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4ND: not determined Table 1. Sialic acid density and distribution in different forms of pathogenic fungi.

Table 1 4ND: not determined Table 1. Sialic acid density and distribution in different forms of pathogenic fungi.

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Figure 1. Staining with colloidal iron hydroxide of anionogenic groups at the cell surface of S. schenckii yeast forms and F. pedrosoi hyphae and conidia. A, Bilayer reactivity in untreated yeasts; B and C, partial and complete removal of anionic groups reacting with CIH after sialidase treatment, magnification <x 16000; D and E, CIH staining of anionic groups on F. pedrosoi hyphae before (D) and after (E) sialidase treatment. Virtually no particles were seen on several sites of treated cells (arrow heads); F and G, similar staining and enzymatic treatment of F. pedrosoi conidia with few or no particles seen on treated (G) cells (arrow heads); magnification x 25000. Figure 1 is from Alviano CS, et al. (1982) FEMS Microbiol Lett 15:223-227 and Souza ET, etal. (1986) / Med Vet Myco/ 24:145-153. Reproduced with permission from FEMS Microbiology Letters and J ournal of Medical and Veterinary Mycology.
Figure 1. Staining with colloidal iron hydroxide of anionogenic groups at the cell surface of S. schenckii yeast forms and F. pedrosoi hyphae and conidia. A, Bilayer reactivity in untreated yeasts; B and C, partial and complete removal of anionic groups reacting with CIH after sialidase treatment, magnification <x 16000; D and E, CIH staining of anionic groups on F. pedrosoi hyphae before (D) and after (E) sialidase treatment. Virtually no particles were seen on several sites of treated cells (arrow heads); F and G, similar staining and enzymatic treatment of F. pedrosoi conidia with few or no particles seen on treated (G) cells (arrow heads); magnification x 25000. Figure 1 is from Alviano CS, et al. (1982) FEMS Microbiol Lett 15:223-227 and Souza ET, etal. (1986) / Med Vet Myco/ 24:145-153. Reproduced with permission from FEMS Microbiology Letters and J ournal of Medical and Veterinary Mycology.
Table 2. Electrophoretic mobility (EPM) of F. pedrosoi and P. brasiliensis before and after treatment with sialidase, showing the contribution of sialic acids to the negative surface charge of these human pathogenic fungi. Electrophoretic mobility is determined using the following equation: EPM =(d/) x (D/V), where d is the distance (in um) covered by cells during measurement (usually 16 pm); tis the time (in seconds) required by a cell to cover the distance d; D is the distance between the two electrodes (18 cm); and Vis the potential applied to the electrodes. ‘Treatment with 0.4 units/ml of sialidase from Clostridium perfringens. ‘Treatment with 0.4 units/ml of sialidase from Vibrio cholerae.
Table 2. Electrophoretic mobility (EPM) of F. pedrosoi and P. brasiliensis before and after treatment with sialidase, showing the contribution of sialic acids to the negative surface charge of these human pathogenic fungi. Electrophoretic mobility is determined using the following equation: EPM =(d/) x (D/V), where d is the distance (in um) covered by cells during measurement (usually 16 pm); tis the time (in seconds) required by a cell to cover the distance d; D is the distance between the two electrodes (18 cm); and Vis the potential applied to the electrodes. ‘Treatment with 0.4 units/ml of sialidase from Clostridium perfringens. ‘Treatment with 0.4 units/ml of sialidase from Vibrio cholerae.
Expression of sialic acids at the fungal cell surface Figure 2. Labelling with FITC-LFA agglutinin of untreated (a) and siali- dase-treated (c) P. brasiliensis yeast forms. Binding of lectin to the cells was much reduced by enzyme treatment (c). Corresponding fields ob- served by phase-contrast microscopy are shown in (b) and (d). Bars, 10 um. Figure 2 is from Soares RMA, et al. (1998) Microbiology 144: 309-14. Reproduced with permission from Microbiology. The ability of animal, plant, microbial, and fungal lectins to bind to specific carbohydrates has been used to determine he nature of the carbohydrate constituents expressed on a variety of microorganisms including protozoa and fungi 31-33]. Some sialic acid-binding lectins have been success- fully used for in situ location of glycoconjugates. The most requently used are the agglutinins from wheat germ WGA), Limax flavus (LFA), Limulus polyphemus (LPA), Sambucus nigra (SNA) and Maackia amurensis (MAA). With the latter two agglutinins, sialic acid 02,3- and a2,6- inkages can be distinguished [34]. Whereas some of these ectins recognize both NeuS5Ac and Neu5Gc, others are specific for N-acetylneuraminic acid only. O-Acetylation may influence lectin binding, either in a positive or nega- ive way.
Expression of sialic acids at the fungal cell surface Figure 2. Labelling with FITC-LFA agglutinin of untreated (a) and siali- dase-treated (c) P. brasiliensis yeast forms. Binding of lectin to the cells was much reduced by enzyme treatment (c). Corresponding fields ob- served by phase-contrast microscopy are shown in (b) and (d). Bars, 10 um. Figure 2 is from Soares RMA, et al. (1998) Microbiology 144: 309-14. Reproduced with permission from Microbiology. The ability of animal, plant, microbial, and fungal lectins to bind to specific carbohydrates has been used to determine he nature of the carbohydrate constituents expressed on a variety of microorganisms including protozoa and fungi 31-33]. Some sialic acid-binding lectins have been success- fully used for in situ location of glycoconjugates. The most requently used are the agglutinins from wheat germ WGA), Limax flavus (LFA), Limulus polyphemus (LPA), Sambucus nigra (SNA) and Maackia amurensis (MAA). With the latter two agglutinins, sialic acid 02,3- and a2,6- inkages can be distinguished [34]. Whereas some of these ectins recognize both NeuS5Ac and Neu5Gc, others are specific for N-acetylneuraminic acid only. O-Acetylation may influence lectin binding, either in a positive or nega- ive way.
aln the SNA and MAA systems separation of young and mature cells of P. brasiliensis cells was not evident (NE). ’ND: not determined. able 3. Percentages of fluorescent cells determined by flow cytofluorimetry of untreated and sialidase-treated yeasts of P rasiliensis and C. neoformans incubated with FITC-labelled lectins.
aln the SNA and MAA systems separation of young and mature cells of P. brasiliensis cells was not evident (NE). ’ND: not determined. able 3. Percentages of fluorescent cells determined by flow cytofluorimetry of untreated and sialidase-treated yeasts of P rasiliensis and C. neoformans incubated with FITC-labelled lectins.
Figure 3. Phase-contrast micrographs of C. neoformans yeast cells agglutinated with influenza viruses (magnification, x 200 in A and x 400 inB, C, and D). A, Control; B, C. neoformans incubated with influenza virus A M 1/5 HS8 (specific for «2,3-sialyl-galactosyl sequences); C, Agglutination of yeast cells with influenza virus A M1/5 (specific for a2,6-sialyl-galactosyl sequences); D, Agglutination after incubation with influenza C virus (specific for Neu5,9AC>). To determine the expression of sialic acids in C. neofor- mans in relation to the capsule size, SNA binding to an encapsulated strain was compared with SNA binding to a poorly encapsulated form. The latter was markedly in- creased in comparison with the fully encapsulated strain as The choice of the most suitable procedure for the isolation, purification and characterization of sialic acids is of crucial importance. This is particularly difficult when small amounts of sialic acid are present among other monosaccharides, es-
Figure 3. Phase-contrast micrographs of C. neoformans yeast cells agglutinated with influenza viruses (magnification, x 200 in A and x 400 inB, C, and D). A, Control; B, C. neoformans incubated with influenza virus A M 1/5 HS8 (specific for «2,3-sialyl-galactosyl sequences); C, Agglutination of yeast cells with influenza virus A M1/5 (specific for a2,6-sialyl-galactosyl sequences); D, Agglutination after incubation with influenza C virus (specific for Neu5,9AC>). To determine the expression of sialic acids in C. neofor- mans in relation to the capsule size, SNA binding to an encapsulated strain was compared with SNA binding to a poorly encapsulated form. The latter was markedly in- creased in comparison with the fully encapsulated strain as The choice of the most suitable procedure for the isolation, purification and characterization of sialic acids is of crucial importance. This is particularly difficult when small amounts of sialic acid are present among other monosaccharides, es-
Figure 4. Flow cytometric analysis of binding of FITC-SNA to C. neoformans T 1-444 strain, T;-444 with salt (CDM-NaCl)-induced capsule reduction, and HEC 3393 strain, a sparsely encapsulated form. Analysis of 5,000 yeast cells is shown; (a) unstained cells and (b) cells incubated with FITC-lectin. This figure has been modified from Rodrigues, et al. Infect Immun 65: 4937-42.
Figure 4. Flow cytometric analysis of binding of FITC-SNA to C. neoformans T 1-444 strain, T;-444 with salt (CDM-NaCl)-induced capsule reduction, and HEC 3393 strain, a sparsely encapsulated form. Analysis of 5,000 yeast cells is shown; (a) unstained cells and (b) cells incubated with FITC-lectin. This figure has been modified from Rodrigues, et al. Infect Immun 65: 4937-42.
Table 4. Effect of sialidase treatment of S. schenkii and C. neoformans yeast cells on their ingestion by mouse peritoneal macrophages.
Table 4. Effect of sialidase treatment of S. schenkii and C. neoformans yeast cells on their ingestion by mouse peritoneal macrophages.
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Biochemistry and cell biologySialic Acids
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