Cytosolic phospholipase A2: a member of the signalling pathway of a new G protein α subunit in Sporothrix schenckii

Background  

Sporothrix schenckii is a pathogenic dimorphic fungus, the etiological agent of sporotrichosis, a lymphocutaneous disease that can remain localized or can disseminate, involving joints, lungs, and the central nervous system. Pathogenic fungi use signal transduction pathways to rapidly adapt to changing environmental conditions and S. schenckii is no exception. S. schenckii yeast cells, either proliferate (yeast cell cycle) or engage in a developmental program that includes proliferation accompanied by morphogenesis (yeast to mycelium transition) depending on the environmental conditions. The principal intracellular receptors of environmental signals are the heterotrimeric G proteins, suggesting their involvement in fungal dimorphism and pathogenicity. Identifying these G proteins in fungi and their involvement in protein-protein interactions will help determine their role in signal transduction pathways.     

Antibody raised against extracellular proteinases ofSporothrix schenckii inS. schenkii inoculated hairless mice

Sporothrix schenckii produces two extracellular proteinases, namely proteinase I and II. Proteinase I is a serine proteinase, inhibited by chymostatin, while proteinase II is an aspartic proteinase, inhibited by pepstatin. Studies on substrate specificity and the effect of proteinase inhibitors on cell growth suggest an important role for these proteinases in terms of fungal invasion and growth. There has, however, been no evidence presented demonstrating thatS. schenckii produces 2 extracellular proteinases in vivo. In order to substantiate the in vivo production of proteinases and to attempt a preliminary serodiagnosis of sporotrichosis, serum antibodies against 2 proteinases were assayed usingS. schenckii inoculated hairless mice. Subsequent to an intracutaneous injection ofS. schenckii to the mouse skin, nodules spontaneously formed and disappeared for a period of 4 weeks. Histopathological examination results were in accordance with the microscopic observations. Micro-organisms disappeared during the fourth week. Serum antibody titers against purified proteinases I and II were measured weekly, using enzyme-linked immunosorbent assay (EIA). As a result, the time course of the antibody titers to both proteinases I and II were parallel to that of macroscopic and microscopic observations in an experimental mouse sporotrichosis model. These results suggest thatS. schenckii produces both proteinases I and II in vivo. Moreover, the detection of antibodies against these proteinases can contribute to a serodiagnosis of sporotrichosis.     

Sporothrix schenckii isolated fromdomestic cats with and without sporotrichosis in Rio de Janeiro,Brazil

A total of 148 cats with a clinical and mycologic diagnosis of sporotrichosis and 84 apparently healthy cats with domiciliary contact with the affected animals were studied. Sporothrix schenckii was isolated from 148 (n = 148; 100%) clinical samples of cutaneous lesion (biopsy, swab or aspiration of purulent secretion), 47(n = 71; 66.2%) nasal cavities, 33 (n = 79; 41.8%) oral cavities, and 15 (n = 38; 39.5%) nails of cats with sporotrichosis. Histopathological examination revealed yeast-like structures in 50 (n = 70; 71.4%) of the biopsies studied. S. schenckii was isolated from the blood culture of one cat (n = 5, 20%) with the disseminated cutaneous form of the disease. On another occasion, the fungus was isolated from the testis of one (n = 7; 14.3%) of the animals submitted to sterilization. In the group of cats with domiciliary contacts, 3(n = 84; 3.57%) oral swabs showed positive cultures. Isolation of S. schenckii from different clinical specimens during both the clinical and preclinical phase reinforces the zoonotic potential of feline sporotrichosis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Analysis of restriction profiles of Mitochondrial DNA from Sporothrix schenckii and related fungi

Restriction profiles by HaeIII of mitochondrial DNA were studied for classification and distinction of Sporothrix schenckii (100 strains), S. schenckii var. luriei (1), S. curviconia (1), S. inflata (7), Ceratocystis stenoceras (17) and C. minor (7). These 6 species showed unique restriction profiles which could be discriminated from each other. S. schenckii was further separable into 11 types, S. inflata into 4 types, C. stenoceras into 4 types and C. minor into 7 types based on restriction profile heterogeneity.     

Diagnosis and Treatment of Lymphocutaneous Sporotrichosis: What Are the Options?

Lymphocutaneous sporotrichosis is the most common clinical form of cutaneous sporotrichosis. Caused by a complex of dimorphic fungi called Sporothrix schenckii complex, it is an occupational disease, present especially in tropical and subtropical areas, and has been reported in all continents. Diagnosis is established by isolation of the causative agent. Therapy of choice for lymphocutaneous sporotrichosis is itraconazole, and in developing countries, potassium iodide solution. In general, the lymphocutaneous form is considered a mild benign form of the disease, and the majority of cases respond well to treatment in about 3–4 months of therapy. In this paper, we have made a general review of the disease, especially of the epidemiology, clinical features and diagnosis of the lymphocutaneous form, as well as a brief analysis of the advantages and disadvantages of diverse treatments.     

Ex vivo determination of potentially virulent Sporothrix schenckii

Hyphae from 30 isolants ofSporothrix andOphiostoma species were washed, dried and pyrolyzed at 350°C. Pyrolysis products were separated on a Carbowax column heated 7.5°C/min to and maintained for 50 min at 160°C. Hydrogen flame detector responses were recorded graphically. Fifteen clinical isolants ofS. schenckii from geographically separated sources produced qualitatively identical pyrograms.S. foliorum, 8 avirulentS. schenckii and otherSporothrix species isolants from soils, andSporothrix states of 6Ophiostoma species yielded pyrograms readily distinguished from each other and from those of virulentS. schenckii. Taxonomic and clinical implications of the pyrograms are mentioned.     

Characterization of a novel antimycotic agent,cinnamyl benzoate,using yeast-phase Sporothrix schenckii

Cinnamyl benzoate specifically inhibited the growth of yeast-phase cells of the pathogenic fungus Sporothrix schenckii. A commercially available antimycotic agent, miconazole nitrate, released large amounts of K⁺ and P_i from S. schenckii probably due to it damaging the cell membrane, but no such release was observed with cinnamyl benzoate or with another commercial antimycotic agent, Tolnaftate. The sterol content of cells treated with cinnamyl benzoate and Tolnaftate was decreased and large amounts of squalene accumulated in the cells. Cinnamyl benzoate may therefore inhibit sterol synthesis in S. schenckii.The authors are with the Department of Applied Microbial Technology, Kumamoto Institute of Technology, lkeda 4-22-1, Kumamoto 860, Japan.     

Effects of proteinase inhibitors on the cutaneous lesion ofSporothrix schenckii inoculated hairless mice

Sporothrix schenckii produces two extracellular proteinases, namely proteinase I and II. Proteinase I is a serine proteinase, inhibited by chymostatin. On the other hand, proteinase II is an aspartic proteinase, inhibited by pepstatin. The addition of either pepstatin or chymostatin to the culture medium did not inhibit cell growth, however the addition of both inhibitors strongly inhibited fungal growth. Accordingly, this suggested that extracellular proteinases play an important role in cell growth and that such cell growth may be suppressed if these proteinases are inhibited. In order to substantiate this speculation in sporotrichosis, the effects of proteinase inhibitors on the cutaneous lesions of mice were studied. Ointments containing 0.1% chymostatin, 0.1% pepstatin and 0.1% chymostatin-0.1% pepstatin were applied twice daily on the inoculation sites of hairless mouse skin, and the time courses of the lesions examined. The inhibitory effect in vivo onS. schenckii was similar to that demonstrated in our previous in vitro study. Compared to the control, the time course curve of the number of nodules present after the application of either pepstatin or chymostatin was slightly suppressed. The application of both pepstatin and chymostatin, however, strongly suppressed nodule formation. This study not only confirmed the role of 2 proteinases ofS, schenckii for fungal growth in vivo, but also may lead to their use as new topical therapeutic agents.     

Effects of hyperthermia on phagocytosis and intracellular killing of Sporothrix schenckii by polymorphonuclear leukocytes

The effects of hyperthermia on phagocytosis and killing of Sporothrix schenckii by polymorphonuclear leukocytes (PMNs) were investigated in order to clarify the mechanism of local thermotherapy for sporotrichosis. Yeast cells of S. schenckii, PMNs and serum were incubated at 37°C or 40°C for 2 or 4 hours. Rate of phagocytosis and killing rate (rate of germination) were estimated, and their processes were observed by transmission electron microscopy. There was no effect of hyperthermia on the phagocytosis rate, but the killing rate increased significantly at 40°C. Electron microscopic examination showed an increase of granularity in the yeast cytoplasm, elongation and fragmentation of the cell membrane. The ultrastructural changes were basically identical under both temperatures, but the degree of these changes was higher at 40°C than at 37°C. Although both intact and degenerated yeasts were found in the same conditions, their transient forms were few, suggesting that the PMN-killing process was completed promptly.     

Differences in virulence between isolates of feline Sporotrichosis

Sporotrichosis is a chronic subcutaneous mycosis caused by Sporothrix schenckii. This work aimed to evaluate the virulence of two different isolates of S. schenckii from cutaneous (CUT) and systemic (SYS) forms of feline sporotrichosis. A standard inoculum with 2 × 10³ yeast cells/ml was prepared from each of the isolates. The experimental infection was carried out with 0.1 ml of the inoculum from both isolates and then injected in the paw pads of Swiss albino mice of groups CUT and SYS. The clinical evolution of the disease and the diameter of the lesion at the inoculated sites were evaluated during nine weeks. Four necropsies were done to collect material from the lesions (p < 0.01). Group CUT demonstrated a more evident clinical evolution of the disease from week two to week five; large lesions in the paw pad on week four (p < 0.01); and a higher incidence of lesions in other parts of the body (p < 0.01) than group SYS (p < 0.01). S. schenckii was isolated from the inoculated site in groups SYS and CUT until days 30 and 45, respectively. Granulomas with yeast cells usually localized in the central area were observed in histopathology sections on days 15 and 30 post-inoculations. Those yeast cells decreased on day 45 being absent on day 62 when tissue repair initiated. The results showed that distinct clinical isolates of S. schenckii cause significant differences in the clinical evolution of sporotrichosis.     

Phylogeny and molecular epidemiology of Sporothrix schenckii in Japan

Mitochondrial DNA(mtDNA) diversity was investigated in 257 clinical isolants of Sporothrix schenckii obtained from 4 districts in Japan. S. schenckii was classified into 10 types based on Hae III restriction profiles. Phylogeny of types constructed by the method of Fitch and Margoliash [1] on the estimated sequence divergence within mtDNA using the methods of Nei and Li [2], showed that S. schenckii are grouped into 2 clusters, one group consisting of types 1, 2 and 3, and the other group consisting of the other seven types. In addition, types 1, 2, and 5 were correlated with their geographic origin, whereas type 4 was present throughout Japan.     

Histopathological studies ofSporothrix schenckii-inoculated mice

Defense mechanisms againstSporothrix schenckii were studied using mouse models. After an intracutaneous injection of the yeast form ofS. schenckii to the dorsal skin of the congenitally athymic nude and normal heterozygote littermate mice, nodules were formed. They regressed and disappeared in 10 weeks in the case of normal mice. On the other hand, nodules and then ulceration developed progressively in nude mice until all animals expired by dissemination of microorganisms at the 11th week of inoculation. Histopathologically the migrated cells were similar in both the normal and the nude mice, particularly during the early phase (within 24 h), with infiltration by PMNs being predominant. Fragmentation ofS. schenckii commenced early during the 12–24 h stage of inoculation in the normal mice, while such fragmentation was scarce in nude mice even though numerous PMNs accumulated. Microscopic observations in the early stages (within 24 h of inoculation) suggested that the lack of killing activity by PMNs in nude mice contributes more to the impaired defense than the lack of macrophage activation by T-cells.     

Sporotrichosis Caused By <Emphasis Type="Italic">Sporothrix globosa</Emphasis> in Rio De Janeiro,Brazil: Case Report

This report describes the first isolation of Sporothrix globosa from a Brazilian patient. A 77-year-old woman was examined for sporotrichosis infection. Histopathological examination of skin biopsy revealed chronic granulomatous infiltrate with microabcess. Furthermore, S. schenckii-like yeasts were evident as demonstrated by PAS and Grocott stains. The fungus was identified based on colony morphology on Sabouraud Dextrose Agar slants, Potato Dextrose Agar, and Corn Meal Agar, microscopic morphology on slides cultures, and assimilation of different carbon sources. The species confirmation was made by molecular methodology.     

Correlations among culture times,sugar composition and biological activities of Sporothrix schenckii antigens

Glycoproteins were isolated by ethanol precipitation, Con A-sepharose 4B and DEAE-sephadex A-50 chromatography from culture filtrates of Sporothrix schenckii ATCC 10268 at incubation periods of 2, 7, and 14 days, and their chemical and immunological properties investigated. Sugar composition of the isolated glycoproteins varied with time of culture, i.e. from mostly mannose on the 2nd day of culture to increasing amounts of rhamnose and small amounts of galactose in addition to mannose on the 7th and 14th day. The changes in sugar composition also were observed to be closely related to the growth morphology of the organisms. The isolated glycoproteins showed different serological reactivity in immunodiffusion tests against rabbit anti-S. schenckii antiserum. In addition, they showed varying degree of cross-reaction with rabbit anti Klebsiella pneumoniae K47, anti Cladosporium werneckii and anti Saccharomyces cerevisiae antisera. The immunodiffusion results correlate well with sugar composition and strongly suggest the possibility that rhamnose, galactose and mannose determinants participate in the serological reaction of S. schenckii. In delayed hypersensitivity skin tests in guinea pigs immunized with S. schenckii, only Con A-binding glycoproteins were reactive. These fractions also resembled each other in amino acid content. The results from the present work indicate that the immunochemical properties of S. schenckii glycoproteins vary with incubation period, and suggest the need for standardization of sporotrichin test antigens.     

Disturbances in the production of inteleukin-1 tumor and necrosis factor in disseminated murine sporotrichosis

Production of Interleukin-1 (IL-1) and Tumor Necrosis Factor (TNF) by adherent peritoneal cells from BALB/c mice was measured at week 2, 4, 6, 8 and 10 after intravenous inoculation with 10⁶ Sporothrix schenckii yeasts. As compared with age-matched controls, IL-1 and TNF production by adherent peritoneal cells fromS. schenckii-infected mice was reduced severely at week 4 and 6 of infection and greater than normal at week 8 and 10. Moreover, between week 4 and 6 of infection there was a depression of delayed type hypersensitivity response to a specific whole soluble antigen, and an increase in fungal multiplication in the livers and spleens of infected mice. Thus, the deficits of cell-mediated immunity in mice with systemicS. schenckii infection may derive, in part, from impaired amplification of the immune response consequent to abnormal generation of IL-1 and TNF.     

Variation in Genotype and Higher Virulence of a Strain of <Emphasis Type="Italic">Sporothrix schenckii</Emphasis> Causing Disseminated Cutaneous Sporotrichosis

Sporotrichosis is usually a localized, lymphocutaneous disease, but its disseminated type was rarely reported. The main objective of this study was to identify specific DNA sequence variation and virulence of a strain of Sporothrix schenckii isolated from the lesion of disseminated cutaneous sporotrichosis. We confirmed this strain to be S. schenckii by^® tubulin and chitin synthase gene sequence analysis in addition to the routine mycological and partial ITS and NTS sequencing. We found a 10-bp deletion in the ribosomal NTS region of this strain, in reference to the sequence of control strains isolated from fixed cutaneous sporotrichosis. After inoculated into immunosuppressed mice, this strain caused more extensive system involvement and showed stronger virulence than the control strain isolated from a fixed cutaneous sporotrichosis. Our study thus suggests that different clinical manifestation of sporotrichosis may be associated with variation in genotype and virulence of the strain, independent of effects due to the immune status of the host.     

Effect of amphotericin B on the lipids of yeast cells of Sporothrix schenckii

Yeast cells of five strains of Sporothrix schenckii were obtained for partial analysis of lipid composition. Quantitative analysis of lipids and sterols were completed, as well as qualitative analysis of sterols by thin layer chromatography and by ultraviolet spectra. These determinations were made on cells cultured in the absence and presence of amphotericin B at sub-MIC (minimum inhibitory concentration) levels. Marked alterations in lipid content were observed in the amphotericin B-treated cells. The major alterations were the reduction of total lipid (18.7–57.6%) and sterols (48.5–96.7%) after exposure to the polyenic antibiotic. It is concluded that amphotericin B altered the lipid profiles, especially sterols of S. schenckii.     

Differences in amino acid sequence of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from two species of Dasycladaceae

In contrast to other plants the plastid genome of Acetabularia is larger in size and shows a high degree of variability. This study on the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase demonstrates that strongly conserved areas also exist in the plastid genome of the Dasycladaceae. Searching for differences in the amino acid sequence of the large subunit from Acetabularia mediterranea and Acicularia schenckii, proteolytic peptides which differ in their elution behaviour in reverse-phase high-performance liquid chromatography were sequenced. Only six amino acids were found to be exchanged in the large subunit from these two species. Since these two species diverged approx. 150 million years ago, these results imply that 0.84 amino-acid exchanges per 100 amino acids have occurred in 10⁸ years, underlining the strong conservatism of the large subunit.Abbreviations A Acetabularia mediterranea - Ac. Acicularia schenckii - HPLC high-performance liquid chromatography - LSU large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase - PAGE polyacrylamide gel electrophoresis - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase - SDS sodium dodecyl sulfate