Yoshimura Fukazawa

Serologic aspects on yeast classification

The classification of living beings is achieved by delimitation based upon their common properties. For this purpose, characteristics of living organisms should refer to those which have been mentioned or published. During such grouping processes, the morphology and growth, as well as biochemical properties reflecting the intermediate metabolism, are first taken into consideration. While there is no contradiction with respect to the classification of higher plants and animals, the criteria mentioned above are not always sufficient to classify microorganisms which are very minute and simple. Therefore, antigenic properties have been considered for the classification of bacteria, mainly pathogenic, but not as yet for yeasts. Furthermore, the serological characteristics of yeasts presented by several investigators (1, 28) have not been routinely adopted as appropriate tools for the classification as have not the GC contents of the DNA of yeasts (14), the coenzyme Q system (35), and the proton magnetic resonance (PMR) spectra of cell wall polysaccharides (7), although the chemical composition of the yeast cell or cell wall is considered to be of prime importance in indicating the relationships among various yeasts. This paper deals with the experimental results of antigen analyses of yeasts, rapid identification recently performed and serological characteristics in the classification of yeasts.

Significance of serological studies on yeasts.

Several methods designed for the classification of yeasts have been published during the century, and among them the most universal method is that of LODDER & KREGER-VAN •IJ (1952), which is really the most distinctive and elaborate work in the field. LODDER & KREGER-VAN RIJ divided the yeasts into families, subfamilies, tribes and genera, depending upon their ability to form ascospores and their morphology. The genera were then subdivided into species mainly based on biochemical characteristics. Since the characteristics are, however, not necessarily as stable, various difficulties are sometimes encountered in the course of classifying and identifying yeasts. This is especially true when the classification is based on the characteristics alone, although they are regarded as fairly stable in outline. On the other hand, living beings as studied in zoology and botany are skillfully assembled into groups principally based upon distinct morphological differences; there is hardly any discrepancy. However, since microorganisms are minute and too simple to be distinguished on the basis of morphological properties alone, their antigenic characteristics are used as a criterion in addition to morphological and biochemical properties. Apart from higher Eumycetes, which has evolved into complicated forms, only the morphological characteristics were relied upon even in the classification of yeasts, which have minute forms and which closely resemble bacteria. Since the data on morphological properties, however, are not too adequate for use in the classification of yeasts, the ability to assimilate potassium nitrate, to ferment glucose strongly and to form a starch-like compound are used as the criteria. YUKAWA et al. (1928, 1929a, 19295), BENItAM (1931) and several other investigators have indicated that serological characteristics can serve in differentiating various species of yeasts. These informations, however, have been taken only as supplementary means for

Chemical Characterization of Capsular Polysaccharide from Cryptococcus neoformans Serotype A-D

During a study of serotyping of Cryptococcus neoformans, we found that the type strain of C. neoformans (CBS 132) was serotype A‐D. This strain agglutinated with both factor 7 serum (specific for serotype A) and factor 8 serum (specific for serotype D) in our serotyping system. Therefore, we investigated the chemical structure of the antigenic capsular polysaccharide of this strain. The soluble capsular polysaccharide was obtained from the culture supernatant fluid by precipitation with ethanol. Column chromatography of the polysaccharide on DEAE‐cellulose yielded three fractions (F‐1 to F‐3). The major antigenic activity was found in the F‐3 fraction. The results obtained by methylation analysis, controlled Smith degradation‐methylation analysis, partial acid hydrolysis, and other structural studies of F‐3 polysaccharide indicated that the polysaccharide contains mannose, xylose, and glucuronic acid at a ratio of 7:2:2, and has a backbone of α(1–3)‐linked D‐mannopyranoside residues with a single branch of β(1–2)‐xylose and glucuronic acid. The ratio of mannose residues with or without a branch in the F‐3 polysaccharide was 4:3 and its molecular weight calculated from the average of the degree of polymerization was 46,500 daltons. These results indicate that the chemical structure of the capsular polysaccharide of serotype A‐D is very similar to those from serotypes A and D, suggesting that small differences in the molar ratio and pattern of linkage of monosaccharides in the branch of the polysaccharides of the three serotypes may be responsible for their different specificities.

A method for the rapid identification of the genus Candida

Serological identification tests were carried out with many strains obtained from an otolaryngologic clinic, from oral cavities of dental patients and from other laboratories. The tests were performed through the slide agglutination method using monospecific or absorbed antisera based on the antigenic structures. The results of serological tests were compared with biological identifications. Since serological identifications alone were found to coincide well with the correlative identifications by biological and serological methods, it is concluded that the identification test using monospecific antisera, based on the antigenic structures of the genusCandida, is a useful and reliable method for a rapid identification of species.

Characterization of Pathogenic Constituents of Cryptococcus neoformans Strains

We examined seven strains, comprising five serotypes, of Cryptococcus neoformans to determine what constituents of the organisms are responsible for pathogenicity and virulence in BALB/c mice. C. neoformans strains were divided into three virulence classes by survival rates after intravenous inoculation of 1 × 105 or 1 × 107 viable cells, and virulence was found not to be correlated with serotype or capsular size. C. neoformans cells resisted phagocytosis in different degrees in the presence of normal serum. Sensitivity of the C. neoformans strains to singlet oxygen ranged from resistance to susceptibility. Histological examination revealed that a weakly encapsulated virulent strain induced inflammatory responses with granuloma formation in the liver, lung, and kidney in addition to formation of cystic foci in the brain. In contrast, although the heavily encapsulated virulent strain produced granulomatous lesions in the liver, this strain preferably produced mucinous cystic foci in the lung, kidney, and brain. Correlation between virulence, and biological, histopathological and physiological evidence suggests that C. neoformans strains are endowed with the implicated multiple pathogenic constituents in various degrees and proportions. The following are suggested as the most important pathogenic constituents: (i) a polysaccharide capsule responsible for resistance to phagocytosis and formation of cystic foci; (ii) a cell surface structure for responsible for resistance to intra‐ or extracellular killing and induction of the granulomatous lesion; (iii) a growth rate suitable for interacting with phagocytic elimination.

Antigenic Relationship between Candida parapsilosis and Candida albicans Serotype B

We examined the antigenic relationship between Candida parapsilosis and C. albicans serotype B with respect to antigenic factors 13 and 13b, specific for the former species and common to both species, respectively. Acetolysis of C. albicans serotype B cell‐wall mannan gave six oligosaccharides. Their chemical structure was determined by 1H‐nuclear magnetic resonance (NMR) spectroscopy, methylation analysis, and partial acid hydrolysis. The structure of the hexasaccharide derived from C. albicans serotype B mannan was α‐D‐Manp‐(1–2)‐α‐D‐Manp‐(1–3)‐α‐D‐Manp‐(1–2)‐α‐D‐Manp‐(1–2)‐α‐D‐Manp‐(1–2)‐D‐Man (M6) which is identical to that from C. parapsilosis mannan. Inhibition of two precipitin reaction systems (anti‐C. albicans serotype B serum and anti‐C. parapsilosis serum to the respective homologous mannan), by oligosaccharides from homologous and heterologous mannans indicated that M6 from either C. albicans serotype B or C. parapsilosis was the most effective inhibitor. Moreover inhibition of the agglutination reaction between factor serum containing anti‐factors 13 and 13b and C. albicans serotype B or C. parapsilosis cells by oligosaccharides from both mannans also indicated that the M6s were the most effective inhibitors. These results suggest that the M6s derived from the two species are identical in their chemical structure, although the structures of the whole mannans of the two species are not identical as demonstrated by gel diffusion precipitation patterns, and that M6s may be involved in the specificities of antigenic factors 13 and 13b. The amount of M6 is larger in C. parapsilosis cell‐wall mannan, suggesting that high repeating frequency of M6 fragment may induce the antibody specific for C. parapsilosis.

Comparison of Serological and Chemical Characteristics of Capsular Polysaccharides of Cryptococcus neoformans var. neoformans Serotype A and Cryptococcus albidus var. albidus

The antigenic formula and chemical structure of capsular polysaccharide (CPS) of Cryptococcus albidus var. albidus (C. albidus) were studied in relation to those of C. neoformans var. neoformans serotype A (C. neoformans A). The results of slide agglutination tests with factor sera and reciprocal adsorption experiments showed that antigenic formula of C. albidus was the same as that of C. neoformans A. The soluble CPSs from the two species were obtained from culture supernatants by precipitation with ethanol followed by purification by chromatography on DEAE‐cellulose column. The structural analyses of such CPSs from the two species showed that the antigenic CPS fractions consisted of a backbone of α(l‐3)‐linked D‐mannopyranosyl residues with a single branch of β(1–2)‐xylose or glucuronic acid, and mostly with O‐acetyl groups, in which side chains and O‐acetyl groups were responsible for antigenic specificity. It was found that there was a minor difference between the CPS of C. neoformans A and that of C. albidus; in the former, unsubstituted mannose residues existed in a low frequency, but in the latter none. Moreover, the 1H‐nuclear magnetic resonance spectra of partially hydrolyzed acidic fragments of the two CPSs indicated that two xylose side chains were present between glucuronic acid side chains. Taken together, it was suggested that these two species of C. neoformans A and C. albidus are closely related to each other in their CPSs.

Serological classification of the genus Torulopsis

Antigenic analyses of 4 species of Torulopsis were carried out by the slide agglutination method using monospecific and absorbed antisera homologous to antigens of the genus Candida. The antigenic structure of each species was confirmed by reciprocal or successive absorption and slide agglutination test. Torulopsis stellata var. cambresieri and T. colliculosa possess the thermostable antigens 1, 3, 4 and 24, but no thermolabile antigen. Their antigenic structures were identical to those of Saccharomyces rosei and S. fermentati, respectively, despite the biological differences among them. Torulopsis holmii has thermostable antigens 1, 3, 4, 5, 6, 10 and 26, but no thermolabile antigen. The antigenic structure of this species is similar to that of S. exiguus. Torulopsis glabrata contains the thermostable antigens 1, 3, 6, 10 and 34, and thermolabile antigen k. In a comparative study, the antigenic structures of many strains coincided with those of the standard when they were analyzed with monospecific antis...

Role of serum factors in the phagocytosis of weakly or heavily encapsulated Cryptococcus neoformans strains by guinea pig peripheral blood leukocytes.

We investigated the opsonic activity of the serum factors affecting phagocytosis of Cryptococcus neoformans in vitro to elucidate the role of humoral factors in the host defense mechanisms against cryptococcosis. Two strains of C. neoformans, one heavily and one weakly encapsulated, were used. Guinea pig peripheral blood leukocytes (PBLs) were used for phagocytosis. The viable weakly encapsulated cells were ingested effectively by PBLs, in the presence of guinea pig normal fresh serum, while the heavily encapsulated cells were not ingested. Neither immune serum, its IgG fraction alone, nor heated serum promoted the phagocytosis of either the weakly or heavily encapsulated strain. On the other hand, immune serum promoted adherence of PBLs to viable cells of the heavily encapsulated strain, forming rosettes in the presence of fresh serum. A substantial amount of C3b component was detected on yeast cells when weakly encapsulated cells were incubated with human fresh serum, or heavily encapsulated cells were incubated with rabbit immune serum together with human fresh serum. Serum chelation experiments also indicated that the factors involved in the alternative complement pathway are opsonins for the weakly encapsulated strain. These results suggest that the alternative pathway plays an important normal opsonic role for weakly encapsulated strains and that specific antibody plays an immune opsonic role for heavily encapsulated strains of C. neoformans via the classical pathway of complement activation.

Reassessment of antigenic determinant of Saccharomyces cerevisiae serotype Ia.

We examined the immunochemical structure of the antigenic determinant of S. cerevisiae serotype Ia. The specific factor serum for S. cerevisiae serotype Ia was obtained either from factor 18 serum by adsorption with heat‐killed cells of Candida glabrata, or from anti‐S. cerevisiae Ia (M 6001) serum by adsorption with heat‐killed cells of S. cerevisiae Ib (IFO 0751). We designated this adsorbed serum as factor 18a. Acetolysis of S. cerevisiae cell wall D‐mannan gave five oligosaccharides. Signals of 1H‐nuclear magnetic resonance spectra of mannooligosaccharides derived from S. cerevisiae mannan were assigned for their linkages by the aid of those of α‐1,3′‐linked mannooligosaccharides derived from glucuronoxylomannan of capsule of Cryptococcus neoformans serotype A‐D. Agglutination‐inhibition experiments revealed that the mannopentaose from S. cerevisiae mannan was the most effective inhibitor. Moreover, inhibitory activities of α‐1,3′‐linked mannotriose, mannotetraose, and mannopentaose which were derived from glucuronoxylomannan of C. neoformans were shown to be higher than those of mannotetraose with one terminal α‐(l‐3) linkage from homologous S. cerevisiae mannan. These results indicate that mannopentaose with terminal two α‐(l‐3) linkages is responsible for the specificity of S. cerevisiae Ia.