RESUMO
BACKGROUND: Cyclic ß-1,2-glucans (CßG) are bacterial cyclic homopolysaccharides with interesting biotechnological applications. These ring-shaped molecules have a hydrophilic surface that confers high solubility and a hydrophobic cavity able to include poorly soluble molecules. Several studies demonstrate that CßG and many derivatives can be applied in drug solubilization and stabilization, enantiomer separation, catalysis, synthesis of nanomaterials and even as immunomodulators, suggesting these molecules have great potential for their industrial and commercial exploitation. Nowadays, there is no method to produce CßG by chemical synthesis and bacteria that synthesize them are slow-growing or even pathogenic, which makes the scaling up of the process difficult and expensive. Therefore, scalable production and purification methods are needed to afford the demand and expand the repertoire of applications of CßG. RESULTS: We present the production of CßG in specially designed E. coli strains by means of the deletion of intrinsic polysaccharide biosynthetic genes and the heterologous expression of enzymes involved in CßG synthesis, transport and succinilation. These strains produce different types of CßG: unsubstituted CßG, anionic CßG and CßG of high size. Unsubstituted CßG with a degree of polymerization of 17 to 24 glucoses were produced and secreted to the culture medium by one of the strains. Through high cell density culture (HCDC) of that strain we were able to produce 4,5 g of pure unsubstituted CßG /L in culture medium within 48 h culture. CONCLUSIONS: We have developed a new recombinant bacterial system for the synthesis of cyclic ß-1,2-glucans, expanding the use of bacteria as a platform for the production of new polysaccharides with biotechnological applications. This new approach allowed us to produce CßG in E. coli with high yields and the highest volumetric productivity reported to date. We expect this new highly scalable system facilitates CßG availability for further research and the widespread use of these promising molecules across many application fields.
Assuntos
Escherichia coli , beta-Glucanas , Escherichia coli/metabolismo , Escherichia coli/genética , beta-Glucanas/metabolismoRESUMO
S-layer (glyco)-proteins (SLPs) form a nanostructured envelope that covers the surface of different prokaryotes and show immunomodulatory activity. Previously, we have demonstrated that the S-layer glycoprotein from probiotic Lactobacillus kefiri CIDCA 8348 (SLP-8348) is recognized by Mincle (macrophage inducible C-type lectin receptor), and its adjuvanticity depends on the integrity of its glycans. However, the glycan's structure has not been described so far. Herein, we analyze the glycosylation pattern of three SLPs, SLP-8348, SLP-8321, and SLP-5818, and explore how these patterns impact their recognition by C-type lectin receptors and the immunomodulatory effect of the L. kefiri SLPs on antigen-presenting cells. High-performance anion-exchange chromatography-pulse amperometric detector performed after ß-elimination showed glucose as the major component in the O-glycans of the three SLPs; however, some differences in the length of hexose chains were observed. No N-glycosylation signals were detected in SLP-8348 and SLP-8321, but SLP-5818 was observed to have two sites carrying complex N-glycans based on a site-specific analysis and a glycomic workflow of the permethylated glycans. SLP-8348 was previously shown to enhance LPS-induced activation on both RAW264.7 macrophages and murine bone marrow-derived dendritic cells; we now show that SLP-8321 and SLP-5818 have a similar effect regardless of the differences in their glycosylation patterns. Studies performed with bone marrow-derived dendritic cells from C-type lectin receptor-deficient mice revealed that the immunostimulatory activity of SLP-8321 depends on its recognition by Mincle, whereas SLP-5818's effects are dependent on SignR3 (murine ortholog of human DC-SIGN). These findings encourage further investigation of both the potential application of these SLPs as new adjuvants and the protein glycosylation mechanisms in these bacteria.
Assuntos
Antígenos CD/metabolismo , Lactobacillus/metabolismo , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Antígenos CD/genética , Cromatografia Líquida de Alta Pressão , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Glicopeptídeos/análise , Glicopeptídeos/química , Glicosilação , Imunização , Interferon gama/metabolismo , Lectinas Tipo C/deficiência , Lectinas Tipo C/genética , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/química , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Polissacarídeos/análise , Polissacarídeos/química , Células RAW 264.7 , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
This study shows the effects of tamoxifen, a known estrogen receptor antagonist used in the treatment of breast cancer, on the sphingolipid pathway of Trypanosoma cruzi, searching for potential chemotherapeutic targets. A dose-dependent epimastigote growth inhibition at increasing concentration of tamoxifen was determined. In blood trypomastigotes, treatment with 10⯵M showed 90% lysis, while 86% inhibition of intracellular amastigote development was obtained using 50⯵M. Lipid extracts from treated and non-treated metabolically labelled epimastigotes evidenced by thin layer chromatography different levels of sphingolipids and MALDI-TOF mass spectrometry analysis assured the identity of the labelled species. Comparison by HPLC-ESI mass spectrometry of lipids, notably exhibited a dramatic increase in the level of ceramide in tamoxifen-treated parasites and a restrained increase of ceramide-1P and sphingosine, indicating that the drug is acting on the enzymes involved in the final breakdown of ceramide. The ultrastructural analysis of treated parasites revealed characteristic morphology of cells undergoing an apoptotic-like death process. Flow cytometry confirmed cell death by an apoptotic-like machinery indicating that tamoxifen triggers this process by acting on the parasitic sphingolipid pathway.
Assuntos
Antiprotozoários/farmacologia , Estágios do Ciclo de Vida/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Esfingolipídeos/antagonistas & inibidores , Tamoxifeno/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Ceramidas/antagonistas & inibidores , Ceramidas/biossíntese , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Modelos Animais de Doenças , Reposicionamento de Medicamentos , Antagonistas de Estrogênios/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Esfingolipídeos/biossíntese , Esfingosina/antagonistas & inibidores , Esfingosina/biossíntese , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismoRESUMO
Brucellaceae are stealthy pathogens with the ability to survive and replicate in the host in the context of a strong immune response. This capacity relies on several virulence factors that are able to modulate the immune system and in their structural components that have low proinflammatory activities. Lipopolysaccharide (LPS), the main component of the outer membrane, is a central virulence factor of Brucella, and it has been well established that it induces a low inflammatory response. We describe here the identification and characterization of a novel periplasmic protein (RomA) conserved in alpha-proteobacteria, which is involved in the homeostasis of the outer membrane. A mutant in this gene showed several phenotypes, such as membrane defects, altered LPS composition, reduced adhesion, and increased virulence and inflammation. We show that RomA is involved in the synthesis of LPS, probably coordinating part of the biosynthetic complex in the periplasm. Its absence alters the normal synthesis of this macromolecule and affects the homeostasis of the outer membrane, resulting in a strain with a hyperinflammatory phenotype. Our results suggest that the proper synthesis of LPS is central to maximize virulence and minimize inflammation.
Assuntos
Proteínas de Bactérias/fisiologia , Brucella/metabolismo , Brucelose/microbiologia , Lipopolissacarídeos/biossíntese , Animais , Brucella/patogenicidade , Gentamicinas , Inflamação/metabolismo , Camundongos , Transporte Proteico , VirulênciaRESUMO
Parasites of the genus Plasmodium responsible for Malaria are obligate intracellular pathogens residing in mammalian red blood cells, hepatocytes, or mosquito midgut epithelial cells. Regarding that detailed knowledge on the sphingolipid biosynthetic pathway of the apicomplexan protozoan parasites is scarce, different stages of Plasmodium falciparum were treated with tamoxifen in order to evaluate the effects of this drug on the glycosphingolipid biosynthesis. Thin layer chromatography, High performance reverse phase chromatography and UV-MALDI-TOF mass spectrometry were the tools used for the analysis. In the ring forms, the increase of NBD-phosphatidyl inositol biosynthesis was notorious but differences at NBD-GlcCer levels were undetectable. In trophozoite forms, an abrupt decrease of NBD-acylated GlcDHCer and NBD-GlcDHCer in addition to an increase of NBD-PC biosynthesis was observed. On the contrary, in schizonts, tamoxifen seems not to be producing substantial changes in lipid biosynthesis. Our findings indicate that in this parasite, tamoxifen is exerting an inhibitory action on Glucosylceramidesynthase and sphingomyelin synthase levels. Moreover, regarding that Plasmodium does not biosynthesize inositolphosphoceramides, the accumulation of phosphatidylinositol should indicate an inhibitory action on glycosylinositol phospholipid synthesis.
Assuntos
Vias Biossintéticas/efeitos dos fármacos , Eritrócitos/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Esfingolipídeos/biossíntese , Tamoxifeno/farmacologia , Apicomplexa , Cromatografia de Fase Reversa , Eritrócitos/metabolismo , Glicoesfingolipídeos/análise , Estágios do Ciclo de Vida , Espectrometria de Massas , Fosfatidilinositóis/análise , Infecções por Protozoários , Esfingolipídeos/análiseRESUMO
In this work, the presence of sulfated N-glycans was studied in a high-mannose-type glycoprotein of Trypanosoma cruzi with serinecarboxipeptidase (TcSCP) activity. The immune cross-reactivity between purified SCP and Cruzipain (Cz) was evidenced using rabbit sera specific for both glycoproteins. Taking advantage that SCP co-purifies with Cz from Concanavalin-A affinity columns, the Cz-SCP mixture was desulfated, ascribing the cross-reactivity to the presence of sulfate groups in both molecules. Therefore, knowing that Cz is a sulfated glycoprotein, with antigenic sulfated epitopes (sulfotopes), SCP was excised from SDS-PAGE and the N-glycosydic chains were analyzed by UV-MALDI-TOF-MS, confirming the presence of short-sulfated high-mannose-type oligosaccharidic chains. Besides, the presence of sulfotopes was analyzed in lysates of the different parasite stages demonstrating that a band with apparent molecular weight similar to SCP was highly recognized in trypomastigotes. In addition, SCP was confronted with sera of infected people with different degrees of cardiac dysfunction. Although most sera recognized it in different groups, no statistical association was found between sera antibodies specific for SCP and the severity of the disease. In summary, our findings demonstrate (1) the presence of sulfate groups in the N-glycosidic short chains of native TcSCP, (2) the existence of immune cross-reactivity between Cz and SCP, purified from epimastigotes, (3) the presence of common sulfotopes between both parasite glycoproteins, and (4) the enhanced presence of sulfotopes in trypomastigotes, probably involved in parasite-host relationship and/or infection. Interestingly, we show for the first time that SCP is a minor antigen recognized by most of chronic Chagas disease patient's sera.
Assuntos
Antígenos de Protozoários/imunologia , Carboxipeptidases/imunologia , Doença de Chagas/imunologia , Glicoproteínas/imunologia , Trypanosoma cruzi/imunologia , Animais , Antígenos de Protozoários/química , Antígenos de Protozoários/metabolismo , Carboxipeptidases/química , Carboxipeptidases/metabolismo , Reações Cruzadas , Cisteína Endopeptidases/imunologia , Glicoproteínas/química , Glicoproteínas/metabolismo , Humanos , Espectrometria de Massas , Processamento de Proteína Pós-Traducional , Proteínas de Protozoários , Coelhos , Sulfatos/análise , Trypanosoma cruzi/enzimologiaRESUMO
Type IV pili (Tfp) are widely distributed adhesins of bacterial surfaces. In plant pathogenic bacteria, Tfp are involved in host colonization and pathogenesis. Xanthomonas citri subsp. citri (Xcc) is the phytopathogen responsible for citrus canker disease. In this work, three Tfp structural genes, fimA, fimA1, and pilA from Xcc were studied. A pilA mutant strain from Xcc (XccΔpilA) was constructed and differences in physiological features, such as motilities, adhesion, and biofilm formation, were observed. A structural study of the purified Tfp fractions from Xcc wild-type and Xcc∆pilA showed that pilins are glycosylated in both strains and that FimA and FimA1 are the main structural components of the pili. Furthermore, smaller lesion symptoms and reduced bacterial growth were produced by Xcc∆pilA in orange plants compared to the wild-type strain. These results indicate that the minor pilin-like gene, pilA, is involved in Tfp performance during the infection process.
Assuntos
Proteínas de Bactérias/metabolismo , Citrus/microbiologia , Proteínas de Fímbrias/metabolismo , Doenças das Plantas/microbiologia , Xanthomonas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Fímbrias/genética , Deleção de Genes , Virulência , Xanthomonas/genética , Xanthomonas/patogenicidadeRESUMO
Ubiquinone 9 (UQ9), the expected product of the long-chain solanesyl diphosphate synthase of Trypanosoma brucei (TbSPPS), has a central role in reoxidation of reducing equivalents in the mitochondrion of T. brucei. The ablation of TbSPPS gene expression by RNA interference increased the generation of reactive oxygen species and reduced cell growth and oxygen consumption. The addition of glycerol to the culture medium exacerbated the phenotype by blocking its endogenous generation and excretion. The participation of TbSPPS in UQ synthesis was further confirmed by growth rescue using UQ with 10 isoprenyl subunits (UQ10). Furthermore, the survival of infected mice was prolonged upon the downregulation of TbSPPS and/or the addition of glycerol to drinking water. TbSPPS is inhibited by 1-[(n-oct-1-ylamino)ethyl] 1,1-bisphosphonic acid, and treatment with this compound was lethal for the cells. The findings that both UQ9 and ATP pools were severely depleted by the drug and that exogenous UQ10 was able to fully rescue growth of the inhibited parasites strongly suggest that TbSPPS and UQ synthesis are the main targets of the drug. These two strategies highlight the importance of TbSPPS for T. brucei, justifying further efforts to validate it as a new drug target.
Assuntos
Alquil e Aril Transferases/metabolismo , Estágios do Ciclo de Vida , Nitrilas/farmacologia , Proteínas de Protozoários/metabolismo , Piridinas/farmacologia , Trypanosoma brucei brucei/enzimologia , Alquil e Aril Transferases/antagonistas & inibidores , Alquil e Aril Transferases/genética , Animais , Doxiciclina/uso terapêutico , Inibidores Enzimáticos/farmacologia , Glicerol/uso terapêutico , Indóis , Maleimidas , Camundongos , Nitrilas/farmacocinética , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Piridinas/farmacocinética , Espécies Reativas de Oxigênio/metabolismo , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/patogenicidade , Tripanossomíase/tratamento farmacológico , Ubiquinona/biossínteseRESUMO
American Trypanosomiasis or Chagas disease (ChD), a major problem that is still endemic in large areas of Latin America, is caused by Trypanosoma cruzi. This agent holds a major antigen, cruzipain (Cz). Its C-terminal domain (C-T) is retained in the glycoprotein mature form and bears several post-translational modifications. Glycoproteins containing sulfated N-linked oligosaccharides have been mostly implicated in numerous specific procedures of molecular recognition. The presence of sulfated oligosaccharides was demonstrated in Cz, also in a minor abundant antigen with serine-carboxypeptidase (SCP) activity, as well as in parasite sulfatides. Sulfate-bearing glycoproteins in Trypanosomatids are targets of specific immune responses. T. cruzi chronically infected subjects mount specific humoral immune responses to sulfated Cz. Unexpectedly, in the absence of infection, mice immunized with C-T, but not with sulfate-depleted C-T, showed ultrastructural heart anomalous pathological effects. Moreover, the synthetic anionic sugar conjugate GlcNAc6SO3-BSA showed to mimic the N-glycan-linked sulfated epitope (sulfotope) humoral responses that natural Cz elicits. Furthermore, it has been reported that sulfotopes participate via the binding of sialic acid Ig-like-specific lectins (Siglecs) to sulfosialylated glycoproteins in the immunomodulation by host-parasite interaction as well as in the parasite infection process. Strikingly, recent evidence involved Cz-sulfotope-specific antibodies in the immunopathogenesis and infection processes during the experimental ChD. Remarkably, sera from chronically T. cruzi-infected individuals with mild disease displayed higher levels of IgG2 antibodies specific for sulfated glycoproteins and sulfatides than those with more severe forms of the disease, evidencing that T. cruzi sulfotopes are antigenic independently of the sulfated glycoconjugate type. Ongoing assays indicate that antibodies specific for sulfotopes might be considered biomarkers of human cardiac ChD progression, playing a role as predictors of stability from the early mild stages of chronic ChD.
Assuntos
Doença de Chagas , Trypanosoma cruzi , Humanos , Animais , Camundongos , Trypanosoma cruzi/metabolismo , Sulfatos/metabolismo , Sulfoglicoesfingolipídeos , Doença de Chagas/parasitologia , Glicoconjugados , Proteínas de Protozoários , Glicoproteínas/metabolismo , OligossacarídeosRESUMO
Xanthomonas axonopodis pv. citri (Xac) causes citrus canker, provoking defoliation and premature fruit drop with concomitant economical damage. In plant pathogenic bacteria, lipopolysaccharides are important virulence factors, and they are being increasingly recognized as major pathogen-associated molecular patterns for plants. In general, three domains are recognized in a lipopolysaccharide: the hydrophobic lipid A, the hydrophilic O-antigen polysaccharide, and the core oligosaccharide, connecting lipid A and O-antigen. In this work, we have determined the structure of purified lipopolysaccharides obtained from Xanthomonas axonopodis pv. citri wild type and a mutant of the O-antigen ABC transporter encoded by the wzt gene. High pH anion exchange chromatography and matrix-assisted laser desorption/ionization mass spectrum analysis were performed, enabling determination of the structure not only of the released oligosaccharides and lipid A moieties but also the intact lipopolysaccharides. The results demonstrate that Xac wild type and Xacwzt LPSs are composed mainly of a penta- or tetra-acylated diglucosamine backbone attached to either two pyrophosphorylethanolamine groups or to one pyrophosphorylethanolamine group and one phosphorylethanolamine group. The core region consists of a branched oligosaccharide formed by Kdo2Hex6GalA3Fuc3NAcRha4 and two phosphate groups. As expected, the presence of a rhamnose homo-oligosaccharide as O-antigen was determined only in the Xac wild type lipopolysaccharide. In addition, we have examined how lipopolysaccharides from Xac function in the pathogenesis process. We analyzed the response of the different lipopolysaccharides during the stomata aperture closure cycle, the callose deposition, the expression of defense-related genes, and reactive oxygen species production in citrus leaves, suggesting a functional role of the O-antigen from Xac lipopolysaccharides in the basal response.
Assuntos
Citrus sinensis/imunologia , Citrus sinensis/microbiologia , Imunidade Inata , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Xanthomonas axonopodis/fisiologia , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Carboidratos , Citrus sinensis/anatomia & histologia , Citrus sinensis/genética , Regulação da Expressão Gênica de Plantas/imunologia , Interações Hospedeiro-Patógeno , Imunidade Inata/genética , Lipopolissacarídeos/biossíntese , Lipopolissacarídeos/isolamento & purificação , Dados de Sequência Molecular , Peróxidos/metabolismo , Folhas de Planta/genética , Folhas de Planta/imunologia , Folhas de Planta/microbiologia , Estômatos de Plantas/anatomia & histologia , Estômatos de Plantas/imunologia , Estômatos de Plantas/microbiologia , Xanthomonas axonopodis/metabolismoRESUMO
RATIONALE: In developing countries, Shigella flexneri (Sf) is the major causative agent of the endemic shigellosis (bacillary dysentery) responsible annually for one million fatalities mostly among infants. Lipopolysaccharides (LPSs) are characteristic components of the outer membrane of the overwhelming majority of Gram-negative bacteria. Since lipid A is essential for the viability of the Gram-negative bacteria, it is subject to extensive chemical studies with new analytical techniques. METHODS: Lipid A was released by mild acid hydrolysis from the lipopolysaccharide which was obtained via the phenol/water extraction, purified and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and matrix-assisted laser desorption/ionization laser-induced dissociation tandem mass spectrometry (MALDI-LID-MS/MS). RESULTS: A detailed structural study of the whole lipid A obtained from S. flexneri variant X was carried out for the first time. Thus, we have shown that lipid A is a heterogeneous mixture having different numbers of acylated and phosphoethanolamine groups attached to the diglucosamine backbone. Furthermore, we found in the phenol phase an unusual hepta-acylated lipid A species, although the abundance was very low. CONCLUSIONS: MALDI-TOF-MS allowed us to unravel the lipid A heterogeneity, which was not previously reported in Sf LPS. It is well known that slight variations of the chemical structure of lipid A may change its biological activity. Thus, the knowledge of the detailed chemical structure represents an essential step for further development of new preventive or therapeutically active compounds.
Assuntos
Lipídeo A/química , Shigella flexneri/química , Conformação Molecular , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
Trypanosoma cruzi cruzipain (Cz) bears a C-terminal domain (C-T) that contains sulfated epitopes "sulfotopes" (GlcNAc6S) on its unique N-glycosylation site. The effects of in vivo exposure to GlcNAc6S on heart tissue ultrastructure, immune responses, and along the outcome of infection by T. cruzi, were evaluated in a murine experimental model, BALB/c, using three independent strategies. First, mice were pre-exposed to C-T by immunization. C-T-immunized mice (C-TIM) showed IgG2a/IgG1 <1, induced the production of cytokines from Th2, Th17, and Th1 profiles with respect to those of dC-TIM, which only induced IL-10 respect to the control mice. Surprisingly, after sublethal challenge, both C-TIM and dC-TIM showed significantly higher parasitemia and mortality than the control group. Second, mice exposed to BSA-GlcNAc6S as immunogen (BSA-GlcNAc6SIM) showed: severe ultrastructural cardiac alterations while BSA-GlcNAcIM conserved the regular tissue architecture with slight myofibril changes; a strong highly specific humoral-immune-response reproducing the IgG-isotype-profile obtained with C-TIM; and a significant memory-T-cell-response demonstrating sulfotope-immunodominance with respect to BSA-GlcNAcIM. After sublethal challenge, BSA-GlcNAc6SIM showed exacerbated parasitemias, despite elevated IFN-γ levels were registered. In both cases, the abrogation of ultrastructural alterations when using desulfated immunogens supported the direct involvement of sulfotopes and/or indirect effect through their specific antibodies, in the induction of tissue damage. Finally, a third strategy using a passive transference of sulfotope-specific antibodies (IgG-GlcNAc6S) showed the detrimental activity of IgG-GlcNAc6S on mice cardiac tissue, and mice treated with IgG-GlcNAc6S after a sublethal dose of T. cruzi, surprisingly reached higher parasitemias than control groups. These findings confirmed the indirect role of the sulfotopes, via their IgG-GlcNAc6S, both in the immunopathogenicity as well as favoring T. cruzi infection.
Assuntos
Doença de Chagas , Trypanosoma cruzi , Animais , Antígenos de Protozoários , Cisteína Endopeptidases , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de ProtozoáriosRESUMO
Glycosylation of the Ab molecule is essential for maintaining the functional structure of Fc region and consequently for Ab-mediated effector functions, such as binding to cells or complement system activation. Alterations in the composition of the sugar moiety can dramatically influence Ab activity; however, it is not completely clear how differences in the N-linked oligosaccharide structure impact the biological function of Abs. We have described that murine IgG1 Abs can be separated according to their ability to elicit in vivo anaphylaxis in a fraction of anaphylactic and other of non-anaphylactic molecules. Furthermore, we showed that the N-linked oligosaccharide chain is essential for the structural conformation of the anaphylactic IgG1, the binding to FcgammaRIII on mast cells, and, consequently, for the ability to mediate anaphylactic reactions. In this study, we evaluated the contribution of individual sugar residues to this biological function. Differences in the glycan composition were observed when we analyzed oligosaccharide chains from anaphylactic or non-anaphylactic IgG1, mainly the presence of more sialic acid and fucose residues in anaphylactic molecules. Interestingly, the enzymatic removal of terminal sialic acid residues in anaphylactic IgG1 resulted in loss of the ability to trigger mast cell degranulation and in vivo anaphylactic reaction, similarly to the deglycosylated IgG1 Ab. In contrast, fucose removal did not affect the anaphylactic function. Therefore, we demonstrated that the ability of murine IgG1 Abs to mediate anaphylaxis is directly dependent on the amount of sialic acid residues associated to the oligosaccharide chain attached to the Fc region of these molecules.
Assuntos
Anafilaxia/imunologia , Anafilaxia/metabolismo , Imunoglobulina G/metabolismo , Ácidos Siálicos/metabolismo , Animais , Sítios de Ligação de Anticorpos , Configuração de Carboidratos , Linhagem Celular , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Ensaio de Imunoadsorção Enzimática , Hibridomas , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/química , Lectinas/química , Lectinas/imunologia , Lectinas/metabolismo , Mastócitos/química , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Oligossacarídeos/química , Oligossacarídeos/imunologia , Oligossacarídeos/metabolismo , Ácidos Siálicos/química , Ácidos Siálicos/fisiologia , Relação Estrutura-AtividadeRESUMO
Pseudomonas veronii 2E, an autochthonous bacterium isolated from sediments associated to a high-polluted watershed, produces a complex matrix of exopolymers with carbohydrates as main components. In this work, four polysaccharides were isolated from the extracellular material. The major acidic polysaccharide named EPO2, was purified and its structure was elucidated using Matrix-assisted laser desorption/ionization and Electrospray ionization mass spectrometry, Infrared spectroscopy, Nuclear magnetic resonance spectroscopy and chemical treatments. This heteropolysaccharide consists in an α(1-4) glucan substituted with N-Acetylglucosamine residues and with a branching α-D-GlcpA-(1-3)-L-Fucp disaccharide. The biosorption capacity of EPO2 and of the whole exopolysaccharide to Pb(II), Zn(II), Cu(II) and Fe(II) was evaluated. EPO2 showed a remarkable sorption capacity for Fe(II) with an efficiency of 70% and for Zn(II) 39%. When the whole exopolysaccharide fraction was tested it showed a significantly lower metal sorption ability than purified EPO2 suggesting the involvement of the distinct acidic branching disaccharide in this interaction.
Assuntos
Cobre/química , Ferro/química , Chumbo/química , Polissacarídeos Bacterianos/química , Pseudomonas/metabolismo , Zinco/química , Adsorção , Matriz Extracelular de Substâncias Poliméricas/química , Espectroscopia de Ressonância Magnética , Polissacarídeos Bacterianos/isolamento & purificação , Solubilidade , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Extracellular polymeric substances (EPS) are bacterial products associated to cell wall or secreted to the liquid media that form the framework of microbial mats. These EPS contain functional groups as carboxyl, amino, hydroxyl, phosphate and sulfhydryl, able to interact with cations. Thus, EPS may be considered natural detoxifying compounds of metal polluted waters and wastewaters. In this work Attenuated Total Reflectance-Fourier Transform Infrared spectroscopy (ATR-FTIR) in combination with multivariate analysis (Principal Component Analysis-PCA-) were used to study the interaction of Cd(II), Cu(II) and Zn(II) and Pseudomonas veronii 2E cells, including bound EPS and cell wall, and its different soluble EPS fractions, previously characterized as Cd(II) ligands of moderate strength. Amino groups present in exopolysaccharide fraction were responsible for Zn(II) and Cu(II) complexation, while carboxylates chelated Cd(II). In lipopolysaccharide fraction, phosphoryl and carboxyl sites were involved in Cd(II) and Cu(II) binding, while Zn(II) interacted with amino groups. Similar results were obtained from cells. These studies confirmed that FTIR-PCA is a rapid analytical tool to provide valuable information regarding the functional groups in biomolecules related to metal interaction. Moreover, a discrimination and identification of functional groups present in both EPS and cells that interacted with Cd(II), Zn(II) and Cu(II) was demonstrated.
Assuntos
Cádmio/química , Cobre/química , Matriz Extracelular de Substâncias Poliméricas/química , Pseudomonas/química , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Zinco/química , Adsorção , Biomassa , Ácidos Carboxílicos/química , Quelantes/química , Lipopolissacarídeos/química , Metais/química , Análise Multivariada , Polímeros/química , Análise de Componente Principal , Ligação ProteicaRESUMO
Bordetella bronchiseptica, known to infect animals and rarely humans, expresses a lipopolysaccharide that plays an essential role in host interactions, being critical for early clearance of the bacteria. On a B. bronchiseptica 9.73 isolate, mutants defective in the expression of genes involved in the biosynthesis of the core region were previously constructed. Herein, a comparative detailed structural analysis of the expressed lipids A by MALDI-TOF mass spectrometry was performed. The Bb3394 LPS defective in a 2-amino-2-deoxy-D-galacturonic acid lateral residue of the core presented a penta-acylated diglucosamine backbone modified with two glucosamine phosphates, similar to the wild-type lipid A. In contrast, BbLP39, resulting in the interruption of the LPS core oligosaccharide synthesis, presented lipid A species consisting in a diglucosamine backbone N-substituted with C14:0(3-O-C12:0) in C-2 and C14:0(3-O-C14:0) in C-2', O-acylated with C14:0(3-O-C10:0(3-OH) in C-3' and with a pyrophosphate in C-1. Regarding Bb3398 also presenting a rough LPS, the lipid A is formed by a hexa-acylated diglucosamine backbone carrying one pyrophosphate group in C-1 and one phosphate in C-4', both substituted with ethanolamine groups. As far as we know, this is the first description of a phosphoethanolamine modification in B. bronchiseptica lipid A. Our results demonstrate that although gene deletions were not directed to the lipid A moiety, each mutant presented different modifications. MALDI-TOF mass spectrometry was an excellent tool to highlight the structural diversity of the lipid A structures biosynthesized during its transit through the periplasm to the final localization in the outer surface of the outer membrane. Graphical Abstract.
Assuntos
Bordetella bronchiseptica/genética , Glicosiltransferases/genética , Lipídeo A/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bordetella bronchiseptica/química , Bordetella bronchiseptica/metabolismo , Difosfatos/química , Glucosamina/química , Glicosiltransferases/química , Lipídeo A/análise , Lipídeo A/genética , Mutação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
Each day, advances in the instrumentation and operating protocols bring new applications and insights into the molecular processes of ultra violet-matrix assisted laser desorption/ionization-mass spectrometry (UV-MALDI MS), increasing its potential use. We report here an approach in which mass spectrometry analysis of sphingolipids has been performed using a fluorescent tag (nitrobenz-2-oxa-1, 3-diazole, NBD) covalently linked to the sphingoid base as matrix. Thus, different labeled-sphingolipids were analyzed: ceramide, dihydroceramide, acetylceramide, glucosylceramide, galactosylceramide, galactosyldihydroceramide. In addition an extract of glycosphingolipids obtained from epimastigote forms of Trypanosoma cruzi metabolically labeled with NBD-ceramide was analyzed. The goal of this work is to show that no matrix needs to be added for the mass spectrometry analysis as the same tag used to label the lipids may generate efficiently analyte ions to obtain high quality signals.
Assuntos
Glicoesfingolipídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrofotometria Ultravioleta/métodos , Animais , Glicoesfingolipídeos/biossíntese , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/instrumentação , Espectrofotometria Ultravioleta/instrumentação , Trypanosoma cruzi/química , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismoRESUMO
Sulfated glycosphingolipids are present on the surface of a variety of cells. They are active participants in adhesion processes in many systems and appear to be involved in the regulation of cell proliferation, differentiation and other developmental cellular events. However, the body of knowledge about synthesis, structure, and function of glycolipids in parasitic protozoa is very limited so far. In this work, we show by metabolic incorporation of [(14)C]palmitic acid, [(14)C]glucose and Na(2)(35)SO(4) that sulfoglycosphingolipids are biosynthesized in the three intraerythrocytic stages of Plasmodium falciparum. After saponification, purification of the labelled acidic components was achieved and two components named SPf1 and SPf2 were characterized. Chemical degradations and TLC analysis pointed out to sulfolipidic structures. Analysis by UV-MALDI-TOF mass spectrometry in the negative ion mode using nor-harmane as matrix showed for SPf2 a structure consisting in a disulfated hexose linked to a 20:1 sphingosine acylated with C18:0 fatty acid. Interestingly, parasite treatment with low concentrations of d,l-threo-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) caused an arrest on parasite development associated to the inhibition of sulfoglycolipid biosynthesis. Taking into account that sulfoglycolipidic structures are currently involved in adhesion processes, our findings open the possibility to study the participation of this type of structures in the described aggregation phenomena in severe malaria and may contribute to clarify the pathogenesis of the disease. This report shows for the first time the synthesis of sulfoglycolipids in Apicomplexa.
Assuntos
Plasmodium falciparum/metabolismo , Sulfoglicoesfingolipídeos/metabolismo , Animais , Antimaláricos/farmacologia , Radioisótopos de Carbono/metabolismo , Cromatografia em Camada Fina , Glucose/metabolismo , Espectrometria de Massas , Morfolinas/farmacologia , Ácido Palmítico/metabolismo , Plasmodium falciparum/química , Plasmodium falciparum/efeitos dos fármacos , Esfingolipídeos/farmacologia , Sulfatos/metabolismo , Sulfoglicoesfingolipídeos/análiseRESUMO
In Gram-positive bacteria, such as lactic acid bacteria, general glycosylation systems have not been documented so far. The aim of this work was to characterize in detail the glycosylation of the S-layer protein of Lactobacillus kefiri CIDCA 83111. A reductive ß-elimination treatment followed by anion exchange high performance liquid chromatography analysis was useful to characterize the O-glycosidic structures. MALDI-TOF mass spectrometry analysis confirmed the presence of oligosaccharides bearing from 5 to 8 glucose units carrying galacturonic acid. Further nanoHPLC-ESI analysis of the glycopeptides showed two O-glycosylated peptides: the peptide sequence SSASSASSA already identified as a signature glycosylation motif in L. buchneri, substituted on average with eight glucose residues and decorated with galacturonic acid and another O-glycosylated site on peptide 471-476, with a Glc5-8GalA2 structure. As ten characteristic sequons (Asn-X-Ser/Thr) are present in the S-layer amino acid sequence, we performed a PNGase F digestion to release N-linked oligosaccharides. Anion exchange chromatography analysis showed mainly short N-linked chains. NanoHPLC-ESI in the positive and negative ion modes were useful to determine two different peptides substituted with short N-glycan structures. To our knowledge, this is the first description of the structure of N-glycans in S-layer glycoproteins from Lactobacillus species. SIGNIFICANCE: A detailed characterization of protein glycosylation is essential to establish the basis for understanding and investigating its biological role. It is known that S-layer proteins from kefir-isolated L. kefiri strains are involved in the interaction of bacterial cells with yeasts present in kefir grains and are also capable to antagonize the adverse effects of different enteric pathogens. Therefore, characterization of type and site of glycosidic chains in this protein may help to understand these important properties. Furthermore, this is the first description of N-glycosidic chains in S-layer glycoprotein from Lactobacillus spp.
Assuntos
Glicoproteínas/análise , Lactobacillus/química , Glicoproteínas de Membrana/química , Proteômica/métodos , Motivos de Aminoácidos , Proteínas de Bactérias/química , Glicosilação , Oligossacarídeos , Polissacarídeos/químicaRESUMO
Brucellaceae are Gram-negative bacteria that cause brucellosis, one of the most distributed worldwide zoonosis, transmitted to humans by contact with either infected animals or their products. The lipopolysaccharide exposed on the cell surface has been intensively studied and is considered a major virulence factor of Brucella. In the last years, structural studies allowed the determination of new structures in the core oligosaccharide and the O-antigen of this lipopolysaccharide. In this work, we have reinvestigated the lipid A structure isolated from B. suis and B. abortus lipopolysaccharides. A detailed study by MALDI-TOF mass spectrometry in the positive and negative ion modes of the lipid A moieties purified from both species was performed. Interestingly, a new feature was detected: the presence of a pyrophosphorylethanolamine residue substituting the backbone. LID-MS/MS analysis of some of the detected ions allowed assurance that the Lipid A structure composed by the diGlcN3N disaccharide, mainly hexa-acylated and penta-acylated, bearing one phosphate and one pyrophosphorylethanolamine residue. Graphical abstract á .