GPI-anchor Synthesis Research Articles

Evolutionary rate covariation is pervasive between glycosylation pathways and points to potential disease modifiers.

Mutations in glycosylation pathways, such as N-linked glycosylation, O-linked glycosylation, and GPI anchor synthesis, lead to Congenital Disorders of Glycosylation (CDG). CDG typically present with seizures, hypotonia, and developmental delay but display large clinical variability with symptoms affecting every system in the body. This variability suggests modifier genes might influence the phenotypes. Because of the similar physiology and clinical symptoms, there are likely common genetic modifiers between CDG. Here, we use evolution as a tool to identify common modifiers between CDG and glycosylation genes. Protein glycosylation is evolutionarily conserved from yeast to mammals. Evolutionary rate covariation (ERC) identifies proteins with similar evolutionary rates that indicate shared biological functions and pathways. Using ERC, we identified strong evolutionary rate signatures between proteins in the same and different glycosylation pathways. Genome-wide analysis of proteins showing significant ERC with GPI anchor synthesis proteins revealed strong signatures with ncRNA modification proteins and DNA repair proteins. We also identified strong patterns of ERC based on cellular sub-localization of the GPI anchor synthesis enzymes. Functional testing of the highest scoring candidates validated genetic interactions and identified novel genetic modifiers of CDG genes. ERC analysis of disease genes and biological pathways allows for rapid prioritization of potential genetic modifiers, which can provide a better understanding of disease pathophysiology and novel therapeutic targets.

The multifunctional roles of the extracellular matrix in the sessile life of the zygnematophyte Penium margaritaceum: stick, glide and cluster.

Adhesion and consequent adoption of a sessile habit is a common feature of many green algae and was likely a key mechanism in terrestrialization by an ancient zygnematophyte (i.e., the Zygnematophyceae, the group of algae ancestral to land plants). Penium margaritaceum is a unicellular zygnematophyte that exhibits a multistep adhesion mechanism, which leads to the establishment of the sessile habit. Based on microscopic and immunological data, a dense aggregate of fibrils containing arabinogalactan-protein (AGP)-like components covers the cell surface and is responsible for initial adhesion. The AGP-like fibrils are 20 μm in diameter and possess chemical profiles similar to land plant AGPs. The fibrils attach to the inner cell wall layers and are very likely connected to the plasma membrane as glycophosphatidylinositol (GPI) lipid-anchored proteins, as they are susceptible to phospholipase C treatment. The presence of GPI-anchored AGPs in Penium is further supported by the identification of putative Penium homologs of land plant AGP genes responsible for GPI-anchor synthesis. After adhesion, cells secrete a complex heteropolysaccharide-containing extracellular polymeric substance (EPS) that facilitates gliding motility and the formation of cell aggregates. Fucoidan-like polymers, major components of brown algal CWs, are a major constituent of both the EPS and the adhesive layer of the CW and their role in the adhesion process is still to be examined.

Identification of emulsification proteins released from the cells by inhibiting the synthesis of GPI-anchor or β-1,3-glucan in Candida albicans

IntroductionA previous study demonstrated a strong emulsification ability of the culture supernatant obtained by cultivation of Candida albicans in a medium containing a β-1,3-glucan synthesis inhibitor and proposed a novel screening method using emulsification as an indicator for β-1,3-glucan synthesis inhibition (Nerome et al., 2021. Evaluating β-1,3-glucan synthesis inhibition using emulsion formation as an indicator. J Microbiol Methods. 190:106327). The emulsification was presumed to be caused by the proteins released from the cells; however, which proteins have a strong emulsification ability was unclear. Furthermore, as many cell wall proteins are connected to β-1,3-glucan via the carbohydrate moiety of the glycosylphosphatidylinositol (GPI)-anchor, which remains when detached from the cell membrane, emulsification might be detected by inhibiting GPI-anchor synthesis. ObjectiveThis study aimed to confirm whether emulsification could be detected by inhibiting GPI-anchor synthesis and identifying emulsification proteins released by inhibiting the synthesis of GPI-anchor or β-1,3-glucan. MethodsC. albicans was cultured in a medium containing a GPI-anchor synthesis inhibitor, and the emulsification by the culture supernatant was evaluated. We identified cell wall proteins released from the cells upon inhibition of β-1,3-glucan or GPI-anchor synthesis by mass spectrometry, their recombinant proteins were prepared, and their emulsification efficacy was evaluated. ResultsIn GPI-anchor synthesis inhibition, a weak emulsification phenomenon was observed compared to the β-1,3-glucan synthesis inhibition. Phr2 protein was released from the cells upon GPI-anchor synthesis inhibition, and recombinant Phr2 showed a strong emulsification activity. Phr2 and Fba1 proteins were released upon β-1,3-glucan synthesis inhibition, and recombinant Fba1 showed a strong emulsification activity. ConclusionsWe concluded that the emulsion phenomenon could be used to screen β-1,3-glucan and GPI-anchor synthesis inhibitors. Also, the two kinds of inhibitors could be distinguished by differences in the growth recovery by osmotic support and strength of emulsification. In addition, we identified the proteins involved in emulsification.

Novel insights from the Plasmodium falciparum sporozoite-specific proteome by probabilistic integration of 26 studies.

Plasmodium species, the causative agent of malaria, have a complex life cycle involving two hosts. The sporozoite life stage is characterized by an extended phase in the mosquito salivary glands followed by free movement and rapid invasion of hepatocytes in the human host. This transmission stage has been the subject of many transcriptomics and proteomics studies and is also targeted by the most advanced malaria vaccine. We applied Bayesian data integration to determine which proteins are not only present in sporozoites but are also specific to that stage. Transcriptomic and proteomic Plasmodium data sets from 26 studies were weighted for how representative they are for sporozoites, based on a carefully assembled gold standard for Plasmodium falciparum (Pf) proteins known to be present or absent during the sporozoite life stage. Of 5418 Pf genes for which expression data were available at the RNA level or at the protein level, 975 were identified as enriched in sporozoites and 90 specific to them. We show that Pf sporozoites are enriched for proteins involved in type II fatty acid synthesis in the apicoplast and GPI anchor synthesis, but otherwise appear metabolically relatively inactive in the salivary glands of mosquitos. Newly annotated hypothetical sporozoite-specific and sporozoite-enriched proteins highlight sporozoite-specific functions. They include PF3D7_0104100 that we identified to be homologous to the prominin family, which in human has been related to a quiescent state of cancer cells. We document high levels of genetic variability for sporozoite proteins, specifically for sporozoite-specific proteins that elicit antibodies in the human host. Nevertheless, we can identify nine relatively well-conserved sporozoite proteins that elicit antibodies and that together can serve as markers for previous exposure. Our understanding of sporozoite biology benefits from identifying key pathways that are enriched during this life stage. This work can guide studies of molecular mechanisms underlying sporozoite biology and potential well-conserved targets for marker and drug development.

Leveraging Whole Genome Sequencing to Define the Mutational Landscape in Paroxysmal Nocturnal Hemoglobinuria

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a prototypic disorder of hematopoietic stem cells leading to recurrent complement-mediated intravascular hemolysis. The development of PNH has been linked to somatic mutations in the PIGA gene, coding for an enzyme critically involved in GPI anchor synthesis. While mutations in PIGA are pathognomonic for PNH, other mutations of GPI biosynthetic pathways have been described in isolated cases. Moreover, additional genetic determinants of clonal advantage and other genetic modifiers of the clinical course have not been sufficiently clarified. Aim: This study was performed to investigate the molecular pathogenesis of PNH in a large patient cohort. Methods: Using a typical myeloid targeted panel, mutations in PIGA gene can be identified 60-70% of cases and thus we performed whole genome sequencing (WGS, mean. coverage 100x). To further clarify the molecular pathogenesis of PIGA WT cases. A cohort of 94 patients (irrespective of PIGA mutations) with a confirmed hemolytic PNH, primary or evolved from antecedent AA was studied. The average clone size at diagnosis was 73.5%, measured by granulocyte flow cytometry. Before extracting all exomic variants, including small variants and large-scale insertions/deletions, primary analysis was performed for initial variant calling and filtering of putative sequencing artifacts (1A). Results: As expected the majority of these patients (73.4%) harbored genetic lesions or mutations in PIGA with a mean variant allele frequency (VAF) of 29% and clone size of 69% by flow. Of these, 45% carried frameshift mutations, while 19% had splice variants and 14.5% carried missense mutations. Lastly, 14.5% of these mutations were based on gained stop codons or in-frame deletions while in 7% large scale genomic deletions in the PIGA locus were found (1B). The majority of these patients (67%) harbored a single genetic lesion, but >1 mutation was found in 23% of patients. Thus flow cytometric clone size may be a result of multiple genetically diverse PIGA mutant clones as indicated by the correlation between PNH clone size and number of PIGA mutants (P=4.14x10-7) (1C). In patients with PNH in whom PIGA mutations were not found, the average clone size was smaller (average 15.0%). We thus subjected them to targeted deep sequencing (coverage mean 1000x) to ascertain that inability to detect mutations was not due to technical sensitivity. Indeed, 8/25 cases turned out to have a PIGA mutation. In addition we studied expression of PIGA in PIGAmut versus WT cases, but no significant decrease in PIGA RNA was found. To further clarify the pathogenesis, we analyzed a gene panel of 33 proteins currently known to be involved in GPI anchor biosynthesis. We found a PIGO nonsense mutation (c.2593C>T) and a PIGV missense variant (c.467G>A) in two WT patients. These two variants were unique in PNH compared to a large cohort of myeloid neoplasias (N=2630) and appeared to be functionally consequential according to stringent in silico predictor and quality filter pipelines. Since the progression of PNH into frank myeloid neoplasia can occur and the dynamics of the expansion of PNH clones is unpredictable and thus may be influenced by additional hits, we queried if additional lesions could explain these two phenomena. We also investigated the concomitant presence of myeloid mutation in these cases. We found additional somatic mutations (panel of 77 genes) in 47.8% of all PNH patients including ASXL1 (N=10), BCOR (N=8), KMT2D (N=6), and RUNX1 (N=4) (1D). Analyzing the VAF revealed that the majority of these events were subclonal in relation to PIGA mutations. Since we studied the patients with already highly expanded clones and due to assertion bias no difference in clone size and additional mutations was found but an ongoing study in early PNH will determine whether subclonal hits predict progression to full blown PNH (1E). Conclusion: Our study shows that WGS can faithfully detect PIGA defects in a majority of PNH patients but this detection may be low in patients with smaller clones. Second, subclonal mutations linked to myeloid neoplasia are frequent events in PNH. Lastly we found two novel mutations in GPI synthetic pathway. Since none of these genes have been implicated in the pathogenesis of PNH, these findings extend our understanding of the pathogenesis of PNH and make the case for a targeted PIG pathway-wide sequencing of affected patients. Disclosures Maciejewski: Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

Molecular analysis of GPI-anchor biosynthesis pathway genes in rat strains used for the Pig-a gene mutation assay

Recent studies support the assumption that mutation of the X-linked Pig-a gene is most likely responsible for the mutant phenotype of the cells deficient in glycosylphosphatidylinositol (GPI)-anchored proteins quantified in the rodent Pig-a gene mutation assay. In humans, however, mutations in both alleles of one of the 30 other genes involved in GPI-anchor synthesis, e.g., PIG-L and PIG-O, cause reduced expression of surface GPI-anchored proteins. Here, we investigated the possibility that the loss of the GPI-anchor detected by the rat Pig-a assay also could be caused by mutation in other GPI-biosynthesis genes. 31 samples were obtained from 8 inbred and outbred rat strains commonly used for genetic toxicology assays. In order to investigate possible sources of variation in the Pig-a assay, variant DNA sequences were evaluated in Cd59 and 24 GPI-biosynthesis genes. In some genes, such as Pig-n and Pig-u, homozygous variations occurred in all animals, suggesting that these variations are due to deviations in the reference genome. Heterozygous Pig-s, Pig-w, Pig-o, Pig-c, Pgap1, Pgap2, Pig-k and Pig-t variations were found, however, indicating that these genes could serve as targets for mutation in the assay. Protein alignment for these altered genes was conducted with possible human, mouse and rat phenotypic mutants from the literature; this analysis demonstrated that many of the variations that we detected were in non-conserved sequences and that no phenotypes for any of these variants could be inferred from known mutants from the literature. All heterozygous variants were in outbred rats. Overall, the findings of this study cannot totally rule out the possibility that mutations in GPI-biosynthesis genes other than Pig-a are detected in the Pig-a assay, but suggest that if it occurs, it must occur only rarely and therefore mutations in genes other than Pig-a have little impact on rat-based experiments.

Homozygous splice-variants in human ARV1 cause GPI-anchor synthesis deficiency

BackgroundMutations in the ARV1 Homolog, Fatty Acid Homeostasis Modulator (ARV1), have recently been described in association with early infantile epileptic encephalopathy 38. Affected individuals presented with epilepsy, ataxia, profound intellectual disability, visual impairment, and central hypotonia. In S. cerevisiae, Arv1 is thought to be involved in sphingolipid metabolism and glycophosphatidylinositol (GPI)-anchor synthesis. The function of ARV1 in human cells, however, has not been elucidated. MethodsMutations were discovered through whole exome sequencing and alternate splicing was validated on the cDNA level. Expression of the variants was determined by qPCR and Western blot. Expression of GPI-anchored proteins on neutrophils and fibroblasts was analyzed by FACS and immunofluorescence microscopy, respectively. ResultsHere we describe seven patients from two unrelated families with biallelic splice mutations in ARV1. The patients presented with early onset epilepsy, global developmental delays, profound hypotonia, delayed speech development, cortical visual impairment, and severe generalized cerebral and cerebellar atrophy. The splice variants resulted in decreased ARV1 expression and significant decreases in GPI-anchored protein on the membranes of neutrophils and fibroblasts, indicating that the loss of ARV1 results in impaired GPI-anchor synthesis. ConclusionLoss of GPI-anchored proteins on our patients' cells confirms that the yeast Arv1 function of GPI-anchor synthesis is conserved in humans. Overlap between the phenotypes in our patients and those reported for other GPI-anchor disorders suggests that ARV1-deficiency is a GPI-anchor synthesis disorder.

Candida albicans; exploring glycosylation pathway in the search of targets for antimicrobial agents and yeast to hyphae transition.

Microbial cell wall is mostly synthesized by the glycosylated proteins with the distinct enzymatic activity. In this review we have concentrated on the description of the certain steps of glycosylation and their effect on the cell wall integrity and yeast to hyphae transition, the process enhancing the pathogenic properties of C.albicans. The glycoproteins play an invaluable role in C. albicans virulence and they modulate adhesive, invasive, morphogenetic and immune stimulating properties of the pathogen as well as its susceptibility to the antifungal agents. Therefore, understanding of C. albicans glycobiology might let us expand the arsenal in the war against fungal enemies. The early stages of N-, O-glycans and GPI-anchor synthesis requires dolichol - the lipid carrier of sugar residues. Diminished supply of dolichol causes series of defects in C. albicans cells, among which aberrant protein glycosylation is the most evident. Furthermore, the relations between the cell wall composition and integrity, resistance to some antifungal and cell wall disturbing agents and dolichol dependent glycosylation are observed. Moreover relevance of these reactions for the morphological differentiation of C.albicans is described.

Nuclear envelope localization of PIG-B is essential for GPI-anchor synthesis in Drosophila.

ABSTRACTMembrane lipid biosynthesis is a complex process that takes place in various intracellular compartments. Glycosylphosphatidylinositol (GPI), a lipid involved in membrane anchoring of some proteins, is synthesized by the PIG enzymes. Most PIGs are localized to the endoplasmic reticulum (ER), but Drosophila PIG-B (DmPIG-B) is localized to the nuclear envelope (NE). To determine whether the NE localization of DmPIG-B is functionally important, we defined the determinants of localization and generated an ER-localized form, denoted DmPIG-B[ER]. The enzymatic activity of DmPIG-B[ER] was comparable to that of NE-localized DmPIG-B[NE]. Expression of DmPIG-B[ER] inefficiently rescued the lethality of the PIG-B mutant, whereas DmPIG-B[NE] rescued this lethality fully. DmPIG-B[ER] was preferentially degraded by lysosomes, suggesting that the NE localization is essential for function and stability of the protein. In addition, we found that the region of the ER proximal to the NE is the site of translation of GPI-anchored proteins and addition of GPI. Thus, the NE and proximal ER may provide a platform for efficient GPI anchoring.

The development of an in vitro Pig-a assay in L5178Y cells.

A recent flow cytometry-based in vivo mutagenicity assay involves the hemizygous phosphatidylinositol class A (Pig-a) gene. Pig-a forms the catalytic subunit of N-acetylglucosaminyltransferase required for glycophosphatidylinositol (GPI) anchor biosynthesis. Mutations in Pig-a prevent GPI-anchor synthesis resulting in loss of cell-surface GPI-linked proteins. The aim of the current study was to develop and validate an in vitro Pig-a assay in L5178Y mouse lymphoma cells. Ethyl methanesulfonate (EMS)-treated cells (186.24-558.72µg/ml; 24h) were used for method development and antibodies against GPI-linked CD90.2 and stably expressed CD45 were used to determine GPI-status by flow cytometry. Antibody concentration and incubation times were optimised (0.18µg/ml, 30min, 4°C) and Zombie Violet™ (viability marker; 0.5%, 30min, RT) was included. The optimum phenotypic expression period was 8 days. The low background mutation frequency of GPI-deficiency [GPI(-)] in L5178Y cells (0.1%) constitutes a rare event, thus flow cytometry acquisition parameters were optimised; 104 cells were measured at medium flow rate to ensure a CV ≤ 30%. Spiking known numbers of GPI(-) cells into a wild-type population gave high correlation between measured and spiked numbers (R2 0.999). We applied the in vitro Pig-a assay to a selection of well-validated genotoxic and non-genotoxic compounds. EMS, N-ethyl-N-nitrosourea and 4-nitroquinoline-N-oxide dose dependently increased numbers of GPI(-) cells, while etoposide, mitomycin C, and a bacterial-specific mutagen did not. Cycloheximide and sodium chloride were negative. Sanger sequencing revealed Pig-a mutations in the GPI(-) clones. In conclusion, this in vitro Pig-a assay could complement the in vivo version, and follow up weak Ames positives and late-stage human metabolites or impurities.

Dynamic Function of DPMS Is Essential for Angiogenesis and Cancer Progression.

Dolichol phosphate mannose synthase (DPMS) is an inverting GT-A-folded enzyme and classified as GT2 by CAZy. DPMS sequence carries a metal-binding DXD motif, a PKA motif, and a variable number of hydrophobic domains. Human and bovine DPMS possess a single transmembrane domain, whereas that from S. cerevisiae and A. thaliana carry multiple transmembrane domains and are superimposable. The catalytic activity of DPMS is documented in all spheres of life, and the 32kDa protein is uniquely regulated by protein phosphorylation. Intracellular activation of DPMS by cAMP signaling is truly due to the activation of the enzyme and not due to increased Dol-P level. The sequence of DPMS in some species also carries a protein N-glycosylation motif (Asn-X-Ser/Thr). Apart from participating in N-glycan biosynthesis, DPMS is essential for the synthesis of GPI anchor as well as for O- and C-mannosylation of proteins. Because of the dynamic nature, DPMS actively participates in cellular proliferation enhancing angiogenesis and breast tumor progression. In fact, overexpression of DPMS in capillary endothelial cells supports increased N-glycosylation, cellular proliferation, and enhanced chemotactic activity. These are expected to be completely absent in congenital disorders of glycosylation (CDGs) due to the silence of DPMS catalytic activity. DPMS has also been found to be involved in the cross talk with N-acetylglucosaminyl 1-phosphate transferase (GPT). Inhibition of GPT with tunicamycin downregulates the DPMS catalytic activity quantitatively. The result is impairment of surface N-glycan expression, inhibition of angiogenesis, proliferation of human breast cancer cells, and induction of apoptosis. Interestingly, nano-formulated tunicamycin is three times more potent in inhibiting the cell cycle progression than the native tunicamycin and is supported by downregulation of the ratio of phospho-p53 to total-p53 as well as phospho-Rb to total Rb. DPMS expression is also reduced significantly. However, nano-formulated tunicamycin does not induce apoptosis. We, therefore, conclude that DPMS could become a novel target for developing glycotherapy treating breast tumor in the clinic.

N-acetylglucosamine affects Cryptococcus neoformans cell-wall composition and melanin architecture.

Cryptococcus neoformans is an environmental fungus that belongs to the phylum Basidiomycetes and is a major pathogen in immunocompromised patients. The ability of C. neoformans to produce melanin pigments represents its second most important virulence factor, after the presence of a polysaccharide capsule. Both the capsule and melanin are closely associated with the fungal cell wall, a complex structure that is essential for maintaining cell morphology and viability under conditions of stress. The amino sugar N-acetylglucosamine (GlcNAc) is a key constituent of the cell-wall chitin and is used for both N-linked glycosylation and GPI anchor synthesis. Recent studies have suggested additional roles for GlcNAc as an activator and mediator of cellular signalling in fungal and plant cells. Furthermore, chitin and chitosan polysaccharides interact with melanin pigments in the cell wall and have been found to be essential for melanization. Despite the importance of melanin, its molecular structure remains unresolved; however, we previously obtained critical insights using advanced nuclear magnetic resonance (NMR) and imaging techniques. In this study, we investigated the effect of GlcNAc supplementation on cryptococcal cell-wall composition and melanization. C. neoformans was able to metabolize GlcNAc as a sole source of carbon and nitrogen, indicating a capacity to use a component of a highly abundant polymer in the biospherenutritionally. C. neoformans cells grown with GlcNAc manifested changes in the chitosan cell-wall content, cell-wall thickness and capsule size. Supplementing cultures with isotopically 15N-labelled GlcNAc demonstrated that the exogenous monomer serves as a building block for chitin/chitosan and is incorporated into the cell wall. The altered chitin-to-chitosan ratio had no negative effects on the mother-daughter cell separation; growth with GlcNAc affected the fungal cell-wall scaffold, resulting in increased melanin deposition and assembly. In summary, GlcNAc supplementation had pleiotropic effects on cell-wall and melanin architectures, and thus established its capacity to perturb these structures, a property that could prove useful for metabolic tracking studies.

The Glycosylphosphatidylinositol Anchor Biosynthesis Genes GPI12, GAA1, and GPI8 Are Essential for Cell-Wall Integrity and Pathogenicity of the Maize Anthracnose Fungus Colletotrichum graminicola.

Glycosylphosphatidylinositol (GPI) anchoring of proteins is one of the most common posttranslational modifications of proteins in eukaryotic cells and is important for associating proteins with the cell surface. In fungi, GPI-anchored proteins play essential roles in cross-linking of β-glucan cell-wall polymers and cell-wall rigidity. GPI-anchor synthesis is successively performed at the cytoplasmic and the luminal face of the ER membrane and involves approximately 25 proteins. While mutagenesis of auxiliary genes of this pathway suggested roles of GPI-anchored proteins in hyphal growth and virulence, essential genes of this pathway have not been characterized. Taking advantage of RNA interference (RNAi) we analyzed the function of the three essential genes GPI12, GAA1 and GPI8, encoding a cytoplasmic N-acetylglucosaminylphosphatidylinositol deacetylase, a metallo-peptide-synthetase and a cystein protease, the latter two representing catalytic components of the GPI transamidase complex. RNAi strains showed drastic cell-wall defects, resulting in exploding infection cells on the plant surface and severe distortion of in planta-differentiated infection hyphae, including formation of intrahyphal hyphae. Reduction of transcript abundance of the genes analyzed resulted in nonpathogenicity. We show here for the first time that the GPI synthesis genes GPI12, GAA1, and GPI8 are indispensable for vegetative development and pathogenicity of the causal agent of maize anthracnose, Colletotrichum graminicola.

SRD5A3‐CDG: Expanding the phenotype of a congenital disorder of glycosylation with emphasis on adult onset features

Increasing numbers of congenital disorders of glycosylation (CDG) have been reported recently resulting in an expansion of the phenotypes associated with this group of disorders. SRD5A3 codes for polyprenol reductase which converts polyprenol to dolichol. This is a major pathway for dolichol biosynthesis for N-glycosylation, O-mannosylation, C-mannosylation, and GPI anchor synthesis. We present the features of five individuals (three children and two adults) with mutations in SRD5A3 focusing on the variable eye and skin involvement. We compare that to 13 affected individuals from the literature including five adults allowing us to delineate the features that may develop over time with this disorder including kyphosis, retinitis pigmentosa, and cataracts. © 2016 Wiley Periodicals, Inc.

The genome of Eimeria falciformis--reduction and specialization in a single host apicomplexan parasite.

BackgroundThe phylum Apicomplexa comprises important unicellular human parasites such as Toxoplasma and Plasmodium. Eimeria is the largest and most diverse genus of apicomplexan parasites and some species of the genus are the causative agent of coccidiosis, a disease economically devastating in poultry. We report a complete genome sequence of the mouse parasite Eimeria falciformis. We assembled and annotated the genome sequence to study host-parasite interactions in this understudied genus in a model organism host.ResultsThe genome of E. falciformis is 44 Mb in size and contains 5,879 predicted protein coding genes. Comparative analysis of E. falciformis with Toxoplasma gondii shows an emergence and diversification of gene families associated with motility and invasion mainly at the level of the Coccidia. Many rhoptry kinases, among them important virulence factors in T. gondii, are absent from the E. falciformis genome. Surface antigens are divergent between Eimeria species. Comparisons with T. gondii showed differences between genes involved in metabolism, N-glycan and GPI-anchor synthesis. E. falciformis possesses a reduced set of transmembrane transporters and we suggest an altered mode of iron uptake in the genus Eimeria.ConclusionsReduced diversity of genes required for host-parasite interaction and transmembrane transport allow hypotheses on host adaptation and specialization of a single host parasite. The E. falciformis genome sequence sheds light on the evolution of the Coccidia and helps to identify determinants of host-parasite interaction critical for drug and vaccine development.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-696) contains supplementary material, which is available to authorized users.

8. Genetic basis of Mabry’s syndrome

We review the discovery of two genes disrupted in Mabry syndrome (hyperphosphatasia with developmental disability; OMIM#239300): a syndrome notable for characteristic facial dysmorphology (hypertelorism, a broad nasal bridge and a tented mouth); subtle hand bone abnormalities (variable shortening of middle and distal phalanges) and nerve abnormalities (plaques disrupting Schwann cells) and persistently elevated serum alkaline phosphatase (ALP). In approximately two thirds of cases, the biochemical abnormalities present in Mabry syndrome result from disruption of the phosphoinositol glycan (GPI) anchor as a result of mutations in the phosphoglycan-inositol biosynthesis type V ( PIGV ) and type O ( PIGO ) genes. The remaining one third of cases have no known mutation in the GPI anchor pathway. These idiopathic cases are reported to accumulate lysosomal storage material that has been putatively identified as a glycolipid material. As twenty genes are integral to GPI anchor synthesis, we hypothesize that idiopathic cases of Mabry syndrome may result from disruptions in other genes encoding GPI anchor biosynthesis enzymes. We are using exome sequencing and candidate gene approaches to identify the gene(s) responsible for idiopathic Mabry syndrome. This work will assist in elucidating the inborn errors of metabolism that underlie the seizures associated with Mabry syndrome.

PIGO mutations in intractable epilepsy and severe developmental delay with mild elevation of alkaline phosphatase levels

Aberrations in the glycosylphosphatidylinositol (GPI)-anchor biosynthesis pathway constitute a subclass of congenital disorders of glycosylation, and mutations in seven genes involved in this pathway have been identified. Among them, mutations in PIGV and PIGO, which are involved in the late stages of GPI-anchor synthesis, and PGAP2, which is involved in fatty-acid GPI-anchor remodeling, are all causative for hyperphosphatasia with mental retardation syndrome (HPMRS). Using whole exome sequencing, we identified novel compound heterozygous PIGO mutations (c.389C>A [p.Thr130Asn] and c.1288C>T [p.Gln430*]) in two siblings, one of them having epileptic encephalopathy. GPI-anchored proteins (CD16 and CD24) on blood granulocytes were slightly decreased compared with a control and his mother. Our patients lacked the characteristic features of HPMRS, such as facial dysmorphology (showing only a tented mouth) and hypoplasia of distal phalanges, and had only a mild elevation of serum alkaline phosphatase (ALP). Our findings therefore expand the clinical spectrum of GPI-anchor deficiencies involving PIGO mutations to include epileptic encephalopathy with mild elevation of ALP.

Transcriptome and Biochemical Analysis Reveals That Suppression of GPI-Anchor Synthesis Leads to Autophagy and Possible Necroptosis in Aspergillus fumigatus

Previously, it has been shown that GPI proteins are required for cell wall synthesis and organization in Aspergillus fumigatus, a human opportunistic pathogen causing life-threatening invasive aspergillosis (IA) in immunocompromised patients. Blocking GPI anchor synthesis leads to severe phenotypes such as cell wall defects, increased cell death, and attenuated virulence. However, the mechanism by which these phenotypes are induced is unclear. To gain insight into global effects of GPI anchoring in A. fumigatus, in this study a conditional expression mutant was constructed and a genome wide transcriptome analysis was carried out. Our results suggested that suppression of GPI anchor synthesis mainly led to activation of phosphatidylinositol (PtdIns) signaling and ER stress. Biochemical and morphological evidence showed that autophagy was induced in response to suppression of the GPI anchor synthesis, and also an increased necroptosis was observed. Based on our results, we propose that activation of PtdIns3K and increased cytosolic Ca2+, which was induced by both ER stress and PtdIns signaling, acted as the main effectors to induce autophagy and possible necroptosis.

Inhibiting GPI Anchor Biosynthesis in Fungi Stresses the Endoplasmic Reticulum and Enhances Immunogenicity

In fungi, the anchoring of proteins to the plasma membrane via their covalent attachment to glycosylphosphatidylinositol (GPI) is essential and thus provides a valuable point of attack for the development of antifungal therapeutics. Unfortunately, studying the underlying biology of GPI-anchor synthesis is difficult, especially in medically relevant fungal pathogens because they are not genetically tractable. Compounding difficulties, many of the genes in this pathway are essential in Saccharomyces cerevisiae. Here, we report the discovery of a new small molecule christened gepinacin (for GPI acylation inhibitor) which selectively inhibits Gwt1, a critical acyltransferase required for the biosynthesis of fungal GPI anchors. After delineating the target specificity of gepinacin using genetic and biochemical techniques, we used it to probe key, therapeutically relevant consequences of disrupting GPI anchor metabolism in fungi. We found that, unlike all three major classes of antifungals in current use, the direct antimicrobial activity of this compound results predominantly from its ability to induce overwhelming stress to the endoplasmic reticulum. Gepinacin did not affect the viability of mammalian cells nor did it inhibit their orthologous acyltransferase. This enabled its use in co-culture experiments to examine Gwt1's effects on host-pathogen interactions. In isolates of Candida albicans, the most common fungal pathogen in humans, exposure to gepinacin at sublethal concentrations impaired filamentation and unmasked cell wall β-glucan to stimulate a pro-inflammatory cytokine response in macrophages. Gwt1 is a promising antifungal drug target, and gepanacin is a useful probe for studying how disrupting GPI-anchor synthesis impairs viability and alters host-pathogen interactions in genetically intractable fungi.

Mutations in PIGO, a Member of the GPI-Anchor-Synthesis Pathway, Cause Hyperphosphatasia with Mental Retardation

Hyperphosphatasia with mental retardation syndrome (HPMRS), an autosomal-recessive form of intellectual disability characterized by facial dysmorphism, seizures, brachytelephalangy, and persistent elevated serum alkaline phosphatase (hyperphosphatasia), was recently shown to be caused by mutations in PIGV, a member of the glycosylphosphatidylinositol (GPI)-anchor-synthesis pathway. However, not all individuals with HPMRS harbor mutations in this gene. By exome sequencing, we detected compound-heterozygous mutations in PIGO, a gene coding for a membrane protein of the same molecular pathway, in two siblings with HPMRS, and we then found by Sanger sequencing further mutations in another affected individual; these mutations cosegregated in the investigated families. The mutant transcripts are aberrantly spliced, decrease the membrane stability of the protein, or impair enzyme function such that GPI-anchor synthesis is affected and the level of GPI-anchored substrates localized at the cell surface is reduced. Our data identify PIGO as the second gene associated with HPMRS and suggest that a deficiency in GPI-anchor synthesis is the underlying molecular pathomechanism of HPMRS.