Leishmania (Viannia) braziliensis Inositol Phosphorylceramide: Distinctive Sphingoid Base Composition.

Inositol phosphorylceramide (IPC), the major sphingolipid in the genus Leishmania but not found in mammals, is considered a potentially useful target for chemotherapy against leishmaniasis. Leishmania (Viannia) braziliensis is endemic in Latin America and causes American tegumentary leishmaniasis. We demonstrated that IPCs are localized internally in parasites, using a specific monoclonal antibody. Treatment with 5 µM myriocin (a serine palmitoyltransferase inhibitor) rendered promastigotes 8-fold less infective than controls in experimental hamster infection, as determined by number of parasites per inguinal lymph node after 8 weeks infection, suggesting the importance of parasite IPC or sphingolipid derivatives in parasite infectivity or survival in the host. IPC was isolated from promastigotes of three L. (V.) braziliensis strains and analyzed by positive- and negative-ion ESI-MS. The major IPC ions were characterized as eicosasphinganine and eicosasphingosine. Negative-ion ESI-MS revealed IPC ion species at m/z 778.6 (d20:1/14:0), 780.6 (d20:0/14:0), 796.6 (t20:0/14:0), 806.6 (d20:1/16:0), and 808.6 (d20:0/16:0). IPCs isolated from L. (V.) braziliensis and L. (L.) major showed significant differences in IPC ceramide composition. The major IPC ion from L. (L.) major, detected in negative-ion ESI-MS at m/z 780.6, was composed of ceramide d16:1/18:0. Our results suggest that sphingosine synthase (also known as serine palmitoyltransferase; SPT) in L. (V.) braziliensis is responsible for synthesis of a long-chain base of 20 carbons (d20), whereas SPT in L. (L.) major synthesizes a 16-carbon long-chain base (d16). A phylogenetic tree based on SPT proteins was constructed by analysis of sequence homologies in species of the Leishmania and Viannia subgenera. Results indicate that SPT gene position in L. (V.) braziliensis is completely separated from that of members of subgenus Leishmania, including L. (L.) major, L. (L.) infantum, and L. (L.) mexicana. Our findings clearly demonstrate sphingoid base differences between L. (V.) braziliensis and members of subgenus Leishmania, and are relevant to future development of more effective targeted anti-leishmaniasis drugs.

Structural diversity and biological significance of glycosphingolipids in pathogenic and opportunistic fungi.

Glycosphingolipids (GSLs) are ubiquitous membrane components and have key roles in biological systems, acting as second messengers or modulators of signal transduction by affecting several events, ranging from cell adhesion, cell growth, cell motility, regulation of apoptosis and cell cycle. Over the last 20 years our laboratory and other research groups determined the glycan and ceramide structures of more than 20 GSLs from several pathogenic/opportunistic fungi, using a combination of gas chromatography, mass spectrometry, nuclear magnetic resonance as well as other immunochemical and biochemical techniques. Fungal GSLs can be divided in two major classes: neutral GSLs, galactosyl- and glucosylceramide (GlcCer), and acidic GSLs, the glycosylinositol-phosphorylceramides (GIPCs). Glycosyl structures in fungal GIPCs exhibited significant structural diversity and distinct composition when compared to mammalian GSLs, e.g., the expression of inositol-mannose and inositol-glucosamine cores and the terminal residue of ß-D-galactofuranose which are absent in mammalian cells. Studies performed by our group demonstrated that GIPC (Galfß 6[Manα3]Manα2InsPCer) elicited in patients with paracoccidioidomycosis an immune response with production of antibodies directed to the terminal residue of ß-D-galactofuranose. Further studies also showed that inhibition of GlcCer biosynthetic pathways affects fungal colony formation, spore germination and hyphal growth, indicating that enzymes involved in GlcCer biosynthesis may represent promising targets for the therapy of fungal infections. Recently, it was shown that GlcCer and GIPCs are preferentially localized in membrane microdomains and monoclonal antibodies directed to these GSLs interfere in several fungal biological processes such as growth and morphological transition. This review focuses on glycan structures carried on sphingolipids of pathogenic/opportunistic fungi, and aspects of their biological significance are discussed.

Glycolipid sensing and innate immunity in paracoccidioidomycosis.

Distinct glycolipid profiles are described in microorganisms, which have been shown to modulate the innate immune system. We tested the hypothesis that glycosphingolipids from Paracoccidioides brasiliensis have immunomodulatory properties on monocytes and dendritic cells of two groups of healthy individuals, one cured of paracoccidioidomycosis in the past (CUR-I) and the other nonexposed to P. brasiliensis (HNE-I). Two classes of glycosphingolipids purified from yeast cells were evaluated: a neutral glycosphingolipid, monohexosylceramide (CMH), and acidic glycosylinositolphosphorylceramides (GIPCs). Both glycosphingolipids affected the functioning of innate immunity cells, interfering with the antigen presenting process: P. brasiliensis yeast cells phagocytosis, IL-10 secretion, and costimulatory molecules and recognition receptors expression by monocytes were altered, while dendritic cell antigen presentation to autologous T cells was markedly down-modulated as shown by reduced T-cell proliferative responses. The mechanisms by which CMH and GIPCs exert their effects differ since the target cells did not always respond similarly to the challenge with the glycosphingolipids. Moreover, CUR-I and HNE-I presented different responses to the glycosphingolipids. Differences not only in the glycosphingolipid structure (such as the polar head group or the ceramide moiety), but also in the innate immunity properties of CUR-I and HNE-I, may underlie these differences and contribute to individual's susceptibility or resistance to develop paracoccidioidomycosis.

Myriocin, a serine palmitoyltransferase inhibitor, blocks cytokinesis in Leishmania (Viannia) braziliensis promastigotes.

We studied the effect of myriocin, an inhibitor of serine palmitoyltransferase, on cultured Leishmania (Viannia) braziliensis promastigotes. Myriocin significantly reduced synthesis of inositol phosphorylceramide, the major sphingolipid expressed in promastigotes as characterized by thin layer chromatography and electrospray ionization mass spectrometry. Log-phase promastigotes treated with 1 µM myriocin showed a 52% reduction in growth rate and morphological alterations such as more rounded shape and shorter flagellum. Promastigotes treated with myriocin also displayed a variety of aberrant cell phenotypes. The percentage of cells with one nucleus and one kinetoplast (1N1K), following treatment with 1 or 5 µM myriocin, decreased from 89% (control value) to 27% or 3%, respectively. The percentage of cells with two nuclei (2N2K) varied from 7% (control value) to 19% and 6% for 1 or 5 µM myriocin-treated parasites, respectively. High percentage of myriocin-treated parasites exhibited large atypical cells presenting three or more nucleus (32% and 89% for 1 or 5 µM myriocin, respectively). Transmission electron microscopy following treatment with 1 µM myriocin showed the presence of 4N parasites possibly as a result of an incomplete cytokinesis. Addition of 3-ketodihidrosphingosine to myriocin-treated promastigotes rescue parasite growth and morphology. Addition of ethanolamine did not rescue the myriocin effect on parasite. Our findings indicate that sphingolipids are essential for the completion of cytokinesis, and may play a major role in cell proliferation in L. (V.) braziliensis, thus, differing from data described for Leishmania major sphingolipid-free mutant, where addition of ethanolamine rescue wild-type parasite characteristics.

Paracoccidioides brasiliensis induces secretion of IL-6 and IL-8 by lung epithelial cells. Modulation of host cytokine levels by fungal proteases.

Paracoccidioides brasiliensis is a pathogenic, dimorphic fungus that causes paracoccidioidomycosis, a systemic human mycosis that is highly prevalent in Latin America. In this study, we demonstrated that P. brasiliensis yeasts induced interleukin (IL)-8 and IL-6 secretion by human lung epithelial A549 cells. However, tumor necrosis factor-α and interferon-γ were undetectable in these cultures. Moreover, P. brasiliensis yeasts induced activation of p38 mitogen-activated protein kinase (MAPK), c-Jun NH(2)-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2 in A549 cells, and IL-8 and IL-6 secretion promoted by this fungus was dependent on activation of p38 MAPK and ERK 1/2. In addition, IL-8 and IL-6 levels were significantly higher in culture supernatants of A549 cells that were incubated with formaldehyde-fixed P. brasiliensis compared to cultures of cells that were infected with live yeasts. Our results indicate that the observed cytokine level differences were due to protease expression, in live yeasts, that degraded these cytokines. Degradation of human recombinant IL-8 and IL-6 by live P. brasiliensis was inhibited by AEBSF and aprotinin, suggesting that these proteases belong to a family of serine proteases. This is the first report showing that P. brasiliensis may modulate host inflammation by expressing proteases that degrade proinflammatory cytokines.

Membrane microdomain components of Histoplasma capsulatum yeast forms, and their role in alveolar macrophage infectivity.

Analysis of membrane lipids of Histoplasma capsulatum showed that ~40% of fungal ergosterol is present in membrane microdomain fractions resistant to treatment with non-ionic detergent at 4°C. Specific proteins were also enriched in these fractions, particularly Pma1p a yeast microdomain protein marker (a plasma membrane proton ATPase), a 30kDa laminin-binding protein, and a 50kDa protein recognized by anti-α5-integrin antibody. To better understand the role of ergosterol-dependent microdomains in fungal biology and pathogenicity, H. capsulatum yeast forms were treated with a sterol chelator, methyl-beta-cyclodextrin (mßCD). Removal of ergosterol by mßCD incubation led to disorganization of ergosterol-enriched microdomains containing Pma1p and the 30kDa protein, resulting in displacement of these proteins from detergent-insoluble to -soluble fractions in sucrose density gradient ultracentrifugation. mßCD treatment did not displace/remove the 50kDa α5-integrin-like protein nor had effect on the organization of glycosphingolipids present in the detergent-resistant fractions. Ergosterol-enriched membrane microdomains were also shown to be important for infectivity of alveolar macrophages; after treatment of yeasts with mßCD, macrophage infectivity was reduced by 45%. These findings suggest the existence of two populations of detergent-resistant membrane microdomains in H. capsulatum yeast forms: (i) ergosterol-independent microdomains rich in integrin-like proteins and glycosphingolipids, possibly involved in signal transduction; (ii) ergosterol-enriched microdomains containing Pma1p and the 30kDa laminin-binding protein; ergosterol and/or the 30kDa protein may be involved in macrophage infectivity.

Role of host glycosphingolipids on Paracoccidioides brasiliensis adhesion.

Binding of yeast forms to human lung fibroblast cultures was analyzed, aiming to better understand the initial steps of Paracoccidioides brasiliensis infection in humans. A significant P. brasiliensis adhesion was observed either to fibroblasts or to their Triton X-100 insoluble fraction, which contains extracellular matrix and membrane microdomains enriched in glycosphingolipids. Since human lung fibroblasts express at cell-surface gangliosides, such as GM1, GM2, and GM3, the role of these glycosphingolipids on P. brasiliensis adhesion was analyzed by different procedures. Anti-GM3 monoclonal antibody or cholera toxin subunit B (which binds specifically to GM1) reduced significantly fungal adhesion to fibroblast cells, by 35% and 33%, respectively. Direct binding of GM1 to yeast forms of P. brasiliensis was confirmed using cholera toxin subunit B conjugated to AlexaFluor(®)488. It was also demonstrated that P. brasiliensis binds to polystyrene plates coated with galactosylceramide, lactosylceramide, trihexosylceramide, GD3, GM1, GM3, and GD1a, suggesting that glycosphingolipids presenting residues of beta-galactose or neuraminic acid at non-reducing end may act as adhesion molecules for P. brasiliensis. Conversely, no binding was detected when plates were adsorbed with glycosphingolipids that contain terminal residue of beta-N-acetylgalactosamine, such as globoside (Gb4), GM2, and asialo-GM2. In human fibroblast (WI-38 cells), GM3 and GM1 are associated with membrane rafts, which remain insoluble after treatment with Triton X-100 at 4°C. Taken together, these results strongly suggest that lung fibroblast gangliosides, GM3 and GM1, are involved in binding and/or infection by P. brasiliensis.

Changes in cell wall synthesis and ultrastructure during paradoxical growth effect of caspofungin on four different Candida species.

Paradoxical growth (PG) has been described for echinocandins and is characterized by cell growth at drug concentrations above the MIC. In this study, two isolates each of Candida albicans, C. tropicalis, C. orthopsilosis, and C. parapsilosis, all of which displaying PG in response to caspofungin, were subjected to MIC, minimal fungicidal concentration (MFC), and time-kill curve assays to evaluate the levels of PG. Cell wall components and ultrastructural modifications of the PG cells were also investigated. The results showed that when cell growth and survival were evaluated by MFC or time-kill curve assays, high concentrations of caspofungin did not show fungicidal activity against PG cells. Furthermore, for C. parapsilosis and C. orthopsilosis, time-kill curves were more discriminatory than MFCs in detecting the PG effect. The four different Candida species studied demonstrated similar alterations in cell wall components and ultrastructure associated with PG. In PG cells, ß-1,3-glucan content decreased from 2.7- to 7.8-fold, whereas chitin content increased from 4.0- to 6.6-fold. An electron microscopy study of the PG cells revealed morphological alterations, clumping of cells, enlarged cells, the absence of filamentation, abnormal septa, and accumulation of chitin in the cell wall. Also, PG cells basically exhibited a single dark high-density layer in the cell wall, indicating the loss of the ß-1,3-glucan layer. Our results present novel details about the ultrastructural alterations that occur in C. albicans, C. parapsilosis, C. orthopsilosis, and C. tropicalis during PG and show that chitin is the major component of the cell walls of PG cells. Stimulation of chitin synthesis may represent a rescue mechanism against caspofungin activity.

Effect of anti-glycosphingolipid monoclonal antibodies in pathogenic fungal growth and differentiation. Characterization of monoclonal antibody MEST-3 directed to Manpalpha1-->3Manpalpha1-->2IPC.

BACKGROUND: Studies carried out during the 1990's demonstrated the presence of fungal glycoinositol phosphorylceramides (GIPCs) with unique structures, some of them showed reactivity with sera of patients with histoplasmosis, paracoccidioidomycosis or aspergillosis. It was also observed that fungal GIPCs were able to inhibit T lymphocyte proliferation "in vitro", and studies regarding the importance of these molecules to fungal survival showed that many species of fungi are vulnerable to inhibitors of sphingolipid biosynthesis. RESULTS: In this paper, we describe a detailed characterization of an IgG2a monoclonal antibody (mAb), termed MEST-3, directed to the Paracoccidioides brasiliensis glycolipid antigen Pb-2 (Manpalpha1-->3Manpalpha1-->2IPC). mAb MEST-3 also recognizes GIPCs bearing the same structure in other fungi. Studies performed on fungal cultures clearly showed the strong inhibitory activity of MEST-3 on differentiation and colony formation of Paracoccidioides brasiliensis, Histoplasma capsulatum and Sporothrix schenckii. Similar inhibitory results were observed when these fungi where incubated with a different mAb, which recognizes GIPCs bearing terminal residues of beta-D-galactofuranose linked to mannose (mAb MEST-1). On the other hand, mAb MEST-2 specifically directed to fungal glucosylceramide (GlcCer) was able to promote only a weak inhibition on fungal differentiation and colony formation. CONCLUSIONS: These results strongly suggest that mAbs directed to specific glycosphingolipids are able to interfere on fungal growth and differentiation. Thus, studies on surface distribution of GIPCs in yeast and mycelium forms of fungi may yield valuable information regarding the relevance of glycosphingolipids in processes of fungal growth, morphological transition and infectivity.

Current relevance of fungal and trypanosomatid glycolipids and sphingolipids: studies defining structures conspicuously absent in mammals.

Recently, glycosphingolipids have been attracting attention due to their role on biological systems as second messengers or modulators of signal transduction, affecting several events, which range from apoptosis to regulation of the cell cycle. In pathogenic fungi, glycolipids are expressed in two classes: neutral monohexosylceramides (glucosyl-or galactosylceramide) and acidic glycosylinositol phosphorylceramides (the latter class carries longer glycan chains). It is worth to mention that monohexosylceramides exhibit significant structural differences in their lipid moieties compared to their mammalian counterparts, whereas the glycosylinositol phosphorylceramides exhibit remarkable structural differences in their carbohydrate moieties in comparison to mammal glycosphingolipids counterpart. We observed that glycosylinositol phosphorylceramides are capable of promoting immune response in infected humans. In addition, inhibiting fungal glycosphingolipid biosynthetic pathways leads to an inhibition of colony formation, spore germination, cell cycle, dimorphism and hyphal growth. Other pathogens, such as trypanosomatids, also present unique glycolipids, which may have an important role for the parasite development and/or disease establishment. Regarding host-pathogen interaction, cell membrane rafts, which are enriched in sphingolipids and sterols, participate in parasite/fungal infection. In this review, it is discussed the different biological roles of (glyco) (sphingo)lipids of pathogenic/opportunistic fungi and trypanosomatids.

Current relevance of fungal and trypanosomatid glycolipids and sphingolipids: studies defining structures conspicuously absent in mammals

Recently, glycosphingolipids have been attracting attention due to their role on biological systems as second messengers or modulators of signal transduction, affecting several events, which range from apoptosis to regulation of the cell cycle. In pathogenic fungi, glycolipids are expressed in two classes: neutral monohexosylceramides (glucosyl-or galactosylceramide) and acidic glycosylinositol phosphorylceramides (the latter class carries longer glycan chains). It is worth to mention that monohexosylceramides exhibit significant structural differences in their lipid moieties compared to their mammalian counterparts, whereas the glycosylinositol phosphorylceramides exhibit remarkable structural differences in their carbohydrate moieties in comparison to mammal glycosphingolipids counterpart. We observed that glycosylinositol phosphorylceramides are capable of promoting immune response in infected humans. In addition, inhibiting fungal glycosphingolipid biosynthetic pathways leads to an inhibition of colony formation, spore germination, cell cycle, dimorphism and hyphal growth. Other pathogens, such as trypanosomatids, also present unique glycolipids, which may have an important role for the parasite development and/or disease establishment. Regarding host-pathogen interaction, cell membrane rafts, which are enriched in sphingolipids and sterols, participate in parasite/fungal infection. In this review, it is discussed the different biological roles of (glyco) (sphingo) lipids of pathogenic/opportunistic fungi and trypanosomatids.

Recentemente, glicoesfingolipídeos têm atraído atenção devido ao seu papel na biologia celular como segundo-mensageiro ou moduladores da transdução de sinal, afetando vários eventos, desde apoptose até a regulação do ciclo celular. Em fungos patogênicos, existem duas classes de glicolipídeos: monohexosil ceramidas neutras (glucosil-ou galactosilceramida) e glicosilinositol fosforilceramidas (os quais apresentam cadeias de carboidratos mais longas). É importante enfatizar que as monohexosil ceramidas exibem diferenças estruturais nas suas porções lipídicas quando comparadas às de mamíferos, enquanto que glicosilinositol fosforilceramidas exibem diferenças estruturais marcantes em suas porções carboidratos em comparação aos glicoesfingolipídeos de mamíferos. Observamos também que glicosilinositol fosforilceramidas são capazes de promover resposta imune em indíviduos infectados. Além do mais, inibição das vias biossintéticas de glicoesfingolipídeos de fungos acarreta a inibição da formação de colônias, germinação de esporos, ciclo celular, dimorfismo e crescimento de hifas. Outros patógenos, como os tripanosomatídeos, também apresentam glicolipídeos únicos, os quais apresentam um papel importante para o desenvolvimento do parasita e/ou para o estabelecimento da doença. Em relação à interação hospedeiro-patógeno, os "membrane rafts", estruturas da membrana plasmática enriquecidas em esfingolipídeos e esteróis, têm participação fundamental na infecção do parasita/fungo. Nesta revisão, discutimos os diferentes papéis biológicos dos (glico) (esfingo) lipídeos de fungos patogênicos/oportunistas e de tripanosomatídeos.

Modulation of the type I hypersensitivity late phase reaction to OVA by Propionibacterium acnes-soluble polysaccharide.

Late phase reaction (LPR) of immediate hypersensitivity is a Th2 response characterized by eosinophil recruitment and related to allergic asthma pathogenesis. Several strategies were developed trying to control the tissue damage observed in this reaction. Recently, we verified that killed Propionibacterium acnes (P. acnes), a Gram-positive bacillus, immunomodulated LPR in a murine model, potentiating or suppressing it depending on the treatment protocol used. However, the bacterium compounds responsible for this effect are not known, leading us to investigate if P. acnes purified soluble polysaccharide (PS) could be a major component involved on the modulation induced by the bacterium. Recently, we demonstrated that PS, like P. acnes, induces adjuvant effect on DNA vaccine, increases bone marrow dendritic cell precursors in vivo and its maturation in vitro, and modulates in vitro macrophage tumoricidal activity. Herein, we determined the chemical PS composition, which is mainly constituted by galactopyranose, ribopyranose, arabinopyranose, glucopyranose, ribofuranose and mannopyranose, and analyzed its capacity to modulate the immediate hypersensitivity in mice. Animals were subcutaneously implanted with coagulated hen's egg white (HEW) and 14 days later challenged with ovalbumin (OVA) in the footpad, developing a typical LPR after 24h. Similarly to the whole bacterium, Th2 response to OVA was potentiated when PS was administered concomitantly to HEW implantation, by increase in footpad eosinophilia and IL-4-producing spleen cells, and decrease in anti-OVA IgG2a titers and IL-12- or IFN-gamma-producing cells. On the other hand, the reaction was abrogated when HEW implantation was performed 1 week after PS-treatment, by decrease in footpad swelling, eosinophilia and anti-OVA IgG1 levels, and increase in IgG2a titers and IL-12-producing cells. These data suggest that PS seems to be the major P. acnes compound responsible for its effects on the modulation of immediate hypersensitivity reaction in mice.

Interaction of epithelial cell membrane rafts with Paracoccidioides brasiliensis leads to fungal adhesion and Src-family kinase activation.

Membrane rafts are cholesterol- and sphingolipid-enriched cell membrane domains, which are ubiquitous in mammals and play an essential role in different cellular functions, including host cell-pathogen interaction. In this work, by using several approaches, we demonstrated the involvement of epithelial cell membrane rafts in adhesion process of the pathogenic fungus Paracoccidioides brasiliensis. This conclusion was supported by the localization of ganglioside GM1, a membrane raft marker, at P. brasiliensis-epithelial cell contact sites, and by the inhibition of this fungus adhesion to host cells pre-treated with cholesterol-extractor (methyl-beta-cyclodextrin, MbetaCD) or -binding (nystatin) agents. In addition, at a very early stage of P. brasiliensis-A549 cell interaction, this fungus promoted activation of Src-family kinases (SFKs) and extracellular signal-regulated kinase 1/2 (ERK1/2) of these epithelial cells. Whereas SFKs were partially responsible for activation of ERK1/2, membrane raft disruption with MbetaCD in A549 cells led to total inhibition of SFK activation. Taking together, these data indicate for the first time that epithelial cell membrane rafts are essential for P. brasiliensis adhesion and activation of cell signaling molecules.

Trypanosomatid and fungal glycolipids and sphingolipids as infectivity factors and potential targets for development of new therapeutic strategies.

Several (glyco)(sphingo)lipids from different human pathogens have been characterized, and frequently many of these molecules are participating in host-pathogen interaction. In Leishmania (Leishmania) amazonensis, for example, amastigotes present on their surface glycosphingolipids (GSLs) with the structure Galbeta1-3Galalpha, which is recognized by 30 kDa receptor of macrophages. Furthermore, other Leishmania species, such as Leishmania (Leishmania) major and Leishmania (Viannia) braziliensis present glycosylinositolphospholipids (GIPLs) which are involved in Leishmania-macrophage interaction. It is worth to mention that these antigens are not expressed in mammalian cells. Leishmania promastigotes also present inositol phosphorylceramide (IPC), a unique sphingolipid characteristic of fungi and plants. It was observed that IPC synthesis is essential for parasite division, since Aureobasidin A, an inhibitor of IPC synthase, inhibited significantly promastigote and amastigote growths. Recently, it was also demonstrated that GIPLs, IPC and sterols are preferentially present in the parasite membrane microdomains resistant to Triton X-100 at 4 degrees C. The disruption of these microdomains by incubating parasites with methyl-beta-cyclodextrin inhibited significantly macrophage infectivity by Leishmania. Other pathogens, such as fungi, also present unique glycolipids which may have an important role for the fungal development and/or disease establishment. Taking together these results, this review will discuss different biological roles for (glyco)(sphingo)lipids of different pathogens.

Analysis of glycosylinositol phosphorylceramides expressed by the opportunistic mycopathogen Aspergillus fumigatus.

Acidic glycosphingolipid components were extracted from the opportunistic mycopathogen Aspergillus fumigatus and identified as inositol phosphorylceramide and glycosylinositol phosphorylceramides (GIPCs). Using nuclear magnetic resonance sppectroscopy, mass spectrometry, and other techniques, the structures of six major components were elucidated as Ins-P-Cer (Af-0), Manp(alpha1-->3)Manp(alpha1-->2)Ins-P-Cer (Af-2), Manp(alpha1-->2)Manp(alpha1-->3)Manp(alpha1-->2)Ins-P-Cer (Af-3a), Manp(alpha1-->3)[Galf(beta1-->6)]Manp(alpha1-->2)-Ins-P-Cer (Af-3b), Manp(alpha1-->2)-Manp(alpha1-->3)[Galf(beta1-->6)]Manp(alpha1-->2)Ins-P-Cer (Af-4), and Manp(alpha1-->3)Manp(alpha1-->6)GlcpN(alpha1-->2)Ins-P-Cer (Af-3c) (where Ins = myo-inositol and P = phosphodiester). A minor A. fumigatus GIPC was also identified as the N-acetylated version of Af-3c (Af-3c*), which suggests that formation of the GlcNalpha1-->2Ins linkage may proceed by a two-step process, similar to the GlcNalpha1-->6Ins linkage in glycosylphosphatidylinositol (GPI) anchors (transfer of GlcNAc, followed by enzymatic de-N-acetylation). The glycosylinositol of Af-3b, which bears a distinctive branching Galf(beta1-->6) residue, is identical to that of a GIPC isolated previously from the dimorphic mycopathogen Paracoccidioides brasiliensis (designated Pb-3), but components Af-3a and Af-4 have novel structures. Overlay immunostaining of A. fumigatus GIPCs separated on thin-layer chromatograms was used to assess their reactivity against sera from a patient with aspergillosis and against a murine monoclonal antibody (MEST-1) shown previously to react with the Galf(beta1-->6) residue in Pb-3. These results are discussed in relation to pathogenicity and potential approaches to the immunodiagnosis of A. fumigatus.

Inhibition of Leishmania (Leishmania) amazonensis growth and infectivity by aureobasidin A.

OBJECTIVES: To study the effect of aureobasidin A, an inhibitor of inositol phosphorylceramide (IPC) synthase, on Leishmania growth and infectivity. METHODS: Effects of aureobasidin A were determined for: (i) promastigote growth in axenic culture; (ii) promastigote infectivity in macrophage monolayers; (iii) development of footpad lesions in BALB/c mice; (iv) differentiation of amastigotes into promastigotes. RESULTS: Aureobasidin A (20 microM) inhibited 90% of Leishmania (Leishmania) amazonensis promastigote growth in axenic culture, but the parasites remained viable, i.e. growth curves returned to normal after aureobasidin A was removed from culture medium. The aureobasidin A IC50 was determined by MTT assay as 4.1 microM for L. (L.) amazonensis promastigotes, 12.6 microM for Leishmania (Leishmania) major and 13.7 microM for Leishmania (Viannia) braziliensis. There was a significant delay in infection when L. (L.) amazonensis promastigotes pre-treated with aureobasidin A were inoculated into BALB/c mouse footpads. When aureobasidin A was added to cultured macrophages infected with amastigotes, the number of infected macrophages was reduced by >90%. CONCLUSIONS: Aureobasidin A is an interesting pharmacological tool to investigate the effect of lipid metabolism inhibition in Leishmania spp.

Expression of antibodies directed to Paracoccidioides brasiliensis glycosphingolipids during the course of paracoccidioidomycosis treatment.

Paracoccidioidomycosis (PCM) is a granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. The immunoglobulin classes and isotypes of antibodies directed to acidic glycosphingolipids (GSLs) and glucosylceramide of P. brasiliensis were determined by enzyme-linked immunosorbent assay of sera from 31 PCM patients. The reactivities of 38 serum samples were analyzed by considering the stage of treatment: before antifungal treatment (n = 10), during 1 to 4 months of treatment (T1-4; n = 9), during 5 to 12 months of treatment (T5-12; n = 9), and posttreatment (PT; n = 10). Sera from healthy subjects (n = 12) were used as controls. Only the GSL Pb-1 antigen, which presents the carbohydrate structure Galfbeta1-6(Manalpha1-3)Manbeta1, was reactive with the PCM patient sera. The PCM patient sera did not react with Pb-2, which lacks the Galf residue and which is considered the biosynthetic precursor of Pb-1, indicating that the Galf residue is essential for antibody reactivity. The Pb-1 glycolipid from nontreated patients elicited a primary immune response with immunoglobulin M (IgM) production and subsequent switching to IgG1 production. The IgG1 titer increased after the start of antifungal treatment (T1-4 group), and general decreases in the anti-Pb-1 antibody titers were observed after 5 months of treatment (T5-12 and PT groups). The Pb-1 antigen, an acidic GSL with terminal Galf residue, has potential application as an elicitor of the host immune response in patients with PCM.

The Hemolymph of the ascidian Styela plicata (Chordata-Tunicata) contains heparin inside basophil-like cells and a unique sulfated galactoglucan in the plasma.

The hemolymph of ascidians (Chordata-Tunicata) contains different types of hemocytes embedded in a liquid plasma. In the present study, heparin and a sulfated heteropolysaccharide were purified from the hemolymph of the ascidian Styela plicata. The heteropolysaccharide occurs free in the plasma, is composed of glucose ( approximately 60%) and galactose ( approximately 40%), and is highly sulfated. Heparin, on the other hand, occurs in the hemocytes, and high performance liquid chromatography of the products formed by degradation with specific lyases revealed that it is composed mainly by the disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4)) (39.7%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(6SO(4)) (38.2%). Small amounts of the 3-O-sulfated disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4)) (9.8%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4))(6SO(4)) (3.8%) were also detected. These 3-O-sulfated disaccharides were demonstrated to be essential for the binding of the hemocyte heparin to antithrombin III. Electron microscopy techniques were used to characterize the ultrastructure of the hemocytes and to localize heparin and histamine in these cells. At least five cell types were recognized and classified as univacuolated and multivacuolated cells, amebocytes, hemoblasts, and granulocytes. Immunocytochemistry showed that heparin and histamine co-localize in intracellular granules of only one type of hemocyte, the granulocyte. These results show for the first time that in ascidians, a sulfated galactoglucan circulates free in the plasma, and heparin occurs as an intracellular product of a circulating basophil-like cell.

Glycosphingolipid expression in squamous cell carcinoma of the upper aerodigestive tract.

UNLABELLED: Glycosphingolipids are integral constituents of cellular membrane, arranged in rafts, and with neoplastic cell anti-social behavior, like uncontrolled cell growth, invasiveness, and metastatic potential. AIM: However, there are few studies about glycosphingolipids (GSL) expression in squamous cell carcinoma (SCC). Since GSL are known to be tumor-associated markers we decided to perform a prospective study on the GSL profiles of SCC. METHOD: Specimens of 33 SCC and normal mucosa were obtained and GSLs were extracted and purified by reverse-phase chromatography on C18 column and alkaline hydrolysis in methanol. GSLs were quantified using densitometry of orcinol-stained HPTLC plates. RESULT: A significant increase of GSLs in SCC (3.57 microg/mg) was observed as compared to normal mucosa (1.92 microg/mg). In SCC, an increase of 2 to 3 times in the amounts of CDH, CTH, Globoside, and GM3 was observed in comparison to normal mucosa. The identification of GM3 as well as its increased expression in SCC was confirmed unequivocally by HPTLC immunostaining and indirect immunofluorescence using MAb DH2 (anti-GM3). BY analyzing SCC and normal mucosa CMHs by GC/MS, normal mucosa expresses only glucosylceramide whereas SCC cells express both glucosylceramide and galactosylceramide. CONCLUSION: The increase in the amount of GSLs in tumor tissue may represent changes of cell membrane microdomains resulting from the malignant transformation process, which is responsible for greater cell-cell or cell-matrix interaction thereby increasing their potential for infiltration and metastasis.

Characterization of Leishmania (Viannia) braziliensis membrane microdomains, and their role in macrophage infectivity.

Detergent-resistant membranes (DRMs) from Leishmania (Viannia) braziliensis promastigotes, insoluble in 1% Triton X-100 at 4 degrees C, were fractionated by sucrose density gradient ultracentrifugation. They were composed of glycoinositolphospholipids (GIPLs), inositol phosphorylceramide (IPC), phosphatidylinositol (PI), phosphatidylethanolamine (PE), and sterols. In contrast, 1% Triton X-100-soluble fraction was composed of PE, phosphatidylcholine, phosphatidylserine, PI, IPC, sterol, and lyso-PI. High-performance thin-layer chromatography (HPTLC) immunostaining using monoclonal antibody SST-1 showed that 85% of GIPLs are present in DRMs, and immunoelectron microscopic analysis showed that SST-1-reactive components are located in patches along the parasite surface. No difference in GIPL pattern was observed by HPTLC between Triton X-100-soluble versus -insoluble fractions at 4 degrees C. Analysis of fatty acid composition in DRMs by GC-MS showed the presence of GIPLs containing an alkylacylglycerol, presenting mainly saturated acyl and alkyl chains. DRMs also contained sterol, IPC with saturated fatty acids, PI with at least one saturated acyl chain, and PE with predominantly oleic acid. Promastigotes treated with methyl-beta-cyclodextrin to disrupt lipid microdomains showed significantly lower macrophage infectivity, suggesting a relationship between lipid microdomains and the infectivity of these parasites.