RESUMO
Cryptococcosis is a fungal disease of global significance for which new effective treatments are needed. The conjugation of the synthetic antimicrobial peptide fragment UBI 31-38 to a coumarin derivative showed to be an effective approach for the design of a novel anticryptococcal agent. In addition to antifungal activity, the conjugate exhibited intense fluorescence, which could be valuable for mechanistic investigations of this molecule. In this work, we studied the photophysical properties of the conjugate and confocal scanning laser microscopy was used to inspect the distribution of the peptide-coumarin conjugate in Cryptococcus cell. The synergism of this compound with amphotericin B or fluconazole against C. gattii and C. neoformans strains was also investigated. The results indicated that the fluorescent conjugate alone as well as its combination with amphotericin B are promising tools against cryptococcosis.
Assuntos
Anfotericina B/farmacologia , Antifúngicos/farmacologia , Cumarínicos/farmacologia , Criptococose/tratamento farmacológico , Cryptococcus/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Anfotericina B/química , Antifúngicos/síntese química , Antifúngicos/química , Cumarínicos/química , Cryptococcus/citologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Processos Fotoquímicos , Relação Estrutura-AtividadeRESUMO
Background: Salivary gland diseases in dogs have an overall incidence of 0.3%. Sialocele and sialoadenitis are the most common injuries and are usually caused by infectious diseases, especially of bacterial origin. Currently, Cryptococcus neoformans has not been registered as the etiological agent since fungal participation is unusual. This case report describes a cryptococcal sialoadenitis in a dog. Case: A 1.9-year-old male poodle had a swelling of the right submandibular region, combined with dry cough and snoring that persisted for about 30 days. The dog often walked in the town square, which has pigeons, and was seen eating chicken droppings. The dog was diagnosed with sialoadenitis in the right submandibular salivary gland. The following laboratory tests were performed cytology, fungal culture, blood count, search for hemoparasites, total plasma protein (ppt), alanine aminotransferase dosage (ALT), alkaline phosphatase (ALP), urea (U), creatinine (CREA), Albumin (ALB), total protein (TP), globulin (GLOB) and amylase (AML). In addition, the dog underwent cervical, thoracic and abdominal ultrasound, and cervical x-rays. The cytology results showed fungal sialoadenitis caused by Cryptococcus neoformans. The blood test results evidenced thrombocytopenia and increased ppt, TP, GLOB, and AML. The lungs and heart x-rays results showed no alterations. However, the proximal third [...](AU)
Assuntos
Animais , Cães , Glândulas Salivares/lesões , Glândulas Salivares/patologia , Columbidae/microbiologia , Cryptococcus neoformans , Contagem de Células Sanguíneas/veterinária , /uso terapêutico , Cryptococcus/citologiaRESUMO
Background: Salivary gland diseases in dogs have an overall incidence of 0.3%. Sialocele and sialoadenitis are the most common injuries and are usually caused by infectious diseases, especially of bacterial origin. Currently, Cryptococcus neoformans has not been registered as the etiological agent since fungal participation is unusual. This case report describes a cryptococcal sialoadenitis in a dog. Case: A 1.9-year-old male poodle had a swelling of the right submandibular region, combined with dry cough and snoring that persisted for about 30 days. The dog often walked in the town square, which has pigeons, and was seen eating chicken droppings. The dog was diagnosed with sialoadenitis in the right submandibular salivary gland. The following laboratory tests were performed cytology, fungal culture, blood count, search for hemoparasites, total plasma protein (ppt), alanine aminotransferase dosage (ALT), alkaline phosphatase (ALP), urea (U), creatinine (CREA), Albumin (ALB), total protein (TP), globulin (GLOB) and amylase (AML). In addition, the dog underwent cervical, thoracic and abdominal ultrasound, and cervical x-rays. The cytology results showed fungal sialoadenitis caused by Cryptococcus neoformans. The blood test results evidenced thrombocytopenia and increased ppt, TP, GLOB, and AML. The lungs and heart x-rays results showed no alterations. However, the proximal third [...]
Assuntos
Animais , Cães , Columbidae/microbiologia , Cryptococcus neoformans , Glândulas Salivares/lesões , Glândulas Salivares/patologia , Contagem de Células Sanguíneas/veterinária , Cryptococcus/citologiaRESUMO
Capsule production is common among bacterial species, but relatively rare in eukaryotic microorganisms. Members of the fungal Cryptococcus genus are known to produce capsules, which are major determinants of virulence in the highly pathogenic species Cryptococcus neoformans and Cryptococcus gattii. Although the lack of virulence of many species of the Cryptococcus genus can be explained solely by the lack of mammalian thermotolerance, it is uncertain whether the capsules from these organisms are comparable to those of the pathogenic cryptococci. In this study, we compared the characteristic of the capsule from the non-pathogenic environmental yeast Cryptococcus liquefaciens with that of C. neoformans. Microscopic observations revealed that C. liquefaciens has a capsule visible in India ink preparations that was also efficiently labeled by three antibodies generated to specific C. neoformans capsular antigens. Capsular polysaccharides of C. liquefaciens were incorporated onto the cell surface of acapsular C. neoformans mutant cells. Polysaccharide composition determinations in combination with confocal microscopy revealed that C. liquefaciens capsule consisted of mannose, xylose, glucose, glucuronic acid, galactose and N-acetylglucosamine. Physical chemical analysis of the C. liquefaciens polysaccharides in comparison with C. neoformans samples revealed significant differences in viscosity, elastic properties and macromolecular structure parameters of polysaccharide solutions such as rigidity, effective diameter, zeta potential and molecular mass, which nevertheless appeared to be characteristics of linear polysaccharides that also comprise capsular polysaccharide of C. neoformans. The environmental yeast, however, showed enhanced susceptibility to the antimicrobial activity of the environmental phagocytes, suggesting that the C. liquefaciens capsular components are insufficient in protecting yeast cells against killing by amoeba. These results suggest that capsular structures in pathogenic Cryptococcus species and environmental species share similar features, but also manifest significant difference that could influence their potential to virulence.
Assuntos
Acanthamoeba castellanii/microbiologia , Cápsulas Bacterianas/química , Cápsulas Bacterianas/imunologia , Cryptococcus/imunologia , Fagócitos/microbiologia , Acanthamoeba castellanii/citologia , Acanthamoeba castellanii/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Fungos/imunologia , Sequência de Bases , Cryptococcus/citologia , Cryptococcus/crescimento & desenvolvimento , Cryptococcus/isolamento & purificação , Elasticidade , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hidrodinâmica , Cinética , Luz , Dados de Sequência Molecular , Monossacarídeos/análise , Fagócitos/citologia , Fagocitose , Espalhamento de Radiação , Alinhamento de Sequência , Caramujos/microbiologia , ViscosidadeRESUMO
We conducted a retrospective study of 58 cases of cryptococcosis (1986-2008) with urine test positive for Cryptococcus sp, in Mycology Laboratory, Santa Casa-Hospital Complex, Porto Alegre, RS, Brazil. The diagnosis of cryptococcuria was based on microscopic examination and culture of urinary sediment. Cryptococcus was isolated from other clinical specimens such as blood, cerebrospinal fluid, ascitic and pleural fluids, respiratory secretions, biopsies of skin, nasal and bone marrow. Cryptocccus neoformans was present in 55 cases and Cryptocccus gattii in three cases. Males predominated (79.3%); age ranged from 12 to 86 years. Acquired Immune Deficiency Syndrome (AIDS) were present in 60.3%, 31.1% did not have AIDS and 5.2% were apparently immunocompetent patients. The most frequent signs and symptoms were headache (53.4%) and fever (51.7%). The most widely used medication was the amphotericin B (43 patients). The mortality rate was 45%. We conclude that the mycological examination of the urine can be an alternative simple, non-invasive and useful in diagnosis of disseminated cryptococcosis, especially when used in conjunction with techniques for demonstration of the capsule (nigrosine) and/or production of melanin in special culture media (Staib agar).
Assuntos
Criptococose/diagnóstico , Criptococose/microbiologia , Cryptococcus/isolamento & purificação , Meios de Cultura/química , Técnicas Microbiológicas/métodos , Micologia/métodos , Urina/microbiologia , Adolescente , Adulto , Ágar , Distribuição por Idade , Idoso , Idoso de 80 Anos ou mais , Anfotericina B/administração & dosagem , Antifúngicos/administração & dosagem , Brasil , Criança , Criptococose/tratamento farmacológico , Criptococose/patologia , Cryptococcus/citologia , Cryptococcus/crescimento & desenvolvimento , Feminino , Humanos , Masculino , Microscopia , Pessoa de Meia-Idade , Estudos Retrospectivos , Seleção Genética , Distribuição por Sexo , Adulto JovemRESUMO
The yeast Cryptococcus flavus secretes a glycosylated alpha-amylase (Amy1) when grown in a starch-containing medium. The effects of N-glycosylation on secretion, enzyme activity, and stability of this glycoprotein were studied. Addition of tunicamycin (TM) to the medium at a concentration higher than 0.5 microg mL(-1) affected C. flavus growth. Amy1 activity increased by 55% in the intracellular fraction after C. flavus growth in the presence of 0.5 microg mL(-1) TM. SDS-PAGE and gel activity detection showed that native enzyme and deglycosylated enzyme had apparent molecular mass of 68 and 64.5 kDa, respectively. The N-glycosylation process did not affect either optimum pH or optimum temperature. The K(M) values of native and non-glycosylated alpha-amylases were 0.052 and 0.098 mg mL(-1), and V(max) values were 0.038 and 0.047 mg min(-1), respectively. However, the non-glycosylated form was more sensitive to inactivation by both the proteolytic enzyme trypsin and high temperature. Furthermore, the activity of the non-glycosylated enzyme was affected by Hg(2+) and Cu(2+) suggesting that N-glycosylation is involved in the folding of Amy1.