A broadly active fucosyltransferase LmjFUT1 whose mitochondrial localization and activity are essential in parasitic Leishmania.

Glycoconjugates play major roles in the infectious cycle of the trypanosomatid parasite Leishmania While GDP-Fucose synthesis is essential, fucosylated glycoconjugates have not been reported in Leishmania major [H. Guo et al., J. Biol. Chem. 292, 10696-10708 (2017)]. Four predicted fucosyltransferases appear conventionally targeted to the secretory pathway; SCA1/2 play a role in side-chain modifications of lipophosphoglycan, while gene deletion studies here showed that FUT2 and SCAL were not essential. Unlike most eukaryotic glycosyltransferases, the predicted α 1-2 fucosyltransferase encoded by FUT1 localized to the mitochondrion. A quantitative "plasmid segregation" assay, expressing FUT1 from the multicopy episomal pXNG vector in a chromosomal null ∆fut1- background, established that FUT1 is essential. Similarly, "plasmid shuffling" confirmed that both enzymatic activity and mitochondrial localization were required for viability, comparing import-blocked or catalytically inactive enzymes, respectively. Enzymatic assays of tagged proteins expressed in vivo or of purified recombinant FUT1 showed it had a broad fucosyltransferase activity including glycan and peptide substrates. Unexpectedly, a single rare ∆fut1- segregant (∆fut1s ) was obtained in rich media, which showed severe growth defects accompanied by mitochondrial dysfunction and loss, all of which were restored upon FUT1 reexpression. Thus, FUT1 along with the similar Trypanosoma brucei enzyme TbFUT1 [G. Bandini et al., bioRxiv, https://www.biorxiv.org/content/10.1101/726117v2 (2021)] joins the eukaryotic O-GlcNAc transferase isoform as one of the few glycosyltransferases acting within the mitochondrion. Trypanosomatid mitochondrial FUT1s may offer a facile system for probing mitochondrial glycosylation in a simple setting, and their essentiality for normal growth and mitochondrial function renders it an attractive target for chemotherapy of these serious human pathogens.

Symbiont modulates expression of specific gene categories in Angomonas deanei

Trypanosomatids are parasites that cause disease in humans, animals, and plants. Most are non-pathogenic and some harbor a symbiotic bacterium. Endosymbiosis is part of the evolutionary process of vital cell functions such as respiration and photosynthesis. Angomonas deanei is an example of a symbiont-containing trypanosomatid. In this paper, we sought to investigate how symbionts influence host cells by characterising and comparing the transcriptomes of the symbiont-containing A. deanei (wild type) and the symbiont-free aposymbiotic strains. The comparison revealed that the presence of the symbiont modulates several differentially expressed genes. Empirical analysis of differential gene expression showed that 216 of the 7625 modulated genes were significantly changed. Finally, gene set enrichment analysis revealed that the largest categories of genes that downregulated in the absence of the symbiont were those involved in oxidation-reduction process, ATP hydrolysis coupled proton transport and glycolysis. In contrast, among the upregulated gene categories were those involved in proteolysis, microtubule-based movement, and cellular metabolic process. Our results provide valuable information for dissecting the mechanism of endosymbiosis in A. deanei.

Symbiont modulates expression of specific gene categories in Angomonas deanei.

Trypanosomatids are parasites that cause disease in humans, animals, and plants. Most are non-pathogenic and some harbor a symbiotic bacterium. Endosymbiosis is part of the evolutionary process of vital cell functions such as respiration and photosynthesis. Angomonas deanei is an example of a symbiont-containing trypanosomatid. In this paper, we sought to investigate how symbionts influence host cells by characterising and comparing the transcriptomes of the symbiont-containing A. deanei (wild type) and the symbiont-free aposymbiotic strains. The comparison revealed that the presence of the symbiont modulates several differentially expressed genes. Empirical analysis of differential gene expression showed that 216 of the 7625 modulated genes were significantly changed. Finally, gene set enrichment analysis revealed that the largest categories of genes that downregulated in the absence of the symbiont were those involved in oxidation-reduction process, ATP hydrolysis coupled proton transport and glycolysis. In contrast, among the upregulated gene categories were those involved in proteolysis, microtubule-based movement, and cellular metabolic process. Our results provide valuable information for dissecting the mechanism of endosymbiosis in A. deanei.

Mitosomal chaperone modulation during the life cycle of the pathogenic protist Giardia intestinalis.

The mitosome is a double-membrane enveloped organelle that is found in few unicellular eukaryotes, one of which is the human intestinal parasitic protist Giardia intestinalis, which also lacks mitochondria and peroxisomes. This flagellated protist grows in vitro as trophozoites and under some conditions, differentiates into cysts, which are characterized by the absence of externalized flagella, a round shape, and the presence of a cyst wall. The presence and distribution of mitosomal proteins, such as giardial iron-sulfur cluster protein (GiIscU), heat-shock protein 70 (mit-HSP70) and giardial chaperonin 60 (GiCpn60), during the process of trophozoite-to-cyst transformation was tracked using confocal laser scanning microscopy and western blotting. During the early stages of the differentiation process (∼12h), there was a significant decrease in the extent of chaperone labeling in the cells, which disappeared after 21h but was recovered during the cyst stage; IscU labeling remained present throughout the differentiation process. This finding was confirmed by mRNA expression analysis, thus indicating that a process modulates the expression of mitosomal chaperones during the G. intestinalis life cycle. Microscopy techniques, such as structured illumination and electron tomography, revealed a novel profile for central mitosomes, as well as the presence of both rounded and elongated mitosomes.

Expanding the knowledge of the chemical structure of glycoconjugates from Trypanosoma cruzi TcI genotype. Contribution to taxonomic studies.

One of the main obstacles to the treatment of Chagas disease is the genetic and phenotypical variance displayed by T. cruzi strains, resulting in differences in morphology, virulence, pathogenicity and drug susceptibility. To better understand the role of glycoconjungates in Chagas disease, we performed the molecular characterization of the O-linked chains from mucins and glycoinositolphospholipids (GIPLs) of the Silvio X10 clone 1 strain. We demonstrated the presence of a ß-galactofuranose (ß-Galf) unity linked to the O-4 position of the α-N-acetylglucosamine (α-GlcNAc)O-4 in Tc-mucins. GIPLs analysis showed that the lipidic portion is exclusively composed of ceramide and the PI-oligossacharidic portion contains the Man4(AEP)GlcN-Ins-PO4 core, substituted by ethanolamine-phosphate (EtNP) on the third distal mannose from inositol, which may or may not have a terminal ß Galf unity. These results confirm the classification of the Silvio X10/1 strain in group T. cruzi I. Again, it is noted that the study of T. cruzi surface glycoconjugates confirm the molecular results and the hypothesis that surface glycoconjugates may be interesting biomarker for the differentiation of trypanosomatid strains.

Mannoprotein MP84 mediates the adhesion of Cryptococcus neoformans to epithelial lung cells.

The capsule is the most important virulence factor of the fungal pathogen Cryptococcus neoformans. This structure consists of highly hydrated polysaccharides, including glucuronoxylomannan (GXM), and galactoxylomannan (GalXM). It is also composed of mannoproteins (MPs) which corresponds to less than 1% of the capsular weight. Despite MPs being the minority and least studied components, four of these molecules with molecular masses of 115, 98, 88, and 84 kDa were identified and characterized as C. neoformans immunoreactive antigens involved in the pathogenesis, and are potential cryptococcosis vaccine candidates. With the aim to describe the adhesive property of MPs, we cloned and expressed the MP84, a mannoprotein with molecular weight of 84 kDa, on Pichia pastoris yeast, and performed interaction assays of C. neoformans with epithelial lung cells, in the presence or absence of capsule components. Two fungal strains, the wild type, NE-241, and a mutant, CAP67, deficient in GXM production, were used throughout this study. The adhesion assays were completed using epithelial lung cells, A549, and human prostate cancer cells, PC3, as a control. We observed that capsulated wild type (NE-241), and acapsular (CAP67) strains adhered significantly to A549 cells, compared with PC3 cells (p < 0.05). GXM inhibits the NE-241 adhesion, but not the CAP67. In contrast, CAP67 adhesion was only inhibited in the presence of MP84. These results demonstrate the involvement of MP in the adhesion of C. neoformans to epithelial lung cells. We conclude that this interaction possibly involves an adhesion-like interaction between MP on the fungal surface and the complementary receptor molecules on the epithelial cells.

Trypanosoma cruzi Trans-sialidase: structural features and biological implications.

Trypanosoma cruzi trans-sialidase (TcTS) has intrigued researchers all over the world since it was shown that T. cruzi incorporates sialic acid through a mechanism independent of sialyltransferases. The enzyme has being involved in a vast myriad of functions in the biology of the parasite and in the pathology of Chagas' disease. At the structural level experiments trapping the intermediate with fluorosugars followed by peptide mapping, X-ray crystallography, molecular modeling and magnetic nuclear resonance have opened up a three-dimensional understanding of the way this enzyme works. Herein we review the multiple biological roles of TcTS and the structural studies that are slowly revealing the secrets underlining an efficient sugar transfer activity rather than simple hydrolysis by TcTS.

Increase of O-glycosylated oncofetal fibronectin in high glucose-induced epithelial-mesenchymal transition of cultured human epithelial cells.

Growing evidences indicate that aberrant glycosylation can modulate tumor cell invasion and metastasis. The process termed "epithelial-mesenchymal transition" (EMT) provides a basic experimental model to shed light on this complex process. The EMT involves a striking decline in epithelial markers, accompanied by enhanced expression of mesenchymal markers, culminating in cell morphology change and increased cell motility. Few recent studies have established the participation glycosylation during EMT. Studies now come into knowledge brought to light the involvement of a site-specific O-glycosylation in the IIICS domain of human oncofetal fibronectin (onfFN) during the EMT process. Herein we show that high glucose induces EMT in A549 cells as demonstrated by TGF-ß secretion, cell morphology changes, increased cellular motility and the emergence of mesenchymal markers. The hyperglycemic conditions increased onfFN protein levels, promoted an up regulation of mRNA levels for ppGalNAc-T6 and FN IIICS domain, which contain the hexapeptide (VTHPGY) required for onfFN biosynthesis. Glucose effect involves hexosamine (HBP) biosynthetic pathway as overexpression of glutamine: fructose-6-phosphate amidotransferase increases mesenchymal markers, onfFN levels and mRNA levels for FN IIICS domain. In summary, our results demonstrate, for the first time that the metabolism of glucose through HBP promotes O-glycosylation of the oncofetal form of FN during EMT modulating tumorogenesis.

Trans-sialidase from Trypanosoma cruzi enhances the adhesion properties and fibronectin-driven migration of thymocytes.

In experimental Trypanosoma cruzi infections, severe thymic atrophy leads to release of activated CD4(+)CD8(+) double-positive (DP) T cells to the periphery. In humans, activated DP T cells are found in the blood in association with severe cardiac forms of human chronic Chagas disease. The mechanisms underlying the premature thymocyte release during the chagasic thymic atrophy remain elusive. We tested whether the migratory properties of intrathymic thymocytes are modulated by the parasite trans-sialidase (TS). We found that TS affected the dynamics of thymocytes undergoing intrathymic maturation, and these changes were accompanied by an increase in the number of recent DP thymic emigrants in the peripheral lymphoid organs. We demonstrated that increased percentages of blood DP T cell subsets were associated with augmented antibody titers against TS in chagasic patients with chronic cardiomyopathy. In vitro studies showed that TS was able to activate the MAPK pathway and actin filament mobilization in thymocytes. These effects were correlated with its ability to modulate the adhesion of thymocytes to thymic epithelial cells and their migration toward extracellular matrix. These findings point to effects of TS that could influence the escape of immature thymocytes in Chagas disease.

Addition of α-O-GlcNAc to threonine residues define the post-translational modification of mucin-like molecules in Trypanosoma cruzi.

Trypanosoma cruzi, an intracellular protozoan etiologic agent of Chagas disease is covered by a dense coat of mucin-type glycoproteins, which is important to promote the parasite entry and persistence in the mammalian host cells. The O-glycosylation of T. cruzi mucins (Tc-mucins) is initiated by enzymatic addition of α-O-N-acetylglucosamine (GlcNAc) to threonine (Thr) by the UDP-GlcNAc:polypeptide α-N-acetylglucosaminyltransferase (pp-α-GlcNAcT) in the Golgi. The Tc-mucin is characterized by the presence of a high structural diversity of O-linked oligosaccharides found among different parasite strains, comprising two O-glycan Cores. In the Core 1, from strains principally associated with the domestic transmission cycle of Chagas disease, the GlcNAc O-4 is substituted with a ß-galactopyranose (ßGalp) unit, and in the most complex oligosaccharides the GlcNAc O-6 is further processed by the addition of ß1 → 2-linked Galp residues creating a short linear Galp-containing chain. In the Core 2 structures, expressed by strains isolated from T. cruzi sylvatic hosts, the GlcNAc O-4 carries a ß-galactofuranose (ßGalf) unit and the GlcNAc O-6 can carry a branched Galpß1 → 3[Galpß1 → 2]Galpß1 → 6 motif. The O-glycans carrying nonreducing terminal ßGalp are available for sialylation by a surface T. cruzi trans-sialidase activity. Based on structural results, this review summarizes available data on the highly conserved process, which adds the GlcNAc unit in α-linkage to Thr residues the basis of the post-translational modification system in T. cruzi mucins. In addition, a mechanism unique employed by the parasite to transfer exogenous sialic acid residues to Tc-mucins is presented.

Sialic acid: a sweet swing between mammalian host and Trypanosoma cruzi.

Commonly found at the outermost ends of complex carbohydrates in extracellular medium or on outer cell membranes, sialic acids play important roles in a myriad of biological processes. Mammals synthesize sialic acid through a complex pathway, but Trypanosoma cruzi, the agent of Chagas' disease, evolved to obtain sialic acid from its host through a trans-sialidase (TcTS) reaction. Studies of the parasite cell surface architecture and biochemistry indicate that a unique system comprising sialoglycoproteins and sialyl-binding proteins assists the parasite in several functions including parasite survival, infectivity, and host-cell recognition. Additionally, TcTS activity is capable of extensively remodeling host cell glycomolecules, playing a role as virulence factor. This review presents the state of the art of parasite sialobiology, highlighting how the interplay between host and parasite sialic acid helps the pathogen to evade host defense mechanisms and ensure lifetime host parasitism.

Sorting of phosphoglucomutase to glycosomes in Trypanosoma cruzi is mediated by an internal domain.

Trypanosoma cruzi relies on highly galactosylated molecules as virulence factors and the enzymes involved in sugar biosynthesis are potential therapeutic targets. The synthesis of UDP-galactose in T. cruzi requires the activity of phosphoglucomutase (PGM), the enzyme that catalyzes the interconversion of glucose-6-phosphate and glucose-1-phosphate. Several enzymes that participate in carbohydrate metabolism in trypanosomes are confined to specialized peroxisome-like organelles called glycosomes. The majority of glycosomal proteins contain peroxisome-targeting signals (PTS) at the COOH- or at the amino-terminus, which drive their transport to glycosomes. We had previously identified the T. cruzi PGM gene (TcPGM) and demonstrated that it encodes a functional enzyme. Here, we show that, in contrast to yeast and mammalian cells, TcPGM resides in glycosomes of the parasite. However, no classical PTS1 or PTS2 motif is present in its sequence. We investigated glycosomal targeting by generating T. cruzi cell lines expressing different domains of TcPGM fused to the green fluorescent protein (GFP). The analysis of the subcellular localization of fusion proteins revealed that an internal targeting signal of TcPGM, residing between amino acid residues 260 and 380, is capable of targeting GFP to glycosomes. These results demonstrate that, in T. cruzi, PGM import into glycosomes is mediated by a novel non-PTS domain that is located internally in the protein.

Overexpression of the aldose reductase GRE3 suppresses lithium-induced galactose toxicity in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, lithium induces a 'galactosemia-like' phenotype as a consequence of inhibition of phosphoglucomutase, a key enzyme in galactose metabolism. Induced galactose toxicity is prevented by deletion of GAL4, which inhibits the transcriptional activation of genes involved in galactose metabolism and by deletion of the galactokinase (GAL1), indicating that galactose-1-phosphate, a phosphorylated intermediate of the Leloir pathway, is the toxic compound. As an alternative to inhibiting entry and metabolism of galactose, we investigated whether deviation of galactose metabolism from the Leloir pathway would also overcome the galactosemic effect of lithium. We show that cells overexpressing the aldose reductase GRE3, which converts galactose to galactitol, are more tolerant to lithium than wild-type cells when grown in galactose medium and they accumulate more galactitol and less galactose-1-phosphate. Overexpression of GRE3 also suppressed the galactose growth defect of the 'galactosemic'gal7- and gal10-deleted strains, which lack galactose-1-P-uridyltransferase or UDP-galactose-4-epimerase activities, respectively. Furthermore, the effect of GRE3 was independent of the inositol monophosphatases INM1 and INM2. We propose that lithium induces a galactosemic state in yeast and that inhibition of the Leloir pathway before the phosphorylation step or stimulation of galactitol production suppresses lithium-induced galactose toxicity.

Cloning and characterization of the phosphoglucomutase of Trypanosoma cruzi and functional complementation of a Saccharomyces cerevisiae PGM null mutant.

Trypanosoma cruzi is the etiological agent of Chagas' disease, a chronic illness characterized by progressive cardiomyopathy and/or denervation of the digestive tract. The parasite surface is covered with glycoconjugates, such as mucin-type glycoproteins and glycoinositolphospholipids (GIPLs), whose glycans are rich in galactopyranose (Galp) and/or galactofuranose (Galf) residues. These molecules have been implicated in attachment of the parasite to and invasion of mammalian cells and in modulation of the host immune responses during infection. In T. cruzi, galactose (Gal) biosynthesis depends on the conversion of uridine diphosphate (UDP)-glucose (UDP-Glc) into UDP-Gal by an NAD-dependent reduction catalyzed by UDP-Gal 4-epimerase. Phosphoglucomutase (PGM) is a key enzyme in this metabolic pathway catalyzing the interconversion of Glc-6-phosphate (Glc-6-P) and Glc-1-P which is then converted into UDP-Glc. We here report the cloning of T. cruzi PGM, encoding T. cruzi PGM, and the heterologous expression of a functional enzyme in Saccharomyces cerevisiae. T. cruzi PGM is a single copy gene encoding a predicted protein sharing 61% amino acid identity with Leishmania major PGM and 43% with the yeast enzyme. The 59-trans-splicing site of PGM RNA was mapped to a region located at 18 base pairs upstream of the start codon. Expression of T. cruzi PGM in a S. cerevisiae null mutant-lacking genes encoding both isoforms of PGM (pgm1Delta/pgm2Delta) rescued the lethal phenotype induced upon cell growth on Gal as sole carbon source.