Lucia Mendonça-Previato

Common Features of Environmental and Potentially Beneficial Plant-Associated Burkholderia

The genus Burkholderia comprises more than 60 species isolated from a wide range of niches. Although they have been shown to be diverse and ubiquitously distributed, most studies have thus far focused on the pathogenic species due to their clinical importance. However, the increasing number of recently described Burkholderia species associated with plants or with the environment has highlighted the division of the genus into two main clusters, as suggested by phylogenetical analyses. The first cluster includes human, animal, and plant pathogens, such as Burkholderia glumae, Burkholderia pseudomallei, and Burkholderia mallei, as well as the 17 defined species of the Burkholderia cepacia complex, while the other, more recently established cluster comprises more than 30 non-pathogenic species, which in most cases have been found to be associated with plants, and thus might be considered to be potentially beneficial. Several species from the latter group share characteristics that are of use when associating with plants, such as a quorum sensing system, the presence of nitrogen fixation and/or nodulation genes, and the ability to degrade aromatic compounds. This review examines the commonalities in this growing subgroup of Burkholderia species and discusses their prospective biotechnological applications.

Expression of functional TLR4 confers proinflammatory responsiveness to Trypanosoma cruzi glycoinositolphospholipids and higher resistance to infection with T. cruzi.

TLRs function as pattern recognition receptors in mammals and play an essential role in the recognition of microbial components. We found that the injection of glycoinositolphospholipids (GIPLs) from Trypanosoma cruzi into the peritoneal cavity of mice induced neutrophil recruitment in a TLR4-dependent manner: the injection of GIPL in the TLR4-deficient strain of mice (C57BL/10ScCr) caused no inflammatory response. In contrast, in TLR2 knockout mice, neutrophil chemoattraction did not differ significantly from that seen in wild-type controls. GIPL-induced neutrophil attraction and MIP-2 production were also severely affected in TLR4-mutant C3H/HeJ mice. The role of TLR4 was confirmed in vitro by testing genetically engineered mutants derived from TLR2-deficient Chinese hamster ovary (CHO)-K1 fibroblasts that were transfected with CD14 (CHO/CD14). Wild-type CHO/CD14 cells express the hamster TLR4 molecule and the mutant line, in addition, expresses a nonfunctional form of MD-2. In comparison to wild-type cells, mutant CHO/CD14 cells failed to respond to GIPLs, indicating a necessity for a functional TLR4/MD-2 complex in GIPL-induced NF-κB activation. Finally, we found that TLR4-mutant mice were hypersusceptible to T. cruzi infection, as evidenced by a higher parasitemia and earlier mortality. These results demonstrate that natural resistance to T. cruzi is TLR4 dependent, most likely due to TLR4 recognition of their GIPLs.

Incorporation of sialic acid into Trypanosoma cruzi macromolecules. A proposal for a new metabolic route

Sialo- and asialoglycoconjugates were isolated from Trypanosoma cruzi epimastigotes and their composition determined. Sialoglycoconjugates bound to wheat germ agglutinin (WGA)-Sepharose and were precipitated by concanavalin A, Wistaria floribunda hemagglutinin and WGA. Asialoglycoconjugate bound to concanavalin A-Sepharose and precipitated with concanavalin-A and W. floribunda hemagglutinin but not with WGA. Cells grown in the presence of fetal calf serum were agglutinated by WGA but not by peanut agglutinin. The reverse was true for cells grown without fetal calf serum. Neuraminidase-treated cells incorporated sialic acid or its 7-carbon analog, 5-acetamido-3,5-dideoxy-L-arabino-2-heptulosonic acid (AcNeu7) from sialylated compounds such as fetuin or sialyl-lactose but did not incorporate free sialic acid. Restoration of the WGA sialylreceptors in neuraminidase-treated cells, as determined by cell agglutination with WGA, was also obtained by incubation with fetuin or sialyl-lactose but not with free sialic acid. Moreover, restoration of agglutinability by WGA in neuraminidase-treated cells or cells grown in medium without fetal calf serum occurred equally well in energy-rich or energy-depleted cells. A transglycosilase reaction for sialic acid incorporation in T. cruzi epimastigotes is suggested.

Capsular polysaccharides galactoxylomannan and glucuronoxylomannan from Cryptococcus neoformans induce macrophage apoptosis mediated by Fas ligand.

The effects of capsular polysaccharides, galactoxylomannan (GalXM) and glucuronoxylomannan (GXM), from acapsular (GXM negative) and encapsulate strains of Cryptococcus neoformans were investigated in RAW 264.7 and peritoneal macrophages. Here, we demonstrate that GalXM and GXM induced different cytokines profiles in RAW 264.7 macrophages. GalXM induced production of TNF‐α, NO and iNOS expression, while GXM predominantly induced TGF‐β secretion. Both GalXM and GXM induced early morphological changes identified as autophagy and late macrophages apoptosis mediated by Fas/FasL interaction, a previously unidentified mechanism of virulence. GalXM was more potent than GXM at induction of Fas/FasL expression and apoptosis on macrophages in vitro and in vivo. These findings uncover a mechanism by which capsular polysaccharides from C. neoformans might compromise host immune responses.

Biosynthesis ofO-N-Acetylglucosamine-linked Glycans inTrypanosoma cruzi CHARACTERIZATION OF THE NOVEL URIDINE DIPHOSPHO-N-ACETYLGLUCOSAMINE:POLYPEPTIDE N-ACETYLGLUCOSAMINYLTRANSFERASE-CATALYZING FORMATION OFN-ACETYLGLUCOSAMINE α1→O-THREONINE

In this study, we have characterized the activity of a uridine diphospho-N-acetylglucosamine:polypeptide-α-N-acetylglucosaminyltransferase (O-α-GlcNAc-transferase) from Trypanosoma cruzi. The activity is present in microsomal membranes and is responsible for the addition of O-linked α-N-acetylglucosamine to cell surface proteins. This preparation adds N-acetylglucosamine to a synthetic peptide KPPTTTTTTTTKPP containing the consensus threonine-rich dodecapeptide encoded by T. cruzi MUC gene (Di Noia, J. M., Sánchez D. O., and Frasch, A. C. C. (1995) J. Biol. Chem. 270, 24146–24149). Incorporation ofN-[3H]acetylglucosamine is linearly dependent on incubation time and concentration of enzyme and substrate. The transferase activity has an optimal pH of 7.5- 8.5, requires Mn2+, is unaffected by tunicamycin or amphomycin, and is strongly inhibited by UDP. The optimized synthetic peptide acceptor for the cytosolic O-GlcNAc-transferase (YSDSPSTST) (Haltiwanger, R. S., Holt, G. D., and Hart, G. W. (1990)J. Biol. Chem. 265, 2563–2568) is not a substrate for this enzyme. The glycosylated KPPTTTTTTTTKPP product is susceptible to base-catalyzed β-elimination, and the presence ofN-acetylglucosamine α-linked to threonine is supported by enzymatic digestion and nuclear magnetic resonance data. These results describe a unique biosynthetic pathway for T. cruzi surface mucin-like molecules, with potential chemotherapeutic implications.

Costimulation of Host T Lymphocytes by a Trypanosomal trans-Sialidase: Involvement of CD43 Signaling

Trans-sialidase is a membrane-bound and shed sialidase from Trypanosoma cruzi, the protozoan parasite responsible for Chagas disease. We investigated the role of soluble trans-sialidase on host CD4+ T cell activation. Trans-sialidase activated naive CD4+ T cells in vivo. Both enzymatically active and inactive recombinant trans-sialidases costimulated CD4+ T cell activation in vitro. Costimulation resulted in increased mitogen-activated protein kinase activation, proliferation, and cytokine synthesis. Furthermore, active and inactive trans-sialidases blocked activation-induced cell death in CD4+ T cells from T. cruzi-infected mice. By flow cytometry, inactive trans-sialidase bound the highly sialylated surface Ag CD43 on host CD4+ T cells. Both costimulatory and antiapoptotic effects of trans-sialidases required CD43 signaling. These results suggest that trans-sialidase family proteins are involved in exacerbated host T lymphocyte responses observed in T. cruzi infection.

STRUCTURAL VARIATION IN THE GLYCOINOSITOLPHOSPHOLIPIDS OF DIFFERENT STRAINS OF TRYPANOSOMA CRUZI

The structures of the glycoinositolphospholipids (GIPLs) from five strains of the protozoan parasiteTrypanosoma cruzi have been determined. Two series of structures were identified, all but one containing the same Man4(AEP)GlcN-Ins-PO4 core. Series 1 oligosaccharides are substituted at the third mannose distal to inositol (Man 3) by ethanolamine-phosphate or 2-aminoethylphosphonic acid, as are some glycosyl-phosphatidylinositol-protein anchors ofT. cruzi. The core can be further substituted by terminal (1–3)-linked β-galactofuranose units. In contrast, Series 2 oligosaccharides do not have additional phosphorus-containing groups attached to Man 3, the latter being substituted instead by a single side chain unit of β-galactofuranose. Series 1 oligosaccharides are present in all strains (G, G-645, Tulahuen CL, and Y) whereas Series 2 structures are present mainly in CL and Y strains. The lipid moiety in the GIPLs from the G, G-645 and Tulahuen strains is predominantly ceramide, as reported for the Y strain, whilst that from the CL strain is a mixture of ceramide and alkylacylglycerol species. The lipid moiety of the GIPLs, and probably also the phosphoinositol-oligosaccharide structures may play an important immunomodulatory role in infection byT. cruzi.

Enzymatically Inactive trans-Sialidase from Trypanosoma cruzi Binds Sialyl and β-Galactopyranosyl Residues in a Sequential Ordered Mechanism

Host/parasite interaction mediated by carbohydrate/lectin recognition results in the attachment to and invasion of host cells and immunoregulation, enabling parasite replication and establishment of infection. Trypanosoma cruzi, the protozoan responsible for Chagas disease, expresses on its surface a family of enzymatically active and inactive trans-sialidases. The parasite uses the active trans-sialidase for glycoprotein sialylation in an unusual trans-glycosylation reaction. Inactive trans-sialidase is a sialic acid-binding lectin that costimulates host T cells through leucosialin (CD43) engagement. The co-mitogenic effect of trans-sialidase can be selectively abrogated by N-acetyllactosamine, suggesting the presence of an additional carbohydrate binding domain for galactosides, in addition to that for sialic acid. Here we investigated the interaction of inactive trans-sialidase in the presence of β-galactosides. By using NMR spectroscopy, we demonstrate that inactive trans-sialidase has a β-galactoside recognition site formed following a conformational switch induced by sialoside binding. Thus prior positioning of a sialyl residue is required for the β-galactoside interaction. When an appropriate sialic acid-containing molecule is available, both sialoside and β-galactoside are simultaneously accommodated in the inactive trans-sialidase binding pocket. This is the first report of a lectin recognizing two distinct ligands by a sequential ordered mechanism. This uncommon binding behavior may play an important role in several biological aspects of T. cruzi/host cell interaction and could shed more light into the catalytic mechanism of the sialic acid transfer reaction of enzymatically active trans-sialidase.

Further studies on the rhamnomannans and acidic rhamnomannans of Sporothrix schenckii and Ceratocystis stenoceras.

Abstract Studies were continued on the 13 C and proton nuclear magnetic resonance spectra of a series of mannose-containing polysaccharides formed by various strains of the closely related fungal species, Sporothrix schenckii and Ceratocystis stenoceras , and the criteria for their differentiation redefined. Exocellular filtrates of C . stenoceras 1099.40 contain mainly a galactan, with smaller proportions of a rhamnomannan having single-unit α- l -rhamnopyranosyl side-chains ( 1 ), an acidic rhamnomannan ( 2 ) containing glucuronic acid, and amylose. The mono-rhamnomannan was characterized by comparison of its 13 C spectrum with that of authentic polysaccharide. The acidic rhamnomannan was characterized partly with the aid of 13 C n.m.r spectroscopy; in the process, fragments obtained by partial hydrolysis were used to assign signals of C-1 and other O -glycosylated 13 C nuclei. Other 13 C signals in spectra of polysaccharides from C. stenoceras and S. schenckii were assigned following tracer experiments that used media containing glucoses labeled with deuterium and 13 C, and by analogy with 13 C signal-displacement effects that are known to occur on O -glycosylation and O -methylation of hydroxyl groups. Evidence was obtained for the presence of structures 3 , 4 , and 5 in exocellular polysaccharide of S. schenckii . These structures give rise to C-1 signals that were assigned by reference to those of (1→4) linked α- d -mannopyranose di- and tri-saccharides.

Involvement of Fungal Cell Wall Components in Adhesion of Sporothrix schenckii to Human Fibronectin

ABSTRACT Systemic sporotrichosis is an emerging infection potentially fatal for immunocompromised patients. Adhesion to extracellular matrix proteins is thought to play a crucial role in invasive fungal diseases. Here we report studies of the adhesion of Sporothrix schenckii to the extracellular protein fibronectin (Fn). Both yeast cells and conidia of S. schenckii were able to adhere to Fn as detected by enzyme-linked immunosorbent binding assays. Adhesion of yeast cells to Fn is dose dependent and saturable.S. schenckii adheres equally well to 40-kDa and 120-kDa Fn proteolytic fragments. While adhesion to Fn was increased by Ca2+, inhibition assays demonstrated that it was not RGD dependent. A carbohydrate-containing cell wall neutral fraction blocked up to 30% of the observed adherence for the yeast cells. The biochemical nature of this fraction suggests the participation of cell surface glycoconjugates in binding by their carbohydrate or peptide moieties. These results provide new data concerning S. schenckii adhesion mechanisms, which could be important in host-fungus interactions and the establishment of sporotrichosis.