Dacryocystitis due to Sporothrix brasiliensis: a case report of a successful clinical and serological outcome with low-dose potassium iodide treatment and oculoplastic surgery.

Sporothrix brasiliensis is the main species of the S. schenckii complex implicated in the zoonotic epidemics of sporotrichosis in Rio de Janeiro, Brazil. Epidemiological features have been already described, such as zoonotic transmission by cats and increased frequency of atypical clinical aspects. The involvement of the face by contact with cats is common in childhood; as a result, ophthalmic manifestations have increased. We report a case of acute dacryocystitis in a 9-year-old girl. A calmodulin-based molecular phylogeny was used to identify the agent as S. brasiliensis. This is a rare type of presentation, usually complicated with nasolacrimal duct occlusion. The patient was cured without sequelae after treatment with a low dose of saturated solution of potassium iodide and decompressive oculoplastic surgery. Therapeutic options and considerations of aetiological agents and serology are discussed.

New posology of potassium iodide for the treatment of cutaneous sporotrichosis: study of efficacy and safety in 102 patients.

BACKGROUND: The first therapeutic choice for the treatment of cutaneous sporotrichosis is oral itraconazole; however, the increase in cases of zoonotic transmission outbreak necessitates a search for effective and safe treatment alternatives. OBJECTIVE: To evaluate a new potassium iodide (KI) posology as an alternative for the treatment of limited cutaneous forms of sporotrichosis. METHODS: One hundred and two patients with sporotrichosis diagnosed by isolation of Sporothrix sp. were included and were divided into 2 groups that received different doses of KI: group A received the conventional dose, and group B received the reduced dose. The cure criteria were based on clinical and serological data. RESULTS: Seventy-nine patients (77.4%) reached clinical cure: 70.6% and 84.3% of groups A and B respectively. Sixteen patients (15.6%) were lost during follow-up, and seven changed drug therapy: five in group A and two in group B. The incidence of adverse events was similar for both groups (64.7%): predominantly metallic taste (44%), followed by mild gastrointestinal intolerance and acneiform eruption (10.7% each). No serious adverse events occurred, and there were no recurrences. Analysis of the results showed no statistically significant difference between groups (P = 0.9255). The improvement in serologic titres was significant in both treatment groups. CONCLUSION: Through statistical analysis, the usual posology was not shown to be superior to the one proposed in this study. Serology for sporotrichosis may be used as a valuable tool in the clinical monitoring of these patients.

Arthritis as a hypersensitivity reaction in a case of sporotrichosis transmitted by a sick cat: clinical and serological follow up of 13 months.

Sporotrichosis is a subacute or chronic fungal infection caused by Sporothrix schenckii, which is commonly acquired by traumatic inoculation of the fungus carried in a contaminated material into the skin. Joint involvement is the most frequent extracutaneous manifestation in immunosuppressed patients. We report the case of an immunocompetent woman who acquired sporotrichosis through the scratch of a sick cat. She presented skin lesions and arthritis possibly because of a hypersensitivity reaction. Treatment resulted in complete cure up to 13 months of clinical and serological follow-up.

Susceptibility of cariogenic microorganisms to phytoconstituents.

This study aimed to evaluate the in vitro antibacterial activity of the phytochemicals thymol, linalool, and citronellol against Streptococcus mutans, Streptococcus salivarius and Streptococcus oralis. Disk diffusion screening on solid medium and measurement of the diameter of the bacterial growth inhibition halos was the technique utilized. The Minimum Inhibitory Concentration (MIC) of the substances was determined using serial substance dilutions and microdilution technique in Brain Heart Infusion culture medium. After incubation for 24 hours in an oven at 37 °C, plate reading was completed and confirmed by visual method using 2,3,5 triphenyl tetrazolium chloride dye. The Minimum Bactericidal Concentration (MBC) was determined from MIC subcultures. Assays were performed in triplicate, and chlorhexidine was used as a positive control. The diameters in mm of the growth inhibition halos ranged between 7.3 and 10.7 for S. mutans, 7.3 and 10.0 for S. oralis, and 8.2 and 9.8 for S. salivarius. The MIC and MBC values obtained converged, ranging from maximum values in the presence of Linalool (1,250.0 mg/mL, 2,500.0 mg/mL and 2,500.0 mg/mL, respectively, for S. mutans, S. oralis, and S. salivarius); and minimum values with Thymol (312.5 µg/ml, 156.2 µg/mL and 156.2 µg/ml, respectively for S. mutans, S. oralis, and S. salivarius). All the tested phytochemicals displayed antibacterial activity, thus representing substances with potential applications in preventing tooth decay.

Effect of genetic modifications by selection for immunological tolerance on fungus infection in mice.

Two strains of mice genetically selected for extreme phenotypes of immunological tolerance to ovalbumin, susceptible (TS) and resistant (TR), were experimentally infected with Sporothrix schenckii. The objective was to observe whether the genetic modifications produced by the selection might be associated with interstrain differences in adaptive immune and innate responses to infection. Therefore, we evaluated the LD(50), CFU, phagocytic index, fungicidal activity, pro-inflammatory cytokines, specific antibody titres, and the delayed-type hypersensitivity reactivity. TR mice were tenfold more susceptible to infection than TS mice, as shown by LD(50) (5 x 10(6) conidia i.v.). In TS mice, the resistance was a consequence of the tissue fungal load reduction, consistent specific T-cell-mediated immunity, and tumour necrosis factor (TNF)-alpha activity at onset of infection. In TR mice, these responses were not precociously detected. Therefore, the absence of CD4(+) T-cell response in the first week of infection might explain the non-clearance of pathogen in TR mice. However, TR mice did show an increase in TNF level and delayed-type hypersensitivity response after the first week post-infection; there was also expansion and increase in granulomatous foci and CFU in the spleen. The expansion of granulomatous foci and the increase in TNF-alpha and tissue fungal load to damaging levels induced severe tissue destruction, general failure of the organs, cachexy and death in TR mice. The results show that genetic selection for extreme phenotypes of immunological tolerance also modified the responses to S. schenckii infection.

Endothelial cells, tissue factor and infectious diseases.

Tissue factor is a transmembrane procoagulant glycoprotein and a member of the cytokine receptor superfamily. It activates the extrinsic coagulation pathway, and induces the formation of a fibrin clot. Tissue factor is important for both normal homeostasis and the development of many thrombotic diseases. A wide variety of cells are able to synthesize and express tissue factor, including monocytes, granulocytes, platelets and endothelial cells. Tissue factor expression can be induced by cell surface components of pathogenic microorganisms, proinflammatory cytokines and membrane microparticles released from activated host cells. Tissue factor plays an important role in initiating thrombosis associated with inflammation during infection, sepsis, and organ transplant rejection. Recent findings suggest that tissue factor can also function as a receptor and thus may be important in cell signaling. The present minireview will focus on the role of tissue factor in the pathogenesis of septic shock, infectious endocarditis and invasive aspergillosis, as determined by both in vivo and in vitro models.

Adhesion of the human pathogen Sporothrix schenckii to several extracellular matrix proteins.

The pathogenic fungus Sporothrix schenckii is the causative agent of sporotrichosis. This subcutaneous mycosis may disseminate in immunocompromised individuals and also affect several internal organs and tissues, most commonly the bone, joints and lung. Since adhesion is the first step involved with the dissemination of pathogens in the host, we have studied the interaction between S. schenckii and several extracellular matrix (ECM) proteins. The binding of two morphological phases of S. schenckii, yeast cells and conidia, to immobilized type II collagen, laminin, fibronectin, fibrinogen and thrombospondin was investigated. Poly (2-hydroxyethyl methacrylate) (poly-HEMA) was used as the negative control. Cell adhesion was assessed by ELISA with a rabbit anti-S. schenckii antiserum. The results indicate that both morphological phases of this fungus can bind significantly to type II collagen, fibronectin and laminin in comparison to the binding observed with BSA (used as blocking agent). The adhesion rate observed with the ECM proteins (type II collagen, fibronectin and laminin) was statistically significant (P < 0.05) when compared to the adhesion obtained with BSA. No significant binding of conidia was observed to either fibrinogen or thrombospondin, but yeast cells did bind to the fibrinogen. Our results indicate that S. schenckii can bind to fibronectin, laminin and type II collagen and also show differences in binding capacity according to the morphological form of the fungus.

Discovering the infectome of human endothelial cells challenged with Aspergillus fumigatus applying a mass spectrometry label-free approach.

Blood vessel invasion is a key feature of invasive aspergillosis. This angioinvasion process contributes to tissue thrombosis, which can impair the access of leukocytes and antifungal drugs to the site of infection. It has been demonstrated that human umbilical vein endothelial cells (HUVECs) are activated and assume a prothrombotic phenotype following contact with Aspergillus fumigatus hyphae or germlings, a process that is independent of fungus viability. However, the molecular mechanisms by which this pathogen can activate endothelial cells, together with the endothelial pathways that are involved in this process, remain unknown. Using a label-free approach by High Definition Mass Spectrometry (HDMS(E)), differentially expressed proteins were identified during HUVEC-A. fumigatus interaction. Among these, 89 proteins were determined to be up- or down-regulated, and another 409 proteins were exclusive to one experimental condition: the HUVEC control or HUVEC:AF interaction. The in silico predictions provided a general view of which biological processes and/or pathways were regulated during HUVEC:AF interaction, and they mainly included cell signaling, immune response and hemostasis pathways. This work describes the first global proteomic analysis of HUVECs following interaction with A. fumigatus germlings, the fungus morphotype that represents the first step of invasion and dissemination within the host. BIOLOGICAL SIGNIFICANCE: A. fumigatus causes the main opportunistic invasive fungal infection related to neutropenic hematologic patients. One of the key steps during the establishment of invasive aspergillosis is angioinvasion but the mechanism associated with the interaction of A. fumigatus with the vascular endothelium remains unknown. The identification of up- and down-regulated proteins expressed by human endothelial cells in response to the fungus infection can contribute to reveal the mechanism of endothelial response and, to understand the physiopathology of this high mortality disease. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.

Susceptibility of cariogenic microorganisms to phytoconstituents / Susceptibilidade de microrganismos cariogênicos a fitoconstituintes

Abstract This study aimed to evaluate the in vitro antibacterial activity of the phytochemicals thymol, linalool, and citronellol against Streptococcus mutans, Streptococcus salivarius and Streptococcus oralis. Disk diffusion screening on solid medium and measurement of the diameter of the bacterial growth inhibition halos was the technique utilized. The Minimum Inhibitory Concentration (MIC) of the substances was determined using serial substance dilutions and microdilution technique in Brain Heart Infusion culture medium. After incubation for 24 hours in an oven at 37 °C, plate reading was completed and confirmed by visual method using 2,3,5 triphenyl tetrazolium chloride dye. The Minimum Bactericidal Concentration (MBC) was determined from MIC subcultures. Assays were performed in triplicate, and chlorhexidine was used as a positive control. The diameters in mm of the growth inhibition halos ranged between 7.3 and 10.7 for S. mutans, 7.3 and 10.0 for S. oralis, and 8.2 and 9.8 for S. salivarius. The MIC and MBC values obtained converged, ranging from maximum values in the presence of Linalool (1,250.0 mg/mL, 2,500.0 mg/mL and 2,500.0 mg/mL, respectively, for S. mutans, S. oralis, and S. salivarius); and minimum values with Thymol (312.5 μg/ml, 156.2 μg/mL and 156.2 μg/ml, respectively for S. mutans, S. oralis, and S. salivarius). All the tested phytochemicals displayed antibacterial activity, thus representing substances with potential applications in preventing tooth decay.

Resumo Este estudo objetivou avaliar a atividade antibacteriana in vitro dos fitoquímicos timol, linalol e citronelol sobre Streptococcus mutans, Streptococcus salivaris e Streptococcus oralis. Utilizou-se a técnica de discos de difusão em meio sólido e medição do diâmetro dos halos de inibição. A concentração inibitória mínima (CIM) das substâncias foi determinada utilizando diluições em série das substâncias e técnica de microdiluição em meio de cultura de Brain Heart Infusion. Após incubação durante 24 horas em estufa a 37 °C, a leitura da placa foi confirmada pelo método visual usando o corante 2,3,5 trifenil cloreto de tetrazólio. A concentração bactericida mínima (CBM) foi determinada a partir de subculturas de MIC. Os ensaios foram realizados em triplicata, e clorexidina foi usada como um controle positivo. Os diâmetros dos halos de inibição do crescimento variaram entre 7,3 e 10,7 por S. mutans, 7,3 e 10,0 por S. oralis, e 8,2 e 9,8 para S. salivaris. Os valores de CIM e CBM obtidos variaram de valores máximos na presença de linalol (1.250,0 mg/mL, 2.500.0 mg/mL e 2.500.0 mg/mL, respectivamente, para o S. mutans, S oralis e S. salivaris); a valores mínimos com timol (312,5 μg/ml, 156,2 μg/mL e 156,2 μg/ml, respectivamente para S. mutans, S. oralis e S. salivaris). Todos os fitoquímicos testados apresentaram atividade antibacteriana, representando, assim, substâncias com potencial de aplicações na prevenção da cárie dentária.

Proteomic analysis of cytosolic proteins associated with petite mutations in Candida glabrata.

The incidence of superficial or deep-seated infections due to Candida glabrata has increased markedly, probably because of the low intrinsic susceptibility of this microorganism to azole antifungals and its relatively high propensity to acquire azole resistance. To determine changes in the C. glabrata proteome associated with petite mutations, cytosolic extracts from an azole-resistant petite mutant of C. glabrata induced by exposure to ethidium bromide, and from its azole-susceptible parent isolate were compared by two-dimensional polyacrylamide gel electrophoresis. Proteins of interest were identified by peptide mass fingerprinting or sequence tagging using a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer. Tryptic peptides from a total of 160 Coomassie-positive spots were analyzed for each strain. Sixty-five different proteins were identified in the cytosolic extracts of the parent strain and 58 in the petite mutant. Among the proteins identified, 10 were higher in the mutant strain, whereas 23 were lower compared to the parent strain. The results revealed a significant decrease in the enzymes associated with the metabolic rate of mutant cells such as aconitase, transaldolase, and pyruvate kinase, and changes in the levels of specific heat shock proteins. Moreover, transketolase, aconitase and catalase activity measurements decreased significantly in the ethidium bromide-induced petite mutant. These data may be useful for designing experiments to obtain a better understanding of the nuclear response to impairment of mitochondrial function associated with this mutation in C. glabrata.

Clinical applicability of natural product(s)-containing mouthwashes as adjunctive treatment of biofilm-induced gingivitis: a systematic review / Aplicabilidade clínica de colutórios à base de produtos naturais como tratamento adjuvante da gengivite induzida por biofilme: uma revisão sistemática

Natural products have emerged as an effective and low-cost alternative for treating various diseases of the oral cavity. This study aimed to evaluate, through a systematic literature review, if there is scientific evidence ensuring the safe and effective use of natural product(s)-containing mouthwashes as adjunctive treatment of biofilm-induced gingivitis. Searches were conducted in the databases Medline, SciELO, LILACS and Cochrane Library, by using combinations of the key words gingivitis/natural products/phytotherapy/mouthwash, in English, Portuguese and Spanish. Studies published until September 2010 were considered. Four examiners analyzed independently: study design and phase, methodological quality (Jadad scale - JE), experimental product and its concentration, dosing interval and time of usage, as well as employed statistical analysis and clinical outcome of interest. From the 503 articles found, 08 were included in the final review as phase II, controlled, randomized and blind clinical trials, scoring 4 (25%) and 5 (75%) in JE. The main natural products assessed were: Azadirachta indica, Garcinia mangostana, Lippia sidoides, Salvadora persica and Sesamum indicum whose concentration, dosing interval, time of usage and adverse effects varied according to each study. The Plaque and Gingival Index were most employed, as well as α = 5% and paired t, Student's t, Wilcoxon and Mann-Whitney tests. A total of 62.5% and 50% of the products significantly reduced supragingival biofilm and gingivitis, respectively. Mouthwashes containing the essential oil from the leaves of L. sidoides (1%) and the extract from the leaves of A. indica (25%) can be indicated as adjunctive treatment of biofilm-induced gingivitis.

Os produtos naturais têm surgido como alternativa eficaz e de baixo custo para o tratamento de várias doenças da cavidade oral. Objetivou-se avaliar, a partir de revisão sistemática da literatura, se há evidências científicas garantindo a utilização segura e eficaz de antissépticos bucais contendo produto(s) natural(is) como tratamento adjuvante da gengivite induzida por biofilme. Foram realizadas buscas nas bases de dados Medline, SciELO, LILACS e Cochrane Library, através de combinações usando as palavras-chave gengivite/produtos naturais/ fitoterápicos/bochechos, em Inglês, Português e Espanhol. Consideraram-se os estudos publicados até setembro de 2010. Quatro examinadores analisaram separadamente: desenho e fase do estudo, qualidade metodológica (escala de Jadad - EJ), produto experimental e a concentração, intervalo de administração e tempo de uso, bem como a análise estatística empregada e os resultados clínicos de interesse. Foram encontrados 503 artigos dos quais 08 foram incluídos na revisão final como sendo ensaios clínicos fase II, controlados, randomizados e cegos, marcando 4 (25%) e 5 (75%) na EJ. Os principais produtos naturais avaliados foram Azadirachta indica, Garcinia mangostana, Lippia sidoides, Salvadora persica e Sesamum indicum, cuja concentração, intervalo de administração, tempo de uso, e efeitos adversos, variaram de acordo com cada estudo. Índice de placa e Índice Gengival foram os mais utilizados, bem como α=5% e testes t-pareado, t-Student, Wilcoxon e Mann-Whitney. 62,5% e 50% dos produtos reduziram significativamente a presença de bioflme supragengival e gengivite, respectivamente. Os colutórios contendo o óleo essencial das folhas de L. sidoides (1%) e o extrato das folhas de A. indica (25%) podem ser indicados como tratamento adjuvante da gengivite induzida por biofilme.

Interactions of Aspergillus fumigatus with vascular endothelial cells.

Invasive aspergillosis is characterized by two different types of angioinvasion. During pulmonary aspergillosis, hyphae are initially outside of the pulmonary vasculature and they invade the endothelial cell lining of the blood vessels by passing from the abluminal to the luminal surface. Some of these hyphal fragments can break off and circulate in the bloodstream. In severely immunocompromised hosts, these blood-borne hyphal fragments adhere to the luminal surface of the endothelial cells and they penetrate the endothelial cell lining of the vasculature by passing from the luminal to the abluminal surface. We have set up in vitro models of luminal and abluminal endothelial cell invasion by Aspergillus fumigatus. Luminal invasion by hyphae results in both endothelial cell damage and stimulation of tissue factor expression. Abluminal invasion causes less endothelial cell damage than luminal invasion, but greater induction of endothelial cells genes encoding cytokines, leukocyte adhesion molecules and tissue factor. These differences in the endothelial cell response to luminal versus abluminal infection may indicate significant differences in the pathogenesis of hematogenously disseminated versus locally invasive versus aspergillosis.

Proteomic analysis of cytosolic proteins associated with petite mutations in Candida glabrata

The incidence of superficial or deep-seated infections due to Candida glabrata has increased markedly, probably because of the low intrinsic susceptibility of this microorganism to azole antifungals and its relatively high propensity to acquire azole resistance. To determine changes in the C. glabrata proteome associated with petite mutations, cytosolic extracts from an azole-resistant petite mutant of C. glabrata induced by exposure to ethidium bromide, and from its azole-susceptible parent isolate were compared by two-dimensional polyacrylamide gel electrophoresis. Proteins of interest were identified by peptide mass fingerprinting or sequence tagging using a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer. Tryptic peptides from a total of 160 Coomassie-positive spots were analyzed for each strain. Sixty-five different proteins were identified in the cytosolic extracts of the parent strain and 58 in the petite mutant. Among the proteins identified, 10 were higher in the mutant strain, whereas 23 were lower compared to the parent strain. The results revealed a significant decrease in the enzymes associated with the metabolic rate of mutant cells such as aconitase, transaldolase, and pyruvate kinase, and changes in the levels of specific heat shock proteins. Moreover, transketolase, aconitase and catalase activity measurements decreased significantly in the ethidium bromide-induced petite mutant. These data may be useful for designing experiments to obtain a better understanding of the nuclear response to impairment of mitochondrial function associated with this mutation in C. glabrata.

Development of an enzyme-linked immunosorbent assay for the serodiagnosis of several clinical forms of sporotrichosis.

We performed a serological study with sera from 92 patients with confirmed sporotrichosis registered between 1999 and 2004 in two hospitals in Rio de Janeiro State, Brazil. The clinical presentation of sporotrichosis was distributed as follows: lymphocutaneous, 67%; fixed cutaneous, 23%; disseminated cutaneous, 8%; and extracutaneous, 2%. Sera were assayed by ELISA against a cell wall antigen of Sporothrix schenckii, SsCBF, that we have previously described. The cross-reactivity was determined with 77 heterologous sera. The serological test showed a sensitivity of 90% and a global efficiency of 86%. A group of 55 patients with several clinical presentations of sporotrichosis was clinically and serologically followed-up for at least 6 months. We observed by ELISA data a decrease in the antibody serum titers which correlated with the progress in healing. An HIV-positive patient with meningeal sporotrichosis was serologically followed-up for over 2 years. Serum and cerebrospinal fluid specimens were examined and significant antibodies levels against the antigen SsCBF were detected. Our results strongly suggest that this serological test is valuable for the differential diagnosis and follow-up of all clinical forms of sporotrichosis.

Identification of a concanavalin A-binding antigen of the cell surface of Sporothrix schenckii.

Sporothrix schenckii (1099-18) cell wall peptido-rhamnomannan (CWPR) was fractionated by affinity chromatography with Concanavalin A. The Con A-bound and Con A-unbound fractions were probed with an anti-S. schenckii rabbit serum. We identified within the Con A-bound fraction three main antigens with approximate molecular weights of 84, 70 and 58 kDa. Glycopeptide beta-elimination reduced rabbit antiserum reactivity for the 84 kDa antigen (gp84) with concomittant enhanced reactivity for the 70 kDa antigen (gp70). By Western blot with Con A-HRP conjugate we demonstrated that gp84 strongly reacted with this lectin and this was the predominant antigen identified. The gp84 antigen was also demonstrated to be present on other S. schenckii strains.

Concanavalin A-binding cell wall antigens of Sporothrix schenckii: a serological study.

Sporothrix schenckii cell wall peptido-rhamnomannan (CWPR) was fractionated by affinity chromatography on Sepharose 4B-Concanavalin A giving rise to S. schenckii concanavalin A-binding (SsCBF) and nonbinding (SsNBF) fractions. The CWPR, SsCBF and SsNBF fractions were probed by enzyme-linked immunosorbent assay (ELISA) with the sera of 35 patients with sporotrichosis from Latin America. Both CWPR and SsNBF, although reacting with sera from patients with sporotrichosis, were also cross-reactive with sera from healthy individuals and from patients with other mycoses. In contrast, SsCBF was shown to react specifically with 100% of the sera from patients with sporotrichosis, compared with control sera and sera from patients with other mycoses or cutaneous leishmaniasis. This antigenic fraction was submitted to mild alkaline hydrolysis to remove O-glycosidically linked oligosaccharides and the resulting components had decreased reactivity with patients' sera. The purified SsCBF-derived O-linked pentasaccharide and tetrasaccharide were potent inhibitors of, respectively 76 and 53% of the reaction between SsCBF and sera from patients with sporotrichosis. Our results suggest that SsCBF is a species-specific antigenic fraction recognized by human sera and that this specificity correlates with the presence of O-linked mannose-containing oligosaccharides decorated with the alpha-L-Rha 1-->4 alpha-D-GlcA and alpha-L-Rha 1-->4 [alpha-L-Rha 1-->2] alpha-D-GlcA epitope structures previously described [1].

Virulence of Sporothrix schenckii conidia and yeast cells, and their susceptibility to nitric oxide.

The involvement of nitric oxide (NO) in macrophage (M phi) fungicidal activity against Sporothrix schenckii, and the relationship between NO susceptibility and the differential virulence of conidia and yeast cells, were investigated. Confirming a previously reported correlation between the length of time in culture and virulence of S. schenckii, conidia isolated from 12-day mycelial cultures (Ss-12) were less virulent to mice than conidia from 7-day cultures (Ss-7) or yeast cells. Indicative of NO production, infected animals showed a significant increase in serum levels of nitrite that was lower in mice infected with Ss-12 than in mice infected with Ss-7 or yeast. Stimulation of murine M phi with interferon-gamma (IFN-gamma) induced NO production and inhibition of fungal growth. The cytotoxic activity of M phi against Ss-12 was significantly greater than against Ss-7 or yeast cells, the highly virulent fungal forms. The addition of NO synthase inhibitors abrogated M phi cytotoxic activity against all fungal forms. The phagocytic activity of M phi against Ss-7 was significantly lower than against Ss-12 or yeast cells. Although the ingestion of fungal cells triggered the oxidative burst in M phi, the fungicidal activity was not altered in the presence of superoxide dismutase (SOD) and catalase. In addition, Ss-12 and yeast cells were more susceptible than Ss-7 to the direct fungicidal activity of the NO donors S-nitroso-N-acetyl-DL-penicillamine (SNAP), S-nitrosoglutathione (GSNO) and 3-morpholinosydnonimine (SIN-1). The results of this study indicate that NO is a key cytotoxic mediator involved in the murine M phi defence against S. schenckii, and that the virulence of Ss-7, Ss-12 and yeast cells may be related to a differential susceptibility to NO.

Involvement of fungal cell wall components in adhesion of Sporothrix schenckii to human fibronectin.

Systemic sporotrichosis is an emerging infection potentially fatal for immunocompromised patients. Adhesion to extracellular matrix proteins is thought to play a crucial role in invasive fungal diseases. Here we report studies of the adhesion of Sporothrix schenckii to the extracellular protein fibronectin (Fn). Both yeast cells and conidia of S. schenckii were able to adhere to Fn as detected by enzyme-linked immunosorbent binding assays. Adhesion of yeast cells to Fn is dose dependent and saturable. S. schenckii adheres equally well to 40-kDa and 120-kDa Fn proteolytic fragments. While adhesion to Fn was increased by Ca(2+), inhibition assays demonstrated that it was not RGD dependent. A carbohydrate-containing cell wall neutral fraction blocked up to 30% of the observed adherence for the yeast cells. The biochemical nature of this fraction suggests the participation of cell surface glycoconjugates in binding by their carbohydrate or peptide moieties. These results provide new data concerning S. schenckii adhesion mechanisms, which could be important in host-fungus interactions and the establishment of sporotrichosis.

Characterization of novel structures of mannosylinositolphosphorylceramides from the yeast forms of Sporothrix schenckii.

Novel structures of glycoinositolphosphorylceramide (GIPC) from the infective yeast form of Sporothrix schenckii were determined by methylation analysis, mass spectrometry and NMR spectroscopy. The lipid portion was characterized as a ceramide composed of C-18 phytosphingosine N-acylated by either 2-hydroxylignoceric acid (80%), lignoceric (15%) or 2,3-dihydroxylignoceric acids (5%). The ceramide was linked through a phosphodiester to myo-inositol (Ins) which is substituted on position O-6 by an oligomannose chain. GIPC-derived Ins oligomannosides were liberated by ammonolysis and characterized as: Manpalpha1-->6Ins; Manpalpha1-->3Manpalpha1-->6Ins; Manpalpha1-->6Manpalpha1-->3Manpalpha1-->3Manpalpha1-->6Ins; Manpalpha1-->2Manpalpha1-->6Manpalpha1-->3Manpalpha1-->3Manpalpha1-->6Ins. These structures comprise a novel family of fungal GIPC, as they contain the Manpalpha1-->6Ins substructure, which has not previously been characterized unambigously, and may be acylated with a 2,3 dihydroxylignoceric fatty acid, a feature hitherto undescribed in fungal lipids.