Conchita Toriello

Sporothrix schenckii complex and sporotrichosis, an emerging health problem.

Sporothrix schenckii, now named the S. schenckii species complex, has largely been known as the etiological agent of sporotrichosis, which is an acute or chronic subcutaneous mycosis of humans and other mammals. Gene sequencing has revealed the following species in the S. schenckii complex: Sporothrix albicans, Sporothrix brasiliensis, Sporothrix globosa, Sporothrix luriei, Sporothrix mexicana and S. schenckii. The increasing number of reports of Sporothrix infection in immunocompromised patients, mainly the HIV-infected population, suggests sporotrichosis as an emerging global health problem concomitant with the AIDS pandemic. Molecular studies have demonstrated a high level of intraspecific variability. Components of the S. schenckii cell wall that act as adhesins and immunogenic inducers, such as a 70-kDa glycoprotein, are apparently specific to this fungus. The main glycan peptidorhamnomannan cell wall component is the only O-linked glycan structure known in S. schenckii. It contains an α-mannobiose core followed by one α-glucuronic acid unit, which may be mono- or di-rhamnosylated. The oligomeric structure of glucosamine-6-P synthase has led to a significant advance in the development of antifungals targeted to the enzymes catalytic domain in S. schenckii.

Phenotyping and Genotyping of Sporothrix schenckii Isolates According to Geographic Origin and Clinical Form of Sporotrichosis

ABSTRACT Sporothrix schenckii isolates of fixed and lymphocutaneous clinical forms from Mexico (MX), Guatemala (GT), and Colombia (CO) as well as environmental isolates from MX were studied by analyzing their phenotypic characteristics (conidial length, thermotolerance by percent growth inhibition [GI] at 35 and 37°C, median lethal dose [LD50]) and genotypic characteristics (by random amplified polymorphic DNA [RAPD] analysis-PCR). A significant difference (P < 0.01) in the mean conidial length of S. schenckii clinical isolates from CO (x̅ = 4.03 ± 1.04 μm) compared with those of clinical isolates from MX (x̅ = 2.06 ± 0.53 μm) and GT (x̅ = 2.68 ± 0.83 μm) was observed. The lowest thermotolerance, as determined by measurement of percent GI, was exhibited by isolates from CO at 35°C (x̅ = 50.1% ± 15.9%) and 37°C (x̅ = 72.7% ± 10.9%). In general, the highest virulence, as determined by measurement of the LD50 for mice, was observed for the MX environmental isolates. RAPD analysis-PCR with 10-mer primers OPBG-01, OPBG-14, and OPBG-19 generated 52 reproducible bands. The 44 Sporothrix isolates fell into four major groups by hierarchical cluster analysis. The first group (group I), formed by 25 (of 27) isolates from MX, had two subgroups: subgroup Ia with 10 environmental isolates and subgroup Ib with 14 clinical isolates. The second group (group II) had two subgroups: subgroup IIa, formed by isolates from CO, and subgroup IIb, formed by isolates from GT. Groups III and IV each had only one clinical isolate from MX. A principal-component analysis of the same data yielded three distinct groups, depending on the geographical origins of the isolates, including the isolates in groups III and IV from MX, which were grouped with the isolates from MX by principal-component analysis. This study revealed that isolates from CO had low thermotolerances at 35 and 37°C and could be associated with superficial skin lesions in patients with fixed clinical forms of sporotrichosis, the most frequent form of the disease in CO. Distinct patterns dependent on geographical origins were also revealed by RAPD analysis-PCR, but these had no relation to the clinical form of the disease.

Sporothrix globosa, a pathogenic fungus with widespread geographical distribution.

Sporothrix globosa, reported from the USA, Europe, and Asia, is a recently described pathogenic species morphologically similar to Sporothrix schenckii. In this study, the phylogenetic affinities of 32 clinical and environmental isolates morphologically identified as S. schenckii, from Mexico, Guatemala, and Colombia, were assessed by cladistic analysis of partial sequences of the calmodulin gene using the maximum parsimony and neighbor-joining methods. The study revealed that one out of 25 isolates from Mexico (4%), one out of three isolates from Guatemala (33.3%), and two out of four isolates from Colombia (50%) belonged to S. globosa, while the other isolates belonged to S. schenckii sensu stricto. This is the first record of S. globosa from Mexico, and Central and South America.

Isolation and some properties of a glycoprotein of 70 kDa (Gp70) from the cell wall of Sporothrix schenckii involved in fungal adherence to dermal extracellular matrix

Sporothrix schenckii is the etiological agent of sporotrichosis, a subcutaneous mycosis and an emerging disease in immunocompromised patients. Adherence to target cells is a prerequisite for fungal dissemination and systemic complications. However, information on the cell surface components involved in this interaction is rather scarce. In this investigation, the extraction of isolated cell walls from the yeast phase of S. schenckii with SDS and separation of proteins by SDS-PAGE led to the identification of a periodic acid-Schiff (PAS)-reacting 70 kDa glycoprotein (Gp70) that was purified by elution from electrophoresis gels. The purified glycopeptide exhibited a pI of 4.1 and about 5.7% of its molecular mass was contributed by N-linked glycans with no evidence for O-linked oligosaccharides. Confocal analysis of immunofluorescence assays with polyclonal antibodies directed towards Gp70 revealed a rather uniform distribution of the antigen at the cell surface with no distinguishable differences among three different isolates. Localization of Gp70 at the cell surface was confirmed by immunogold staining. Gp70 seems specific for S. schenckii as no immunoreaction was observed in SDS-extracts from other pathogenic and non-pathogenic fungi. Yeast cells of the fungus abundantly adhered to the dermis of mouse tails and the anti-Gp70 serum reduced this process in a concentration-dependent manner. Results are discussed in terms of the potential role of Gp70 in the host-pathogen interaction.

2D-immunoblotting analysis of Sporothrix schenckii cell wall

We utilized two-dimensional gel electrophoresis and immunoblotting (2D-immunoblotting) with anti-Sporothrix schenckii antibodies to identify antigenic proteins in cell wall preparations obtained from the mycelial and yeast-like morphologies of the fungus. Results showed that a 70-kDa glycoprotein (Gp70) was the major antigen detected in the cell wall of both morphologies and that a 60-kDa glycoprotein was present only in yeast-like cells. In addition to the Gp70, the wall from filament cells showed four proteins with molecular weights of 48, 55, 66 and 67 kDa, some of which exhibited several isoforms. To our knowledge, this is the first 2D-immunoblotting analysis of the S. schenckii cell wall.

Histoplasma capsulatum yeast cells attach and agglutinate human erythrocytes

The ability of yeast cells of Histoplasma capsulatum to attach and agglutinate human erythrocytes has been described. This is the first report involving these yeasts in the hemagglutination phenomenon. Results revealed that the yeast cells were able to bind to erythrocytes irrespective of blood groups and to agglutinate them when a high density of yeast cells was used. Assays on the inhibition of yeast attachment to erythrocytes were also performed, using sugar-treated yeast cells. Results indicate that galactose (Gal), mainly the beta-anomer, specially inhibited yeast attachment. Disaccharides (Gal-derivatives) and glycosaminoglycans containing Gal residues, mainly chondroitin sulfate C, promote this type of inhibition. In addition, preliminary data of inhibition assays also involved a probable ionic strength driven mechanism mediated by sialic acid and heparan sulfate, suggesting that yeast binding to erythrocytes could be associated with negative charges of both molecules.

Detection of 2 immunoreactive antigens in the cell wall of Sporothrix brasiliensis and Sporothrix globosa.

The cell wall of members of the Sporothrix schenckii complex contains highly antigenic molecules which are potentially useful for the diagnosis and treatment of sporotrichosis. In this study, 2 immunoreactive antigens of 60 (Gp60) and 70 kDa (Gp70) were detected in the cell wall of the yeast morphotypes of Sporothrix brasiliensis and Sporothrix globosa.

Entomopathogenic fungi from ‘El Eden’ Ecological Reserve, Quintana Roo, Mexico

Entomopathogenic fungi were isolated and identified from insects collected from the tropical forest and an agricultural area at El Eden Ecological Reserve, Quintana Roo, Mexico. These fungi were studied to determine their potential as biological control agents of greenhouse Trialeurodes vaporariorum (Homoptera: Aleyrodidae), and to contribute to the knowledge of biodiversity of this area. No pest insects were observed in the tropical forest. In contrast, all insects collected in the agricultural area were considered important pests by the local farmers, with the whitefly, as the most relevant, plentiful in Cucurbitaceae plants. From approximately 3400 collected insects in three different surveys, different anamorphic Ascomycetes were recovered. One isolate of Aspergillus sp., two of Penicillium sp., three of Paecilomyces marquandii, and three of Verticillium sp. out of 308 insects (2.9%) from three insect orders, Hymenoptera, Diptera and Isoptera in the tropical forest. In contrast, a higher number of fungal isolates were recovered from the agricultural area: three isolates from Aspergillus parasiticus, 100 of Fusarium moniliforme, one of Aschersonia sp., and 246 of Fusarium oxysporum out of 3100 insects (11.3%) from three insect orders, Homoptera, Coleoptera and Lepidoptera. The results of this study show Fusarium moniliforme and F. oxysporum as highly virulent to infected insects in the agricultural area, with 100 and 246 isolates respectively, out of 350 infected insects of 3100 studied specimens. Laboratory whitefly nymph bioassays with isolates Ed29a of F. moniliforme, Ed322 of F. oxysporum, and Ed22 of P. marquandii showed 96 to 97.5% insect mortality with no significant differences (P < 0.05) among them. F. oxysporum Ed322 produced no mortality when inoculated on tomato, bean, squash and maize seedlings (with and without injuries) compared to the 100% mortality caused by phytopathogenic strains, F. oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicis lycopersici.

Estudio de la patogenicidad de conidiobolus Coronatus en animales de experimentacion

The pathogenicity of a strain of Conidiobolus coronatus isolated from Aenolamia postica was investigated in 152 mice, 42 guinea pigs and 47 hamsters. Four routes of inoculation were used: intraperitoneal, subcutaneous, intratracheal and scarification in the oral mucosa. Each animal was inoculated 3 times at 3 week intervals. Animals were sacrificed at 1, 8, 15, 45 of 225 days after the first inoculation. Observations of macroscopic lesions (nodules, abscesses, adhesions, etc.) direct examinations, cultures and histopathological studies of different tissues were performed. The aforementioned lesions of tissue were the most frequently found and all of them were resolved by spontaneous cure. The animals inoculated intraperitoneally and subcutaneouslly presented the highest number of positive results. The histopathological features at the 1st and 8th days after inoculation showed an acute inflamatory infiltrate with well preserved fungus filaments; at the 15th day granulomas with giant cells were observed containing fragmented and disintegrated fungal hyphae; at the 45th and 225th days a fibrosis reaction was observed and no fungal filaments, could be found. In no animal was rhinoentomophthoromycosis never reproduced, and according to these results it is considered that either this strain is not a pathogen or its pathogenicity is very low.ResumenA partir de una cepa de Conidiobolus coronatus aislada de Aenolamia postica, se inocularon 152 ratones, 42 cobayos y 47 hamsters por vías intraperitoneal (IP), subcutánea (SC), intratraqueal y por escarificación en la mucosa oral; cada animal se inoculó 3 veces a intervalos de una semana. Los animales se sacrificaron a los 1, 8, 15, 45 y 225 días de la primera inoculación, haciéndose estudios de alteraciones macroscópicas (nódulos, abscesos, adherencias), exámenes directos, cultivos e histopatología. Las alteraciones macroscópicas de los tejidos fueron las más frecuentes y todas evolucionaron a la curación espontánea. Los animales presentaron un mayor múmero de estudios positivos cuando se inocularon por vía IP y SC. Los estudios histopatológicos hechos al 1° y 8° día, demostraron un infiltrado inflamatorio agudo y filamentos fúngicos bien conservados; a los 15 días se observaron granulomas con células gigantes y filamentos fragmentados en desintegración; a los 45 y 225 días se observó una reacción de fibrosis sin filamentos fúngicos. Se consideró que en ningún caso se logró reproducir la rinoentomoftoromicosis y de acuerdo a éstos resultados se estima que esta cepa no tiene patogenicidad o bien ésta es muy baja.

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.