gliding motility?

• Does N-glycosylation occur in specific classes of parasite proteins? Are there specific proteins for which N-glycosylation is functionally important? Is site-specific N-glycosylation important for parasite development?

• Are the TSR domains of sporozoites of rodent malaria parasites O-fucosylated as in the case of P. falciparum sporozoites and if so, is the O-fucosylation pattern in the TSR domain of P. berghei sporozoites similar or different compared to P. falciparum sporozoites?

• Is O-GlcNAcylation of parasite proteins a dynamic process in parasites that competes with phosphorylation sites? Are there cytoplasmic- and nuclear-resident N-acetylglucosaminyltransferase (O-GlcNAc transferase, OGT) that transfers single residues of β-O-linked GlcNAc to proteins, and O-linked N-acetylglucosaminidase (OGA) that removes single residues of β-O-linked GlcNAc from proteins present in parasites?

• Considering that functionally important classes of proteins are O-GlcNAcylated what functional roles O-GlcNAcylation of proteins play in parasite biology?

• What is the glycosylation status and its functional relevance in lifecycle stages, such as gamete, ookinete, and oocyst?

• Do malaria sporozoites contain terminal α-Gal-containing glycan epitopes? If α-Gal are found to be present, which individual sporozoite proteins display α-Gal residues and to what extent the proteins are modified with α-Gal residues? Given that α-galactosyltransferase gene is thought to be absent in parasite genome, is it possible that α-Gal are acquired from mosquito glycoproteins by transglycosylation reaction?

• If parasites do not express α-Gal-containing glycans, what is the significance of UDP-Gal pool present in parasites?

Highlights.

• GPIs are crucial for P. falciparum development and survival both in mosquitos and humans.

• The reaction steps of GPI precursor biosynthesis and GPI biosynthesis of malaria parasites are important targets for drug development.

• In P. falciparum, β-O-GlcNAcylation occurs in functionally important protein classes, such as transcription factors, energy metabolism pathway, stress response, cytoskeletal motor assembly and signaling, and thus it may have vital functional relevance.

• Fucosylation and tryptophan C-mannosylation of the TRAP family of proteins of P. falciparum are essential for the protein structural stability, gametocyte egress, ookinete and oocyst development, sporogenesis, and infection transmission.

• In-depth knowledge of structure and functions of parasite glycans may provide important targets for the development of effective vaccines and/or drugs for malaria.

Glossary

Egress

The process of infected erythrocytes or hepatocytes having multinucleated matured parasites rupturing and releasing merozoites into the host blood stream

Invasion

The process of merozoites and sporozoites infecting red blood cells and hepatocytes, respectively

Gametocytes

Parasites that are differentiated into a sexual form inside the infected erythrocytes for subsequent transmission to mosquitos

Glycosyltransferases

Enzymes which transfer sugar residues from nucleotide diphosphate sugar donors to acceptor substrates

Merozoites

Daughter parasites released into the host blood stream after parasites in infected hepatocytes or erythrocytes are developed, replicated, and differentiated into multinucleated parasites, matured, and ruptured. Merozoites specifically invade host erythrocytes for asexual propagation and sexual differentiatio

Micronemes

Tubular shaped secretory organelles present at the apical end of merozoites. During invasion, micronemes secret ligands which are trafficked to the cell surface of parasite and bind receptors on host cells. Microneme secretion is critical for the successful invasion of host cells. Also, egress of merozoites from host cells and motility of sporozoites rely on the secretion of adhesive ligands from micronemes

Motility

Process of parasite moving

Oocysts

The cyst-like parasite stage developed from ookinetes in the mosquito mid gut wall. Ooysts produce thousands of sporozoites, which migrate to the salivary gland for transmission to humans when mosquitos bite the host for blood meal

Ookinetes

Worm-like parasites developed from diploid zygotes produced by the fusion of male and female gametocytes in the mosquito midgut. Ookinetes traverse mid gut epithelium and adhere to the gut wall and then differentiated into oocysts

Rhoptries

Bulb-shaped secretory organelles present at the apical end of merozoites and sporozoites. Like micronemes, rhoptries contain adhesive proteins which bind receptors on host cells during invasion. Rhoptry secretion is critical for the successful invasion of host cells

Schizont

A multinucleated parasite stage produced through nuclear replication inside the infected hepatocytes or erythrocytes that upon further maturation ruptures and releases merozoites into the blood stream

Sporozoites

Parasites produced from the matured oocysts. Sporozoites specifically invade salivary glands of mosquitos. When mosquitos bite the host and inject saliva for a blood meal, sporozoites enter the blood stream, traverse though liver endothelia, and infect hepatocytes. In infected hepatocytes the parasites grow and eventually develop into merozoites, which upon releasing into the blood stream infect erythrocytes

Trophozoites

Metabolically highly active and rapidly developing parasites inside the infected hepatocytes or erythrocytes