Whole transcriptome sequencing for revealing the pathogenesis of sporotrichosis caused by Sporothrix globosa.

This study aimed to investigate the molecular mechanism of sporotrichosis and identify possible novel therapeutic targets. Total RNA was extracted from skin lesion samples from sporotrichosis patients and used to construct a long-chain RNA transcriptome library and miRNA transcriptome library for whole transcriptome sequencing. The differentially expressed genes (DEGs) between the groups were identified, and then Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Gene Set Enrichment Analysis enrichment analyses were performed based on the DEGs. An lncRNA-miRNA-mRNA ceRNA network was constructed. The expressions of JAK/STAT pathway-related proteins were detected in the patient and control tissues using RT-qPCR and Western blot analysis. Enrichment analysis showed that the DEGs were mainly enriched in various infectious diseases and immune response-related signaling pathways. Competing endogenous RNA network analysis was performed and identified the hub lncRNAs, miRNAs, and mRNAs. Compared with the control group, the mRNA expressions of SOCS3, IL-6, and JAK3 were significantly upregulated, while the expression of STAT3 did not change significantly. Also, the protein expressions of SOCS3, IL-6, JAK3, and STAT3, as well as phosphorylated JAK3 and STAT3, were significantly upregulated. We identified 671 lncRNA DEGs, 3281 mRNA DEGs, and 214 miRNA DEGs to be involved in Sporothrix globosa infection. The study findings suggest that the JAK/STAT pathway may be a therapeutic target for sporotrichosis.

Genotyping of intraspecies polymorphisms of Sporothrix globosa using partial sequence of mitochondrial DNA.

Restriction fragment length polymorphism (RFLP) of mitochondrial DNA (mtDNA) had been used for molecular identification of Sporothrix spp., which is the causative fungi of sporotrichosis and the most prevalent deep-seated dermatomycosis. Also, mtDNA-RFLP had been used to investigate the molecular epidemiology of sporotrichosis. While the current standard for molecular diagnosis is performed by sequence analysis of the calmodulin gene (CAL), correspondence between the results from CAL and mtDNA is of diagnostic and epidemiological interest. Here, we investigated the correspondence between CAL and mtDNA used for molecular identification of Sporothrix globosa and S. schenckii, which are two major species. We also investigated and propose molecular markers suitable to describe the epidemiology of S. globosa, which is considered as a species with few intraspecific polymorphisms. Eighty-seven strains morphologically identified as S. schenckii sensu lato were investigated. They were identified as group A (17 types, 17 strains) or B (14 types, 70 strains) by mtDNA-RFLP. Partial sequences of CAL, internal transcribed spacer, and spacer between atp9 and cox2 genes of mtDNA of these strains were determined. All group A strains corresponded to S. schenckii, and group B to S. globosa. The sequences of the amplicons targeted on the spacer region in mtDNA of S. globosa ranged 510-515 bp in length and exhibited 10 molecular variations, whereas CAL indicated seven molecular variations. In conclusion, most of the S. schenckii sensu lato strains isolated from Japanese sporotrichosis patients were confirmed as S. globosa, because group B, which comprised the majority of strains, matched perfectly with S. globosa by the CAL sequencing study. We proposed sequence variations in the spacer between atp9 and cox2 genes of mtDNA as a suitable molecular epidemiological marker for S. globosa.

Sporothrix globosa melanin inhibits antigenpresentation by macrophages and enhances deep organ dissemination.

Melanin is a Sporothrix virulence factor that can inhibit the innate immune functions of macrophages such as phagocytosis and killing. However, no data on melanin's influence on antigen presentation by macrophages are available. In this study, we used conidia, yeasts, and melanin ghosts (MGs) from a black Sporothrix globosa strain (MEL+) and its ultraviolet-induced albino mutant (MEL-), to study the influence of melanin on expression of molecules involved in antigen presentation by mouse macrophages (MHC class II, CD80, CD86), as well as on levels of transcription factors regulating their expression (CIITA and promoters I, III, and IV). A murine infection model was used to assess the virulence of both strains and differences in expression of MHC class II and CD80/86 in vivo. MHC class II, CD86 CIITA, and PIV expressions were lower in macrophages infected with MEL+ than in macrophages infected with MEL- conidia, while CD80 expression was similar. No statistical difference in gene expression was observed between macrophages infected by MEL+ and MEL- yeasts. Infection by MGs alone had no clear effect on expression of antigen presentation-associated molecules. Mice infected with MEL+ S. globosa had significantly higher fungal burdens in the lung, liver, spleen, kidney, and testicle compared with mice infected with MEL- S. globosa 21 days post-infection. MHC class II expression changes in the animal study were similar to those observed in the in vitro experiment. Our results indicate that S. globosa melanin can inhibit expression of antigen presentation-associated molecules during both the early and late stages of infection, representing a new mechanism to evade host immunity and to enhance dissemination. Further investigations of melanin's impact on adaptive immunity will be helpful in understanding this fungal virulence factor.

Coinfection of domestic felines by distinct Sporothrix brasiliensis in the Brazilian sporotrichosis hyperendemic area.

Microbial interactions may impact patient's diagnosis, prognosis and treatment. Sporotrichosis is a hyperendemic neglected zoonosis in Brazil, caused by Sporothrix brasiliensis. Four pairs of clinical isolates of Sporothrix were recovered from four diseased cats (CIM01-CIM04, two isolates per animal) raising the possibility of coinfection in a sporotrichosis hyperendemic area, Brazil. Each isolate of the pair had distinct pigmentation in mycological culture, and was designated as "Light" or "Dark", for low and high pigmentation, respectively. Dark isolates reacted strongly with monoclonal antibodies to melanin (p ≤ 0.05) by both ELISA and FACS quantitation, and displayed a ring pattern with some regions exhibiting higher punctuated labeling at cell wall by immunofluorescence. In turn, Light isolates reacted less intensely, with few and discrete punctuated labeling at the cell wall. PCR identified all isolates as S. brasiliensis, MAT1-2 idiomorph. Sequencing of ß-tubulin and calmodulin genes followed by phylogenetic analysis placed all eight isolates within the same cluster as others from the Brazilian hyperendemic area. The ability of these strains to stimulate cytokine production by human PBMCs (Peripheral blood mononuclear cells) was also analyzed. CIM01 and CIM03 Light and Dark isolates showed similar cytokine profiles to the control strain, while CIM02 and CIM04 behaved differently (p < 0.001), suggesting that differences in the surface of the isolates can influence host-fungus interaction. MICs for amphotericin B, terbinafine, caspofungin, micafungin, itraconazole, fluconazole, and voriconazole were obtained (CLSI M38-A2/M27-A3). Pairwise comparisons showed distinct MICs between Sporothrix Light and Dark isolates, higher than at least two-fold dilutions, to at least one of the antifungals tested. Isolates from the same pair displayed discrepancies in relation to fungistatic or fungicidal drug activity, notably after itraconazole exposure. Since S. brasiliensis Light and Dark isolates show disparate phenotypic parameters it is quite possible that coinfection represents a common occurrence in the hyperendemic area, with potential clinical implications on feline sporotrichosis dynamics. Alternatively, future studies will address if this specie may have, as reported for other fungi, broad phenotypic plasticity.

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.

Mannose-Binding Lectin and Mannose-Binding Lectin-Associated Serine Protease-2 Genotypes and Serum Levels in Patients with Sporotrichosis.

Mannose-binding lectin (MBL) and MBL-associated serine protease-2 (MASP-2) are important proteins in the lectin pathway of the immune system. Mannose-binding lectin and MASP-2 deficiencies have been reported to be responsible for various fungal infections. We investigated the association of MBL and MASP-2 variants with sporotrichosis in a Chinese population and revealed one rare heterozygous mutation in a disseminated cutaneous patient without immunosuppressive conditions (MASP2, p.156_159dupCHNH). We also found that sporotrichosis patients had decreased levels of MBL and MASP-2 in their serum samples compared with controls. Our findings linked, for the first time, MASP-2 deficiencies with susceptibility to Sporothrix sp.

Two­component histidine kinase DRK1 is required for pathogenesis in Sporothrix schenckii.

Sporothrix schenckii is a pathogenic dimorphic fungus with a global distribution. It grows in a multicellular hyphal form at 25˚C and a unicellular yeast form at 37˚C. The morphological switch from mold to yeast form is obligatory for establishing pathogenicity in S. schenckii. Two­component signaling systems are utilized by eukaryotes to sense and respond to external environmental changes. DRK1is a hybrid histidine kinase, which functions as a global regulator of dimorphism and virulence in Blastomyces dermatitidis and Histoplasma capsulatum. An intracellular soluble hybrid histidine kinase, homologous to DRK1 in B. dermatitidis, has previously been identified in S. schenckii and designated as SsDRK1. In the present study, the function of SsDRK1 was investigated using double stranded RNA interference mediated by Agrobacterium tumefaciens. SsDRK1 was demonstrated to be required for normal asexual development, yeast­phase cell formation, cell wall composition and integrity, melanin synthesis, transcription of the morphogenesis­associated gene Ste20 that is involved in the high osmolarity glycerol/mitogen­activated protein kinase pathway, and pathogenicity of S. schenckii in a murine model of cutaneous infection. Further investigations into the signals SsDRK1 responds to, and the interactions of upstream transmembrane hybrid histidine kinases with SsDRK1, are required to uncover novel targets for anti­fungal therapies.

Immune Response Induced by an Immunodominant 60 kDa Glycoprotein of the Cell Wall of Sporothrix schenckii in Two Mice Strains with Experimental Sporotrichosis.

Cell wall (CW) components of fungus Sporothrix schenckii are the major inductors antigens of immune responses. The immunodominant 60 kDa glycoprotein (gp60) has been shown to be associated with the virulence of this fungus but its role in experimental sporotrichosis is unknown. In this work, the immunological effects of CW-purified gp60 were investigated in a model of experimental subcutaneous sporotrichosis in normal and gp60-preimmunized C57BL/6 and BALB/c mice strains which were then infected with S. schenckii conidia. Results showed that both mice strains use different cytokine profiles in order to fight S. schenckii infection; C57BL/6 mice seem to use a Th17 response while BALB/c mice tend to depend on a Th1 profile. Preimmunization with gp60 showed a downregulatory effect on the immune response since cytokines levels were diminished in both strains. There were no significant differences in the magnitude of dorsoplantar inflammation between gp60-preimmunized and nonimmunized mice of both strains. However, skin lesions due to the infection in gp60-preimmunized mice were more severe in BALB/c than in C57BL/6 mice, suggesting that the antigen exerts a higher downregulatory effect on the Th1 response.

Chromosomal polymorphism in the Sporothrix schenckii complex.

Sporotrichosis is a polymorphic disease caused by a complex of thermodimorphic fungi including S. brasiliensis, S. schenckii sensu stricto (s. str.), S. globosa and S. luriei. Humans and animals can acquire the disease through traumatic inoculation of propagules into the subcutaneous tissue. Despite the importance of sporotrichosis as a disease that can take epidemic proportions there are just a few studies dealing with genetic polymorphisms and genomic architecture of these pathogens. The main objective of this study was to investigate chromosomal polymorphisms and genomic organization among different isolates in the S. schenckii complex. We used pulsed field gel electrophoresis (PFGE) to separate chromosomal fragments of isolated DNA, followed by probe hybridization. Nine loci (ß-tubulin, calmodulin, catalase, chitin synthase 1, Internal Transcribed Spacer, Pho85 cyclin-dependent kinase, protein kinase C Ss-2, G protein α subunit and topoisomerase II) were mapped onto chromosomal bands of Brazilian isolates of S. schenckii s. str. and S. brasiliensis. Our results revealed the presence of intra and interspecies polymorphisms in chromosome number and size. The gene hybridization analysis showed that closely related species in phylogenetic analysis had similar genetic organizations, mostly due to identification of synteny groups in chromosomal bands of similar sizes. Our results bring new insights into the genetic diversity and genome organization among pathogenic species in the Sporothrix schenckii complex.

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.

Caracterização da glicoproteína de 70 kDa de Sporothrix schenckii e sua participação na regulação da resposta imune na esporotricose experimental / Characterization of the glycoprotein of 70 kDa of Sporothrix schenkii and its participation in the immune response in experimental sporotrichosis

A esporotricose é uma micose subcutânea de caráter crônico e de ampla distribuição mundial, cujo agente patogênico é o fungo dimórfico térmico, Sporothrix schenckii. Esse trabalho teve como principal objetivo a caracterização da atividade biológica da glicoproteína de 70 kDa (gp70), secretado pelas células leveduriformes de S. schenckii, visando à produção de anticorpos monoclonais e estudos de imunização passiva. Para atingir tais objetivos, foi produzido um hibridoma secretor de anticorpos monoclonais contra a fração de 70 kDa, denominado de AcMo P6E7. Através de ensaios de imunização passiva, foi observado que a administração do AcMo P6E7 em camundongos BALB/c foi capaz de modificar o curso da infecção experimental por S. schenckii, protegendo esses animais contra a infecção. Através de ensaios de imunofluorescência foi verificado que a gp70 está presente na superfície das células leveduriformes de S. schenckii e é uma adesina putativa para fibronectina e laminina...

Polimorfismo fenotípico de cepas autóctonas de sporothrix schenkii / Phenotypic polimorphism in indigenous strains of sporothrix schenkii

Se presentan los resultados macroscópicos, de asimilación de azúcares y de virulencia de 55 cepas de Sporothrix schenckii aisladas a partir de lesiones de pacientes con esporotricosis cútanea, que consultaron al laboratorio de micología de la Facultad de Medicina de la Universidad de Antioquia en Medellín, Colombia, y que fueron coleccionadas en el transcurso de 10 años. La morfología macroscópica de las colonias y su pigmentación se evaluaron tanto en agar mycosel como en extracto de malta. La mayoría de las cepas en los dos medios presentaban colonias con 2 ó 3 colores diferentes. En mycosel 5 cepas (9.1 por ciento) fueron monocromáticas y éste fue el medio más estable para definir las características de pigmentación. El 85 por ciento de las cepas en mycosel fueron café claras, café oscuras, plisadas o plisadas y umbilicadas. Todas las cepas asimilaron D-glucosa, glicerol y D-xilosa en el sistema Api 20C y 25 cepas se clasificaron en 9 biotipos de asimilación de la A a la I. La mayoría de las cepas tanto pigmentadas como albinas, resultaron virulentas para ratones. En éstos predominaron los cuerpos en cigarro en forma de naveta y no se visualizaron cuerpos asteroides en los exudados testiculares. Se demuestra así la gran heterogeneidad fenotípica de las cepas autóctonas de S. schenckii, se plantea la importancia de correlacionar estos hallazgos con los patrones de heterogeneidad gen ética informados por investigadores Japoneses y quizás explicar por esta diversidad fenotípica y genotípica, el polimorfismo clínico de la enfermedad y establecer mapas de distribución de los diferentes biotipos o genotipos en Colombia y América Latina. Incluso el cruzar cepas distantes en su biotipo o genotipo podría facilitar la obtención de la forma de reproducción sexual del microorganismo

We studied macroscopic colony findings, sugar assimilation patterns and virulence of 55 Sporothrix schenckij strains obtained from patients with cutaneous sporothrichosis. They were collected during a 10-year period at the Mycology Laboratory, University of Antioquia, School of Medicine, Medellín, Colombia. Pigmentation types and macroscopic morphological characteristics' were studied on mycosel agar and malt extract. In most cases 2 or 3 colony colors were present In both media. In mycosel agar only 5 strains (9.1%) were monochromatic. Pigmentation was very stable in that medium. Eighty five percent of the mycosel agar colonies were beige, brown, pleated or pleated and umbilicated. All strains assimilated D-glucosa, glycerol and D-xylosa. We established 9 patterns of assimilation (blotypes), from A to I In 25 strains. Both pigmented and albino strains were virulent for mice. We emphasize the diversity of our Indigenous strains, and the importance of genotypic characterization and of the correlation studies of phenotypic and genotypic variation with the clinical and geographical patterns of the disease

Karyotyping by PFGE of clinical isolates of Sporothrix schenckii.

From October 1991 to December 1992 we had eight patients with sporotrichosis at Tsukuba University Hospital in Japan. With 8 strains isolated from these patients, PFGE (pulsed-field gel electrophoresis) analyses were carried out to examine whether the karyotype of S. schenckii is distinguished by our method and whether this molecular approach is a useful means of biotyping of S. schenckii strains. Chromosomes were separated by contour-clamped homogeneous electric field (CHEF) gel electrophoresis. The strains had six to eight chromosomes and a total genome size was approx. 28 Mbp. Although these karyotypes of all the isolates looked closely similar to each other, they were grouped into three types.

[Occupational and familial sporotrichosis]. / Esporotricose ocupacional e familial.

The incidence of occupational mycoses and the main criteria to make a correct diagnosis are commented. The sources of infection of the superficial and deep professional mycotic infections are analysed. The occurrence of an epidemic outbreak of sporotrichosis in 13 members of one family is reported. The parents and 11 sibling were infected with the grass utilized for the filling of mattress, in a small domestic craftsmanship.

A familial epidemic of cutaneous sporotrichosis occurring in north Texas.

A small familial epidemic of cutaneous sporotrichosis occurring in North Texas is reported. The disease occurred in four out of six children in two separate families of paternal cousins. The presumed source of infection was old prairie hay used for mulching. Trauma at the time of exposure seemed minimal or absent. Response to oral potassium iodide therapy was prompt and satisfactory.