Discrimination between Sporothrix schenckii and Ceratocystis stenoceras rhamnomannans by proton and carbon-13 magnetic resonance spectroscopy

The proton and (13)C magnetic resonance (CMR) spectra of 10 Sporothrix schenckii and three Ceratocystis stenoceras rhamnomannans were studied. On the basis of their CMR spectra, the rhamnomannans from S. schenckii strains could be classified into two major types, I and II, readily distinguishable from the polysaccharides formed at the same temperature (25 C) by C. stenoceras. Rhamnomannans from S. schenckii synthesized at 37 C gave CMR spectra (type III) differing from those of polysaccharides of types I and II and from those of C. stenoceras polysaccharides. The contention that C. stenoceras might be the perfect form of S. schenckii is not supported by the present data. A presumptive pathogenic mutant of C. stenoceras synthesized a type I rhamnomannan characteristic of S. schenckii, clearly differing from the rhamnomannans from C. stenoceras synthesized in the same conditions. Nuclear magnetic resonance spectroscopy revealed differences in the structures of the polysaccharides from these species not previously recognized by methylation analysis.     

Comparison of the rhamnomannans from the human pathogen Sporothrix schenckii with those from the Ceratocystis species

Rhamnomannans from several isolates of Sporothrix schenckii and Ceratocystis species were compared. No major differences emerged in the analysis of the carbohydrate composition of polysaccharides from either species. Methylation analysis for the identification of the glycosidic linkages present in the polysaccharides showed that they all were very similar with minor differences being observed among strains and between polysaccharides obtained at 25 and 37 C. Partially methylated derivatives were identified by gas-liquid chromatography and mass spectrometry. Their proportions suggest that polysaccharides from S. schenckii and C. stenoceras have a general structure similar to that of C. ulmi: a (1 --> 6)-linked alpha-d-mannopyranosyl main chain substituted in the 3-positions by alpha-l-rhamnopyranosyl and in many cases by alpha-l-rhamnopyranosyl (1-->2)-l-rhamnopyranosyl side chains. Differences among polysaccharides resided in the proportions of the dirhamnosyl side chains and of 4-O-substituted and 2, 4-di-O-substituted mannose units. Increased proportions of dirhamnosyl side chains were observed in the polysaccharides from S. schenckii strains and from a pathogenic variant of C. stenoceras grown at 25 C. Proton magnetic resonance spectra (H-1 region) of the rhamnomannans showed that S. schenckii strains could be placed in two different groups similar but not identical to any Ceratocystis species. These spectra were very close to those of the polysaccharides from the C. clavata and C. ambrosia groups of Ceratocystis species.     

Delayed hypersensitivity cross-reactions between Sporothrix schenckii and Ceratocystis species in sporotrichotic patients.

Cutaneous delayed hypersensitivity to antigens prepared from Sporothrix schenckii and several Ceratocystis species, including C. stenoceras, C. ulmi, C. ips, and C. minor, was tested in 14 patients with known cutaneous sporotrichosis. Extensive cross-reactions were observed. Nonsporotrichotic people (controls) did not react to these antigens. The correlation coefficient between antigens of S. schenckii and each Ceratocystis species was calculated from the areas of the cutaneous reactions. Among the Ceratocystis species tested, the correlation coefficient between S. schenckii and C. stenoceras was 0.91.     

RFLP analysis of the internal transcribed spacer regions of Sporothrix schenckii.

Restriction fragment length polymorphism (RFLP) analysis was performed on the internal transcribed spacer regions of 204 Sporothrix schenckii isolates and on one strain each of the related fungi, S. schenckii var. luriei, S. curviconia, S. inflata and Ceratocystis stenoceras. S. schenckii isolates, which have been collected from around the world, have already been typed according to their mitochondrial DNA (mtDNA), and are kept in the Department of Dermatology, Kanazawa Medical University, Japan. Approximately 600 bp of the internal transcribed spacer region 1 (ITS1) of their nuclear ribosomal RNA gene (rDNA), 5.8S rDNA and ITS2 was amplified by PCR. From ITS-RFLP analysis of the PCR products, S. schenckii isolates comprised 4 types, rDNA types I - IV. The rDNA type I - III strains corresponded to the Group A strains (mtDNA types 1 - 3, 11, 14 - 19, 22 and 23), while the rDNA type IV strains corresponded to the Group B strains (mtDNA types 4 - 10, 12, 13, 20 and 21), as previously categorized according to their mtDNA-RFLP. The ITS-RFLP patterns of the above 4 related fungi all differed from those of the 4 rDNA types of S. schenckii. Furthermore, only 22 (3.5%) out of a sequence of about 620 bases of the ITS regions of the rDNA differed among representatives of the mtDNA types 1 - 5, 7, 11, 14 - 19, 22 and 23. This difference in the ITS region is smaller than the 10% difference among isolates when estimated by mtDNA-RFLP. From the phylogenetic tree based on the base sequences, rDNA type I - III strains belong to Group I, while rDNA type IV strains belong to Group II which correspond with Groups A and B based on their mtDNA. The Group I strains are predominant in South America and Africa, while Group II are predominant in Australia and Asia. ITS-RFLP analysis is better than mtDNA-RFLP in allowing faster discrimination and identification, and for its ability to divide the 4 types into groups, which is useful in clinical diagnosis and epidemiological investigations of S. schenckii.     

Use of a mouse model to evaluate clinical and environmental isolates of Sporothrix spp. from the largest U.S. epidemic of sporotrichosis.

Five clinical and 69 environmental isolates from the largest U.S. epidemic of sporotrichosis were evaluated in NYLAR male mice following intravenous injection of 5 x 10(6) to 2 x 10(8) conidia per mouse. The clinical isolates and eight environmental isolates produced 100% mortality in groups of three mice each between 12 and 24 days after injection. These virulent isolates grew at 37 degrees C, were dematiaceous by virtue of melanin (melanized) on permissive media (e.g., potato dextrose agar), produced ovoid conidia borne sympodially on lateral conidiophores and pleurogenously about the main hyphal axis, and were identified as Sporothrix schenckii. Two melanized environmental isolates that grew at 35 degrees C but not at 37 degrees C were not virulent and had subtle morphological differences from S. schenckii. The remaining environmental isolates were not melanized, were not virulent, and were not S. schenckii; five were identified as Ophiostoma stenoceras and the remainder were identified as Sporothrix spp. Quantitative organ cultures revealed that clinical isolates grew exponentially in livers and testes, in contrast to an isolate of O. stenoceras that was eliminated from liver, lung, and spleen but that persisted in the testes throughout the 14-day sample period. This model helped to confirm the identification of S. schenckii isolates obtained from the environment.     

Molecular analysis of DNA polymorphism of Sporothrix schenckii.

Partial cDNA cloning of a putative membrane transporter protein gene expressed in Sporothrix schenckii and DNA polymorphism of the isolated gene are described here. DNA fragments were isolated from S. schenckii, and the deduced amino acid sequence from one of the fragments contained a region homologous to the conserved sequence of the membrane transporter protein family. 188-bp fragments encoding the homologous region were amplified from many strains of S. schenckii, and were subjected to polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) analysis. The results demonstrated that the strains of S. schenckii were divided into three groups, and several base substitutions among these groups were observed. This finding agreed with the classification of S. schenckii strains based on the mitochondrial DNA diversity, because the three groups were clearly located on the branches of the phylogenetic tree constructed by digestion profiles of mitochondrial DNA with restriction enzymes. The correlation of the results of PCR-SSCP analysis with the mitochondrial DNA diversity might indicate linkage of the mutation of the membrane transporter protein gene with the evolution of S. schenckii, suggesting the protein plays an essential role in S. schenckii.     

Ingestion of yeast forms of Sporothrix schenckii by mouse peritoneal macrophages.

The ingestion by thioglycolate-elicited mouse peritoneal macrophages of yeast forms of two strains of Sporothrix schenckii was studied. Yeast forms opsonized with concanavalin A (ConA) were extensively phagocytized, and the phagocytic indexes depended on the concentration of ConA and apparently on the number of lectin receptors at the yeast surface as well. Neuraminidase treatment of S. schenckii increased the ingestion of unopsonized yeasts 7.7-fold. The addition of monosaccharides and derivatives partially inhibited phagocytosis. Mannose, rhamnose, and galactose, which are major constituents of S. schenckii surface antigens, reduced the phagocytic indexes by 40 to 50%. Glucosamine, N-acetylglucosamine, and N-acetylneuraminic acid were equally effective as inhibitors of phagocytosis. A mixture of five neutral sugars and glucosamine inhibited phagocytosis by 73%. The inhibitory effect of simple sugars could be amplified by using neuraminidase-treated yeast cells. Pentoses and glucose were inactive or slightly inhibitory. A purified rhamnomannan inhibited phagocytosis of the homologous strain, whereas partially purified peptidopolysaccharides were toxic to peritoneal macrophages. A partially purified galactomannan from S. schenckii was inhibitory (62% inhibition), and a peptidopolysaccharide fraction in which the O-linked carbohydrate chains had been removed neither was toxic to macrophages nor inhibited phagocytosis. Pretreatment of macrophages with simple sugars under conditions inhibiting ingestion or binding of S. schenckii did not affect phagocytosis of latex particles or sensitized sheep erythrocytes. The presence of receptors at the peritoneal macrophages which bind S. schenckii cell surface components is suggested.     

Isolation and characterization of Sporothrix schenckii from clinical and environmental sources associated with the largest U.S. epidemic of sporotrichosis.

The largest recorded epidemic of sporotrichosis in the United States occurred in 1988 and involved a total of 84 cases in 15 states. All cases were associated with Wisconsin-grown sphagnum moss. Twenty-one clinical isolates of Sporothrix schenckii and 69 environmental isolates of Sporothrix spp. from the epidemic were characterized and compared. The environmental isolates were recovered from 102 samples of sphagnum moss and other material by using direct plating techniques. Characteristics examined included macroscopic and microscopic morphology, conversion to a yeast phase, exoantigen reactions, and virulence in mice. On the basis of these studies, eight environmental isolates were identified as S. schenckii, five were identified as Ophiostoma stenoceras, and the remainder were identified as Sporothrix species. The environmental isolates of S. schenckii were recovered from moss samples from one Pennsylvania nursery and from three New York State Soil and Water Conservation districts, but none were recovered from moss directly from the bogs in Wisconsin.     

C-13-NMR-spektroskopische untersuchungen an polymerhomologen reihen von α,1→6- und α,1→4-glucanen

The ¹³C-NMR spectra of malto- and isomalto-oligosaccharides, amylose and dextrane were analysed. It was observed that in both series of oligosaccharides and polysaccharides the resonances of the central glucose units are independent of the chain length with the exception of the C-atoms 1 and 4 of amylose which show deviations of 0,4 and 0,5 ppm. These small effects can possibly be explained by a definite polysaccharide chain conformation in solution.     

Immunodiffusion studies of various structural preparations from mycobacterial cells.

Various structures and other preparations from mycobacterial cells were analyzed by immunodiffusion. The preparations were obtained from four strains referred to the species Mycobacterium bovis (BCG), Mycobacterium fortuitum, Mycobacterium phlei and Mycobacterium smegmatis. They represented cell walls (CW), culture filtrates (CF), artificially disintegrated cell material (XP), protoplasms (PP), crude ribosomes (CR), ribosomal 50S subunits (50S), ribosomal 30S subunits (30S), ribosomal 16S core particles (16S) and ribosomal-free fractions (UCS). The preparations were analyzed by reference precipitation systems based on CF, CR and 30S preparations. Serological comparisons were made with material from the same species and cross-testing between material from different species was not performed. It was shown that from the precipitinogenic point of view the CF and XP materials were very similar and that they both contained protoplasmic material to a large extent. Furthermore, it was found that ribosomal precipitinogens constitute an important part of the precipitinogens in PP as well as in the CF and the XP preparations. The ribosomal precipitinogens were found to represent the two subunits as well as the 16S core particle. One ribosomal precipitinogen, designated beta, was shown in all preparations except the CW and the UCS.     

Immunodiffusion studies of ribosomes in classification of mycobacteria and related taxa.

Ribosomal preparations consisting of crude ribosomes (CR), 30S subunits (30S) and 16S core particles (16S) from four strains of the species Mycobacterium bovis (BCG), Mycobacterium fortuitum, Mycobacterium phlei and Mycobacterium smegmatis were analyzed by immunodiffusion technique for taxonomical purposes. The ribosomal preparations tested contained several interspecies cross-reacting precipitinogens. The number of precipitinogens demonstrated at the homologous reactions was generally larger than the number of precipitinogens shown at the heterologous reactions indicating a probable presence of species-specific antigens in ribosomes. The largest number of possible species-specific precipitinogens was demonstrated when crude ribosomal preparations were studied. However, such precipitinogens were also shown in the 30S subunits and they were individually analyzed. The 16S core particles were dominated by cross-reacting precipitinogens. The number of ribosomal precipitinogens shared by M. phlei and M. smegmatis was large indicating a close taxonomical relationship between these two species. Apart from the four mycobacterial strains studied, 15 other strains representing the genera Mycobacterium, Arthrobacterium, Corynebacterium, Kurthia, Nocardia and Rhodococcus were included in the study. The presence of intergenerically cross-reacting precipitinogens in the ribosomal preparations was demonstrated.     

Deoxyribonucleic Acid Base Composition and Hybridization Studies on the Human Pathogen Sporothrix schenckii and Ceratocystis Species

Deoxyribonucleic acid (DNA) base composition, as measured by guanine plus cytosine (G + C) content, was determined in 17 strains of Sporothrix schenckii and Ceratocystis species. The average G + C content for S. schenckii was 54.7 +/- 0.2 mol%. No strain of Ceratocystis, with the exception of C. minor, gave values for G + C content similar to those obtained for S. schenckii. All DNA samples from strains of S. schenckii cross-hybridized with high percentages of relative binding. DNA samples from C. stenoceras and C. pilifera showed low degrees of homology with S. schenckii DNA. The DNA from C. minor hybridized with 75% binding with that from a strain of S. schenckii, as compared with the homologous reaction.     

Binding studies of a monoclonal antibody specific for 3-deoxy-D-manno-octulosonic acid with a panel of Klebsiella pneumoniae lipopolysaccharides representing all of the O serotypes.

A monoclonal antibody (MAb) raised against Salmonella minnesota R595 and specific for alpha-3-deoxy-D-manno-octulosonic acid (alpha-Kdo) of the inner core was tested for binding to lipopolysaccharides (LPS) of Klebsiella pneumoniae. The MAb was tested in several assay systems (enzyme-linked immunosorbent assay, passive hemolysis, and inhibition of passive hemolysis) with a large panel (n = 23) of K. pneumoniae LPS representing all nine currently known O serotypes. MAb 20 showed reactivity with almost all O serotypes of K. pneumoniae LPS, and this reactivity could be inhibited by synthetic Kdo. This suggests an epitope in the cores of these Klebsiella LPS much like that in the inner core of LPS of S. minnesota. Large differences in reactivity between LPS of different strains belonging to the same O serotype were observed. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis of LPS followed by immunoblotting, reactivity of MAb 20 was observed only with the fast-moving fraction possibly representing the nonsubstituted core. No binding was seen with the high-molecular-weight fraction that contained core material substituted with several units of O-antigen building blocks. The chemical basis for these differences in reactivity remains to be established. As far as we know, this is the first report containing comprehensive immunochemical data on the LPS core of K. pneumoniae.     

Detection of Sporothrix schenckii in clinical samples by a nested PCR assay

Cutaneous sporotrichosis is a chronic granulomatous fungal infection caused by Sporothrix schenckii with worldwide distribution. Its traditional diagnosis is time-consuming and difficult to differentiate from that of a clinical sporotrichoid lesion caused by various pathogens. In this study, a nested PCR assay for the detection of S. schenckii was evaluated by using a sequence of 18S rRNA gene as a target. For the examination of specificity and sensitivity, five clinical isolates with 1 ATCC 10213 strain of S. schenckii, 10 strains of clinical common fungi, 3 strains of Mycobacterium spp., Staphylococcus aureus, and normal human skin tissue were used. The expected fragment was amplified from six S. schenckii isolates in the first round and nested PCR but not from other microorganisms and human DNA. Their sequences were 100% identical to the S. schenckii 18S rRNA gene sequence deposited in GenBank. A detection limit of 40 fg of S. schenckii DNA extract was determined with ethidium bromide staining. Serial dilution studies demonstrated that the nested PCR could detect a DNA amount of 1 CFU of S. schenckii in tissue samples. We further investigated the nested PCR assay for the detection of S. schenckii from the tail tissues of 5 experimentally infected mice and from the clinical biopsy specimens of 12 patients with sporotrichosis confirmed by culture or histochemical staining. The nested PCR assay was positive in all 5 infected mice and in 11 of the 12 clinical specimens. The high sensitivity and specificity of this nested PCR indicate that the assay can provide rapid diagnosis with sufficient accuracy to be clinically useful for patients with sporotrichosis.     

Heterogeneity of the rhamnomannans from one strain of the human pathogen Sporothrix schenckii determined by 13C nuclear magnetic resonance spectroscopy

The synthesis of different rhamnomannans in a strain of Sporothrix schenckii (1099.12) was shown by use of 13C nuclear magnetic resonance spectroscopy. Fractionation of a polysaccharide preparation from cells grown at 25 degrees C provided a neutral monorhamnosyl rhamnomannan and an acidic rhamnomannan containing 4-O-substituted glucuronic acid units and also (1 leads to 2)-linked dirhamnosyl side chains.     

Analysis of peptidogalactomannans from the mycelial surface of Aspergillus fumigatus.

Peptidogalactomannans (pGMs) from mycelium of two strains of Aspergillus fumigatus were fractionated by Cetavlon precipitation and size exclusion chromatography and their carbohydrate structures analysed using methylation-fragmentation analysis, partial acetolysis and 13C-nuclear magnetic resonance spectroscopy. The most significant difference between the pGMs of the two strains was the degree of branching and the proportion of non-reducing ends of alpha-D-Manp and beta-D-Galf units. Methylation data showed that the pGM from AF 2109 contained alpha-D-Manp and beta-D-Galf non-reducing end units in a proportion of 3:1 while, in contrast, the proportion of these structures in pGM from AF 2140 was 7:1, resulting in a highly branched structure. The immunoreactivity of the pGM fractions was tested by indirect immunofluorescence. The fractions were also tested in an ELISA system with rabbit antiserum raised to whole cells of A. fumigatus NCPF 2140 and with serum from patients with either proven aspergilloma or ABPA. The carbohydrate moiety of the pGM appears to be responsible for the antigenicity. Periodate treatment, partial acid hydrolysis and beta-elimination removed most of the antibody binding capacity.     

Synthesis of melanin-like pigments by Sporothrix schenckii in vitro and during mammalian infection

Melanin has been implicated in the pathogenesis of several important human fungal pathogens. Existing data suggest that the conidia of the dimorphic fungal pathogen Sporothrix schenckii produce melanin or melanin-like compounds; in this study we aimed to confirm this suggestion and to demonstrate in vitro and in vivo production of melanin by yeast cells. S. schenckii grown on Mycosel agar produced visibly pigmented conidia, although yeast cells grown in brain heart infusion and minimal medium broth appeared to be nonpigmented macroscopically. However, treatment of both conidia and yeast cells with proteolytic enzymes, denaturant, and concentrated hot acid yielded dark particles similar in shape and size to the corresponding propagules, which were stable free radicals consistent with identification as melanins. Melanin particles extracted from S. schenckii yeast cells were used to produce a panel of murine monoclonal antibodies (MAbs) which labeled pigmented conidia, yeast cells, and the isolated particles. Tissue from hamster testicles infected with S. schenckii contained fungal cells that were labeled by melanin-binding MAbs, and digestion of infected hamster tissue yielded dark particles that were also reactive. Additionally, sera from humans with sporotrichosis contained antibodies that bound melanin particles. These findings indicate that S. schenckii conidia and yeast cells can produce melanin or melanin-like compounds in vitro and that yeast cells can synthesize pigment in vivo. Since melanin is an important virulence factor in other pathogenic fungi, this pigment may have a similar role in the pathogenesis of sporotrichosis.     

Antifungal susceptibilities of Sporothrix albicans, S. brasiliensis, and S. luriei of the S. schenckii complex identified in Brazil

We studied 40 strains of the species complex formerly classified as the single species Sporothrix schenckii to identify new species within this complex and evaluate their antifungal susceptibility profiles. Based on phenotypic tests (ability to grow at 37°C, colony diameters, and pigmentation of the colonies, as well as assimilation of sucrose and raffinose) and molecular assays (amplification of a fragment of the calmodulin gene), here we report the identification of S. albicans, S. brasiliensis, S. luriei, and S. schenckii; two isolates of these species were detected as itraconazole-resistant strains.     

Characterization of the antigenic lipopolysaccharide O chain and the capsular polysaccharide produced by Actinobacillus pleuropneumoniae serotype 13

Serotyping of Actinobacillus pleuropneumoniae, the etiologic agent of porcine pleuropneumonia, is important for epidemiological studies and for the development of homologous vaccine cell preparations. The serology is based on the specific chemical structures of capsular polysaccharides (CPSs) and lipopolysaccharide (LPS) antigenic O-polysaccharide moieties (O-PSs), and knowledge of these structures is required for a molecular-level understanding of their serological specificities. The structures of A. pleuropneumoniae serotype 1 to 12 CPSs and O-PSs have been elucidated; however, the structures associated with three newly proposed serotypes (serotypes 13, 14, and 15) have not been reported. Herein we described the structures of the antigenic O-PS and CPS of A. pleuropneumoniae serotype 13. The O-PS of the A. pleuropneumoniae serotype 13 LPS is a polymer of branched tetrasaccharide repeating units composed of l-rhamnose, 2-acetamido-2-deoxy-d-galactose, and d-galactose residues (1:1:2). By use of hydrolysis, methylation, and periodate oxidation chemical methods together with the application of one- and two-dimensional 1H and 13C nuclear magnetic resonance spectroscopy and mass spectrometry, the structures of the O chain and CPS were determined. The CPS of A. pleuropneumoniae serotype 13 was characterized as a teichoic-acid type polymer. The LPS O antigen was identical to the O-PS produced by A. pleuropneumoniae serotype 7. The CPS has the unique structure of a 1,3-poly(glycerol phosphate) teichoic acid type I polymer and constitutes the macromolecule defining the A. pleuropneumoniae serotype 13 antigenic specificity.     

Different virulence levels of the species of Sporothrix in a murine model

A comparative study on the experimental pathogenicity of five species of Sporothrix of clinical interest, Sporothrix albicans , Sporothrix brasiliensis , Sporothrix globosa , Sporothrix mexicana , and Sporothrix schenckii sensu stricto, was performed using an immunocompetent murine model. Two strains of each species and two levels of inoculum for each strain (2 × 10⁷ and 2 × 10⁴ conidia/animal) were tested by intravenous inoculation of mice (ten per group). Mortality was caused by the low inoculum of one strain of S. brasiliensis only, and the high inocula of S. brasiliensis and S. schenckii strains. Other inocula and other species tested did not kill any of the experimental animals. Tissue burden studies showed fungal spread to kidneys, lungs, spleen, brain, and testicles. S. brasiliensis was recovered extensively from all of the studied organs, and S. schenckii and S. globosa were recovered in lower amounts. Histopathological studies revealed differences in the lesions, which ranged from local inflammation with a low number of fungal cells at the injection site in mice infected with S. globosa , to massive infiltration of fungal cells in organs of those infected with S. brasiliensis . Our findings showed that S. brasiliensis and S. schenckii were the most virulent species, and suggest that lesional mechanisms could be species-specific.