Immunochemical study on the mannans of Candida albicans NIH A-207, NIH B-792, and J-1012 strains prepared by fractional precipitation with cetyltrimethylammonium bromide

The mannans of Candida albicans NIH A-207 (A strain, serotype A), C. albicans NIH B-792 (B strain, serotype B), and C. albicans J-1012 (J strain, serotype C) prepared by fractional precipitation with cetyltrimethylammonium bromide (Cetavlon) were investigated for their immunochemical properties. Upon treatment with 10 mM HCl at 100 degrees C for 60 min, the mannans of A and B strains each released a mixture of manno-oligosaccharides ranging from hexaose to mannose together with (for each one) an acid-modified mannan, while J-strain mannan released lower oligosaccharides, tetraose to mannose. The acid-modified mannan of B strain did not show antibody-precipitating activity against homologous antiserum, whereas acid-modified A- and J-strain mannans retained most of this activity. The acid-released oligosaccharides were assumed to consist of beta-1,2-linked D-mannopyranosyl residues from the results of specific rotation and proton magnetic resonance studies.     

Secretion of inducible proteinase by pathogenic Candida species

The ability of three isolates each of seven pathogenic Candida species to grow in a liquid medium containing bovine serum albumin (BSA) as a nitrogen source was determined. All three strains of C. albicans, two strains of C. guilliermondii and one strain of C. tropicalis grew well. At any time proteinase activity was detected in the culture filtrates of only the most virulent species--C. albicans, C. tropicalis and C. parapsilosis and this observation was related to complete hydrolysis of BSA. Serologically, cross reactions were demonstrated between anti-proteinase antiserum and C. albicans and C. tropicalis culture filtrates. These results further emphasise the role of the inducible proteinase of Candida in the pathogenesis of candidosis.     

几种念珠菌DNA总含量的流式细胞分析

应用流式细胞术(FCM)对处于稳定生长阶段的念珠菌属(Candida)的7种8株念珠菌进行了DNA总含量的流式细胞(FCM)分析。这8株念珠菌是:白念珠菌(C.albicans)2株,热带念珠菌(C.tropicalis),克柔念珠菌(C.krusei),近平滑念珠菌(C.parapsiolosis),乳酒念珠菌(C.kefyr),白念珠菌星形变种(C.stellatoidea),即血清B型白念珠菌,季也蒙念珠菌(C.guilliermondii)各一株。应用EB一步插入法染色,用鸡红细胞(CRBC)作为内参标准进行DNA总含量测定。分析结果表明:稳定生长阶段的组方图上,大多数念珠菌细胞处于DNA合成周期的G_0/G_1期;DNA总含量有明显的种间和种内差异。     

Serotyping of Candida albicans isolated from clinical specimens

A specific antiserum to Candida albicans serotype A was prepared absorbing a total antiserum with Candida albicans serotype B cells. This specific antiserum was used for serotyping C. albicans strains obtained from patients in different hospitals of Havana City, Cuba. Two hundred strains (95.2%) were serotype A, the remaining 10 (4.8%) were serotype B. Results were also correlated with strains isolated from the specimen origin, sex and race of the patient. The usefulness of this specific antiserum to determine C. albicans serotypes and its therapeutic value are pointed out.     

Antigenicity of Candida tropicalis strain cells cultured at 27 and 37 degrees C

All cells of four Candida tropicalis strains IFO 0199 (Ct-0199), IFO 0587 (Ct-0587), IFO 1400 (Ct-1400), and IFO 1647 (Ct-1647), obtained by cultivation at 27 and 37 degrees C for 48 h in yeast extract-added Sabouraud liquid medium, showed the shapes of typical budding yeast and the same agglutination patterns against factor sera 1, 4, 5 and 6 in the commercially available kit 'Candida Check'. The cells of the C. tropicalis IFO 0589 strain display the same properties at 27 degrees C but formed hyphae at 37 degrees C. The cell wall mannan (Ct-0589-37-M) obtained from the strain cells cultured at 37 degrees C had lost most of its reactivity against factor sera 4, 5 and 6 in an enzyme-linked immunosorbent assay, in contrast to the mannan (Ct-0589-27-M) at 27 degrees C. The 1H-nuclear magnetic resonance patterns of the mannans obtained from the cells of the four C. tropicalis strains IFO 0199, IFO 0587, IFO 1400, and IFO 1647, obtained by cultivation at 37 degrees C, did not change compared to those at 27 degrees C. By contrast, the Ct-0589-37-M had significantly lost the beta-1,2-linked mannopyranose units, corresponding to the serum factors 5 and 6. These results show that the IFO 0589 strain is an unusual strain among the general C. tropicalis strains studied.     

Relationship between phosphate content and serological activities of the mannans of Candida albicans strains NIH A-207, NIH B-792, and J-1012.

The mannans from Candida albicans strains NIH A-207 (serotype A), NIH B-792 (serotype B), and J-1012 (serotype C) were fractionated on a column of diethylaminoethyl-Sephadex into five subfractions containing different amounts of phosphate. Antibody-precipitating activities of the mannan subfractions of strains NIH A-207 and NIH B-792 were proportional to their phosphate content, while those of strain J-1012 did not show regularly proportional precipitin activity. A similar tendency was also observed in the cross-reaction between the mannan su,fractions of strains NIH A-207 and J-1012 and their heterologous antisera. The mannans of strain NIH B-792 showed lower cross-reactivities against antisera of strains NIH A-207 and NIH B-792, i.e., only two subfractions containing larger amounts of phosphate were able to react with these antisera.     

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 alpha-D-Manp-(1-2)-alpha-D-Manp-(1-3)-alpha-D-Manp-(1- 2)-alpha-D-Manp-(1-2)- alpha-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.     

Application of Agglutinins for the Rapid and Accurate Identification of Medically Important Candida Species

Agglutinins have been prepared against the medically important Candida species. Crude antisera to the various species demonstrated intense cross-reactions with heterologous yeastlike fungi as well as with many true yeasts. However, carefully monitored adsorptions of selected antisera allowed the production of six factor sera that proved useful in a slide agglutination test. These six sera permitted the rapid and specific identification of C. guilliermondii, C. krusei, C. parapsilosis, and C. pseudotropicalis. They also allowed the delineation of two groups: (i) C. albicans (type A)-C. tropicalis and (ii) C. albicans (type B)-C. stellatoidea. C. albicans type A could be readily distinguished from C. tropicalis by its ability to form germ tubes in serum. C. stellatoidea could be distinguished from C. albicans type B by its predominantly filamentous growth on a nutritionally deficient medium. The medically important Candida species could be identified within 24 hr by the combined use of serological and morphological procedures.     

Purification and characterization of secretory proteinase of Candida albicans.

Proteolytic activity of medically important yeasts was tested in both YCB-BSA agar and medium. All of 134 strains of Candida albicans, 13 of 18 strains of Candida tropicalis and 11 of 18 strains of Candida parapsilosis had this activity, while none of 52 Candida glabrata strains or of 11 Cryptococcus neoformans strains tested had proteolytic activity. Strains of C. albicans fell into five groups based on the level and time-course of in vitro proteinase productivity. Five strains randomly selected from each group were tested for pathogenicity in mice. The strain possessing the strongest pathogenicity was used to purify proteinase. The molecular weight of the proteinase was approximately 44,000 daltons and its isoelectric point was pH 4.2. Optimal pH of the proteinase was 3.2 and the enzyme was stable below pH 7.0 and lost its activity above pH 8.0 at 37 C in a 60-min incubation. The 23 amino acid sequence of the proteinase N-terminus was determined.     

Immunologic significance of diverse specificity of monoclonal antibodies against mannans of Candida albicans

Two agglutinating IgM mAb against mannan Ag of Candida albicans strains were investigated for their specificity. The agglutinating patterns of both mAb with a panel of stationary phase of yeast cells of standard strains did not match those of any known polyclonal antibody (PAb) factors. The reactive patterns of both mAb for a given panel of 202 isolates of seven Candida species and the mode of competitive binding between mAb and a PAb factor, as determined by a combination of direct and indirect immunofluorescence staining, demonstrated that the two mAb were a part of PAb factor 4 (a prescribed reactive pattern of adsorbed PAb) but were different from each other in their specificity. The thereby designated mAb 4b and 4c were tested under PAb factor 4-positive Candida strains and further division into two to three serotypes of each species were made. 1H-Nuclear magnetic resonance spectra (500 MHz) of purified neutral mannans from strains of each serotype showed that although the 1H-nuclear magnetic resonance spectra of mannans from two subtypes of C. albicans serotype A and from each of two serotypes of Candida guilliermondii and Candida glabrata were identical or similar, the two mAb were still able to distinguish their fine determinant structures. Our findings suggest that mAb with entirely identical specificity cannot be produced against even the same determinant groups of mannan. In addition, the fact that microheterogeneity may occur without limit in the mannans of the strains suggests that antibodies with unlimited diverse specificities are produced directed against these antigenic varieties as well.     

口腔黏膜真菌鉴定方法的比较

比较常见用于黏膜真菌菌种鉴别的多种方法,探寻最佳的鉴别方法。采集230例普通人群口腔黏膜样本,分别用玉米吐温-80培养观察厚膜孢子法、糖发酵生化反应法、CHROMagar假丝酵母菌显色培养基法、ITS基因的PCR-RFLP(聚合酶链反应-限制性片段长度多态性)法、ITS测序菌种鉴定法,鉴别真菌各菌株。结果显示:有56例菌株至少通过1种方法检出真菌;玉米吐温-80分离培养假丝酵母菌37株;50例菌株ITS基因测序共鉴定出8个菌种,白假丝酵母菌(C.albicans)29株,近平滑假丝酵母菌(C.parapsilosis)10株,热带假丝酵母菌(C.tropicalis)5株,Candida metapsilosis 1株,Lodderomyces elongisporus 1株,克柔假丝酵母菌(Candida krusei)1株,乙醇假丝酵母菌(C.ethanolica)1株,季也蒙毕赤酵母菌(Pichia guilliermondii)2株;CHROMagar假丝酵母菌显色培养基法鉴定出3种菌株,分别是白假丝酵母菌、热带假丝酵母菌、近平滑假丝酵母菌;PCR-RFLP法检出5种菌株,分别是白假丝酵母菌、热带假丝酵母菌、近平滑假丝酵母菌、季也蒙毕赤酵母菌、克柔假丝酵母菌,与基因的测序鉴定一致率为91%;糖发酵生化反应法阳性标本占被检出真菌例数的46.4%(26/56)。结果表明:ITS基因的测序法可以准确鉴定真菌各个菌种;PCR-RFLP法能鉴定常见的菌种,但操作繁琐;CHROMagar假丝酵母菌显色培养基法能快速准确鉴别3种常见假丝酵母菌菌种;玉米吐温-80可以准确培养鉴别白假丝酵母菌;糖发酵生化反应法,缺乏足够的敏感度和特异性,难以准确鉴别各个菌种。     

Immunochemical characterization of Candida albicans cell wall antigens: specific determinant of Candida albicans serotype A mannan

We investigated the chemical structure of the specific determinant in the mannan of Candida albicans M-1012 (serotype A) strain. Acetolysis of the mannan, obtained by alkali extraction and purified as the copper complex, gave mannose and six oligosaccharides (from di- to hexasaccharide) and a small amount of a heptasaccharide. We examined the inhibition by these oligosaccharides up to hexaose of the precipitin reaction between anti-factor 6 serum specific for serotype A and homologous mannan, and found that the mannohexaose was the most effective inhibitor. These, and results obtained by proton magnetic resonance (PMR) spectroscopy, methylation analysis, and other structural studies, suggest that the main component of this hexaose consists of one terminal alpha (1-3) linkage in addition to four alpha (1-2) linkages, and that this alpha (1-3)-containing mannohexaose may be responsible for the specificity of antigenic factor 6. Further results obtained by analyses of polarimetry, PMR spectroscopy, and chromium trioxide oxidation-methylation of C. albicans M-1012 mannan has a beta-linkage in addition to alpha-linkages, and that the mode of the beta-linkage is mainly (1-6) linkage. Further evidence obtained by Smith degradation-methylation analysis and by quantitative precipitin reactions of intact and acid-degraded mannan suggests that the antigenic determinant of antigenic factor 6 may be bound, via the beta (1-6) linkage, to C-6 of mannose residues involved in oligosaccharide side chains of serotype A mannan.     

Surface hydrophobicity changes of two Candida albicans serotype B mnn4delta mutants

Cell surface hydrophobicity (CSH) of Candida species enhances virulence by promoting adhesion to host tissues. Biochemical analysis of yeast cell walls has demonstrated that the most significant differences between hydrophobic and hydrophilic yeasts are found in the acid-labile fraction of Candida albicans phosphomannoprotein, suggesting that this fraction is important in the regulation of the CSH phenotype. The acid-labile fraction of C. albicans is unique among fungi, in that it is composed of an extended polymer of beta-1,2-mannose linked to the acid-stable region of the N-glycan by a phosphodiester bond. C. albicans serotype A and B strains both contain a beta-1,2-mannose acid-labile moiety, but only serotype A strains contain additional beta-1,2-mannose in the acid-stable region. A knockout of the C. albicans homolog of the Saccharomyces cerevisiae MNN4 gene was generated in two serotype B C. albicans patient isolates by using homologous gene replacement techniques, with the anticipation that they would be deficient in the acid-labile fraction and, therefore, demonstrate perturbed CSH. The resulting mnn4delta-deficient derivative has no detectable phosphate-linked beta-1,2-mannose in its cell wall, and hydrophobicity is increased significantly under conditions that promote the hydrophilic phenotype. The mnn4delta mutant also demonstrates an unanticipated perturbation in the acid-stable mannan fraction. The present study reports the first genetic knockout constructed in a serotype B C. albicans strain and represents an important step for dissecting the regulation of CSH.     

Antigenic cell wall mannoproteins in Candida albicans isolates and in other Candida species.

Polyclonal antibodies (pAbs) and monoclonal antibodies (mAbs), raised against mannoprotein components from Candida albicans ATCC 26555 (serotype A) blastoconidia and mycelial cell walls, were used to investigate antigenic similarities among wall mannoproteins from other C. albicans serotype A and B strains, and from C. tropicalis and C. guilliermondii. Radioactively labelled walls isolated from cells grown at either 28 degrees C or 37 degrees C were digested with a beta-glucanase complex (Zymolyase 20T) to release cell-wall-bound mannoproteins. Numerous molecular species with different electrophoretic mobilities were released from the various isolates. Differences appeared to be related to both the organism and the growth temperature. Among the major protein components solubilized were mannoproteins larger than 100 kDa (high molecular mass mannoproteins), heterogeneous in size in most cases. Antigenic homology was detected among the cell wall high molecular mass mannoproteins of the two C. albicans serotype A isolates, whereas significant qualitative and quantitative differences were detected between serotype A and serotype B cell-wall-bound antigenic profiles. Moreover, C. tropicalis and C. guilliermondii wall antigenic determinants were not recognized by the preparations of pAbs and mAbs raised against C. albicans walls. A mannoprotein with a molecular mass of 33-34 kDa was present in the enzymic wall digests of all the organisms studied. When probed with pAbs raised against the protein moiety of the 33 kDa cell wall mannoprotein of Saccharomyces cerevisiae, antigenic cross-reactivity was observed in all cases except C. tropicalis. There appear to be significant antigenic differences between the mannoproteins of different isolates of C. albicans, and between those of C. albicans and other Candida species.     

Existence of novel beta-1,2 linkage-containing side chain in the mannan of Candida lusitaniae, antigenically related to Candida albicans serotype A.

The antigenicity of Candida lusitaniae cells was found to be the same as that of Candida albicans serotype A cells, i.e. both cell wall mannans react with factors 1, 4, 5, and 6 sera of Candida Check. However, the structure of the mannan of C. lusitaniae was significantly different from that of C. albicans serotype A, and we found novel beta-1,2 linkages among the side-chain oligosaccharides, Manbeta1-->2Manbeta1--> 2Manalpha1-->2Manalpha1-->2Man (LM5), and Manbeta1-->2Man-beta1-->2Manbeta1-->2Manalpha1-->2Manalpha1-->2Man (LM6). The assignment of these oligosaccharides suggests that the mannoheptaose containing three beta-1,2 linkages obtained from the mannan of C. albicans in a preceding study consisted of isomers. The molar ratio of the side chains of C. lusitaniae mannan was determined from the complete assignment of its H-1 and H-2 signals and these signal dimensions. More than 80% of the oligomannosyl side chains contained beta-1,2-linked mannose units; no alpha-1,3 linkages or alpha-1,6-linked branching points were found in the side chains. An enzyme-linked immunosorbent inhibition assay using oligosaccharides indicated that LM5 behaves as factor 6, which is the serotype A-specific epitope of C. albicans. Unexpectedly, however, LM6 did not act as factor 6.     

Distribution of antigenic oligomannosyl side chains in the cell wall mannans of several strains of<Emphasis Type="Italic"> Candida tropicalis</Emphasis>

In order to clarify the distribution of antigenic oligomannosyl side chains in the cell wall mannans of the pathogenic yeast Candida tropicalis, the chemical structure of mannans isolated from four C. tropicalis strains was investigated using nuclear magnetic resonance, two-dimensional homonuclear Hartmann-Hahn (2D-HOHAHA) spectroscopy. Two-dimensional maps of the 2D-HOHAHA clearly showed the distribution of oligomannosyl side chains in the mannans. The linear side chain Manalpha1-3Manalpha1-(2Manalpha1-)(n)2Man [n> or =2] is present in the mannans from C. tropicalis IFO 0589 and IFO 1400, but not in the mannans from IFO 0199 and IFO 1647. The mannan of IFO 0589 is the only mannan with the branched side chains, Manalpha1-3[Manalpha1-6]Manalpha1-(2Manalpha1-)(n)2Man and Manalpha1-2Manalpha1-3[Manalpha1-6]Manalpha1-(2Manalpha1-)(n)2Man [n> or =2]. However, this mannan lacked the phosphate group and the beta-1,2-linked oligomannosyl side chain which are features of this group. The mannans of the C. tropicalis strains IFO 0589 and IFO 1400 possessed the side chains containing an alpha-1,3-linked mannose residue previously observed in Candida albicans.     

Diabetes mellitus and candidiases

Patients in various clinical states of diabetes mellitus (according to the recommendation of the American Diabetes Association) as a primary diagnosis were examined for fungal infections by Candida species. Candida spp. were detected in urine, in the material taken from the mouth cavity, nails, skin lesions, ears and eyes, by cultivation on the Sabouraud agar, CHROMagar Candida, and by saccharide assimilation. In the group of diabetics with symptoms of oral candidiasis and denture stomatitis C. albicans was identified in 8 cases, C. tropicalis in 3, C. parapsilosis in 2; 1 strain of C. guilliermondii was also isolated. In patients with urinary tract infections the presence of C. albicans was shown in 12 cases; C. parapsilosis was detected in 6 cases and two strains of each C. tropicalis and C. krusei were also isolated. In patients with leg ulcers C. albicans (25 cases), C. parapsilosis (5), C. tropicalis (3) and one strain of each C. krusei and C. robusta were isolated. Otomycosis was associated with one strain of C. albicans, C. parapsilosis, C. tropicalis and C. guilliermondii. C. albicans was most frequently associated with onychomycosis, paronychia and endophthalmitis; C. parapsilosis was the second most rated yeast.     

血培养分离出212株念珠菌的菌种分布及耐药性分析

目的分析血培养分离出的念珠菌的菌种分布及耐药分析,为临床念珠菌血症的诊治提供理论依据。方法回顾性分析2017年1月—2020年12月期间,本院血培养分离出的念珠菌的菌种分布、药敏试验结果及患者血培养分离出念珠菌前后96 h内的G试验结果。结果血培养中分离出念珠菌314例,阳性率2.1%,其中非重复分离株212例。检出率最高的是近平滑念珠菌(72株,34.0%),其次是白念珠菌(55株,25.9%)和光滑念珠菌(28株,13.2%)。念珠菌检出率最高的科室为ICU(62株,29.2%),其次是新生儿科(39株,18.4%)和血液科(20株,9.4%)。检出的212株念珠菌除一株近平滑念珠菌为两性霉素B的非野生株,其余均为两性霉素B的野生株。白念珠菌对唑类药物的敏感率超过90%。但光滑念珠菌和热带念珠菌对唑类药物的敏感性较低。血培养分离出念珠菌的前后96 h内,G试验的阳性率为73.7%。结论本院念珠菌检出率前3位分别是近平滑念珠菌、白念珠菌和光滑念珠菌。光滑念珠菌、热带念珠菌对唑类药物敏感性比较低,在经验用药时需要合理选择抗真菌药物。G试验对念珠菌血症有较高的价值,需要对结果进行动态监测。     

Characterization of Candida antigens by crossed-immunoaffinoelectrophoresis

The antigens of three Candida albicans strains (3153 A, 3156 B and CBS 1905) and one C. tropicalis strain were studied by means of crossed-immunoaffinoelectrophoresis with the corresponding polyvalent antisera. Most antigens (from 63.8% to 77.7% depending on the strain) were bound to concanavalin A-sepharose and about 20% to blue cibacron-sepharose for all the strains tested. Free concanavalin A, wheat germ lectin-sepharose and Helix pomatia lectin-sepharose revealed differences between C. albicans 3153 A and C. albicans CBS 1905 on the one hand and C. albicans 3156 B and C. tropicalis on the other, since affinity percentages were from 4.2 to 10.2 and from 14.2 to 20.0 respectively. Among 10 previously described species-specific antigens of C. albicans, 4 were never bound and 5 were bound to concanavalin A-sepharose which was considered an unsuitable agent for antigen purification since it retained 77% of C. albicans antigens. One important species-specific antigen was bound to blue cibacron sepharose and the corresponding purification could be undertaken. Similar results were found for 12 species-specific antigens of C. tropicalis. Blue cibacron-sepharose as well as wheat germ lectin or Helix pomatia lectin-sepharose were found suitable agents for purification of some of them.     

Candida albicans serotype A strains grow in yeast extract-added Sabouraud liquid medium at pH 2.0, elaborating mannans without beta-1,2 linkage and phosphate group

Cultivation of three Candida albicans strains, NIH A-207, J-1012, and NIH B-792, abbreviated as A-, J-, and B-strains, respectively, in yeast extract-enrich Sabouraud liquid medium at pH 2.0 provided the following findings, i.e., the two former strains belonging to serotype A were able to grow in this medium in almost the same rates as those in the same medium of pH 5.9, while B-strain cells did not proliferate under the former condition. The cells of A- and J-strains cultivated at pH 2.0 did not undergo agglutination with the factor serum 6 in a commercially available factor serum kit, Candida Check, corresponding to C. albicans serotype A-specific epitope. It was also revealed by 1H-13C correlation spectra of the mannans isolated from the cells of A- and J-strains contained neither phosphate group nor beta-1,2-linked mannopyranose unit, although these mannans retained non-reducing terminal alpha-1,3 linked mannopyranose units, providing a substantiating evidence that the serotype A-specific epitope contains a non-reducing terminal beta-1,2-linked mannopyranose unit.