Errol Reiss

A galactoxylomannan antigen of Cryptococcus neoformans serotype A

Abstract The capsular polysaccharide of Cryptococcus neoformans serotype A was fractionated into two chemically and serologically distinct heteroglycans by differential precipitation with cetyltrimethylammonium bromide (CTAB). The major, viscous, acidic glucuronoxylomannan (GXM, 88% w/w) was precipitated with CTAB, while a previously undetected galactoxylomannan (GalXM, 12% w/w) remained in solution. GalXM is characterized by ( i ) molar ratios of galactose:mannose: xylose:glucuronic acid of 1.9:1.8:1.0.2 and 2% of O -acetyl; ( ii ) a molecular weight of 275,000 ± 25,000, estimated by gel-permeation chromatography; ( iii ) extensive degradation by NaIO 4 ; ( iv ) precipitation in gel by a lectin, concanavalin A, indicating nonreducing mannosyl termini; and ( v ) a distinct, immunoprecipitin arc in counterimmunoelectrophoresis. GalXM was further purified by gel-permeation or ion-exchange chromatography.

Sequence Analysis of the Internal Transcribed Spacer 2 (ITS2) from Yeast Species Within the Genus Candida

Abstract. Nucleotide sequences of the internal transcribed spacer 2 (ITS2) regions were determined for 13 species within the genus Candida, representing a collection of those species pathogenic for humans. No two species had identical sequences and the sizes of ITS2 varied fourfold, representing an apparent continuous gradient of nucleotides. When present, sequence homologies were observed in the 5′ end of ITS2, and many species exhibited more limited homologies within three known conserved domains found in other yeasts. Cluster analysis of primary sequence revealed a concordance with a known taxonomic subfamily and suggests that certain species within the genus form a similar grouping. A majority of species exhibited similar presumptive RNA secondary structures, consistent with the hypothesis that these spacer regions are essential for correct processing of the 5.8S and 28S subunits.

An electrophoretic karyotype for Candida albicans reveals large chromosomes in multiples

SummaryUsing field-inversion gel electrophoresis we defined an electrophoretic karyotype for the yeast, Candida albicans. The karyotype is distinct from other species of Candida and is species specific. A total of five distinct chromosomal mobility groups were observed, at least four of which are composed of a minimum of two fragments each. From the apparent sizes of these fragments relative to the large chromosomes of the morphologically related yeast Saccharomyces cerevisiae, together with the known genome size of this organism, we conclude that the karyotype is the result of the migration of intact chromosomes.

Analytical isoelectric focusing of secreted dermatophyte proteins applied to taxonomic differentiation of Microporum and Trichophyton species (preliminary studies)

Culture filtrates were prepared from dermatophytes under standard conditions and adapted for analytical isoelectric focusing in thin layer polyacrylamide gels over the pH range 3.5-9.5. Dermatophytes grown in trypticase soy broth secreted a large number of proteins displaying a wide range of isoelectric points (pIs). Trichophyton megninii extracts contained a triplet of proteins focusing in the pH 8.0-8.5 range that were absent in taxonomically related T. kuryangei isolates. Single ascospore isolates and standard tester strains of Nannizzia otae (+) mating type were differentiated from the (-) mating type by proteins focusing at pH 6.5 and 8.4. These were markedly reduced in the (+) type. The isofocused pattern of Microsporum canis conformed closely to the (-) mating type of N. otae. The protein patterns of T. megninii and T. kuryangei were distinct from those obtained with M. canis and M. equinum because of an intense-staining broad protein band, pI 7.2, and three periodic acid-Schiff-positive glycoproteins focusing in the acidic range which were absent in the Microsporum species. A characteristic protein or doublet (pI 8.7) was present in the Microsporum species and absent in the Trichophyton species. Analytical isoelectric focusing is a potentially useful method to distinguish inter- and intra-species differences in the pattern of secreted dermatophyte proteins present in culture filtrates and in trichophytins. The information derived may be useful in the classification of species.

Antigenemia in rabbits infected with Candida albicans serotype B: detection by enzyme immunoassay and preliminary characterization of the antigen

Peroxidase-labeled IgG against Candida albicans serotype A cell walls failed to detect antigenemia in rabbits infected with C. albicans type B. Therefore type B antisera were produced in rabbits immunized with either type B cell walls; whole blastoconidia heated and killed at 60 degrees C (HK); a sublethal intravenous dosage of 10(6) colony forming units (c.f.u.); or a normally lethal dose of 10(7) c.f.u. injected into subcutaneously implanted plastic chambers. The four antisera were conjugated to peroxidase for a double antibody sandwich enzyme immunoassay. Sixteen rabbits, immunosuppressed with cortisone, received 10(7) type B blastoconidia intravenously. On day 4 their kidneys contained between 3.4 X 10(8) and 1.4 X 10(11) c.f.u. Antigenemia was detected only with the conjugated anti-HK-IgG. A mean concentration of 246 ng ml-1 of circulating antigen was detected only when serum-antigen complexes were dissociated by boiling in EDTA. The properties of the circulating antigen were as follows: relative molecular weight between 50 000 and 100 000 da; stable to boiling and proteinases, labile to sodium periodate oxidation, and bound by concanavalin A. These properties are consistent with a polysaccharide or glycoprotein, probably the mannoprotein of the cell wall. Antigenic specificity was probed further using IgG produced against heat-killed C. albicans serotype A. Antigenemia resulting from infection with serotype B was detected in 5 of 5 rabbits tested with the anti-HK-serotype B IgG but only with 3 of 5 tested with the anti-HK-serotype A IgG. In addition, the concentrations detected with serotype B reagents were 31 times higher.

Effect of periodate oxidation on the detection of antibodies against the M-antigen of histoplasmin by enzyme immunoassay (EIA) inhibition

An enzyme immunoassay (EIA) inhibition method was devised that detects antibodies against the M-factor, a major protein antigen in histoplasmin. Purified M-factor still contains a large amount of a carbohydrate antigen impurity, but this can be inactivated by periodate oxidation. The resulting product improves the performance of the EIA to detect anti-M by (a) giving a more specific measurement, (b) increasing the percent inhibition in sera that contain antibodies to M-factor, and (c) increasing the precision in measuring negative reactions. An inhibition of 20% or greater is recommended for recording a result as positive.

Immunoelectronmicroscopic characterization of monoclonal antibodies (MAbs) against Cryptococcus neoformans

Three monoclonal antibodies (MAbs) (BA4, BD1, CD6) reacted with Cryptococcus neoformans capsular glucuronoxylomannan (GXM) polysaccharide showing distinctive patterns against four serotypes as revealed by enzyme immunoassay (EIA), dot EIA, and immunofluorescence. Immunoelectron microscopy (IEM) was used to characterize binding sites for the MAbs on the C. neoformans capsule. All three MAbs bound to the capsule of serotype A strains 9104 and 9759. Differences in the intensity of binding to the two serotype A strains could not be explained by capsule diameter. The MAb BA-4 IgM bound well to 9759 (large capsule) and poorly to 9104 (small capsule), whereas MAb BD-1 (IgG-1) bound well to strain 9104 and poorly to strain 9759. Spurrs embedment inactivated the BA-4-binding epitopes in the C. neoformans 9759 capsule, but did not inactivate the ones that bound to BD-1. The epitopes recognized by BA-4 were different than the BD-1-binding determinants. The MAb CD-6 bound to a cytoplasmic precursor of capsular GXM. CD-6 (IgG) stained the capsule, cell wall, and cytoplasm of both C. neoformans tester strains. Competitive binding experiments were conducted. Single immunogold labelling showed that BD-1 inhibited the binding of BA-4, but not vice versa. The interaction between CD-6 and BA-4 resulted in a reciprocal inhibition. Double-labelling experiments showed reciprocal inhibition between BA-4 and each of the IgG MAbs. These MAbs are directed against capsular polysaccharide or its intracellular precursor. None of the MAbs stained C. neoformans cap 67, an acapsular mutant that does not contain GXM.

Ultrastructure of acapsular mutant Cryptococcus neoformans cap 67 and monosaccharide composition of cell extracts

Acapsular mutant Cryptococcus neoformans cap 67 was grown in Pines citrate broth medium for 3 days and the cells then transferred to a nitrogen-free medium for 6 days. The cells were subjected to a four stage extraction with buffered Triton-X1OO, cold dilute alkaline borohydride, hot dilute acetic acid, and a second alkaline extraction. Galactoxylomannan antigens were recovered from the culture supernates of both 3 day old and 9 day-old yeast cells. The alkaline extracts contained water-soluble galactoxylomannan and a water-insoluble glucan. Dilute acid treatment released a minor amount of carbohydrate from the cells. The second alkaline extraction yielded increased amounts of glucan and galactoxylomannan from the 9 day-old cells. Soluble non-dialyzable cell extracts were antigenically identical in immunodiffusion with the culture supernate antigens. After the extraction sequence, all of the galactose, xylose, and mannose were removed from the cells. The walls retained their shape after extraction but their layers were loosened. Cells resuspended in nitrogen-free medium for six days developed thickened walls with alternating electron-dense and electronlucent layers. The major constituent of the thickened 9 day-old cell walls was glucose, only 5% glucosamine was detected.

Immunochemical Analysis of Histoplasmin Proteins and Polysaccharide

Histoplasmin is the supernatant fluid obtained after static cultivation of the mycelial form of Histoplasma capsulatum for 3 to 6 months at ambient temperature (22 to 25°C) on asparagine-glucose-glycerol-salts medium (Smith et al, 1948). Three types of histoplasmin exist — skin test reagents from the mycelial and yeast forms and serological histoplasmin. The method of preparing skin test histoplasmin was established long ago (Shaw et al, 1950) and single lots were used widely for many years. The skin test antigen was standardized by its ability to elicit a reproducible cutaneous hypersensitivity response in sensitized humans. The antigens in histoplasmin responsible for the skin test have been inferred but no direct structure-activity evidence exists, because monomolecular antigens have not been available. Recently, histoplasmin from yeast form cultures has been approved for human use as a skin test antigen (Scalarone et al, 1985). The bulk of accumulated information concerns mycelial histoplasmin and there are far fewer data about the characteristics of the yeast form product. For that reason, and because of space limitation, the following discussion applies primarily to mycelial histoplasmin.