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.

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.

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.

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.

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.

Terpinen-4-ol inhibits the growth of Sporothrix schenckii complex and exhibits synergism with antifungal agents.

Aim: This study investigated the effect of terpinen-4-ol against Sporothrix schenckii complex and its interactions with antifungals. Materials & methods: The antifungal activity of terpinen-4-ol was evaluated by broth microdilution. The potential effect on cellular ergosterol concentration was evaluated by spectrophotometry. The antibiofilm activity was evaluated by violet crystal staining and XTT reduction assay. The potential pharmacological interactions with antifungals were evaluated by the checkerboard assay. Results: terpinen-4-ol (T-OH) showed minimal inhibitory concentrations ranging from 4 to 32 mg/l decreasing cellular ergosterol content and presented a SMIC ranging from 64 to 1024 mg/l for Sporothrix spp. The combinations of T-OH with itraconazole or terbinafine were synergistic. Conclusion: T-OH has antifungal activity against Sporothrix spp. and acts synergistically with standard antifungals.

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.

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.

Synthesis and Antifungal Activity of Coumarins Derivatives Against Sporothrix spp.

Sporotrichosis is a serious public health problem in Brazil that affects human patients and domestic animals, mainly cats. Thus, the search for new antifungal agents is required also due to the emergence and to the lack of effective drugs available in the therapeutic arsenal. The aim of this study was to evaluate the in vitro antifungal profile of two synthetic series of coumarin derivatives against Sporothrix schenckii and Sporothrix brasiliensis. The three-components synthetic routes used for the preparation of coumarin derivatives have proved to be quite efficient and compounds 16 and 17 have been prepared in good yields. The inhibitory activity of nineteen synthetic coumarins derivatives 16a-i and 17a-j were evaluated against Sporothrix spp. yeasts and the most potent compounds were 16b and 17i. However, according to concentrations able to inhibit (minimum inhibitory concentrations) and kill (minimum fungicidal concentrations) the cells, 17i was more effective than 16b against Sporothrix spp. Thus, 17i exhibited good antifungal activity against S. brasiliensis and S. schenckii, suggesting that it is an important scaffold for the development of novel antifungal agents.

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.

Caracterização e avaliação da capacidade protetora dos peptí­deos imunogênicos de Sporothrix brasiliensis / Characterization and evaluation of the protective capacity of the immunogenic peptides of Sporothrix brasiliensis

A esporotricose é uma doença crônica que envolve o tecido subcutâneo afetando seres humanos e animais, causada pelo fungo termodimórfico Sporothrix spp.. A esporotricose é endêmica na América latina, principalmente no Brasil que teve o maior surto zoonótico já registrado, ocorrendo na cidade do Rio de Janeiro. A espécie Sporothrix brasiliensis é a mais diagnosticada no surto e a mais virulenta entre as especies de Sporothrix spp., causando formas mais graves da doença. A esporotricose em gatos é endêmica, fatal e um dos principais fatores pelo alto número de casos no Rio de Janeiro. O tratamento é longo e não vem sendo o suficiente para conter o número de casos da doença. Uma vacina contra a esporotricose poderia mudar esse paradigma no Brasil. O presente trabalho obteve o proteoma da cepa S. brasiliensis 5110 por meio de uma eletroforese 2D, e caracterizou e identificou as possíveis proteínas imunogênicas do fungo por espectrometria de massa. Por meio de programas de predição, foi avaliado e sintetizado 7 sequências de aminoácidos,das proteínas identificadas com maiores chances de se acoplar a molécula MHC de classe II. Apenas 3 foram capazes de induzir proliferação in vitro, os peptídeos ZR3, ZR4 e ZR8, que foram utilizados como vacina na esporotricose subcutânea e avaliados sua eficácia por meio da carga fúngica, diâmetro das lesões, perfil celular e níveis de citocinas. Neste trabalho concluímos que o peptídeo ZR8 foi o melhor candidato à vacina na esporotricose, pois foi capaz de diminuir o diâmetro das lesões, aumentar os níveis de citocinas protetoras (IFN-γ, IL-17A e IL-1ß) e aumentar o número de células TCD4+ e CD3-/CD19+, sendo assim induzindo uma resposta imunológica protetora na esporotricose subcutânea

Sporotrichosis is a chronic disease, which involves the subcutaneous tissue affecting humans and animals caused by the thermodymorphic fungus Sporothrix spp. Sporotrichosis is endemic in Latin America, mainly in Brazil that had the largest zoonotic outbreak ever recorded, occurring in the city of Rio de Janeiro. The Sporothrix brasiliensis is the species more diagnosed in the outbreak and most virulent, causing severe forms of the disease. Sporotrichosis in cats is endemic, fatal and the main factors due to the high number of cases of the disease in Rio de Janeiro. The treatment is long, and has not been enough to contain the number of cases of sporotrichosis. A vaccine against sporotrichosis could change this paradigm in Brazil. The present work obtained the proteome of S. brasiliensis 5110 strain by 2D electrophoresis, and characterized and identified possible immunogenic proteins by mass spectrometry. By prediction programs were evaluated and synthesized 7 peptide sequence from antigenic proteins that have the highest chances of coupling to the MHC class II molecule. From these 7 peptides only 3 were able to induce proliferation in vitro, called ZR3, ZR4 and ZR8 peptides, that were used as a vaccine in subcutaneous sporotrichosis and evaluated their efficacy through fungal load, lesion diameter, cell profile and cytokine levels. We conclude that ZR8 peptide was the best candidate for sporotrichosis vaccine, since it was able to decrease the lesion diameter, increase the levels of protective cytokines (IFN-γ, IL-17A and IL-1ß) and increase the number of CD4+ T cells and CD3-/CD19+ inducing a protective immune response in subcutaneous sporotrichosis

Molecular identification and phenotypic characterisation of Sporothrix globosa from clinical cases of Eastern Assam, North-east India.

Sporotrichosis is known to be endemic in the state of Assam, North-east India, which is situated in the Sub-Himalayan region. This disease is an acute or chronic infection caused by Sporothrix schenckii species complex which currently includes several species of clinical relevance such as Sporothrix brasiliensis, Sporothrix globosa, Sporothrix schenckii sensu stricto, Sporothrix albicans, Sporothrix mexicana, Sporothrix pallida and Sporothrix luriei. S. globosa is the prevalent species in India. Eight culture-positive patients were diagnosed from suspected consecutive cases of two lymphocutaneous and six fixed cutaneous forms over a period of 4 years in a clinical mycology laboratory of a tertiary care centre in Eastern Assam. Phenotypic speciation was inconclusive using the criteria of Marimon et al. because of atypical growth pattern shown by the isolates. Our isolates showed good growth at 37°C ranging from 6 to 27 mm; four of the isolates showed growth of 11-27 mm unlike S. globosa strains reported earlier. Molecular identification was done by sequencing both the internal transcribed spacer (ITS) region and the calmodulin (CAL) protein encoding gene (partial). All the isolates were identified as S. globosa. Molecular confirmation of species using ITS region and CAL protein encoding gene (partial) is necessary for isolates of S. globosa showing atypical biopatterns.

Antifungal and immunomodulatory activity of a novel cochleate for amphotericin B delivery against Sporothrix schenckii.

INTRODUCTION: Sporotrichosis is an emergent subcutaneous mycoses caused by species of the Sporothrix schenckii complex. Amphotericin B (AmB) remains the main antifungal drug for the treatment of systemic infections, but its use is limited by toxicity reasons. AFCo3 is a novel cochleate containing detoxified LPS, which exhibits drug delivery and immunomodulating properties. Here, AFCo3 was used as the vehicle for AmB to evaluate the immunomodulatory and antifungal efficacy against S. schenckii in vitro and in vivo. METHODS AND RESULTS: The minimum inhibitory concentrations of AFCo3-AmB and AmB were 0.25 and 1µg/mL respectively. The minimum fungicidal concentration was 0.5µg/mL for AFCo3-AmB and 2µg/mL for AmB. AFCo3-AmB was less cytotoxic than AmB for peritoneal macrophages, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and reduced the AmB-induced hemolysis in murine erythrocytes. AFCo3-AmB improved the intracellular killing of phagocytized yeast and it enhanced the in vitro production of IL-1ß, TNF-α and NO in peritoneal macrophages. Moreover, AFCo3-AmB was more effective than AmB in reducing spleen and liver fungal burden after repeated (five days) intraperitoneal administration of 5mg/kg of AmB, in a Balb/c model of systemic infection, associated to a significant induction of Th1/Th17 response. Finally, blood chemistry revealed that AFCo3-AmB did not cause changes suggestive of nephrotoxicity, such as increases in total proteins, albumin, creatinine and blood urea nitrogen that were caused by free AmB. CONCLUSIONS: AFCo3-AmB exhibited a significant immunomodulator action, reduced toxicity and improved antifungal action against S. schenckii, suggesting a potential use as AmB delivery for systemic sporotrichosis treatment.

Studies in Phylogeny, Development of Rapid IdentificationMethods, Antifungal Susceptibility, and Growth Rates of Clinical Strains of Sporothrix schenckii Complex in Japan.

Sporotrichosis is a fungal infection caused by the Sporothrix species, which have distinct virulence profiles and geographic distributions. We performed a phylogenetic study in strains morphologically identified as Sporothrix schenckii from clinical specimens in Japan, which were preserved at the Medical Mycology Research Center, Chiba University. In addition, we examined the in vitro antifungal susceptibility and growth rate to evaluate their physiological features. Three hundred strains were examined using sequence analysis of the partial calmodulin gene, or polymerase chain reaction（PCR）method using newly designed species-specific primers; 291 strains were Sporothrix globosa and 9 strains were S. schenckii sensu stricto (in narrow sense, s. s.). S. globosa strains were further clustered into two subclades, and S. schenckii s. s. strains were divided into three subclades. In 38 strains of S. globosa for which antifungal profiles were determined, 4 strains (11%) showed high minimal inhibitory concentration (MIC) value for itraconazole. All tested strains of S. schenckii s. s. and S. globosa showed low sensitivity for amphotericin B. These antifungals are used for treatment of sporotrichosis when infection is severe. S. schenckii s. s. grew better than S. globosa; wherein S. globosa showed restricted growth at 35℃ and did not grow at 37℃. Our molecular data showed that S. globosa is the main causal agent of sporotrichosis in Japan. It is important to determine the antifungal profiles of each case, in addition to accurate species-level identification, to strategize the therapy for 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.

Analysis of Sporothrix schenckii sensu stricto and Sporothrix brasiliensis virulence in Galleria mellonella.

The study of the host-pathogen interaction is essential to understand the mechanisms underlying adhesion, colonization and tissue damage by pathogens. This is usually achieved by performing in vivo studies using small mammals, such as rats, mice and guinea pigs. Nowadays, the mouse models of systemic or subcutaneous infection are the gold standard assays to analyze the virulence of members of the Sporothrix schenckii complex. There are, however, invertebrates that have been recently used as alternative hosts to assess the virulence of both bacteria and fungi, and among them, larvae of Galleria mellonella are popular because they are easy to breed, and require non-specialized facilities to maintain the colony. Here, we assessed the use of G. mellonella larvae to test the virulence of S. schenckii sensu stricto and Sporothrix brasiliensis strains, and found that infection with yeast-like cells, but not with conidia or germlings, reproduces the virulence data generated in the mouse model of infection. Furthermore, with this insect model we could classify the virulence of some strains as low, intermediate or high, in line with the observations in the mammalian model. Therefore, G. mellonella is suitable, and a new alternative, to test virulence of both S. schenckii sensu stricto and S. brasiliensis.

Construction and analysis of the cDNA subtraction library of yeast and mycelial phases of Sporothrix globosa isolated in China: identification of differentially expressed genes.

Species included in the Sporothrix schenckii complex are temperature-dependent with dimorphic growth and cause sporotrichosis that is characterized by chronic and fatal lymphocutaneous lesions. The putative species included in the Sporothrix complex are S. brasiliensis, S. globosa, S. mexicana, S. pallida, S. schenckii, and S. lurei. S. globosa is the causal agent of sporotrichosis in China, and its pathogenicity appears to be closely related to the dimorphic transition, i.e. from the mycelial to the yeast phase, it adapts to changing environmental conditions. To determine the molecular mechanisms of the switching process that mediates the dimorphic transition of S. globosa, suppression subtractive hybridization (SSH) was used to prepare a complementary DNA (cDNA) subtraction library from the yeast and mycelial phases. Bioinformatics analysis was performed to profile the relationship between differently expressed genes and the dimorphic transition. Two genes that were expressed at higher levels by the yeast form were selected, and their differential expression levels were verified using a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). It is believed that these differently expressed genes are involved in the pathogenesis of S. globosa infection in China.