Sugar nucleotide quantification by liquid chromatography tandem mass spectrometry reveals a distinct profile in Plasmodium falciparum sexual stage parasites
Abstract:The obligate intracellular lifestyle of Plasmodium falciparum and the difficulties in obtaining sufficient amounts of biological material have hampered the study of specific metabolic pathways in the malaria parasite. Thus, for example, the pools of sugar nucleotides required to fuel glycosylation reactions have never been studied in-depth in well-synchronized asexual parasites or in other stages of its life cycle. These metabolites are of critical importance, especially considering the renewed interest in the… Show more
“…However, besides glycosylphosphatidylinositol (GPI) anchors 9 , the immunogenicity of carbohydrates allegedly located in the surface of this parasite is largely underappreciated since the parasite seems to have lost many of the genes required to elaborate complex carbohydrates 10 . Nevertheless, recent works showed the presence of precursors involved in glycoconjugate biosynthesis 11 , 12 and identified new glycosylations in the parasite surface 13 – 15 . Some of these sugars modify important antigens in the fight against malaria, such as the circumsporozoite surface protein (CSP), which is the main component of the RTS,S vaccine 16 and is O-fucosylated in malaria sporozoites.…”
Naturally-acquired antibody responses to malaria parasites are not only directed to protein antigens but also to carbohydrates on the surface of Plasmodium protozoa. Immunoglobulin M responses to α-galactose (α-Gal) (Galα1-3Galβ1-4GlcNAc-R)-containing glycoconjugates have been associated with protection from P. falciparum infection and, as a result, these molecules are under consideration as vaccine targets; however there are limited field studies in endemic populations. We assessed a wide breadth of isotype and subclass antibody response to α-Gal in children from Mozambique (South East Africa) and Ghana (West Africa) by quantitative suspension array technology. We showed that anti-α-Gal IgM, IgG and IgG1–4 levels vary mainly depending on the age of the child, and also differ in magnitude in the two sites. At an individual level, the intensity of malaria exposure to P. falciparum and maternally-transferred antibodies affected the magnitude of α-Gal responses. There was evidence for a possible protective role of anti-α-Gal IgG3 and IgG4 antibodies. However, the most consistent findings were that the magnitude of IgM responses to α-Gal was associated with protection against clinical malaria over a one-year follow up period, especially in the first months of life, while IgG levels correlated with malaria risk.
“…However, besides glycosylphosphatidylinositol (GPI) anchors 9 , the immunogenicity of carbohydrates allegedly located in the surface of this parasite is largely underappreciated since the parasite seems to have lost many of the genes required to elaborate complex carbohydrates 10 . Nevertheless, recent works showed the presence of precursors involved in glycoconjugate biosynthesis 11 , 12 and identified new glycosylations in the parasite surface 13 – 15 . Some of these sugars modify important antigens in the fight against malaria, such as the circumsporozoite surface protein (CSP), which is the main component of the RTS,S vaccine 16 and is O-fucosylated in malaria sporozoites.…”
Naturally-acquired antibody responses to malaria parasites are not only directed to protein antigens but also to carbohydrates on the surface of Plasmodium protozoa. Immunoglobulin M responses to α-galactose (α-Gal) (Galα1-3Galβ1-4GlcNAc-R)-containing glycoconjugates have been associated with protection from P. falciparum infection and, as a result, these molecules are under consideration as vaccine targets; however there are limited field studies in endemic populations. We assessed a wide breadth of isotype and subclass antibody response to α-Gal in children from Mozambique (South East Africa) and Ghana (West Africa) by quantitative suspension array technology. We showed that anti-α-Gal IgM, IgG and IgG1–4 levels vary mainly depending on the age of the child, and also differ in magnitude in the two sites. At an individual level, the intensity of malaria exposure to P. falciparum and maternally-transferred antibodies affected the magnitude of α-Gal responses. There was evidence for a possible protective role of anti-α-Gal IgG3 and IgG4 antibodies. However, the most consistent findings were that the magnitude of IgM responses to α-Gal was associated with protection against clinical malaria over a one-year follow up period, especially in the first months of life, while IgG levels correlated with malaria risk.
“…Although highly divergent at the sequence level, GNAT proteins are well-conserved in structure and catalytic mechanism 26 , 32 , 33 and GNA1 enzymes have been identified and characterized throughout the eukaryote kingdom 26 , 27 , 33 . However, the identification of GNA1 in the genome of P. falciparum , or any other apicomplexan, has remained elusive 29 despite the fact that the presence of UDP-GlcNAc was verified in different parasite stages 34 – 36 . Figure 1 Biosynthesis of UDP-GlcNAc in Apicomplexa.…”
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.
“…2A ). Rapamycin-treated cultures showed an ∼2.5-fold invasion rate reduction in the first IDC, indicating the expansion of a small proportion of viable parasites that likely reflects the kinetics of DiCre-mediated excision ( 19 ), GNA1 turnover, and/or the dynamics of sugar nucleotide pools ( 24 ) ( Fig. 2B ).…”
Section: Resultsmentioning
confidence: 99%
“…Sugar nucleotides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and multiple-reaction monitoring (MRM) in a QTRAP 6500 System (AB Sciex). The metabolites, separated using a Hypercarb PGC column (5 μm, 2.1 × 100 mm; Thermo Fisher Scientific) with a mobile phase composed of 0.1% formic acid (pH 9) and an acetonitrile gradient ( 24 ), were identified by their diagnostic MRM transitions ( 38 ).…”
UDP-N-acetylglucosamine (UDP-GlcNAc), the main product of the hexosamine biosynthetic pathway, is an important metabolite in protozoan parasites since its sugar moiety is incorporated into glycosylphosphatidylinositol (GPI) glycolipids and N- and O-linked glycans. Apicomplexan parasites have a hexosamine pathway comparable to other eukaryotic organisms, with the exception of the glucosamine-phosphate N-acetyltransferase (GNA1) enzymatic step that has an independent evolutionary origin and significant differences from nonapicomplexan GNA1s. By using conditional genetic engineering, we demonstrate the requirement of GNA1 for the generation of a pool of UDP-GlcNAc and for the development of intraerythrocytic asexual Plasmodium falciparum parasites. Furthermore, we present the 1.95 Å resolution structure of the GNA1 ortholog from Cryptosporidium parvum, an apicomplexan parasite which is a leading cause of diarrhea in developing countries, as a surrogate for P. falciparum GNA1. The in-depth analysis of the crystal shows the presence of specific residues relevant for GNA1 enzymatic activity that are further investigated by the creation of site-specific mutants. The experiments reveal distinct features in apicomplexan GNA1 enzymes that could be exploitable for the generation of selective inhibitors against these parasites, by targeting the hexosamine pathway. This work underscores the potential of apicomplexan GNA1 as a drug target against malaria.
IMPORTANCE Apicomplexan parasites cause a major burden on global health and economy. The absence of treatments, the emergence of resistances against available therapies, and the parasite’s ability to manipulate host cells and evade immune systems highlight the urgent need to characterize new drug targets to treat infections caused by these parasites. We demonstrate that glucosamine-6-phosphate N-acetyltransferase (GNA1), required for the biosynthesis of UDP-N-acetylglucosamine (UDP-GlcNAc), is essential for P. falciparum asexual blood stage development and that the disruption of the gene encoding this enzyme quickly causes the death of the parasite within a life cycle. The high-resolution crystal structure of the GNA1 ortholog from the apicomplexan parasite C. parvum, used here as a surrogate, highlights significant differences from human GNA1. These divergences can be exploited for the design of specific inhibitors against the malaria parasite.
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