RESUMO
The majority of therapeutic strategies for mycosis require the protracted administration of antifungals, which can result in significant toxicities and have unacceptable failure rates. Hence, there is an urgent need for the development of improved therapeutic approaches, and monoclonal antibody-based drugs are potentially a powerful alternative to standard antifungals. To develop a broad antibody-like reagent against mycosis, wheat germ agglutinin (WGA) was linked to the effector Fc region of murine IgG2a. The resultant WGA-Fc displayed high affinity to purified chitin and bound efficiently to fungal cell walls, co-localizing with chitin, in patterns ranging from circular (Histoplasma capsulatum) to punctate (Cryptococcus neoformans) to labeling at the bud sites (Candida albicans and Saccharomyces cerevisiae). WGA-Fc directly inhibited fungal growth in standard cultures. WGA-Fc opsonization increased fungal phagocytosis, as well augmented the antifungal functions by macrophages. Prophylactic administration of WGA-Fc fully protected mice against H. capsulatum, correlating with a reduction in lung, spleen and liver fungal burdens. Administration of WGA-Fc also dramatically diminished pulmonary inflammation. Hence, the opsonic activity of WGA-Fc effectively modulates fungal cell recognition and promotes the elimination of fungal pathogens. Therefore, we propose WGA-Fc as a potential "pan-fungal" therapeutic that should be further developed for use against invasive mycoses.
Assuntos
Antifúngicos/farmacologia , Fungos/efeitos dos fármacos , Imunoconjugados/farmacologia , Infecções Fúngicas Invasivas/tratamento farmacológico , Proteínas Recombinantes de Fusão/farmacologia , Animais , Antifúngicos/uso terapêutico , Células CHO , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Quitina/metabolismo , Cricetulus , Modelos Animais de Doenças , Fungos/metabolismo , Humanos , Hibridomas , Imunoconjugados/genética , Imunoconjugados/uso terapêutico , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/farmacologia , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Imunoglobulina G/genética , Imunoglobulina G/farmacologia , Imunoglobulina G/uso terapêutico , Infecções Fúngicas Invasivas/microbiologia , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/uso terapêutico , Aglutininas do Germe de Trigo/genética , Aglutininas do Germe de Trigo/farmacologia , Aglutininas do Germe de Trigo/uso terapêuticoRESUMO
The principal capsular component of Cryptococcus neoformans, glucuronoxylomannan (GXM), interacts with surface glycans, including chitin-like oligomers. Although the role of GXM in cryptococcal infection has been well explored, there is no information on how chitooligomers affect fungal pathogenesis. In this study, surface chitooligomers of C. neoformans were blocked through the use of the wheat germ lectin (WGA) and the effects on animal pathogenesis, interaction with host cells, fungal growth and capsule formation were analyzed. Treatment of C. neoformans cells with WGA followed by infection of mice delayed mortality relative to animals infected with untreated fungal cells. This observation was associated with reduced brain colonization by lectin-treated cryptococci. Blocking chitooligomers also rendered yeast cells less efficient in their ability to associate with phagocytes. WGA did not affect fungal viability, but inhibited GXM release to the extracellular space and capsule formation. In WGA-treated yeast cells, genes that are involved in capsule formation and GXM traffic had their transcription levels decreased in comparison with untreated cells. Our results suggest that cellular pathways required for capsule formation and pathogenic mechanisms are affected by blocking chitin-derived structures at the cell surface of C. neoformans. Targeting chitooligomers with specific ligands may reveal new therapeutic alternatives to control cryptococcosis.
Assuntos
Cryptococcus neoformans/patogenicidade , Cápsulas Fúngicas/metabolismo , Fagocitose/efeitos dos fármacos , Polissacarídeos/metabolismo , Aglutininas do Germe de Trigo/farmacologia , Animais , Encéfalo/microbiologia , Quitina/metabolismo , Criptococose/tratamento farmacológico , Criptococose/patologia , Cryptococcus neoformans/crescimento & desenvolvimento , Cryptococcus neoformans/metabolismo , Cápsulas Fúngicas/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Aglutininas do Germe de Trigo/metabolismoRESUMO
An efficient affinity chromatographic matrix based on chitosan for wheat germ agglutinin (WGA) purification was developed. The matrices assayed consisted of chitosan mini-spheres cross-linked with epichlorhydrin 45, 250 or 500 mM. The maximum adsorption capacity of pure WGA - calculated from the corresponding isotherms - was between 43.2 and 48.9 mg/g at pH 5.0 and between 16.6 and 27.6 mg/g at pH 8.5. However, the adsorption of agglutinin from wheat germ extract was higher at pH 8.5. In addition, 0.5 g of mini-spheres cross-linked with epichlorhydrin 250 mM adsorbed 94.5% of the WGA present in 5 mL of the concentrated extract. Acetic acid was able to elute 100% of the adsorbed WGA. The purity of the WGA obtained was greater than 95% and the purification factor was 56.8. The matrix was able to maintain an efficient performance of the purification process for three consecutive cycles. A new method to monitor the purification process by RP-HPLC was developed.
Assuntos
Cromatografia de Afinidade/métodos , Aglutininas do Germe de Trigo/isolamento & purificação , Adsorção , Marcadores de Afinidade , Quitosana , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Reutilização de Equipamento , Hemaglutinação/efeitos dos fármacos , Humanos , Técnicas In Vitro , Microscopia Eletrônica de Varredura , Aglutininas do Germe de Trigo/farmacologiaRESUMO
For the rhizobacterium Azospirillum brasilense, the optimal nutritional range of C:N ratios corresponds to the presence of malate (ca. 3 to 5 g l(-1) of its sodium salt) and ammonium (ca. 0.5 to 3 g l(-1) of NH4Cl) as preferred carbon and nitrogen sources, respectively. This microaerophilic aerotactic bacterium is known to have a narrow optimal oxygen concentration range of ca. 3 to 5 microM, which is 1.2% to 2% of oxygen solubility in air-saturated water under normal conditions. In this work, the effects of stress conditions (bound-nitrogen deficiency related to a high C:N ratio in the medium; excess of oxygen) on aerobically grown A. brasilense Sp245, a native wheat-associated endophyte, were investigated in the absence and presence of wheat germ agglutinin (WGA, plant stress protein and a molecular host-plant signal for the bacterium) using FTIR spectroscopy of whole cells in the diffuse reflectance mode (DRIFT). The nutritional stress resulted in the appearance of prominent spectroscopic signs of poly-3-hydroxybutyrate (PHB) accumulation in the bacterial cells; in addition, splitting of the amide I band related to bacterial cellular proteins indicated some stress-induced alterations in their secondary structure components. Similar structural changes were observed in the presence of nanomolar WGA both in stressed A. brasilense cells and under normal nutritional conditions. Comparative analysis of the data obtained and the relevant literature data indicated that the stress conditions applied (which resulted in the accumulation of PHB involved in stress tolerance) and/or the presence of nanomolar concentrations of WGA induced synthesis of bacterial cell-surface (glyco)proteins rich in beta-structures, that could be represented by hemagglutinin and/or porin.
Assuntos
Azospirillum brasilense/efeitos dos fármacos , Nitrogênio/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Aglutininas do Germe de Trigo/farmacologia , Aerobiose , Azospirillum brasilense/crescimento & desenvolvimento , Azospirillum brasilense/metabolismo , Hidroxibutiratos/metabolismo , Nitrogênio/farmacologia , Poliésteres/metabolismoRESUMO
In order to better understand the role played by surface glycoconjugates during cell adhesion and endocytosis by the dematiaceous fungi Fonsecaea pedrosoi, we analyzed the interaction between this microorganism and five mutants of Chinese Hamster Ovary (CHO) cells, which differ from each other in the exposition of carbohydrate residues on the cell surface. Five clones (Gat-2 parental, and the clones: Lec1, Lec2, Lec8 and ldlLec1) were tested and the adhesion and endocytic indexes were determined after 2 hours of interaction. The Lec1 and ldlLec1 clones, which present exposed mannose residues, showed the greater adhesion index (AI). On the other hand, the Lec8 clone, which exposes N-acetylglucosamine on the cell surface, presented the greater endocytic index. The role played by surface-exposed carbohydrate residues was further analyzed by addition of mannose or N-acetylglucosamine to the interaction medium and by previous incubation of the cells in the presence of the lectins Concanavalin A (ConA) and wheat germ agglutinin (WGA). The results obtained suggest that mannose residues are involved in the first step of adhesion of F. pedrosoi to the cell surface, while N-acetylglucosamine residues are involved on its ingestion process.
Assuntos
Ascomicetos/fisiologia , Carboidratos/fisiologia , Acetilglucosamina/farmacologia , Animais , Ascomicetos/efeitos dos fármacos , Ascomicetos/ultraestrutura , Células CHO , Carboidratos/farmacologia , Adesão Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Concanavalina A/farmacologia , Cricetinae , Endocitose/efeitos dos fármacos , Glicosilação , Manose/farmacologia , Microscopia Eletrônica , Aglutininas do Germe de Trigo/farmacologiaRESUMO
With the aim of identifying and differentiating Trypanosoma cruzi from Trypanosoma rangeli, culture epimastigotes from 30 Honduran trypanosomatid isolates were analyzed by susceptibility to complement lysis, reactivity to lectins, reactivity to monoclonal antibodies specific for T. cruzi, and isoenzymatic electrophoretic patterns. Using these four methodologies, 27 of the 30 trypanosomatid isolates, as well as 5 clones, were identified as T. cruzi, whereas the remaining three isolates were classified as T. rangeli. None of the isolates presented mixed trypanosome species. Results indicate that both trypanosomatid species circulate in Honduras and that any of the four methods employed may be used to reliably differentiate T. cruzi from T. rangeli.
Assuntos
Doença de Chagas/parasitologia , Insetos Vetores/parasitologia , Triatominae/parasitologia , Trypanosoma cruzi/classificação , Trypanosoma/classificação , Tripanossomíase/parasitologia , Testes de Aglutinação , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antiprotozoários/imunologia , Doença de Chagas/veterinária , Proteínas do Sistema Complemento/imunologia , Doenças do Cão/parasitologia , Cães , Honduras , Humanos , Isoenzimas/análise , Gambás/parasitologia , Trypanosoma/enzimologia , Trypanosoma/imunologia , Trypanosoma/isolamento & purificação , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/imunologia , Trypanosoma cruzi/isolamento & purificação , Tripanossomíase/veterinária , Aglutininas do Germe de Trigo/farmacologiaRESUMO
In previous reports we have shown that phytohemagglutinin (PHA)-activated human lymphocytes had positive inotropic effects on spontaneously beating isolated rat atria. In this study, we demonstrated that the stimulatory effect on heart contractility induced by lymphocytes was linked to early events of lymphocyte activation by lectins. Active soluble factors were gradually released to the fluid phase. Similar results were obtained with both mitogenic (PHA) and nonmitogenic (WGA) lectins indicating that the stimulatory action of activated lymphocytes did not require cell division. Absence of Ca2+ inhibited both the generation of the stimulatory activity and lymphocyte proliferation. In contrast, verapamil, dexamethasone and low concentrations of cycloheximide eliminated only the appearance of the stimulatory effect.