RESUMO
Psd1 is a pea plant defensin which can be actively expressed in Pichia pastoris and shows broad antifungal activity. This activity is dependent on fungal membrane glucosylceramide (GlcCer), which is also important for its internalization, nuclear localization, and endoreduplication. Certain cancer cells present a lipid metabolism imbalance resulting in the overexpression of GlcCer in their membrane. In this work, in vitroassays using B16F10 cells showed that labeled fluorescein isothiocyanate FITC-Psd1 internalized into live cultured cells and targeted the nucleus, which underwent fragmentation, exhibiting approximately 60% of cells in the sub-G0/G1 stage. This phenomenon was dependent on GlcCer, and the participation of cyclin-F was suggested. In a murine lung metastatic melanoma model, intravenous injection of Psd1 together with B16F10 cells drastically reduced the number of nodules at concentrations above 0.5 mg/kg. Additionally, the administration of 1 mg/kg Psd1 decreased the number of lung inflammatory cells to near zero without weight loss, unlike animals that received melanoma cells only. It is worth noting that 1 mg/kg Psd1 alone did not provoke inflammation in lung tissue or weight or vital signal losses over 21 days, inferring no whole animal cytotoxicity. These results suggest that Psd1 could be a promising prototype for human lung anti-metastatic melanoma therapy.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Defensinas/farmacologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Pisum sativum/química , Proteínas de Plantas/farmacologia , Animais , Antineoplásicos Fitogênicos/química , Apoptose/efeitos dos fármacos , Biópsia , Linhagem Celular , Permeabilidade da Membrana Celular , Proliferação de Células/efeitos dos fármacos , Defensinas/química , Modelos Animais de Doenças , Feminino , Imunofluorescência , Glucosilceramidas/metabolismo , Imuno-Histoquímica , Neoplasias Pulmonares/tratamento farmacológico , Melanoma Experimental , Camundongos , Modelos Moleculares , Proteínas de Plantas/química , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
C8-desaturated and C9-methylated glucosylceramide (GlcCer) is a fungal-specific sphingolipid that plays an important role in the growth and virulence of many species. In this work, we investigated the contribution of Aspergillus nidulans sphingolipid Δ8-desaturase (SdeA), sphingolipid C9-methyltransferases (SmtA/SmtB) and glucosylceramide synthase (GcsA) to fungal phenotypes, sensitivity to Psd1 defensin and Galleria mellonella virulence. We showed that ΔsdeA accumulated C8-saturated and unmethylated GlcCer, while gcsA deletion impaired GlcCer synthesis. Although increased levels of unmethylated GlcCer were observed in smtA and smtB mutants, ΔsmtA and wild-type cells showed a similar 9,Me-GlcCer content, reduced by 50% in the smtB disruptant. The compromised 9,Me-GlcCer production in the ΔsmtB strain was not accompanied by reduced filamentation or defects in cell polarity. When combined with the smtA deletion, smtB repression significantly increased unmethylated GlcCer levels and compromised filamentous growth. Furthermore, sdeA and gcsA mutants displayed growth defects and raft mislocalization, which were accompanied by reduced neutral lipids levels and attenuated G. mellonella virulence in the ΔgcsA strain. Finally, ΔsdeA and ΔgcsA showed increased resistance to Psd1, suggesting that GlcCer synthesis and fungal sphingoid base structure specificities are relevant not only to differentiation but also to proper recognition by this antifungal defensin.
Assuntos
Aspergillus nidulans/metabolismo , Glucosilceramidas/metabolismo , Glucosiltransferases/metabolismo , Microdomínios da Membrana/metabolismo , Antifúngicos/química , Aspergillus nidulans/genética , Aspergillus nidulans/crescimento & desenvolvimento , Defensinas/metabolismo , Glucosilceramidas/química , Glucosilceramidas/genética , Glucosiltransferases/química , Glucosiltransferases/genética , Metilação , Metiltransferases/genética , Oxirredutases/metabolismo , Esfingolipídeos/química , Esfingolipídeos/metabolismoRESUMO
Glucosylceramides (GlcCer) are the main neutral glycosphingolipids expressed in fungal cells. In this work, glucosylceramides (GlcCer) were extracted from three strains of Scedosporium (Pseudallescheria) boydii, one strain of Pseudallescheria ellipsoidea and one strain of Pseudallescheria angusta and purified by several chromatographic steps. Using high-performance thin layer chromatography (HPTLC), we found a similarity between GlcCer obtained from all of the analysed strains. A detailed structural analysis of the P. ellipsoidea GlcCer was performed via electrospray ionization mass spectrometry (ESI-MS) and confirmed in 1- and 2-D heteronuclear NMR experiments ((1)H-(13) C HSQC). GlcCer species produced by mycelial forms of these strains displayed the same structure previously demonstrated by our group for P. boydii, Cryptococcus neoformans, Pseudallescheria minustipora, Fusarium solani, and Colletotrichum gloesporioides. A monoclonal antibody (mAb) against GlcCer was used for immunofluorescence experiments. Our results revealed that GlcCer is present on the surface of these fungi, and no difference was observed in the GlcCer structure of the present set of strains in terms of geographic or clinical origin, suggesting a conserved GlcCer structure similar to those previously described for Scedosporium apiospermum, Scedosporium aurantiacum, and P. minutispora. The surface distribution of GlcCer in these fungi is suggestive of the involvement of this molecule in fungal growth.
Assuntos
Glucosilceramidas/química , Micoses/microbiologia , Pseudallescheria/metabolismo , Scedosporium/metabolismo , Glucosilceramidas/metabolismo , Humanos , Estrutura Molecular , Pseudallescheria/química , Pseudallescheria/isolamento & purificação , Scedosporium/química , Scedosporium/isolamento & purificação , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Gaucher disease (GD) is an orphan disease characterized by the lack or incapacity of glucocerebrosidase (hGCase) to properly process glucosylceramide, resulting in its accumulation in vital structures of the human body. Enzyme replacement therapy supplies hGCase to GD patients with a high-cost recombinant enzyme produced in vitro in mammalian or plant cell culture. In this study, we produced hGCase through the direct injection of recombinant adenovirus in the mammary gland of a non-transgenic goat. The enzyme was secreted in the milk during six days at a level up to 111.1 ± 8.1 mg/L, as identified by mass spectrometry, showing high in vitro activity. The milk-produced hGCase presented a mass correspondent to the intermediary high-mannose glycosylated protein, which could facilitate its delivery to macrophages through the macrophage mannose receptor. Further studies are underway to determine the in vivo delivery capacity of milk-hGCase, but results from this study paves the way toward the generation of transgenic goats constitutively expressing hGCase in the milk.
Assuntos
Terapia de Reposição de Enzimas , Doença de Gaucher/genética , Glucosilceramidase/biossíntese , Proteínas Recombinantes/administração & dosagem , Adenoviridae/genética , Animais , Feminino , Doença de Gaucher/enzimologia , Doença de Gaucher/patologia , Glucosilceramidase/administração & dosagem , Glucosilceramidase/genética , Glucosilceramidas/metabolismo , Cabras/genética , Humanos , Glândulas Mamárias Animais/enzimologia , Leite/metabolismoRESUMO
Psd1 is a plant defensin that has antifungal activity against several pathogenic and nonpathogenic fungi. Previous analysis of Psd1 chemical shift perturbations by nuclear magnetic resonance (NMR) spectroscopy demonstrated that this defensin interacts with phospholipids and the sphingolipid glucosylceramide isolated from Fusarium solani (GlcCer(Fusarium solani)). In this study, these interactions were evaluated by real-time surface plasmon resonance (SPR) analysis. The data obtained demonstrated that Psd1 could bind more strongly to small unilamellar vesicles (SUV)-containing GlcCer(Fusarium solani) than to SUV that was composed of phosphatidylcholine (PC) alone or was enriched with GlcCer that had been isolated from soybeans. An increase in the SPR response after cholesterol or ergosterol incorporation in PC-SUV was detected; however, SUV composed of PC:Erg (7:3; molar:molar) became unstable in the presence of Psd1, suggesting membrane destabilization. We also observed a lack of Psd1 internalization in Candida albicans strains that were deficient in the glucosyl ceramide synthase gene. Together, these data indicate that GlcCer is essential for Psd1 anchoring in the fungal plasma membrane as well as internalization.
Assuntos
Candida albicans/fisiologia , Defensinas/metabolismo , Glucosilceramidas/metabolismo , Lipossomos/metabolismo , Proteínas de Plantas/metabolismo , Ressonância de Plasmônio de Superfície , Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Defensinas/farmacologia , Endocitose/efeitos dos fármacos , Cinética , Testes de Sensibilidade Microbiana , Microscopia Confocal , Fosfatidilcolinas/metabolismo , Proteínas de Plantas/farmacologiaRESUMO
Scedosporium apiospermum is an emerging fungal pathogen that causes both localized and disseminated infections in immunocompromised patients. Glucosylceramides (CMH, GlcCer) are the main neutral glycosphingolipids expressed in fungal cells. In this study, glucosylceramides (GlcCer) were extracted and purified in several chromatographic steps. Using high-performance thin layer chromatography (HPTLC) and electrospray ionization mass spectrometry (ESI-MS), N-2'-hydroxyhexadecanoyl-1-ß-D-glucopyranosyl-9-methyl-4,8-sphingadienine was identified as the main GlcCer in S. apiospermum. A monoclonal antibody (Mab) against this molecule was used for indirect immunofluorescence experiments, which revealed that this CMH is present on the surface of the mycelial and conidial forms of S. apiospermum. Treatment of S. apiospermum conidia with the Mab significantly reduced fungal growth. In addition, the Mab also enhanced the phagocytosis and killing of S. apiospermum by murine cells. In vitro assays were performed to evaluate the CMHs for their cytotoxic activities against the mammalian cell lines L.929 and RAW, and an inhibitory effect on cell proliferation was observed. Synergistic in vitro interactions were observed between the Mab against GlcCer and both amphotericin B (AmB) and itraconazole. Because Scedosporium species develop drug resistance, the number of available antifungal drugs is limited; our data indicate that combining immunotherapy with the available drugs might be a viable treatment option. These results suggest that in S. apiospermum, GlcCer are most likely cell wall components that are targeted by antifungal antibodies, which directly inhibit fungal development and enhance macrophage function; furthermore, these results suggest the combined use of monoclonal antibodies against GlcCer and antifungal drugs for antifungal immunotherapy.
Assuntos
Glucosilceramidas/metabolismo , Macrófagos/microbiologia , Scedosporium/crescimento & desenvolvimento , Scedosporium/metabolismo , Anfotericina B/farmacologia , Animais , Anticorpos Monoclonais/imunologia , Linhagem Celular , Meios de Cultivo Condicionados/metabolismo , Glucosilceramidas/química , Glucosilceramidas/imunologia , Itraconazol/farmacologia , Masculino , Camundongos , Scedosporium/efeitos dos fármacos , Scedosporium/fisiologiaRESUMO
We show that RsAFP2, a plant defensin that interacts with fungal glucosylceramides, is active against Candida albicans, inhibits to a lesser extent other Candida species, and is nontoxic to mammalian cells. Moreover, glucosylceramide levels in Candida species correlate with RsAFP2 sensitivity. We found RsAFP2 prophylactically effective against murine candidiasis.
Assuntos
Antifúngicos , Candida/efeitos dos fármacos , Candidíase/tratamento farmacológico , Defensinas , Proteínas de Plantas , Animais , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Candida/classificação , Candida/isolamento & purificação , Candida albicans/efeitos dos fármacos , Candida albicans/isolamento & purificação , Candidíase/microbiologia , Defensinas/metabolismo , Defensinas/farmacologia , Defensinas/uso terapêutico , Glucosilceramidas/metabolismo , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Proteínas de Plantas/metabolismo , Proteínas de Plantas/farmacologia , Proteínas de Plantas/uso terapêutico , Resultado do TratamentoRESUMO
The mechanisms by which macromolecules are transported through the cell wall of fungi are not known. A central question in the biology of Cryptococcus neoformans, the causative agent of cryptococcosis, is the mechanism by which capsular polysaccharide synthesized inside the cell is exported to the extracellular environment for capsule assembly and release. We demonstrate that C. neoformans produces extracellular vesicles during in vitro growth and animal infection. Vesicular compartments, which are transferred to the extracellular space by cell wall passage, contain glucuronoxylomannan (GXM), a component of the cryptococcal capsule, and key lipids, such as glucosylceramide and sterols. A correlation between GXM-containing vesicles and capsule expression was observed. The results imply a novel mechanism for the release of the major virulence factor of C. neoformans whereby polysaccharide packaged in lipid vesicles crosses the cell wall and the capsule network to reach the extracellular environment.
Assuntos
Parede Celular/metabolismo , Cryptococcus neoformans/metabolismo , Polissacarídeos Bacterianos/metabolismo , Vesículas Secretórias/metabolismo , Animais , Cápsulas Bacterianas/biossíntese , Transporte Biológico , Linhagem Celular , Centrifugação com Gradiente de Concentração , Espaço Extracelular/metabolismo , Glucosilceramidas/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Polissacarídeos/metabolismo , Esteróis/metabolismoRESUMO
An IgG2a monoclonal antibody anti-glucosylceramide was established and termed MEST-2. High performance thin layer chromatography immunostaining, and solid-phase radioimmunoassay showed that MEST-2 reacts with glucosylceramide from yeast and mycelium forms of Paracoccidioides brasiliensis, Histoplasma capsulatum, and Sporothrix schenckii; from hyphae of Aspergillus fumigatus; and from yeast forms of Candida albicans, Cryptococcus neoformans, Cryptococcus laurentii, and Cryptococcus albidus. Studies on the fine specificity of MEST-2 showed that it recognizes the beta-D-glucose residue, and that the 2-hydroxy group present in the fatty acid is an important auxiliary feature for the antibody binding. It was also demonstrated that phosphatidylcholine and ergosterol modulate MEST-2 reactivity to glucosylceramide, by solid-phase radioimmunoassay. Indirect immunofluorescence showed that MEST-2 reacts with the surface of yeast forms of P. brasiliensis, H. capsulatum and S. schenckii. Weak staining of mycelial forms of P. brasiliensis and hyphae of A. fumigatus was also observed. The availability of a monoclonal antibody specific to fungal glucosylceramide, and its potential use in analyzing biological roles attributed to glucosylceramide in fungi are discussed.
Assuntos
Anticorpos Monoclonais/imunologia , Fungos/metabolismo , Glucosilceramidas/imunologia , Plantas/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Glucosilceramidas/metabolismo , Metabolismo dos Lipídeos , TemperaturaRESUMO
BACKGROUND/AIMS: Undernutrition reduces the hypothalamic ganglioside concentration. This may be attributed to some modifications in the contents of precursors of sphingolipid biosynthesis in undernourished rats. The present study evaluated the serine palmitoyl transferase activity (SPT; EC 2.3.1.50) during the development of the rat hypothalamus. This work also shows the L-[3-(14)C]serine metabolic labeling of hypothalamic sphingolipids in normal and undernourished rats at weaning. METHODS: The SPT activity was determined in microsomal fractions obtained from the hypothalamus of normal rats (diet: 25% protein) and pre- and postnatally undernourished rats (diet: 8% protein since pregnancy) at 21 days of gestational age and at 7, 14, and 21 days of postnatal life. RESULTS: The enzymatic activity was lower in the hypothalamus of undernourished than in the hypothalamus of control rats since the 7th postnatal day. Incorporation of the precursor L-[3-(14)C]serine into sphingolipid fraction was lower in the hypothalamus of undernourished rats than in the hypothalamus of control rats on the 21st postnatal day which coincided with the age of the highest difference in SPT activity between normal and undernourished rats. CONCLUSION: These results indicate that undernutrition reduces the biosynthesis of the main sphingolipids during the period of brain growth spurt.