Virulence Factors in Sporothrix schenckii, One of the Causative Agents of Sporotrichosis.

Sporothrix schenckii is one of the etiological agents of sporotrichosis, a fungal infection distributed worldwide. Both, the causative organism and the disease have currently received limited attention by the medical mycology community, most likely because of the low mortality rates associated with it. Nonetheless, morbidity is high in endemic regions and the versatility of S. schenckii to cause zoonosis and sapronosis has attracted attention. Thus far, virulence factors associated with this organism are poorly described. Here, comparing the S. schenckii genome sequence with other medically relevant fungi, genes involved in morphological change, cell wall synthesis, immune evasion, thermotolerance, adhesion, biofilm formation, melanin production, nutrient uptake, response to stress, extracellular vesicle formation, and toxin production are predicted and discussed as putative virulence factors in S. schenckii.

Unbalanced inflammatory reaction could increase tissue destruction and worsen skin infectious diseases - a comparative study of leishmaniasis and sporotrichosis.

The clinical presentations of skin diseases produced by different pathogens, as American tegumentary leishmaniasis (ATL) and sporotrichosis can be similar and possibly influenced by the skin immune system (SIS). The aim of the study was to understand the underlying mechanisms of skin inflammation produced by different pathogens. We used immunohistochemistry to analyze 96 patients: a- localized cutaneous leishmaniasis (LCL-ATL); b- sporotrichoid cutaneous leishmaniasis (SCL-ATL); c-lymphocutaneous (LC-SP); d- fixed (F-SP) sporotrichosis. LCL-ATL and SCL-ATL had a significantly higher percentage of CD8, FasL and NOS2 than sporotrichosis. In contrast, LC-SP had a substantially higher percentage of CD4, BCl2 and neutrophils than ATL lesions. These results indicated some differences in the profile of the in situ immune response suggesting that SIS is a complex, adaptable system capable of different responses to intracellular or extracellular pathogens. However, regardless of the etiological agents, the inflammatory reaction and clinical manifestations can be similar. SCL-ATL and LC-SP presented similarities in both clinical presentation and in situ inflammatory profile (CD3, CD22, neutrophils, macrophages). The clinical presentation of ATL and sporotrichosis could be explained by a combination of factors both of the host SIS and the etiological agent. The unbalanced host parasite relationship could result in atypical manifestations of skin disease.

Sporothrix brasiliensis produces the highest levels of oxidative stress in a murine model among the species of the Sporothrix schenckii complex.

INTRODUCTION:: We compared indicators of oxidative stress in the tissue of mice infected with strains from Sporothrix schenckii complex. METHODS:: Mice were inoculated with Sporothrix brasiliensis, Sporothrix schenckii sensu stricto, Sporothrix globosa, Sporothrix mexicana or Sporothrix albicans. The activity of catalase and glutathione were accessed in the liver and spleen. RESULTS:: Animals infected with S. brasiliensis exhibited splenomegaly and significant decrease in catalase activity, and protein and non-protein thiol content compared to animals infected with the other species. CONCLUSIONS:: Sporothrix brasiliensis exhibits higher pathogenicity compared to other species of the Sporothrix schenckii complex by increasing oxidative stress in animal tissue.

Sporothrix brasiliensis produces the highest levels of oxidative stress in a murine model among the species of the Sporothrix schenckii complex

Abstract INTRODUCTION: We compared indicators of oxidative stress in the tissue of mice infected with strains from Sporothrix schenckii complex. METHODS: Mice were inoculated with Sporothrix brasiliensis, Sporothrix schenckii sensu stricto, Sporothrix globosa, Sporothrix mexicana or Sporothrix albicans. The activity of catalase and glutathione were accessed in the liver and spleen. RESULTS: Animals infected with S. brasiliensis exhibited splenomegaly and significant decrease in catalase activity, and protein and non-protein thiol content compared to animals infected with the other species. CONCLUSIONS: Sporothrix brasiliensis exhibits higher pathogenicity compared to other species of the Sporothrix schenckii complex by increasing oxidative stress in animal tissue.

Oxidative stress in rats experimentally infected by Sporothrix schenckii.

The aim of this study was to evaluate whether oxidative stress occurs in rats experimentally infected by Sporothrix schenckii, and its possible effect on disease pathogenesis. Thirty rats were divided into two groups: the group A (uninfected, n = 18) and the group B (infected by S. schenckii, n=21). Blood samples were collected on days 15, 30 and 40 post-infection (PI). At each sampling time, six rats of the group A, and seven of the group B were bled. TBARS (thiobarbituric acid reactive substances) levels in serum samples were measured to evaluate lipid peroxidation. In addition, catalase (CAT) and superoxide dismutase (SOD) activities, known as biomarkers of antioxidants levels, were verified in whole blood. Seric pro-inflammatory cytokine levels were measured (IFN-γ, TNF-α, and IL-6), which showed that these inflammatory mediators were at higher levels in the infected rats (P < 0.001). In comparison to uninfected animals, rats with sporotrichosis showed significantly higher (p < 0.01) levels of TBARS on day 40 PI; CAT activity was significantly increased (p < 0.01) on days 30 and 40 PI; and SOD activity was increased (p < 0.01) on day 40 PI. Infected rats showed larger testicles and granulomas in the testicular capsule, as well as hepatic granulomas and splenic follicular hyperplasia. All tissues (testicle, spleen, and liver) showed inflammation associated with numerous fungal structures. These results demonstrated that the intense inflammatory response (seric and tissue) in sporotrichosis is a likely mechanism for redox imbalance, and consequently cause the oxidative stress in experimentally infected rats.

Anti-inflammatory activity of Vismia guianensis (Aubl.) Pers. extracts and antifungal activity against Sporothrix schenckii.

ETHNOPHARMACOLOGICAL RELEVANCE: Vismia guianensis (Aubl.) Pers. is traditionally used in North and Northeast of Brazil for the treatment of dermatomycoses. Since the strategy associating immunomodulators with antifungal drugs seems to be promissory to improve the treatment efficacy in fungal infections, we aimed to investigate the antifungal activity of V. guianensis ethanolic extract of leaves (VGL) and bark (VGB) against Sporothrix schenckii ATCC 16345 and their antinflammatory activities. MATERIAL AND METHODS: The extracts were analyzed by HPLC-DAD-IT MS/MS for in situ identification of major compounds. Antifungal activity was evaluated in vitro (microdilution test) and in vivo using a murine model of S. schenckii infection. The production of TNF-α, IFN-γ, IL-4, IL-10 and IL-12 by measured by ELISA, as well as measured the production and inhibition of the NO after treatment with the plant extracts or itraconazole (ITR). RESULTS: Two O-glucosyl-flavonoids and 16 prenylated benzophenone derivatives already described for Vismia were detected. Both VGL and VGB showed significant antifungal activity either in in vitro assay of microdilution (MIC=3.9µg/mL) and in vivo model of infection with reduction of S. schenckii load in spleen. It was also observed a predominance of reduction in the production of NO and the proinflammatory cytokines evaluated except TNFα, but with stimulation of IL-10, as evidence of a potential anti-inflammatory effect associated. CONCLUSION: The results showed that both VGL and VGB have a significant antifungal against S. schenckii and an anti-inflammatory activity. These results can support the use of these extracts for alternative treatment of sporotrichosis.

[Presence of lactoferrin (LF) in lymphocutaneous sporotrichosis. Yeast-bound antimicrobial peptide]. / Presencia de lactoferrina (LF) en la esporotricosis linfocutánea. Péptido antimicrobiano unido a levaduras.

Sporotrichosis is a common subcutaneous mycosis in Latin America, produced by dimorphic fungi belong to Sporothrix schenckii complex of cryptic species. Infection is acquired by traumatic inoculation with contaminated organic material. Host immune response includes polymorphonuclear neutrophils chemotaxis and release of granular components. Lactoferrin is a protein member of the transferrin family of iron-binding proteins, present inside polymorphonuclear granular structure, and has been reported to affect growth and development of infectious agents, including fungal organisms. Nevertheless, lactoferrin expression in sporotrichosis infections has not been reported yet. OBJECTIVE: To determine the expression of lactoferrin using immunohistochemical staining in sporotrichosis human infection. MATERIAL AND METHODS: The dermatology department's files during a period of five years were reviewed; cases with a diagnosis of sporotrichosis were selected and lactoferrin immunostaining was performed when enough biological material was available. RESULTS: Three cases with a diagnosis of sporotrichosis and adequate biological material on paraffin block were identified. In all cases, lactoferrin immunostaining was positive around yeast cell.

Phenotypic characteristics associated with virulence of clinical isolates from the Sporothrix complex.

The Sporothrix complex members cause sporotrichosis, a subcutaneous mycosis with a wide spectrum of clinical manifestations. Several specific phenotypic characteristics are associated with virulence in many fungi, but studies in this field involving the Sporothrix complex species are scarce. Melanization, thermotolerance, and production of proteases, catalase, and urease were investigated in 61 S. brasiliensis, one S. globosa, and 10 S. schenckii strains. The S. brasiliensis strains showed a higher expression of melanin and urease compared with S. schenckii. These two species, however, presented similar thermotolerances. Our S. globosa strain had low expression of all studied virulence factors. The relationship between these phenotypes and clinical aspects of sporotrichosis was also evaluated. Strains isolated from patients with spontaneous regression of infection were heavily melanized and produced high urease levels. Melanin was also related to dissemination of internal organs and protease production was associated with HIV-coinfection. A murine sporotrichosis model showed that a S. brasiliensis strain with high expression of virulence factors was able to disseminate and yield a high fungal burden in comparison with a control S. schenckii strain. Our results show that virulence-related phenotypes are variably expressed within the Sporothrix complex species and might be involved in clinical aspects of sporotrichosis.

Immunoproteomic analysis reveals a convergent humoral response signature in the Sporothrix schenckii complex.

Sporotrichosis is a polymorphic disease that affects both humans and animals worldwide. The fungus gains entry into a warm-blooded host through minor trauma to the skin, typically by contaminated vegetation or by scratches and bites from a diseased cat. Cellular and humoral responses triggered upon pathogen introduction play important roles in the development and severity of the disease. We investigated molecules expressed during the host-parasite interplay that elicit the humoral response in human sporotrichosis. For antigenic profiling, Sporothrix yeast cell extracts were separated by two-dimensional (2D) gel electrophoresis and probed with pooled sera from individuals with fixed cutaneous and lymphocutaneous sporotrichosis. Thirty-five IgG-seroreactive spots were identified as eight specific proteins by MALDI-ToF/MS. Remarkable cross-reactivity among Sporothrix brasiliensis, Sporothrix schenckii, and Sporothrix globosa was noted and antibodies strongly reacted with the 70-kDa protein (gp70), irrespective of clinical manifestation. Gp70 was successfully identified in multiple spots as 3-carboxymuconate cyclase. In addition, 2D-DIGE characterization suggested that the major antigen of sporotrichosis undergoes post-translational modifications involving glycosylation and amino acid substitution, resulting in at least six isoforms and glycoforms that were present in the pathogenic species but absent in the ancestral non-virulent Sporothrix mexicana. Although a primary environmental function related to the benzoate degradation pathway of aromatic polymers has been attributed to orthologs of this molecule, our findings support the hypothesis that gp70 is important for pathogenesis and invasion in human sporotrichosis. We propose a diverse panel of new putative candidate molecules for diagnostic tests and vaccine development. BIOLOGICAL SIGNIFICANCE: Outbreaks due to Sporothrix spp. have emerged over time, affecting thousands of patients worldwide. A sophisticated host-pathogen interplay drives the manifestation and severity of infection, involving immune responses elicited upon traumatic exposure of the skin barrier to the pathogen followed by immune evasion. Using an immunoproteomic approach we characterized proteins of potential significance in pathogenesis and invasion that trigger the humoral response during human sporotrichosis. We found gp70 to be a cross-immunogenic protein shared among pathogenic Sporothrix spp. but absent in the ancestral environmental S. mexicana, supporting the hypothesis that gp70 plays key roles in pathogenicity. For the first time, we demonstrate with 2D-DIGE that post-translational modifications putatively involve glycosylation and amino acid substitution, resulting in at least six isoforms and glycoforms, all of them IgG-reactive. These findings of a convergent humoral response highlight gp70 as an important target serological diagnosis and for vaccine development among phylogenetically related agents of sporotrichosis.

Influence of TLR-2 in the immune response in the infection induced by fungus Sporothrix schenckii.

Toll-like receptors (TLRs) play an important role in immunity, since they bind to pathogen surface antigens and initiate the immune response. However, little is known about the role of TLR-2 in the recognition of S. schenckii and in the subsequent immune response. Therefore, the aim of this study was to evaluate the involvement of TLR-2 in the immune response induced by S. schenckii. C57BL/6 mice (WT) and C57BL/6 TLR-2 knockout (TLR-2-/-) were used to evaluate, over a period of 10 weeks of sporotrichotic infection, the influence of TLR-2 over macrophages production of IL-1ß, IL-12 and TNF-α, their stimulation level by NO release and the production of IFN -γ, IL-6, IL-17 and TGF-ß by spleen cells. The results showed that the production of pro-inflammatory mediators and NO, TLR-2 interference is striking, since its absence completely inhibited it. IL-17 production was independent of TLR-2. The absence of Th1 response in TLR2-/- animals was concomitant with IL-17 production. Therefore, it can be suggested that TLR-2 absence interferes with the course of the infection induced by the fungus S. schenckii.

Differences in cell morphometry, cell wall topography and gp70 expression correlate with the virulence of Sporothrix brasiliensis clinical isolates.

Sporotrichosis is a chronic infectious disease affecting both humans and animals. For many years, this subcutaneous mycosis had been attributed to a single etiological agent; however, it is now known that this taxon consists of a complex of at least four pathogenic species, including Sporothrix schenckii and Sporothrix brasiliensis. Gp70 was previously shown to be an important antigen and adhesin expressed on the fungal cell surface and may have a key role in immunomodulation and host response. The aim of this work was to study the virulence, morphometry, cell surface topology and gp70 expression of clinical isolates of S. brasiliensis compared with two reference strains of S. schenckii. Several clinical isolates related to severe human cases or associated with the Brazilian zoonotic outbreak of sporotrichosis were genotyped and clustered as S. brasiliensis. Interestingly, in a murine subcutaneous model of sporotrichosis, these isolates showed a higher virulence profile compared with S. schenckii. A single S. brasiliensis isolate from an HIV-positive patient not only showed lower virulence but also presented differences in cell morphometry, cell wall topography and abundant gp70 expression compared with the virulent isolates. In contrast, the highly virulent S. brasiliensis isolates showed reduced levels of cell wall gp70. These observations were confirmed by the topographical location of the gp70 antigen using immunoelectromicroscopy in both species. In addition, the gp70 molecule was sequenced and identified using mass spectrometry, and the sequenced peptides were aligned into predicted proteins using Blastp with the S. schenckii and S. brasiliensis genomes.

Dendritic cell are able to differentially recognize Sporothrix schenckii antigens and promote Th1/Th17 response in vitro.

Sporotrichosis is a disease caused by the dimorphic fungus Sporothrix schenckii. The main clinical manifestations occur in the skin, however the number of systemic and visceral cases has increased, especially in immunocompromised patients. Dendritic cells (DCs) are highly capable to recognize the fungus associated data and translate it into differential T cells responses both in vivo and in vitro. Although, the mechanisms involved in the interaction between DCs and S. schenckii are not fully elucidated. The present study investigated the phenotypic and functional changes in bone marrow dendritic cells (BMDCs) stimulated in vitro with the yeast form of S. schenckii or exoantigen (ExoAg) and its ability to trigger a cellular immune response in vitro. Our results demonstrated that the live yeast of S. schenckii and its exoantigen, at a higher dose, were able to activate BMDCs and made them capable of triggering T cell responses in vitro. Whereas the yeast group promoted more pronounced IFN-γ production rather than IL-17, the Exo100 group generated similar production of both cytokines. The exoantigen stimulus suggests a capability to deviate the immune response from an effector Th1 to an inflammatory Th17 response. Interestingly, only the Exo100 group promoted the production of IL-6 and a significant increase of TGF-ß, in addition to IL-23 production. Interestingly, only Exo100 group was capable to promote the production of IL-6 and a significant increase on TGF-ß, in addition with IL-23 detection. Our results demonstrated the plasticity of DCs in translating the data associated with the fungus S. schenckii and ExoAg into differential T cell responses in vitro. The possibility of using ex vivo-generated DCs as vaccinal and therapeutic tools for sporotrichosis is a challenge for the future.

Immunohistochemical localization of activated and mature CD83+ dendritic cells in granulomas of sporotrichosis.

Dendritic cells (DC) are considered to be the most potent antigen-presenting cells (APC) and CD83 is expressed at a high level on immune-competent, activated and mature DC. In order to obtain insight into the immune response in sporotrichosis, we searched immunohistochemically for CD83+ DC in the granulomatous skin lesions of patients. A small but significant subpopulation of CD83+ DC (approximately 0.2 to 0.8% of inflammatory cells) was found. The most common distribution pattern of CD83+ DC was as clusters with mononuclear lymphoid cells within the granulomas. In addition, CD83+ DC were occasionally scattered around the granulomas. High-intensity staining of CD83 antigens was detected not only on the surface, but also in the cytoplasm of DC. No CD83+ DC were observed in the epidermis. A considerable amount of CD1a+ cells were detected in the epidermis, papillary dermis and around the granulomas, but CD1a+ cells were rarely found within the granulomas. There were large numbers of CD68+ monocytes and macrophages in the granulomas. These results indicate that activated and mature DC may play a role in the immune response to sporotrichosis.

Comparison between histochemical and immunohistochemical methods for diagnosis of sporotrichosis.

AIMS: To compare the efficacy of histochemical and immunohistochemical methods in detecting forms of Sporothrix schenckii in tissue. METHODS: Thirty five cutaneous biopsy specimens from 27 patients with sporotrichosis were stained by histochemical haematoxylin and eosin, periodic acid Schiff, and Gomori's methenamine silver methods and an immunohistochemical (avidin-biotin complex immunoperoxidase) (ABC) technique associated with a newly produced rabbit polyclonal antibody anti-Sporothrix schenckii. RESULTS: A total of 29 (83%) cases were positive by the ABC method used in association with anti-Sporothrix schenckii rabbit polyclonal antibodies. Histochemical methods, using silver staining, periodic acid Schiff, and conventional haematoxylin and eosin detected 37%, 23%, and 23% of forms of S schenckii, respectively. The ABC technique was significantly more reliable than periodic acid Schiff and silver staining techniques. CONCLUSIONS: It is concluded that immunostaining is an easy and rapid method which can efficiently increase the accuracy of the diagnosis of sporotrichosis in human tissue.

Metabolic activity of phagocytes in experimental sporotrichosis.

Phagocytosis plays an important role as a protective mechanism against infections, since polymorphonuclear leukocytes (PMN) and macrophages are the first cellular lines opposed to aggressive microorganisms. In patients with sporotrichosis a diminished capability of killing engulfed yeast by their PMN has been described, but the origin of this deficiency remains unknown. In this work, partial aspects of the oxidative metabolism of PMN leukocytes and peritoneal macrophages of mongolian gerbils experimentally infected with sporotrichosis were studied. For this purpose the nitroblue tetrazolium (NBT) test as described by Baehner and Nathan (1) and myeloperoxidase activity measured according to Kaplow's method were utilized. The PMN and macrophages of mongolian gerbils infected with sporotrichosis showed increased reduction of NBT when compared with the phagocytic cells of normal ones, as is usually observed in most infections. Myeloperoxidase activity was diminished in both PMN and macrophages, but this diminution was statistically significant only in PMN leukocytes. These results show that part of the oxidative mechanisms of phagocytic cells can be impaired in experimental sporotrichosis, and could be correlated with the diminished fungicidal activity of PMN leukocytes obtained from patients infected with sporotrichosis.