Giulia Bandini

Biosynthesis of GDP-fucose and other sugar nucleotides in the blood-stages of Plasmodium falciparum

Background: GDP-fucose and other sugar nucleotide biosynthetic pathways are conserved in the P. falciparum genome. Results: These pathways are active in the intraerythrocytic life cycle of the parasite. Conclusion: The parasite biosynthesizes GDP-fucose and other sugar nucleotides not related to the glycosylphosphatidylinositol structures Significance: Their presence strongly suggests that they are involved in the biosynthesis of glycans not yet characterized. Carbohydrate structures play important roles in many biological processes, including cell adhesion, cell-cell communication, and host-pathogen interactions. Sugar nucleotides are activated forms of sugars used by the cell as donors for most glycosylation reactions. Using a liquid chromatography-tandem mass spectrometry-based method, we identified and quantified the pools of UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, GDP-mannose, and GDP-fucose in Plasmodium falciparum intraerythrocytic life stages. We assembled these data with the in silico functional reconstruction of the parasite metabolic pathways obtained from the P. falciparum annotated genome, exposing new active biosynthetic routes crucial for further glycosylation reactions. Fucose is a sugar present in glycoconjugates often associated with recognition and adhesion events. Thus, the GDP-fucose precursor is essential in a wide variety of organisms. P. falciparum presents homologues of GDP-mannose 4,6-dehydratase and GDP-l-fucose synthase enzymes that are active in vitro, indicating that most GDP-fucose is formed by a de novo pathway that involves the bioconversion of GDP-mannose. Homologues for enzymes involved in a fucose salvage pathway are apparently absent in the P. falciparum genome. This is in agreement with in vivo metabolic labeling experiments showing that fucose is not significantly incorporated by the parasite. Fluorescence microscopy of epitope-tagged versions of P. falciparum GDP-mannose 4,6-dehydratase and GDP-l-fucose synthase expressed in transgenic 3D7 parasites shows that these enzymes localize in the cytoplasm of P. falciparum during the intraerythrocytic developmental cycle. Although the function of fucose in the parasite is not known, the presence of GDP-fucose suggests that the metabolite may be used for further fucosylation reactions.

Phosphoglucomutase is absent in Trypanosoma brucei and redundantly substituted by phosphomannomutase and phospho‐N‐acetylglucosamine mutase

The enzymes phosphomannomutase (PMM), phospho‐N‐acetylglucosamine mutase (PAGM) and phosphoglucomutase (PGM) reversibly catalyse the transfer of phosphate between the C6 and C1 hydroxyl groups of mannose, N‐acetylglucosamine and glucose respectively. Although genes for a candidate PMM and a PAGM enzymes have been found in the Trypanosoma brucei genome, there is, surprisingly, no candidate gene for PGM. The TbPMM and TbPAGM genes were cloned and expressed in Escherichia coli and the TbPMM enzyme was crystallized and its structure solved at 1.85 Å resolution. Antibodies to the recombinant proteins localized endogenous TbPMM to glycosomes in the bloodstream form of the parasite, while TbPAGM localized to both the cytosol and glycosomes. Both recombinant enzymes were able to interconvert glucose‐phosphates, as well as acting on their own definitive substrates. Analysis of sugar nucleotide levels in parasites with TbPMM or TbPAGM knocked down by RNA interference (RNAi) suggests that, in vivo, PGM activity is catalysed by both enzymes. This is the first example in any organism of PGM activity being completely replaced in this way and it explains why, uniquely, T. brucei has been able to lose its PGM gene. The RNAi data for TbPMM also showed that this is an essential gene for parasite growth.

O-fucosylated glycoproteins form assemblies in close proximity to the nuclear pore complexes of Toxoplasma gondii.

Significance We describe here the discovery that assemblies of O-fucosylated proteins localize to the nuclear membrane of Toxoplasma gondii, particularly in proximity to the nuclear pore complexes (NPCs). O-fucose is added to Ser and Thr residues found in some of the Phe-Gly (FG) domain-containing proteins that characterize the NPC channel as well as in Ser-rich sequences in many proteins predicted to have roles in transcription, mRNA processing, and cell signaling. O-fucosylation of nucleocytosolic proteins has not been described previously in any eukaryote and appears to be unique to T. gondii and closely related apicomplexans. Toxoplasma gondii is an intracellular parasite that causes disseminated infections in fetuses and immunocompromised individuals. Although gene regulation is important for parasite differentiation and pathogenesis, little is known about protein organization in the nucleus. Here we show that the fucose-binding Aleuria aurantia lectin (AAL) binds to numerous punctate structures in the nuclei of tachyzoites, bradyzoites, and sporozoites but not oocysts. AAL also binds to Hammondia and Neospora nuclei but not to more distantly related apicomplexans. Analyses of the AAL-enriched fraction indicate that AAL binds O-linked fucose added to Ser/Thr residues present in or adjacent to Ser-rich domains (SRDs). Sixty-nine Ser-rich proteins were reproducibly enriched with AAL, including nucleoporins, mRNA-processing enzymes, and cell-signaling proteins. Two endogenous SRDs-containing proteins and an SRD-YFP fusion localize with AAL to the nuclear membrane. Superresolution microscopy showed that the majority of the AAL signal localizes in proximity to nuclear pore complexes. Host cells modify secreted proteins with O-fucose; here we describe the O-fucosylation pathway in the nucleocytosol of a eukaryote. Furthermore, these results suggest O-fucosylation is a mechanism by which proteins involved in gene expression accumulate near the NPC.

The disruption of GDP-fucose de novo biosynthesis suggests the presence of a novel fucose-containing glycoconjugate in Plasmodium asexual blood stages

Glycosylation is an important posttranslational protein modification in all eukaryotes. Besides glycosylphosphatidylinositol (GPI) anchors and N-glycosylation, O-fucosylation has been recently reported in key sporozoite proteins of the malaria parasite. Previous analyses showed the presence of GDP-fucose (GDP-Fuc), the precursor for all fucosylation reactions, in the blood stages of Plasmodium falciparum. The GDP-Fuc de novo pathway, which requires the action of GDP-mannose 4,6-dehydratase (GMD) and GDP-L-fucose synthase (FS), is conserved in the parasite genome, but the importance of fucose metabolism for the parasite is unknown. To functionally characterize the pathway we generated a PfGMD mutant and analyzed its phenotype. Although the labelling by the fucose-binding Ulex europaeus agglutinin I (UEA-I) was completely abrogated, GDP-Fuc was still detected in the mutant. This unexpected result suggests the presence of an alternative mechanism for maintaining GDP-Fuc in the parasite. Furthermore, PfGMD null mutant exhibited normal growth and invasion rates, revealing that the GDP-Fuc de novo metabolic pathway is not essential for the development in culture of the malaria parasite during the asexual blood stages. Nonetheless, the function of this metabolic route and the GDP-Fuc pool that is generated during this stage may be important for gametocytogenesis and sporogonic development in the mosquito.

Differential Roles for Inner Membrane Complex Proteins across Toxoplasma gondii and Sarcocystis neurona Development

The inner membrane complex (IMC) is a defining feature of apicomplexan parasites key to both their motility and unique cell division. To provide further insights into the IMC, we analyzed the dynamics and functions of representative alveolin domain-containing IMC proteins across developmental stages. Our work shows universal but distinct roles for IMC1, -3, and -7 during Toxoplasma asexual division but more specialized functions for these proteins during gametogenesis. In addition, we find that IMC15 is involved in daughter formation in both Toxoplasma and Sarcocystis tachyzoites, bradyzoites, and sporozoites. IMC14 and IMC15 function in limiting the number of Toxoplasma offspring per division. Furthermore, IMC7, -12, and -14, which are recruited in the G1 cell cycle stage, are required for stress resistance of extracellular tachyzoites. Thus, although the roles of the different IMC proteins appear to overlap, stage- and development-specific behaviors indicate that their functions are uniquely tailored to each life stage requirement. ABSTRACT The inner membrane complex (IMC) of apicomplexan parasites contains a network of intermediate filament-like proteins. The 14 alveolin domain-containing IMC proteins in Toxoplasma gondii fall into different groups defined by their distinct spatiotemporal dynamics during the internal budding process of tachyzoites. Here, we analyzed representatives of different IMC protein groups across all stages of the Toxoplasma life cycle and during Sarcocystis neurona asexual development. We found that across asexually dividing Toxoplasma stages, IMC7 is present exclusively in the mother’s cytoskeleton, whereas IMC1 and IMC3 are both present in mother and daughter cytoskeletons (IMC3 is strongly enriched in daughter buds). In developing macro- and microgametocytes, IMC1 and -3 are absent, whereas IMC7 is lost in early microgametocytes but retained in macrogametocytes until late in their development. We found no roles for IMC proteins during meiosis and sporoblast formation. However, we observed that IMC1 and IMC3, but not IMC7, are present in sporozoites. Although the spatiotemporal pattern of IMC15 and IMC3 suggests orthologous functions in Sarcocystis, IMC7 may have functionally diverged in Sarcocystis merozoites. To functionally characterize IMC proteins, we knocked out IMC7, -12, -14, and -15 in Toxoplasma. IMC14 and -15 appear to be involved in switching between endodyogeny and endopolygeny. In addition, IMC7, -12, and -14, which are all recruited to the cytoskeleton outside cytokinesis, are critical for the structural integrity of extracellular tachyzoites. Altogether, stage- and development-specific roles for IMC proteins can be discerned, suggesting different niches for each IMC protein across the entire life cycle. IMPORTANCE The inner membrane complex (IMC) is a defining feature of apicomplexan parasites key to both their motility and unique cell division. To provide further insights into the IMC, we analyzed the dynamics and functions of representative alveolin domain-containing IMC proteins across developmental stages. Our work shows universal but distinct roles for IMC1, -3, and -7 during Toxoplasma asexual division but more specialized functions for these proteins during gametogenesis. In addition, we find that IMC15 is involved in daughter formation in both Toxoplasma and Sarcocystis. IMC14 and IMC15 function in limiting the number of Toxoplasma offspring per division. Furthermore, IMC7, -12, and -14, which are recruited in the G1 cell cycle stage, are required for stress resistance of extracellular tachyzoites. Thus, although the roles of the different IMC proteins appear to overlap, stage- and development-specific behaviors indicate that their functions are uniquely tailored to each life stage requirement.

Leishmania major UDP-sugar pyrophosphorylase salvages galactose for glycoconjugate biosynthesis.

Graphical abstract

Ethanol and Isopropanol in Concentrations Present in Hand Sanitizers Sharply Reduce Excystation of Giardia and Entamoeba and Eliminate Oral Infectivity of Giardia Cysts in Gerbils

ABSTRACT Enteric protozoan parasites, which are spread by the fecal-oral route, are important causes of diarrhea (Giardia duodenalis) and amebic dysentery (Entamoeba histolytica). Cyst walls of Giardia and Entamoeba have a single layer composed of fibrils of β-1,3-linked GalNAc and β-1,4-linked GlcNAc (chitin), respectively. The goal here was to determine whether hand sanitizers that contain ethanol or isopropanol as the active microbicide might reduce transmission of these parasites. We found that treatment with these alcohols with or without drying in a rotary evaporator (to model rapid evaporation of sanitizers on hands) kills 85 to 100% of cysts of G. duodenalis and 90 to 100% of cysts of Entamoeba invadens (a nonpathogenic model for E. histolytica), as shown by nuclear labeling with propidium iodide and failure to excyst in vitro. Alcohols with or without drying collapsed the cyst walls of Giardia but did not collapse the cyst walls of Entamoeba. To validate the in vitro results, we showed that treatment with alcohols eliminated oral infection of gerbils by 1,000 G. duodenalis cysts, while a commercial hand sanitizer (Purell) killed E. invadens cysts that were directly applied to the hands. These results suggest that expanded use of alcohol-based hand sanitizers might reduce the transmission of Giardia and Entamoeba.

O-fucosylation of thrombospondin-like repeats is required for processing of MIC2 and for efficient host cell invasion by Toxoplasma gondii tachyzoites

Toxoplasma gondii is an intracellular parasite that causes disseminated infections which can lead to neurological damage in fetuses and immunocompromised individuals. Microneme protein 2 (MIC2)2, a member of the thrombospondin-related anonymous protein (TRAP) family, is a secreted protein important for motility, host cell attachment, invasion, and egress. MIC2 contains six thrombospondin type I repeats (TSRs) that are modified by C-mannose and O-fucose in Plasmodium spp. and mammals. Here we used mass spectrometry to show that the four TSRs in T. gondii MIC2 with protein O-fucosyltransferase 2 (POFUT2) acceptor sites are modified by a dHexHex disaccharide, while Trp residues within three TSRs are also modified with C-mannose. Disruption of genes encoding either pofut2 or nucleotide sugar transporter 2 (nst2), the putative GDP-fucose transporter, results in loss of MIC2 O-fucosylation, as detected by an antibody against the GlcFuc disaccharide, and markedly reduced cellular levels of MIC2. Furthermore, in 10-15% of the Δpofut2 or Δnst2 vacuoles, MIC2 accumulates earlier in the secretory pathway rather than localizing to micronemes. Dissemination of tachyzoites in human foreskin fibroblasts is reduced in these knockouts, which both show defects in attachment to and invasion of host cells comparable to the phenotype observed in the Βmic2. These results, which show O-fucosylation of TSRs is required for efficient processing of MIC2 and for normal parasite invasion, are consistent with the recent demonstration that P. falciparum Δpofut2 has decreased virulence and support a conserved role for this glycosylation pathway in quality control of TSR-containing proteins in eukaryotes.

Enabling tools for Toxoplasma glycobiology

Infection by the protozoan parasite Toxoplasma gondii is a major health risk owing to its chronic nature, ability to reactivate to cause blindness and encephalitis, and high prevalence in human populations. Like nearly all eukaryotes, Toxoplasma glycosylates many of its proteins and lipids and assembles polysaccharides. Unlike most eukaryotes, Toxoplasma divides and differentiates in vacuoles within host cells. While their glycans resemble canonical models, they exhibit species-specific variations that have inhibited deeper investigations into their roles in parasite biology and virulence. The genome of Toxoplasma encodes a suite of likely glycogenes expected to assemble a range of N-glycans, O-glycans, a C-glycan, GPI-anchors, and polysaccharides, along with their requisite precursors and membrane transporters. To facilitate genetic approaches to investigate the roles of specific glycans, we mapped probable connections between 59 glycogenes, their enzyme products, and the glycans to which they contribute. We adapted a double-CRISPR/Cas9 strategy and a mass spectrometry-based glycomics workflow to test these relationships, and conducted infection studies in fibroblast monolayers to probe cellular functions. Through the validated disruption of 17 glycogenes, we also discovered novel Glc0-2-Man6-GlcNAc2-type N-glycans, GalNAc2- and Glc-Fuc-type O-glycans, and a nuclear O-Fuc type glycan. We describe the guide sequences, disruption constructs, and mutant strains, which are freely available to practitioners for application in the context of the relational map to investigate the roles of glycans in their favorite biological processes.

The Apicomplexa-specific glucosamine-6-phosphate N -acetyltransferase gene family encodes a key enzyme for glycoconjugate synthesis with potential as therapeutic target

Apicomplexa form a phylum of obligate parasitic protozoa of great clinical and veterinary importance. These parasites synthesize glycoconjugates for their survival and infectivity, but the enzymatic steps required to generate the glycosylation precursors are not completely characterized. In particular, glucosamine-phosphate N-acetyltransferase (GNA1) activity, needed to produce the essential UDP-N-acetylglucosamine (UDP-GlcNAc) donor, has not been identified in any Apicomplexa. We scanned the genomes of Plasmodium falciparum and representatives from six additional main lineages of the phylum for proteins containing the Gcn5-related N-acetyltransferase (GNAT) domain. One family of GNAT-domain containing proteins, composed by a P. falciparum sequence and its six apicomplexan orthologs, rescued the growth of a yeast temperature-sensitive GNA1 mutant. Heterologous expression and in vitro assays confirmed the GNA1 enzymatic activity in all lineages. Sequence, phylogenetic and synteny analyses suggest an independent origin of the Apicomplexa-specific GNA1 family, parallel to the evolution of a different GNA1 family in other eukaryotes. The inability to disrupt an otherwise modifiable gene target suggests that the enzyme is essential for P. falciparum growth. The relevance of UDP-GlcNAc for parasite viability, together with the independent evolution and unique sequence features of Apicomplexa GNA1, highlights the potential of this enzyme as a selective therapeutic target against apicomplexans.