Transcriptome Analysis of Dimorphic Fungus Sporothrix schenckii Exposed to Temperature Stress.

PURPOSE: Sporothrix schenckii is a thermally dimorphic fungus. In a saprotrophic environment or culturing at 25 °C, it grows as mycelia, whereas in host tissues or culturing at 37 °C, it undergoes dimorphic transition and division into pathogenic yeast cells. S. schenckii can cause serious disseminated sporotrichosis in immunocompromised hosts and presents an emerging global health problem. The mycelium-to-yeast transition was a consequence of the adaptive process to different environment. Some studies showed that the transition was significantly related to the virulence and pathogenesis of dimorphic fungi. However the genetic mechanisms of this complicated biological process are poorly understood. METHOD: Our study presented a comparative transcriptomic analysis perspective on temperature stress in a visceral isolates of S. schenckii, obtaining more genetic information related to dimorphic transition. RESULTS: The 9.38 Gbp dataset was generated and assembled into 14,423 unigenes. Compared with gene and protein databases, 9561 unigenes were annotated. Comparative analysis identified 1259 genes expressed differentially in mycelium and yeast phase, and were categorized into a number of important biological processes, such as synthesis and metabolism, transmembrane transport, biocatalysis, oxidation reduction, and cellular signal transduction. CONCLUSIONS: The findings suggested that temperature-dependent transition was tightly associated with stress adaptation, growth and development, signal regulation, adhesion, and colonization, which was predicted to be related with virulence and pathogenesis. Collection of these data should offer fine-scale insights into the mechanisms of dimorphism and pathogenesis of S. schenckii, and meanwhile facilitate the evolutionary and function studies of other dimorphic fungi.

Analysis of biofilm formation by Sporothrix schenckii.

The development of mature biofilms is an aid in numerous aspects of the life cycle of fungi. It is well known that Sporothrix schenckii complex causes a benign subcutaneous mycosis, but recent studies have suggestedthat biofilm formation may be one of the important factors involved in its virulence. Here we report the study of the biomass organization and a model of the stages of S. schenckii biofilm development: adsorption, active adhesion, microcolony formation, maturation, and dispersal of biofilm fragments. During the development, the biofilm is surrounded by extracellular matrix, which contains glycoprotein (mannose rich), carbohydrates, lipids, and nucleic acid. In addition, the extracellular DNA increases in extracellular matrix as a key component to structural integrity and antifungal resistance. The study of S. schenckii biofilm contributes to a better understanding of growth biofilm and physiology, adding new insights into the mechanisms of virulence and persistence of pathogenic microorganisms.

Influence of iron on growth and on susceptibility to itraconazole in Sporothrix spp.

We evaluated the growth and the susceptibility to oxidative stress of Sporothrix spp., exposed to different iron concentrations in culture medium, and the susceptibility of Sporothrix spp. to itraconazole, alone and in combination with to the iron chelator deferasirox. The results showed that the growth of S. brasiliensis isolates was more affected by iron availability in comparison to S. schenckii, but both fungal species conidia became more prone to oxidative stress when iron was added to culture medium. Conversely, the combination of itraconazole and deferasirox only resulted in synergism against a minority of S. schenckii isolates.

Morphological changes and phagocytic activity during the interaction of human neutrophils with Sporothrix schenckii: An in vitro model.

Histopathological studies of human sporotrichosis lesions show pyogenic and granulomatous processes in which polymorphonuclear neutrophils (PMNs) play a central role. Few studies regarding the events associated with the interaction of human PMNs with Sporothrix schenckii have been made despite their importance in the clinical manifestations of the disease. In this study, human PMNs were co-cultured with conidia or yeast cells of S. schenckii to compare the phagocytic activity and morphological changes that could provide a clearer insight into the role of these phagocytes in the initial phase of sporotrichosis. PMNs showed increased cell size and separation of the nuclear lobes after phagocytosis. Through Scanning Electron Microscopy (SEM) analysis, an increase in cells with flattened filaments and vesicles on their surface was observed. Phagocytosed conidia showed a significant increase in width and size. The phagocytic activity was greater against yeasts than with conidia, but the viability of both S. schenckii cellular morphotypes was not drastically affected even after 2 h of co-culture. In conclusion, morphological changes in PMNs suggest that S. schenckii induces processes that may favor proinflammatory events. These phagocytes show a high ability to bind or ingest S. schenckii cells without affecting their viability. Morphological changes recorded in ingested conidia, suggest that this fungus could make the dimorphic switching in PMNs.

Congo Red affects the growth, morphology and activity of glucosamine-6-phosphate synthase in the human pathogenic fungus Sporothrix schenckii.

Sporothrix schenckii is the etiological agent of sporotrichosis, a mycosis of humans and other mammals. Little is known about the responses of this thermodimorphic pathogen to perturbations in the cell wall (CW) by different stress conditions. Here we describe the effect of Congo Red (CR) on the fungal growth, morphogenesis and activity of glucosamine-6-phosphate (GlcN-6-P) synthase. Under conditions of yeast development, 15 µM CR abolished conidia (CN) germination, but when yeast cells were first obtained in the absence of the dye and then post-incubated in its presence, yeasts rapidly differentiated into mycelial cells. On the other hand, under conditions of mycelium development, 150 µM CR did not affect CN germination, but filamentous cells underwent structural changes characterized by a distorted CW contour, the loss of polarity and the formation of red-pigmented, hyphal globose structures. Under these conditions, CR also induced a significant and transient increase in the activity of GlcN-6-P synthase, an essential enzyme in CW biogenesis.

Interaction with Pantoea agglomerans Modulates Growth and Melanization of Sporothrix brasiliensis and Sporothrix schenckii.

Sporothrix brasiliensis and Sporothrix schenckii stand as the most virulent agents of sporotrichosis, a worldwide-distributed subcutaneous mycosis. The origin of Sporothrix virulence seems to be associated with fungal interactions with organisms living in the same environment. To assess this hypothesis, the growth of these two species in association with Pantoea agglomerans, a bacterium with a habitat similar to Sporothrix spp., was evaluated. Growth, melanization, and gene expression of the fungus were compared in the presence or absence of the bacterium in the same culture medium. Both S. brasiliensis and S. schenckii grew in contact with P. agglomerans yielding heavily melanized conidia after 5 days of incubation at 30 °C in Sabouraud agar. This increased melanin production occurred around bacterial colonies, suggesting that fungal melanization is triggered by a diffusible bacterial product, which is also supported by a similar pattern of melanin production during Sporothrix spp. growth in contact with heat-killed P. agglomerans. Growth of P. agglomerans was similar in the presence or absence of the fungus. However, the growth of S. brasiliensis and S. schenckii was initially inhibited, but further enhanced when these species were co-cultured with P. agglomerans. Moreover, fungi were able to use killed bacteria as both carbon and nitrogen sources for growth. Representational difference analysis identified overexpressed genes related to membrane transport when S. brasiliensis was co-cultured with the bacteria. The down-regulation of metabolism-related genes appears to be related to nutrient availability during bacterial exploitation. These findings can lead to a better knowledge on Sporothrix ecology and virulence.

Efficacy of a poly-aggregated formulation of amphotericin B in treating systemic sporotrichosis caused by Sporothrix brasiliensis.

In severe cases of sporotrichosis, it is recommended to use amphotericin B deoxycholate (D-AMB) or its lipid formulations and/or in association with itraconazole (ITC). Our aim was to evaluate the antifungal efficacy of a poly-aggregated amphotericin B (P-AMB), a nonlipid formulation, compared with D-AMB on systemic sporotrichosis caused by Sporothrix brasiliensis. In vitro assays showed that Sporothrix schenckii sensu stricto and S. brasiliensis yeast clinical isolates were susceptible to low concentrations of P-AMB and D-AMB. Although P-AMB presented a higher minimal inhibitory concentration (MIC) compared to D-AMB, its cytotoxic effect on renal cells and erythrocytes was lower. For the in vivo assays, male BALB/c mice were intravenously infected with S. brasiliensis yeasts, and P-AMB or D-AMB was administered 3 days post-infection. The efficacy of five therapeutic regimens was tested: intravenous monotherapy with P-AMB or D-AMB, intravenous pulsed-therapy with P-AMB or D-AMB, and intravenous therapy with P-AMB, followed by oral ITC. These treatments increased murine survival and controlled the fungal burden in the liver, spleen, lungs, and kidneys. However, only D-AMB monotherapy or the pulsed-therapies with D-AMB or P-AMB led to 100% survival of the mice 45 days post-infection; only pulsed administration of D-AMB was able to control the fungal load in all organs 45 days post-infection. Accordingly, the histopathological findings showed reductions in the fungal burden and inflammatory reactions in these treatment regimens. Together, our results suggest that the P-AMB formulation could be considered as an alternative drug to D-AMB for treating disseminated sporotrichosis.

Cholinergic enzymes and inflammatory markers in rats infected by Sporothrix schenckii.

The aim of this study was to evaluate the cholinesterase activity in serum, whole blood, and lymphocytes, as well as to verify its relation to immune response in rats experimentally infected by Sporothrix schenckii. For this study, 63 Wistar rats (Rattus norvegicus), male, adult were divided into three groups: the negative control group (GC: n = 21), the group infected subcutaneously (GSC: n = 21), and the group infected intraperitoneally (GIP: n = 21). The groups were divided into subgroups and the following variables were evaluated at 15, 30, and 40 days post-infection (PI): acetylcholinesterase (AChE) activity in lymphocytes and whole blood, butyrylcholinesterase (BChE) activity in serum, cytokines levels (IL-1, IL-6, TNFα, and INF-γ), immunoglobulins levels (IgA, IgG, IgM, and IgE), and protein profile by electrophoresis. Both infected groups showed increased levels of inflammatory parameters (P < 0.05) in tissue and inflammatory infiltrates. The activities of AChE in lymphocytes and BChE in serum increased (P < 0.05) significantly in animals from the GSC group on day 40 PI compared to the GC group. Regarding the GIP, there was a marked increase in the AChE activity in lymphocytes on days 30 and 40 PI, and in whole blood on days 15, 30, and 40 PI compared to GC. Furthermore, IL-10, an anti-inflammatory cytokine, was also present in high levels during chronic systemic S. schenckii infections in animals. Therefore, it is concluded that cholinesterase has an important modulatory role in the immune response during granulomatous infection by S. schenckii.

In Vitro Studies of Chromone-Tetrazoles against Pathogenic Protozoa, Bacteria, and Fungi.

In vitro studies to fourteen previously synthesized chromone-tetrazoles and four novel fluorine-containing analogs were conducted against pathogenic protozoan (Entamoeba histolytica), pathogenic bacteria (Pseudomonas aeruginosa, and Staphylococcus aureus), and human fungal pathogens (Sporothrix schenckii, Candida albicans, and Candida tropicalis), which have become in a serious health problem, mainly in tropical countries.

Susceptibility of species within the Sporothrix schenckii complex to a panel of killer yeasts.

The Sporothrix schenckii complex is the etiologic agent of sporotrichosis, a subacute or chronic mycosis which can affect humans and animals. Killer yeasts have been used in the medical field for development of novel antimycotics and biotyping of pathogenic fungi. The action of 18 killer yeasts on the growth of 88 characterized S. schenckii, Sporothrix globosa, Sporothrix brasiliensis, and Sporothrix mexicana clinical and environmental isolates was evaluated. Killer studies were performed on Petri dishes containing cheese black starch agar. The yeasts Candida catenulata (QU26, QU31, QU127, LV102); Trichosporon faecale (QU100); Trichosporon japonicum (QU139); Kluyveromyces lactis (QU30, QU99, QU73); Kazachstania unispora (QU49), Trichosporon insectorum (QU89), and Kluyveromyces marxianus (QU103) showed activity against all strains of the S. schenckii complex tested. Observation by optical microscopy of S. brasiliensis 61 within the inhibition haloes around the colonies of the killer yeasts QU100, QU139, and LV102 showed that there was no conidiation, but there was hyphal proliferation. The toxins were fungistatic against S. brasiliensis 61. There was no difference in susceptibility to the toxins among the S. schenckii species complex. Further investigations are necessary to clearly establish the mechanism of action of the toxins.

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

BACKGROUND: Adaptive 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. RESULTS: A new receptor, SsPAQR1 (Sporothrix schenckii progesterone-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. CONCLUSION: This 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.

Cell-free antigens of Sporothrix brasiliensis: antigenic diversity and application in an immunoblot assay.

Sporotrichosis is a subcutaneous mycosis diagnosed by isolation of the fungus in culture. Serological tests for help in diagnosis in general do not use purified or recombinant antigens, because there is a paucity of described immunoreactive proteins, especially for the new described Sporothrix species, such as Sporothrix brasiliensis. This study aims to characterise antigens from S. brasiliensis and verify their application in serodiagnosis of sporotrichosis. An immunoblot assay allied with computer-based analysis was used to identify putative antigenic molecules in a cell-free extracts of both morphological phases of this fungus, and to delineate antigenic polymorphism among seven S. brasiliensis isolates and one S. schenckii Brazilian strain. The mycelial and yeast phase of the fungus originated 14 and 23 reactive bands, respectively, which were variable in intensity. An 85 kDa antigen, verified in the yeast phase of the fungus, was observed in all strains used and the immunodominant protein was identified. This protein, however, cross-react with serum samples from patients infected with other pathogens. The results show that the S. brasiliensis cell-free antigen extract is a single and inexpensive source of antigens, and can be applied on the sporotrichosis serodiagnosis.

Calcium/calmodulin kinase1 and its relation to thermotolerance and HSP90 in Sporothrix schenckii: an RNAi and yeast two-hybrid study.

BACKGROUND: Sporothrix schenckii is a pathogenic dimorphic fungus of worldwide distribution. It grows in the saprophytic form with hyaline, regularly septated hyphae and pyriform conidia at 25°C and as the yeast or parasitic form at 35°C. Previously, we characterized a calcium/calmodulin kinase in this fungus. Inhibitors of this kinase were observed to inhibit the yeast cell cycle in S. schenckii. RESULTS: The presence of RNA interference (RNAi) mechanism in this fungus was confirmed by the identification of a Dicer-1 homologue in S. schenckii DNA. RNAi technology was used to corroborate the role of calcium/calmodulin kinase I in S. schenckii dimorphism. Yeast cells were transformed with the pSilent-Dual2G (pSD2G) plasmid w/wo inserts of the coding region of the calcium/calmodulin kinase I (sscmk1) gene. Transformants were selected at 35°C using resistance to geneticin. Following transfer to liquid medium at 35°C, RNAi transformants developed as abnormal mycelium clumps and not as yeast cells as would be expected. The level of sscmk1 gene expression in RNAi transformants at 35°C was less than that of cells transformed with the empty pSD2G at this same temperature. Yeast two-hybrid analysis of proteins that interact with SSCMK1 identified a homologue of heat shock protein 90 (HSP90) as interacting with this kinase. Growth of the fungus similar to that of the RNAi transformants was observed in medium with geldanamycin (GdA, 10 µM), an inhibitor of HSP90. CONCLUSIONS: Using the RNAi technology we silenced the expression of sscmk1 gene in this fungus. RNAi transformants were unable to grow as yeast cells at 35°C showing decreased tolerance to this temperature. The interaction of SSCMK1 with HSP90, observed using the yeast two-hybrid assay suggests that this kinase is involved in thermotolerance through its interaction with HSP90. SSCMK1 interacted with the C terminal domain of HSP90 where effector proteins and co-chaperones interact. These results confirmed SSCMK1 as an important enzyme involved in the dimorphism of S. schenckii, necessary for the development of the yeast phase of this fungus. Also this study constitutes the first report of the transformation of S. schenckii and the use of RNAi to study gene function in this fungus.

Gamma radiation effects on Sporothrix schenckii yeast cells.

Sporotrichosis is a subcutaneous mycosis caused by Sporothrix schenckii. Zoonotic transmission to man can occur after scratches or bites of animals, mainly cats. In this study, the gamma radiation effects on yeast of S. schenckii were analyzed with a view of developing a radioattenuated vaccine for veterinary use. The cultures were irradiated at doses ranging from 1.0 to 9.0 kGy. The reproductive capacity was measured by the ability of cells to form colonies. No colonies could be recovered above 8.0 kGy, using inocula up to 10(7) cells. Nevertheless, yeast cells irradiated with 7.0 kGy already were unable to produce infection in immunosuppressed mice. Evaluation by the FungaLight™ Kit (Invitrogen) indicated that yeast cells remained viable up to 9.0 kGy. At 7.0 kGy, protein synthesis, estimated by the incorporation of [L-(35)S] methionine, continues at levels slightly lower than the controls, but a significant decrease was observed at 9.0 kGy. The DNA of 7.0 kGy irradiated cells, analyzed by electrophoresis in agarose gel, was degraded. Cytoplasmic vacuolation was the main change verified in these cells by transmission electron microscopy. The dose of 7.0 kGy was considered satisfactory for yeast attenuation since irradiated cells were unable to produce infection but retained viability, metabolic activity, and morphology.

In vitro antifungal susceptibility of Sporothrix globosa isolates from Jilin Province, northeastern China: comparison of yeast and mycelial phases.

The dimorphic fungus Sporothrix globosa is the predominant etiologic agent causing sporotrichosis in China, particularly in the northeast. It has been demonstrated that the incubation temperature and growth phase can influence in vitro antifungal susceptibility profiles of S. schenckii sensu stricto and S. brasiliensis (sibling species of S. globosa). Few studies have reported on the antifungal susceptibility of S. globosa, especially using large numbers of isolates. In this study, we assessed the susceptibility of 80 isolates of S. globosa originating from Jilin Province, northeastern China, to six antifungal agents (itraconazole, terbinafine, voriconazole, posaconazole, fluconazole, and amphotericin B), at varying incubation temperatures and in different fungal growth phases. The isolates were most sensitive to terbinafine (geometric mean [GM] of the minimum inhibitory concentration [MIC]: 0.0356 µg/ml for the mycelial phase at 30 °C, 0.0332 µg/ml for the mycelial phase at 35 °C, and 0.031 µg/ml for the yeast phase, respectively), followed by posaconazole (GM of the MIC: 4.2501 µg/ml for the mycelial phase at 30 °C, 1.4142 µg/ml for the mycelial phase at 35 °C, and 0.7195 µg/ml for the yeast phase, respectively) and itraconazole (GM of the MIC: 6.8448 µg/ml for the mycelial phase at 30 °C, 3.1383 µg/ml for the mycelial phase at 35 °C, and 1.0263 µg/ml for the yeast phase, respectively). The isolates were relatively resistant to fluconazole (GM of the MIC: 76.7716 µg/ml for the mycelial phase at 30 °C, 66.2570 µg/ml for the mycelial phase at 35 °C, and 24.4625 µg/ml for the yeast phase, respectively) and voriconazole (GM of the MIC: 26.2183 µg/ml for the mycelial phase at 30 °C, 13.6895 µg/ml for the mycelial phase at 35 °C, and 1.3899 µg/ml for the yeast phase, respectively). For all the tested azole drugs, the MICs at 30 °C were significantly higher than those at 35 °C (P < 0.001); for all agents except terbinafine, the MICs of S. globosa in the yeast phase were significantly lower than those of the strains in the mycelial phase (P < 0.001). These results show that the sensitivities of S. globosa to antifungal compounds are dependent on incubation temperature and growth phase. To the best of our knowledge, this is the largest study of antifungal susceptibility of S. globosa isolates reported to date. To establish epidemiological cutoff values for S. globosa, further antifungal susceptibility testing studies by independent laboratories located in different regions and using uniform conditions are required.

Sporothrix brunneoviolacea and Sporothrix dimorphospora, two new members of the Ophiostoma stenoceras-Sporothrix schenckii complex.

Sporothrix inflata is a saprobic member of the Ophiostoma stenoceras-Sporothrix schenckii species complex, reported mainly from soil. Ophiostoma bragantinum, an ascomycete described from Brazil, has been proposed as its possible teleomorph. Previous studies revealed that Sporothrix inflata is phenotypically and genetically variable, suggesting the existence of cryptic species. During a continued survey on the biodiversity of microfungi from different countries, seven isolates morphologically similar to S. inflata were obtained from soil samples collected in Spain and USA. In this study their phenotypic features and phylogenetic relationships were assessed. DNA sequence data of two nuclear loci revealed that these isolates correspond to two unnamed clades in S. inflata s.l., one of which also included the type strain of Humicola dimorphospora, a species that traditionally has been considered a synonym of S. inflata. These two groups are proposed herein as Sporothrix brunneoviolacea sp. nov. and Sporothrix dimorphospora comb. nov. S. brunneoviolacea is characterized phenotypically by the production of a diffusible violet-brown pigment in culture and mostly globose, pigmented, lateral blastoconidia. On the other hand S. dimorphospora lacks diffusible pigments and shows mostly subglobose to obovoid pigmented lateral blastoconidia. In contrast to the type strain of S. inflata S. brunneoviolacea and S. dimorphospora assimilate raffinose. The phylogenetic analysis suggested that the proposed anamorph-teleomorph connection between S. inflata and O. bragantinum might not be correct.

Antifungal activity of different molecular weight chitosans against planktonic cells and biofilm of Sporothrix brasiliensis.

Sporotrichosis, caused by Sporothrix schenckii complex species, is the most prevalent subcutaneous mycosis in many areas of Latin America. Chitosan has been used as an antifungal agent; however the effects of the molecular weight (MW) of chitosan (i.e. high (HMW), medium (MMW) and low (LMW) molecular weight chitosan) on S. brasiliensis has not been well described, particularly on biofilms. Effects on the planktonic form activity of S. brasiliensis were quantified by broth microdilution, while anti-biofilm activity was quantified by measuring metabolic activity via XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide and biomass formation (crystal violet). The molecular weight of chitosan modulated its effect on the planktonic form of S. brasiliensis, presenting lower MIC values for LMW chitosan. With regards both the adhesive and mature phases of biofilm, the LMW chitosan reduced biomass and metabolic activity most effectively. This study confirms the effects of the molecular weight and deacetylation degree of chitosan on its antifungal properties for potentially pathogenic fungi.

Antifungal activity of silver salts of Keggin-type heteropolyacids against Sporothrix spp.

Sporotrichosis is a chronic and subacute mycosis causing epidemiological outbreaks involving sick cats and humans in southeastern Brazil. The systemic disease prevails in cats, and in humans, the symptoms are restricted to skin in immunocompetent individuals. Under these conditions, the prolonged treatment of animals and cases of recurrence justify the discovery of new treatments for sporotrichosis. This work addresses the antifungal activity of silver salts of Keggin-type heteropolyacid salts (Ag-HPA salts) such as Ag3[PW12O40], Ag6[SiW10V2O40], Ag4[SiW12O40] and Ag3[PMo12O40] and interactions with the antifungal drugs itraconazole (ITC), terbinafine (TBF) and amphotericin B (AMB) on the yeast and mycelia forms of Sporothrix spp. Sporothrix spp. yeast cells were susceptible to Ag-HPA salts at minimum inhibitory concentration (MIC) values ranging from 8 to 128 µg/mL. Interactions between Ag3[PW12O40] and Ag3[PMo12O40] with itraconazole and amphotericin B resulted in higher antifungal activity with a reduction in growth and melanization. Treated cells showed changes in cell membrane integrity, vacuolization, cytoplasm disorder, and membrane detachment. Promising antifungal activity for treating sporotrichosis was observed for the Ag-HPA salts Ag3[PMo12O40] and Ag3[PW12O40], which have a low cost, high yield and activity at low concentrations. However, further evaluation of in vivo tests is still required.

Heterogeneity of proteins expressed by Brazilian Sporothrix schenckii isolates.

The profiles of proteins present in the exoantigens of Brazilian Sporothrix schenckii isolates were studied and compared by electrophoresis (SDS-PAGE). Thirteen isolates from five different regions of Brazil (1,000 to 2,000 km apart) and ten from a more limited region (200 to 400 km apart within the state of São Paulo) were cultured in Sabouraud, M199 and minimum (MM) media. Qualitative and quantitative differences in the expression of proteins, which varied according to the medium and the isolate, were observed. Fractions with the same MW but varying in intensity were detected, as well as fractions present in 1 isolate but absent in others. Dendrograms were constructed and isolates grouped based on the fractions obtained, irrespective of the intensity. The results showed that Brazilian S. schenckii isolates express different protein profiles, a feature also present in isolates from a more restricted region. The exoantigens were found to have a maximum of 15 protein fractions, ranging in MW from 19-220 KDaltons depending on the medium used for the cultures. These data show the great heterogeneity of Brazilian S. schenckii protein expression.