Schenckii Yeast Cells Research Articles

Proteases of Sporothrix schenckii: Cytopathological effects on a host-cell model

BackgroundSporotrichosis is a fungal infection caused by the Sporothrix schenckii complex. The adhesion of the fungus to the host tissue has been considered the key step in the colonization and invasion, but little is known about the early events in the host–parasite interaction. AimsTo evaluate the proteolytic activity of S. schenckii on epithelial cells. MethodsThe proteolytic system (at pH 5 and 7) was evaluated using azocoll and zymograms. The host–parasite interaction and epithelial cell response were also analyzed by examining the microfilament cytoskeleton using phalloidin-FITC and transmission electron microscopy. Finally, the metabolic activity was determined using an XTT assay. ResultsThe zymograms showed that S. schenckii yeast cells possess high intracellular and extracellular proteolytic activities (Mr≥200, 116, 97, and 70kDa) that are pH dependent and are inhibited by PMSF and E64, which act on serine and cysteine-type proteases. During the epithelial cell–protease interaction, the cells showed alterations in the microfilament distribution, as well as in the plasma membrane structure. Moreover, the metabolic activity of the epithelial cells decreased 60% without a protease inhibitor. ConclusionsOur data demonstrate the complexity of the cellular responses during the infection process. This process is somehow counteracted by the action of proteases inhibitors. Furthermore, the results provide critical information for understanding the nature of host–fungus interactions and for searching a new effective antifungal therapy, which includes protease inhibitors.

Characterization and ligand identification of a membrane progesterone receptor in fungi: existence of a novel PAQR in Sporothrix schenckii

BackgroundAdaptive responses in fungi result from the interaction of membrane receptors and extracellular ligands. Many different classes of receptors have been described in eukaryotic cells. Recently a new family of receptors classified as belonging to the progesterone-adiponectin receptor (PAQR) family has been identified. These receptors have the seven transmembrane domains characteristic of G-protein coupled receptors, but their activity has not been associated directly to G proteins. They share sequence similarity to the eubacterial hemolysin III proteins.ResultsA new receptor, SsPAQR1 (Sporothrixschenckiiprogesterone-adiponectinQ receptor1), was identified as interacting with Sporothrix schenckii G protein alpha subunit SSG-2 in a yeast two-hybrid assay. The receptor was identified as a member of the PAQR family. The cDNA sequence revealed a predicted ORF of 1542 bp encoding a 514 amino acids protein with a calculated molecular weight of 57.8 kDa. Protein domain analysis of SsPAQR1 showed the 7 transmembrane domains (TM) characteristic of G protein coupled receptors and the presence of the distinctive motifs that characterize PAQRs. A yeast-based assay specific for PAQRs identified progesterone as the agonist. S. schenckii yeast cells exposed to progesterone (0.50 mM) showed an increase in intracellular levels of 3′, 5′ cyclic adenosine monophosphate (cAMP) within the first min of incubation with the hormone. Different progesterone concentrations were tested for their effect on the growth of the fungus. Cultures incubated at 35°C did not grow at concentrations of progesterone of 0.05 mM or higher. Cultures incubated at 25°C grew at all concentrations tested (0.01 mM-0.50 mM) with growth decreasing gradually with the increase in progesterone concentration.ConclusionThis work describes a receptor associated with a G protein alpha subunit in S. schenckii belonging to the PAQR family. Progesterone was identified as the ligand. Exposure to progesterone increased the levels of cAMP in fungal yeast cells within the first min of incubation suggesting the connection of this receptor to the cAMP signalling pathway. Progesterone inhibited the growth of both the yeast and mycelium forms of the fungus, with the yeast form being the most affected by the hormone.

L-DOPA accessibility in culture medium increases melanin expression and virulence ofSporothrix schenckiiyeast cells

Melanin is a complex polymer widely distributed in nature and has been described as an important virulence factor in several pathogenic fungi, including Sporothrix schenckii. The aim of the present work was to investigate the presence of melanin on the surface of S. schenckii yeast cells which showed differences in their virulence depending on the culture conditions under which they were grown. Yeast cells were cultivated in brain heart infusion (BHI) broth from Difco and Oxoid. BHI from these two vendors are different in their brain and heart infusion contents. Yeasts cultivated in the medium containing the higher brain infusion content were highly virulent as ascertained by the mice mortality rate, CFU and histopathology. Transmission electron microscopy revealed a higher expression of electron dense granules on the fungal cell wall of the most virulent yeast cells. Flow cytometry analysis, with anti-melanin antibodies, confirmed that this pigment was melanin. Furthermore, spectrophotometric analysis showed a higher concentration of this polymer on NaOH and cell wall extracts of the most virulent yeast cells. These results suggest that differences in the relative content of brain and heart infusion in the culture medium modulated melanin expression on the surface of S. schenckii yeast cells and, as a consequence, virulence. A new pathway of melanin biosynthesis in S. schenckii is proposed which involves the use of phenolic compounds from rich brain medium as melanin substrate.

Response of macrophage Toll‐like receptor 4 to a Sporothrix schenckii lipid extract during experimental sporotrichosis

Toll-like receptors have been implicated in the recognition of various pathogens, including bacteria, viruses, protozoa and fungi. However, no information is available about Toll-like receptor 4 (TLR4) participation in Sporothrix schenckii recognition and the consequent triggering of the immune response to this fungal pathogen. Following activation of TLRs by ligands of microbial origin, several responses are provoked, including reactions in immune cells that may lead them to produce signalling factors that trigger inflammation. The present study was designed to elucidate the role of TLR4 during the host response to S. schenckii. TLR4-deficient (C3H/HeJ) and control mice (C3H/HePas) were infected with S. schenckii yeast cells and immune response was assessed over 10 weeks by assaying production of pro-inflammatory mediator (nitric oxide and tumour necrosis factor-alpha) and anti-inflammatory cytokine (interleukin-10) by peritoneal macrophages and their correlation with apoptosis in peritoneal exudate cells. We found that both pro-inflammatory and anti-inflammatory mediators are reduced in TLR4-deficient mice, suggesting the involvement of this receptor in the recognition of this infectious agent. Translocation into the nucleus of nuclear transcription factor, nuclear factor-kappaB, was also evaluated and showed higher levels in TLR-4 normal mice, consistent with the results found for cytokine production. We are showing here, for the first time, the involvement of TLR4 in S. schenckii recognition. Taken together, our results demonstrate that the activation of peritoneal macrophages in response to S. schenckii lipid extracts has different responses in these two mouse strains which differ in TLR4 expression, suggesting an important role for TLR4 in governing the functions of macrophages in this fungal infection.

Cytosolic phospholipase A2: a member of the signalling pathway of a new G protein α subunit in Sporothrix schenckii

BackgroundSporothrix schenckii is a pathogenic dimorphic fungus, the etiological agent of sporotrichosis, a lymphocutaneous disease that can remain localized or can disseminate, involving joints, lungs, and the central nervous system. Pathogenic fungi use signal transduction pathways to rapidly adapt to changing environmental conditions and S. schenckii is no exception. S. schenckii yeast cells, either proliferate (yeast cell cycle) or engage in a developmental program that includes proliferation accompanied by morphogenesis (yeast to mycelium transition) depending on the environmental conditions. The principal intracellular receptors of environmental signals are the heterotrimeric G proteins, suggesting their involvement in fungal dimorphism and pathogenicity. Identifying these G proteins in fungi and their involvement in protein-protein interactions will help determine their role in signal transduction pathways.ResultsIn this work we describe a new G protein α subunit gene in S. schenckii, ssg-2. The cDNA sequence of ssg-2 revealed a predicted open reading frame of 1,065 nucleotides encoding a 355 amino acids protein with a molecular weight of 40.9 kDa. When used as bait in a yeast two-hybrid assay, a cytoplasmic phospholipase A2 catalytic subunit was identified as interacting with SSG-2. The sspla2 gene, revealed an open reading frame of 2538 bp and encoded an 846 amino acid protein with a calculated molecular weight of 92.62 kDa. The principal features that characterize cPLA2 were identified in this enzyme such as a phospholipase catalytic domain and the characteristic invariable arginine and serine residues. A role for SSPLA2 in the control of dimorphism in S. schenckii is suggested by observing the effects of inhibitors of the enzyme on the yeast cell cycle and the yeast to mycelium transition in this fungus. Phospholipase A2 inhibitors such as AACOCF3 (an analogue of archidonic acid) and isotetrandrine (an inhibitor of G protein PLA2 interactions) were found to inhibit budding by yeasts induced to re-enter the yeast cell cycle and to stimulate the yeast to mycelium transition showing that this enzyme is necessary for the yeast cell cycle.ConclusionA new G protein α subunit gene was characterized in S. schenckii and protein-protein interactions studies revealed this G protein alpha subunit interacts with a cPLA2 homologue. The PLA2 homologue reported here is the first phospholipase identified in S. schenckii and the first time a PLA2 homologue is identified as interacting with a G protein α subunit in a pathogenic dimorphic fungus, establishing a relationship between these G proteins and the pathogenic potential of fungi. This cPLA2 homologue is known to play a role in signal transduction and fungal pathogenesis. Using cPLA2 inhibitors, this enzyme was found to affect dimorphism in S. schenckii and was found to be necessary for the development of the yeast or pathogenic form of the fungus.

Comparison of virulence of differentSporothrix schenckiiclinical isolates using experimental murine model

The virulence of two strains of Sporothrix schenckii isolated from patients with lymphocutaneous or disseminated sporotrichosis were examined in BALB/c mice (Group 1 and 2, respectively). The mice were inoculated subcutaneously into the left hind footpad with 4 x 10(6) S. schenckii yeast cells in order to evaluate (i) the development of cutaneous lesions, (ii) signs of inactivity, (iii) weight loss, (iv) survival rates, (v) number of viable yeast cells in the lungs and spleen, (vi) splenic index, (vii) extent of organ lesions, and (viii) immunological responses. Comparison of the two groups showed more severe disease in Group 2 mice that developed significant weight and hair loss associated with inactivity and left hind footpad lesions that extended close to the testicular area. The histopathology and large number of viable microorganisms isolated from the spleen confirmed the higher invasive ability of this strain. Moreover, a decrease of an in vitro specific lymphoproliferative response and IFN-gamma production were observed over time in Group 2 mice. As a result, at the end of the experiment, the S. schenckii-antigen (Ss-Ag) response was considered negative with a stimulation index (SI) = 2. In contrast, Group 1 mice presented a positive response to Ss-Ag (SI = 14.1). These results confirm the existence of different virulence profiles in S. schenckii strains. In addition, the use of subcutaneous inoculation as a suitable route for verification of the pathogenicity of this fungus in the murine model was confirmed.

The in vitro interaction of Sporothrix schenckii with human endothelial cells is modulated by cytokines and involves endothelial surface molecules

Sporothrix schenckii is the etiological agent of sporotrichosis, a subcutaneous mycosis that can evolve to systemic complications in immunocompromised patients. Interactions with endothelium are thought to be essential for systemic infections. In the present work, we studied the interaction between S. schenckii and human umbilical vein endothelial cells (HUVECs). S. schenckii interacts with HUVECs in a time-dependent manner. Morphological analysis showed that yeasts locate to interendothelial junctions. Ultrastructural studies showed that internalized yeasts were found inside endocytic vacuoles as early as 2 h, without causing any detectable damage to HUVECs after 24 h of infection. The viability of infected HUVECs was confirmed by the MTT assay. When HUVECs were treated with different concentrations of Interleukin-1β or transforming growth factor-beta, a significant dose-dependent increase in cell-associated yeasts was observed. The preliminary analysis of the endothelial surface ligands for S. schenckii cells revealed two major molecules, with M r of ≈90 and 135 kDa. The interaction of endothelial cell surface molecules with S. schenckii yeast cells was modulated by divalent cations. This is the first demonstration that S. schenckii is able to adhere and invade endothelial cells without significantly affect cellular integrity. Our results suggest the contribution of cytokine-modulated calcium-dependent molecules to this process.

Synthesis of melanin-like pigments by Sporothrix schenckii in vitro and during mammalian infection.

Melanin has been implicated in the pathogenesis of several important human fungal pathogens. Existing data suggest that the conidia of the dimorphic fungal pathogen Sporothrix schenckii produce melanin or melanin-like compounds; in this study we aimed to confirm this suggestion and to demonstrate in vitro and in vivo production of melanin by yeast cells. S. schenckii grown on Mycosel agar produced visibly pigmented conidia, although yeast cells grown in brain heart infusion and minimal medium broth appeared to be nonpigmented macroscopically. However, treatment of both conidia and yeast cells with proteolytic enzymes, denaturant, and concentrated hot acid yielded dark particles similar in shape and size to the corresponding propagules, which were stable free radicals consistent with identification as melanins. Melanin particles extracted from S. schenckii yeast cells were used to produce a panel of murine monoclonal antibodies (MAbs) which labeled pigmented conidia, yeast cells, and the isolated particles. Tissue from hamster testicles infected with S. schenckii contained fungal cells that were labeled by melanin-binding MAbs, and digestion of infected hamster tissue yielded dark particles that were also reactive. Additionally, sera from humans with sporotrichosis contained antibodies that bound melanin particles. These findings indicate that S. schenckii conidia and yeast cells can produce melanin or melanin-like compounds in vitro and that yeast cells can synthesize pigment in vivo. Since melanin is an important virulence factor in other pathogenic fungi, this pigment may have a similar role in the pathogenesis of sporotrichosis.

Calcium uptake and efflux during the yeast to mycelium transition inSporothrix schenckii

A study was made of calcium metabolism during germ tube formation in Sporothrix schenckii yeast cells. A net efflux of calcium was observed very early in the transformation process and remained constant thereafter. The efflux of calcium in yeast cells induced to form germ tubes was twice that observed in yeast cells not induced to form germ tubes. Two peaks of calcium uptake were observed in germ tube forming yeast cells at 30 and 300 minutes following inoculation, while non-induced yeast cells, a continuous increase in uptake was observed which ultimately reached higher values than the ones obtained in germ tube forming cells. Substances which affect calcium metabolism in other cells such as cobalt ions, ionophore A23187 and compound R24571 were observed to inhibit germ tube formation and calcium uptake. In addition, ionophore A23187 was found to increase calcium efflux to approximately twice the control values. The inhibition of germ tube formation brought about by substances which inhibit calcium uptake or increase efflux suggests that the intracellular calcium concentration in these cells must be precisely regulated for the yeast to mycelium transition to occur.

Ultrastructural localization of specific surface antigens in the dimorphic fungusSporothrix schenckii

The ultrastructural localization of antigens recognized by an antiserum raised against formaline-killed Sporothrix schenckii yeast cells was investigated on yeast and mycelial phases of the fungus. Immunogold procedures revealed that these antigens were located on the cell surface of both growth phases. Labelling was heterogeneous and involved areas of Concanavalin A-binding sugar residues.

Activation of the alternative complement pathway by Sporothrix schenckii.

We studied the activation of complement by Sporothrix schenckii yeast cells. Total complement activity, and the effect of various activators on this activity, were assayed on aliquots of fresh nonimmune human serum with and without prior treatment with chelators. Both total hemolytic complement and C3 were consumed (activated) in serum chelated with magnesium ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, which blocks the classical pathway but leaves the alternative pathway intact. Further, C3 was consumed, but C4 (exclusively a component of the classical pathway) was preserved, in nonchelated serum after challenge by S. schenckii yeast cells. Absorption of serum with S. schenckii yeast cells to deplete antibodies did not alter these results. Furthermore, immunofluorescence studies demonstrated that C3, but not immunoglobulin G, was deposited on yeast cells during incubation with nonimmune serum. These data indicate that S. schenckii yeast cells activate the alternative complement pathway in vitro independently of antibody. These data do not define a role for the alternative pathway in in vivo host defenses against infection with this organism but provide a foundation for studies to evaluate such a role.

Estudo da ocorrência da esporotricose-infecção em animais da espécie bovina

Bovines of three countries in the State of Sao Paulo, Brasil, show a high incidence of Sporotrichosis infection on according to the results obtained with intradermic test: 28,36%. The antigen chosen for this study was a suspension of S. schenckii yeast cells.

Purification and isolation of a biologically active peptido-rhamnogalactan from Sporothrix schenckii.

ABSTRACTA biologically active peptido‐rhamnogalactan was isolated from disrupted Sporothrix schenckii yeast cells by sodium deoxycholate extraction and was further purified by DEAE‐Sephadex A‐50 chromatography. This fraction, 60.6% sugar and 3.0% nitrogen, was active in delayed hypersensitivity skin tests in sporotrichosis patients and also cross‐reacted serologically with a peptido‐rhamnomannan isolated from Sporothrix schenckii culture filtrates. These results suggest strong that L‐rhamnose, α‐1, 2 linked, is more important in determining the serological specificity of Sporothrix schenckii than the α‐1, 3 linked L‐rhamnose or the mannan backbone of the peptido‐rhamnomannans so far studied by others.