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.

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.