Serological cross-reactivity betweenKlebsiella pneumoniae K47 andSporothrix species

The serological cross-reactivity ofKlebsiella pneumoniae K47 antiserum with antigens of 11Sporothrix species was investigated by use of immunodiffusion. Cross-reactions occurred withK. pneumoniae K47 and theSporothrix speciesS. schenckii, S. schenckii var.luriei, S. curviconia, andS. inflata.     

Antigenic relationships between Aracoccidioides loboi and other pathogenic fungi determined by immunofluorescence

Summary The fluorescent antibody technique was used to study antigenic relationships betweenParacoccidioides loboi and other pathogenic fungi. The findings suggest thatP. loboi is more closely related antigenically to certainP. brasiliensis strains than to others and that it has antigens in common with the yeast form ofHistoplasma capsulatum, H. duboisii, Blastomyces dermatitidis, Candida albicans and also the mycelial form ofCoccidioides immitis. Serum globulins from 3 cases of keloidal blastomycosis were labelled with fluorescein isothiocyanate. These conjugates showed slight or no reactivity withP. loboi, the yeast forms ofP. brasiliensis, H. capsulatum, H. duboisii andB. dermatitidis, However, they stained brightlyC. albicans, serotypes A and B, the tissue form ofC. immitis and the yeast form ofSporotrichum schenckii. Adsorption of these reagents withC. albicans eliminated all staining except that forS. schenckii. These patients had no history of clinical sporotrichosis.Deceased. Last address: Fundacão Gonçalo Moniz, Salvador, Bahia, Brazil. Requests for reprints should be sent to Dr.William Kaplan.Dr.Miranda is in private practice in Rio de Janeiro, Brazil.     

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.     

Identification of three chitin synthase genes in the dimorphic fungal pathogenSporothrix schenckii

Degenerate PCR primers were used to amplify a 600-bp conserved gene region for chitin synthases from genomic DNA ofSporothrix schenckii, a dimorphic fungal pathogen of humans and animals. Three chitin synthase gene homologs were amplified as shown by DNA sequence analysis and by Southern blotting experiments. Based on differences among the predicted amino acid sequences of these homologs, each was placed within one of three different chitin synthase classes. Phylogenies constructed with the sequences and the PAUP 3.1.1. program showed thatS. schenckii consistently clustered most closely withNeurospora crassa in each of the three chitin synthase classes. These findings are significant because the phylogenies support by a new method the grouping of the imperfect fungusS. schenckii with the Pyrenomycetes of the Ascomycota.     

A comparison of some Ceratocystis species with sporothrix schenckii

Sympodulosporogenous states ofC. minor, C. montia, C. multiannulata, C. narcissi, C. nigrocarpa, C. perparvispora andC. pilifera were compared with typical and atypical strains ofS. schenckii from the aspects of some culture, morphological, serological characteristics, and mouse virulence. With a possible exception, all the former characteristics demonstrated among theCeratocystis species were either the same as, or varied in the same way as those observed amongS. schenckii strains. None of the former hydrolyzed starch, although all of the latter did. All species and strains cross reacted serologically in agglutination and Arthus-rype reactions, and produced similar gross pathological signs when injected with gastric mucin intraperitoneally into mice. Except for some variations in sizes and shapes of sympodulospores and of yeast-like budding forms observed in tissue smears and in cultures incubated at 37°C, theCeratocystis species were not significantly different from typical strains ofS. schenckii.

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Zusammenfassung Der sympodulosporogene Zustand vonC. minor, C. montia, C. multiannulata, C. narcissi, C. nigrocarpa, C. perparvispora undC. pilifera wurde mit typischen und atypischen Stämmen vonS. schenckii betreffs kultureller, morphologischer, serologischer Charakteristik und auf Virulenz für Mäuse untersucht. Mit einer möglichen Ausnahme waren alle Charakteristiken unter denCeratocystis-Arten dieselben oder sie wechselten wie diejenigen unter den Stämmen vonS. schenckii. Keine Stämme derCeratocystis hydrolysierten Stärke, während die Stämme vonS. schenckii getan haben. Alle die Arten und Stämme zeigten Kreuzreaktionen serologisch und in der Arthus-reaktion und zeigten dieselben groß-pathologischen Veränderungen nach intraperitonealen Injektionen mit Magenmuzin in der Maus. Außer etlicher Abwechslungen in Größe und Gestalt der Sympodulosporen und der hefe-ähnlichen Keimung, die in Gewebeausstrichen und in Kulturen bei 37°C beobachtet worden sind, waren dieCeratocystis-Arten nicht wesentlich unterschiedlich von typischen Stämmen vonS. schenckii.

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The author, Dr.R. W. Davidson (Colorado State University, Ft. Collins, Colorado), and Dr.F. Mariat (Institut Pasteur, Paris) have, in collaboration, identified the ascigerous state ofSporothrix schenckii strain G-118 isolated by the latter to beCeratocystis stenoceras (Robak)C. Moreau.     

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.     

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.     

Protein Profiling of the Dimorphic Pathogenic Fungus, Sporothrix schenckii

Sporotrichosis is a common cutaneous mycosis caused by the dimorphic fungus Sporothrix schenckii, which exhibits a temperature-dependent dimorphic switch. At 25°C, it grows in a mycelial phase, while at 37°C, it forms unicellular yeast cells. The formation of yeast cells was thought to be a requisite for the pathogenicity of S. schenckii. To identify fragments that might be related to morphogenesis, whole-cell proteins from the mold and early yeast stages of S. schenckii were analyzed using 2DE. Among thousands of protein molecules displayed, more than 300 showed a differential expression between the two phases. In particular, 24 yeast-specific proteins were identified using MALDI-TOF/MS. One of the most interesting proteins was a hybrid histidine kinase, DRK1, a global regulator of dimorphism and virulence in Blastomyces dermatitidis and Histoplasma capsulatum that was abundant in the yeast phase. Our study introduced a new approach to study dimorphism in S. schenckii, and the data may help us better understand the molecular mechanisms of phase transition.     

Cell Wall Glycoproteins Participate in the Adhesion of <Emphasis Type="Italic">Sporothrix schenckii</Emphasis> to Epithelial Cells

Sporothrix schenckii is the etiologic agent of sporotrichosis. This fungal infection is an emerging disease potentially fatal in immunocompromised patients. The adhesion to host cells is a crucial early event related with the dissemination of pathogens. In order to clarify the mechanisms of adhesion of S. schenckii yeast cell to epithelial cells, we studied the biochemical basis of this process. The electrophoretic analysis of cell wall protein from S. schenckii coupled at ConA and stained with HRP, revealed nine different proteins with MW ≥ 180, 115, 90, 80, 58, 40, 36, 22 and 18 kDa. Using ligand-like assay with biotinylated S. schenckii surface proteins, five proteins with MW ≥ 190, 180, 115, 90 and 80 kDa which have affinity to epithelial cells were identified. The adhesion of yeast to epithelial monolayer was significantly inhibited when S. schenckii was pretreated with concanavalinA (ConA) and wheat germ agglutinin (WGA) lectins, alkali, periodate, trypsin, endoglycosidase H (EndoH), salt solutions and detergents. The ability of adhesion of S. schenckii yeast was recovered by blocking the lectin with sugar complementary. These data suggest that surface glycoprotein with mannose and glucose residue could be participate in the process of fungal adhesion to epithelial cells.     

Chemical composition and immunological properties of glycoproteins of Sporothrix species

Glycoproteins of 11Sporothrix species were purified from their respective culture filtrates by use of DEAE-Sephadex A-50 and QAE-Sephadex A-25 column chromatography and investigated for their chemical and immunological properties. On the basis of sugar composition, the glycoproteins of the 11Sporothrix species could be divided into two groups, i.e., rhamnose containing (i.e., Rha⁺), and non rhamnose containing (i.e., Rha^–) groups. The species in the former group wereS. curviconia, S. inflata, S. schenckii andS. schenckii var. luriei, and those in the latter group wereS. cyanescens, S. foliorum, S. fungorum, S. ghanensis, S. imectorum, S. luteoalba andS. ramosissima. The glycoproteins of four of the (Rha⁺) species were relatively similar in elution patterns of DEAE-Sephadex A-50 chromatograms, sugar and amino acid compositions, serological reactivity with rabbit andS. schenckii serum and rabbit antiKlebsiella pneumoniae K47 serum, and cutaneous delayed hypersensitivity. In the case of the (Rha^–) species, the glycoproteins of five species cross-reacted with rabbit antiS. schenckii serum and all, but theS. cyanescens, glycoprotein were reactive to some degree in skin tests in sporotrichotic patients. These results strongly suggest that the chemical and immunological properties of these glycoproteins correspond with the morphological observations amongSporothrix species.     

Nitrogen source effects on rhizosphere pH and nutrient accumulation by Pacific Northwest conifers

Growth responses ofCasuarina cunninghamiana to inoculation withFrankia are described in unsterilized field soils at three sites. At Mt Crawford, South Australia, seedlings of three provenances ofC. cunninghamiana were inoculated with a singleFrankia source just prior to planting out. Forty-four months after planting, inoculation had more than doubled wood production by twoC. cunninghamiana provenances, whilst a third provenance grew poorly and did not respond to inoculation. In Zimbabwe, seedlings of one provenance ofC. cunninghamiana were inoculated in the nursery with one of four differentFrankia strains. In an N deficient soil at Kadoma, three of theseFrankia increased tree height 14 months after planting by between 50% and 70% in comparison to the uninoculated seedlings. The fourthFrankia strain resulted in increased tree height to three times that of the uninoculated controls and up to double that of the other threeFrankia strains. At Gympie, Queensland, Australia, seedlings ofC. cunninghamiana raised open-rooted in a nursery bed were inoculated withFrankia seventeen weeks before planting out. During the 22 months following planting in the field, tree growth was limited by soil P status and there was no response in tree height or stem diameter to inoculation withFrankia or to N fertilizer unless P was applied. In the presence of added P there was a significant response both toFrankia inoculation and to N fertilizer. This positive interaction between P application and N treatment was reflected in wood volumes-inoculated trees and those trees supplied N fertilizer produced 34% and 95% more wood volume than did the uninoculated trees. These results demonstrate the potential to increase the productivity of Casuarina plantings by inoculation withFrankia and by alleviation of P deficiency.     

Photosynthesis of Grass Species Differing in Carbon Dioxide Fixation Pathways : VI. DIFFERENTIAL EFFECTS OF TEMPERATURE AND LIGHT INTENSITY ON PHOTORESPIRATION IN C(3), C(4), AND INTERMEDIATE SPECIES

The effects of temperature and photosynthetically active radiation levels on photorespiration were investigated in Panicum milioides Nees ex Trin. and Panicum schenckii Hack., species known to have low photorespiration rates and other characteristics intermediate between C₃ and C₄ species. Comparisons were made with the C₃ grass species tall fescue (Festuca arundinacea Schreb.). An increase in temperature from 20 to 35 C raised photorespiration from 7.3 to 10.2 milligrams per square decimeter per hour in tall fescue, but the increase in P. schenckii was less than 1 milligram per square decimeter per hour. Increases in temperature caused much less change in CO₂ compensation concentration in P. milioides and P. schenckii than in tall fescue, values of 160 microliters per liter being obtained in tall fescue at 40 C compared to about 40 microliters per liter for P. milioides and P. schenckii. Photorespiration in P. schenckii increased by only about 1 milligram CO₂ per square decimeter per hour as the photosynthetically active radiation level was raised from 100 to 2,000 microEinsteins per square meter per second. Loss of CO₂ into CO₂-free air actually decreased from 2.2 to 1.0 milligrams per square decimeter per hour as the radiation level was raised from 100 to 1,100 microEinsteins per square meter per second but tended to rise again at 2,000 microEinsteins per square meter per second. In contrast, photorespiration in tall fescue tripled with radiation level over the same range, reaching a maximum value of 7.2 milligrams per square decimeter per hour as determined by extrapolation of the CO₂ response curves to zero CO₂. The CO₂ compensation concentration in tall fescue was nearly insensitive to photosynthetically active radiation above 140 microEinsteins per square meter per second but, in P. milioides and P. schenckii, it decreased from values of 69 and 62 microliters per liter, respectively, to values of 21 and 16 as the radiation level was increased from 50 to 1075 microEinsteins per square meter per second. Interpolation of CO₂-response curves showed that an increase in photosynthetically active radiation level from 100 to 2,000 microEinsteins per square meter per second reduced the CO₂ compensation value of P. schenckii from 38 to 19 microliters per liter. Data from these experiments indicate reduced photorespiration or a CO₂-recycling mechanism in P. milioides and P. schenckii which causes apparent photorespiration to be nearly insensitive to temperature in the 20 to 35 C range and to decrease at high radiation intensities.     

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.     

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.     

Gastrointestinal inoculation of Sporothrix schenckii in mice

Antibiotic-decontaminated and untreated conventional mice were inoculated intragastrically with 10⁷ viable cells of Sporothrix schenckii to compare the incidence of gastrointestinal (GI) colonization. In control mice, S. schenckii was completely eliminated from the GI tract by 12 h post-inoculation. Antibiotictreated mice also failed to become colonized with this fungus, however, higher population levels of Sporothrix cells remained in the GI tract for a longer period of time before being eliminated. The ability of S. schenckii to disseminate from the lumen of the bowel to infect other organs was also tested. Results indicate that the gastrointestinal tract is not a portal of entry into the host for S. schenckii.     

Ribosomal DNA Sequencing and Phylogenetic Analysis of Environmental <Emphasis Type="Italic">Sporothrix schenckii</Emphasis> Strains: Comparison with Clinical Isolates

The authors reported the isolation and genetic characterization of Sporothrix schenckii strains from natural environmental samples and commercial amended and garden soils. Twenty-six isolates were recovered and identified as S. schenckii by using both phenotypic and molecular methods. The majority of the strains were isolated from commercial amended and garden soils, indicating that these products represent an important reservoir of the fungus. Sequencing and phylogenetic analysis of the D1-D2 region of the 28S rRNA gene of environmental isolates, including S. schenckii ATCC 10268 and two Italian clinical strains, revealed a degree of difference sufficient to justify the separation of the examined isolates in two principal groups (environmental and clinical). Such separation in two groups is further supported by two well-conserved nucleotide polymorphisms, caused by single-base transitions, in the D1-D2 domain of rDNA. In this study, the presence in nature and/or in commercial products of S. schenckii is discussed. To our knowledge, this is the first study that reports the environmental isolation of S. schenckii from southern Italy.     

Candida steatolytica sp.n.

A new yeast species is described,C. steatolytica, which strongly hydrolizes fat and grows withi-inositol as the sole source of carbon. OtherCandida species capable of usingi-inositol are compared and their classification discussed.C. nivalis andC. frigida are considered to be synonymous withC. curiosa andC. curvata withC. humicola. C. punicea produces ballistospores and must be rejected from the genusCandida.I would like to express my thanks to Dr. J. P. van der Walt for the opportunity of studying this species and for suggesting the specific epithet.     

Novel antigenic determinants from peptidorhamnomannans of Sporothrix schenckii

Antisera were raised in rabbits against acetone-dried yeast-likeand mycelium forms of Sporothrix schenckii. These antisera weretested for immunoprecipitation of peptidorhamnomannans isolatedfrom both cell types. Both antisera reacted strongly with S.schenckiipeptidorhamnomannans, but the reactions were weak with ß-eliminatedpeptidopolysaccharides. These antisera did not recognize theSaccharomyces cerevisiae mannoprotein, and reacted poorly withCeratocystis (Ophiostoma) stenoceras cell wall glycopeptides.Since ß-eliminated glycopeptides were poorly reactive,we investigated the activity of O-glycosidically linked oligosaccharideswhich were liberated from the peptidorhamnomannans by mild alkalinehydrolysis, using a hapten inhibition test. The rates of inhibitionshowed that the immunodominant epitopes in O-linked tetra- andpentasaccharides had the following novel structures:     

Growth of dimorphic human pathogenicfungi on media containing cycloheximide and chloramphenicol

Summary Studies of 24 strains ofBlastomyces dermatitidis confirmed previously published results that the yeast-phase of this fungus is more sensitive than the mycelial-phase to cycloheximide and chloramphenicol.Studies of 5 strains each ofHistoplasma capsulatum, Paracoccidioides brasiliensis andSporotrichum schenckii show that that these species also have a similar yeast-phase mycelial -phase sensitivity differential in regard to these antibiotics.A cycloheximide resistant strain ofB. dermatitidis was developed from a sensitive strain.The experimental results support the general practice of using 0.5 mg/ml cycloheximide and 0.05 mg/ml chloramphenicol in media for the isolation of the four fungi at 25° C. The results indicate, however, that some strains would not be recovered at 37° C with similar concentrations of these antibiotics.It is recommended that a concentration of not more than 0.2 mg/ml chloramphenicol should be used to preserve sputum which is subsequently to be cultured forB. dermatitidis, Histoplasma capsulatum, Paracoccidioides brasiliensis orS. schenckii.