Candida Albicans Serotype Research Articles

Determination of <i>α</i>-1,3-Linked Mannose Residue in the Cell Wall Mannan of <i>Candida tropicalis</i> NBRC 1400 Strain

To investigate the chemical structure of cell wall mannan obtained from pathogenic yeast, Candida tropicalis NBRC 1400 (former antigenic standard strain, IFO 1400). As a result of two-dimensional NMR analysis, it was shown that the mannan of this strain is composed of α-1,6-, α-1,3-, α-1,2- and β-1,2-linked mannose residues. In this research, the mannan was subjected to three degradation procedures, acid-treatment, α-mannosidase, and acetolysis under two conditions in order to determine the chemical structure of the antigenic oligomannosyl side chains in this molecule. The 1H-nuclear magnetic resonance spectra of resultant oligosaccharides, pentaose and hexaose, demonstrated the existence of the oligomannosyl side chains corresponding to Manα1-3Manα1-2Manα1-2Manα1-2Man and Manα1-3Manα1-2Manα1-2Manα1-2Manα1-2Man, respectively, which have previously also been found in Candida albicans serotype A strain mannans. These findings indicate that C. tropicalis and C. albicans serotype A have no significant difference in the chemical structure of these cell wall mannans. Therefore, it can be interpreted that it is extremely difficult to distinguish both species by targeting the antigenic group in these mannans.

Mannoproteins from yeast and hyphal form of Candida albicans considerably differ in mannan and protein content

Significant differences in carbohydrate composition of mannoproteins obtained from yeast and hyphal cell walls of Candida albicans (serotypes A and B) were found. Yeast mannoproteins from both serotypes consisted up to 46% of mannan while the same parts from hyphal cells contained only about 14% of mannan. Another difference was in protein content, 47–53% for yeasts, 3–4.5% for hyphae, respectively. Moreover, HPLC profiles of yeast mannoproteins were more complex compared to those of hyphal form. Subsequently, mannans were prepared from yeast and hyphal mannoproteins using cetavlon fractionation. Mannans from both yeast serotypes contained higher amounts of mannose (91.4% serotype A; 92.8% serotype B) than mannans from hyphae (66.4% serotype A; 76.3% serotype B). Unlike mannans from serotype B, mannans from serotype A contained β-(1→2)-linked mannopyranosyl units in acid-stable moiety. Further, hyphal mannans were less branched than yeast mannans. The shift from yeast to hyphal form probably led to simplification of mannan structure.

Ultrasonic and free-radical degradation of mannan from Candida albicans

Mannan from pathogenic Candida albicans serotype A was degraded by means of ultrasound and/or OH generated in situ by Fenton reaction. The kinetics of degradation was monitored by HPLC analysis and the weight-average molecular weights (Mw) and index of polydispersity (PDI) were compared. A well-defined low-molecular-weight mannan (∼30kDa) with narrowed PDI of 1.8 was obtained after 120min of ultrasonication. Similar or even lower Mw (up to 16kDa) was achieved upon free-radical exposure depending on Fe2+ concentration used; however, this was accompanied by overall broadening of PDI and distinct changes in polymer structure as indicated by NMR analysis.

Comments on "Biofilms of Candida albicans serotypes A and B differ in their sensitivity to photodynamic therapy".

Dear Editor, In the April 2014 issue of the online-first article, Rossoni et al. [1] reported that Candida albicans serotype B is more sensitive to photodynamic treatment than serotype A. As we know, significant variation among particular strains within the same species in response to the photodynamic treatment exists [2]. The differences can reach several logs in survival of viable counts. We have previously shown that taking into consideration the genetic background of microorganisms, i.e., Staphylococcus aureus, within the same closely related strains, assigned as a single epidemic clone, we can identify strains that are highly resistant as well as highly sensitive to photodynamic inactivation (PDI) [3]. Thus, just having a sufficiently large database of strains, one can properly infer about the importance of a particular feature in response to PDI. Unfortunately, as far as I am concerned, study of Rossoni et al. failed to include other than single reference strains of C. albicans. On the basis of single isolates, one must not drew any relevant conclusions. Moreover, switching the sensitizer can totally reverse the observed response to photodynamic treatment [4]. Consequently, such a general conclusion proposed by the authors stating that C. albicans serotype B is more sensitive to photoinactivation is not supported by the evidence, as it requires studies concerning different sensitizing agents. However, the authors have not provided any details about the effectiveness of photoinactivation against both C. albicans serotypes based on other photosensitizers. Assessing the influence of microorganism’s features on susceptibility to photoinactivation is always a complex issue. Of course, studying one sensitizer and single isolates can give us some information, but the problems in study design should not be ignored, as statistical analysis requires more numerous groups to be compared and to draw relevant conclusions. Those limitations reduce the reliability of presented results.

Biofilms of Candida albicans serotypes A and B differ in their sensitivity to photodynamic therapy.

Candida albicans is classified into different serotypes according to cell wall mannan composition and cell surface hydrophobicity. Since the effectiveness of photodynamic therapy (PDT) depends on the cell wall structure of microorganisms, the objective of this study was to compare the sensitivity of in vitro biofilms of C. albicans serotypes A and B to antimicrobial PDT. Reference strains of C. albicans serotype A (ATCC 36801) and serotype B (ATCC 36802) were used for the assays. A gallium-aluminum-arsenide laser (660 nm) was used as the light source and methylene blue (300 μM) as the photosensitizer. After biofilm formation on the bottom of a 96-well microplate for 48 h, each Candida strain was submitted to assays: PDT consisting of laser and photosensitizer application (L + P+), laser application alone (L + P-), photosensitizer application alone (L-P+), and application of saline as control (L-P-). After treatment, biofilm cells were scraped off and transferred to tubes containing PBS. The content of the tubes was homogenized, diluted, and seeded onto Sabouraud agar plates to determine the number of colony-forming units (CFU/mL). The results were compared by analysis of variance and Tukey test (p < 0.05). The two strains studied were sensitive to PDT (L + P+), with a log reduction of 0.49 for serotype A and of 2.34 for serotype B. Laser application alone only reduced serotype B cells (0.53 log), and the use of the photosensitizer alone had no effect on the strains tested. It can be concluded that in vitro biofilms of C. albicans serotype B were more sensitive to PDT.

Yeast mannans protect liposomes against peroxidation but do not scavenge free radicals

Abstract The mannans from Candida albicans serotype A, Candida dubliniensis , Candida tropicalis and Saccharomyces cerevisiae protected liposomes against peroxidation by OH radicals in a concentration-dependent manner. The most efficacious antioxidant was mannan from C. albicans serotype A with antioxidant activity ∼49% comparable with known antioxidant carboxymethylated glucan (∼46%). The natural antioxidant α-tocopherol exhibited 90% protection of liposomes. The mannans and glucans scavenged negligible amount of free radicals in the common 2,2-diphenyl-1-picrylhydrazyl test. It seems likely that protection of liposomes against OH radicals by polysaccharides is not due to their scavenging properties, but may be caused by a sterical hindrance. The polysaccharides containing flexible β-linkages are more effective protectors of liposome peroxidation than these polysaccharides that contain α-linkages only.

Influence of oxidative and osmotic stresses on the structure of the cell wall mannan of Candida albicans serotype A

In this study, we investigated the structural changes in the cell wall mannan of Candida albicans serotype A strain cells cultured under various stress conditions, that is, oxidative stress of 3.5 mM H 2O 2, osmotic stress of 1.5 M NaCl, and heat stress at 37 °C, compared with the normal condition of 30 °C in yeast extract-added Sabouraud liquid medium (YSLM). Based on the 1H nuclear magnetic resonance (NMR) and fluorophore-assisted carbohydrate electrophoresis (FACE) analyses of the mannans, we showed that the proportion of the terminal β-1,2-linked mannose side chain unit in the mannan increased in the cell proliferation process under both the normal condition and the oxidative stress condition. The osmotic stress induced a slight decrease in the proportion of the β-1,2-linked mannose unit in the acid-labile fraction. The heat stress induced a significant decrease in the proportions of the β-1,2-linked mannose unit in both the acid-labile and acid-stable fractions. Based on these results, we propose that C. albicans significantly changes the mannan structures under various stress conditions and that sufficient attention to the cultural conditions is needed to perform an accurate diagnosis of candidiasis.

Growth Inhibition of Candida albicans in the Presence of Antiserum Elicited in Rabbits by Mannan-Protein Conjugate

Antifungal properties of rabbit antiserum prepared by immunization are reported. The immunization was done by a chemically prepared conjugate consisting of Candida albicans (serotype A) surface mannan and human serum albumin. Addition of rabbit antiserum to D-glucose medium inoculated with C. albicans effectively inhibited its growth. Moreover, C. albicans cells treated with rabbit antiserum revealed the entire loss of viability (expressed as decreased mitochondrial dehydrogenase activity). No growth of treated cells on an agar plate was observed. The results confirmed that the mannan-protein conjugate could be considered as an effective component of perspective vaccine.

Structural analysis of cell wall mannan of Candida sojae, a new yeast species isolated from defatted soybean flakes

We investigated the structural and immunochemical characteristics of cell wall mannan obtained from Candida sojae JCM 1644, which is a new yeast species isolated from defatted soybean flakes. The results of a slide-agglutination test and of an enzyme-linked immunosorbent assay using anti-factor sera to the pathogenic Candida species indicated that the cells and the C. sojae mannan were cross-reactive to the specific anti-factor sera against Candida albicans serotype A (FAb 6) and Candida guilliermondii (FAb 9). Two-dimensional homonuclear Hartmann-Hahn analysis indicated that the mannan consisted of various linked oligomannosyl side chains containing alpha-1,2-, alpha-1,3-, alpha-1,6- and beta-1,2-linked mannose residues. However, although the determinants of antigenic factors 6 and 9 could be not found in this mannan, branched side chains, Manbeta1-2Manalpha1-3[Manalpha1-6]Manalpha1-(2Manalpha1-)n2Man and a linear alpha-1,6-linked polymannosyl backbone, which are cross-reacted by FAbs 6 and 9, respectively, were identified. The mannan was subjected to acetolysis in order to determine the polymerization length of the alpha-1,2-linked oligomannosyl residue in the side chains. The result of (1)H-nuclear magnetic resonance analysis of the released oligosaccharides showed that the remarkable regularity in the length of alpha-1,2-linked oligomannosyl side chains, which were previously found in mannans of other Candida species, is not observed in this mannan.

Amended structure of side chains in a cell wall mannan from Candida albicans serotype A strain grown in yeast extract-Sabouraud liquid medium under acidic conditions: detection of the branched side chains corresponding to antigenic factor 4

In a previous study, we reported the excess production of alpha-1,3-linked mannose residues with the complete disappearance of beta-1,2-linked mannose residues in cell wall mannans of Candida albicans serotype A strain cells, which were grown in yeast extract-Sabouraud liquid medium at pH 2.0. In the present study, we examined the immunochemical reactivity of the same mannan of NIH A-207 with an enzyme-linked immunosorbent assay (ELISA) using several antisera to antigenic factors of the genus Candida (FAbs) and the structure of the mannan by two-dimensional homonuclear Hartmann-Hahn analysis. The ELISA showed that the mannan reacts to FAb 4 but not to FAbs 13b and 34, which are reported to be antibody factors against linear side chains containing an alpha-1,3-linked mannose residue. In the Hartmann-Hahn analysis, we found two branched side chains, Man alpha 1-2Man alpha 1-3[Man alpha 1-6]Man alpha 1-(2Man alpha 1-)(2)2Man and Man alpha 1-3[Man alpha 1-6]Man alpha 1-(2Man alpha 1-)(2)2Man, instead of the previously reported linear side chains. The branched side chains are oversynthesized under acidic conditions.

Preparation and Characterization of a Monoclonal Antibody Against Mannoprotein ofCandida albicans

BALB/c mice were immunized via injection with whole cell of Candida albicans serotype A. The spleens were fused with myeloma cells of SP2/0 origin. A mannoprotein-reactive monoclonal antibody (MAb) was selected and characterized by ELISA technique. This MAb reacted with strains of Candida such as C. albicans, C. tropicalis, and C. albicans of the Persian Type Culture Collection (PTCC). However, our antibody did not react with other Candida species such as C. parapsilosis, C. glabrata, C. stellatoidae, C. lusitania, C. krusei, and S. cervisiae. These antibodies also did not recognize extracts of other fungal species such as Aspergillus fumigatus and Aspergillus flavus, and bacterial strains such as Staphylococcus aureus and Pseudomonas aeruginosa. Polyclonal antibody produced in this study could not differentiate the above species and was reactive towards all fungal species mentioned above except bacterial strains of S. aureus and P. aeruginosa. Western blot analysis of ligand affinity-purified mannoproteins of C. albicans wall protein using this MAb showed reactivity toward a single protein band in the region of 55-65 kDa molecular weight. The same antibody, when examined with unpurified C. albicans extract, reacted with a broad band in the region of 55-105 kDa, which we concluded was due to a possible different glycosylation pattern of mannoprotein in crude extract in which the higher molecular weight protein was eliminated by ligand-binding affinity purification.

Contribution of cell surface hydrophobicity protein 1 (Csh1p) to virulence of hydrophobic Candida albicans serotype A cells

The CSH1 gene product is the first protein implicated to affect the phenotype of cell surface hydrophobicity in Candida albicans. Ablation of expression of CSH1 resulted in a 75% loss of the cell surface hydrophobicity (CSH) phenotype. When the C. albicans csh1 knockout derivative was cultured from frozen stocks, it had reacquired CSH levels similar to the parent strain and isogenic reintegrant in the absence of Csh1p re-expression through an unknown mechanism. Prior to reacquisition of CSH, the knockout was less adherent to fibronectin than the parent. Comparison of the csh1 knockout and CSH1 reintegrant in a hematogenous dissemination model allows analysis of Csh1p contribution to virulence using matched strains with similar levels of CSH. No statistical significance between the knockout and reintegrant was found in virulence based on median day of survival, although a reproducible delay in onset of lethal infection for the knockout was observed. A modest difference in mucosal colonization in a vaginal infection model was also observed between the knockout and reintegrant. The present study demonstrates that Csh1p contributes to virulence of C. albicans in mice, but other gene products also contribute to the CSH phenotype and virulence.

Statistical surveillance of mucocutaneous candidiasis in Showa University Fujigaoka Hospital in the past 5 years

We investigated the epidemiology of 618 patients of mucocutaneous candidiasis who visited our outpatient clinic between 1993 and 1997. Compared with previous reports in Japan, the incidence of candida intertrigo increased and that of erythema mycoticum infantile decreased. The incidence of "others", such as nail candidiasis and candidiasis developed under plaster increased. We identified the species of Candida from patients in 496 recent cases, and these species were cultured successfully in 79.2%. As reported by previous authors, a majority or our results were Candida albicans serotype A. We also investigated the risk factors such as diseases or conditions which might be related to immunodeficiency (neoplasm, AIDS) and that might force a patient to restrict himself to bed for a long period (neurological disease). The former was found in 22.7%, and the latter in 23.3% of patients.

Macrophage phagocytosis of Candida albicans. An in vitro study

Considering the role of macrophages in relation to fungi and the various utilized methodologies, the authors established an in vitro model to evaluate macrophage phagocytosis of Candida albicans. Activated macrophages were obtained from the peritoneal cavity of isogenic mice (A/Sn). Two different strains of Candida albicans serotype A and serotype B with different levels of pathogenicity in vivo and other similar characteristics were utilized in the study. Several microscopic fields containing about 200 macrophages were counted. The percentage of macrophages phagocytizing at least one viable or nonviable yeast cell determined an average number of phagocytized yeasts. Neutral red and fluorescein diacetate plus ethidium bromide were used for staining. It is possible to conclude that this is an efficient model related to the used methodology. The average number of yeasts in both strains were similar when inside macrophages, and there was a higher percentage of C. albicans serotype A phagocytosis, which was not experimentally pathogenic in vivo.

Serotype distribution and secretory acid proteinase activity of Candida albicans isolated from the oral mucosa of patients with denture stomatitis.

Denture stomatitis is the most common form of oral Candida infection in humans. In the current study, the distribution of Candida albicans serotype A and B as well as the activity of the secreted acid proteinase were determined in clinical isolates from patients with denture stomatitis. It was found that 70% of individuals with clinical signs of denture stomatitis exhibited fungal growth, with C. albicans representing the most frequently isolated species (75%). Of the C. albicans isolates, 75% were serotype A and 25% were serotype B, representing a significant increase of serotype B compared to a control group of non-denture-wearing HIV-seronegative individuals with oral candidiasis, but no significant difference compared with isolates from HIV-seropositive patients, who also exhibited a high percentage of serotype B. The mean secretory acid proteinase activity of C. albicans isolates from denture stomatitis patients (2796 +/- 819 U/l) was statistically not different from the mean secretory acid proteinase activity in non-denture-wearing HIV-seronegative individuals (2324 +/- 1487 U/l). Both values were significantly lower than the mean secretory acid proteinase activity of C. albicans from HIV-seropositive individuals (4256 +/- 2372 U/l). No correlation exists between the C. albicans serotype and the amount of secreted acid proteinase, indicating that serotype and secretory acid proteinase expression are two independent pathogenetic factors in oral candidiasis. These results indicate that there seems to be strain selection for C. albicans serotype B in denture stomatitis. These results further indicate that increased secretion of the acid proteinase seems to be of pathogenetic significance in the candidiasis of HIV-seropositive patients but not in denture stomatitis. Nevertheless, the secretory acid proteinase is likely to be an important pathogenetic factor also in denture stomatitis, where an increased secretion of the acid proteinase may not be required because of decreased salivary flow and a low pH under the denture, which will result in a high enzymatic activity.

Definitive chemical evidence for the constitutive ability of Candida albicans serotype A strains to synthesize β-1,2 linked oligomannosides containing up to 14 mannose residues

We have previously reported the presence of phosphate bound β-1,2 linked oligomannosides with unusually high degrees of polymerization (DP>7) in the mannan of Candida albicans strain VW32. To confirm this observation, we have prepared these oligomannosides from the mannan of C. albicans strain NIH A 207. Gel filtration chromatography and TLC analysis revealed DP up to 14. For both strains, NMR analysis confirmed the exclusive presence of β-1,2 linkages in the pools of oligomannosides with a DP higher than 6 which presented an average DP of 10.6 (VW32) and 10.4 (NIH A 207). These results are important to consider in relation with the ability of these C. albicans derived oligomannosides to trigger TNFα synthesis according to their DP.

Amended structure of side chains in a cell wall mannan from Candida albicans serotype A strain grown in yeast extract-Sabouraud liquid medium under acidic conditions: detection of the branched side chains corresponding to antigenic factor 4

In a previous study, we reported the excess production of α-1,3-linked mannose residues with the complete disappearance of β-1,2-linked mannose residues in cell wall mannans of Candida albicans serotype A strain cells, which were grown in yeast extract-Sabouraud liquid medium at pH 2.0. In the present study, we examined the immunochemical reactivity of the same mannan of NIH A-207 with an enzyme-linked immunosorbent assay (ELISA) using several antisera to antigenic factors of the genus Candida (FAbs) and the structure of the mannan by two-dimensional homonuclear Hartmann-Hahn analysis. The ELISA showed that the mannan reacts to FAb 4 but not to FAbs 13b and 34, which are reported to be antibody factors against linear side chains containing an α-1,3-linked mannose residue. In the Hartmann-Hahn analysis, we found two branched side chains, Manα1–2Manα1–3[Manα1–6]Manα1-(2Manα1-) 22Man and Manα1–3[Manα1–6]Manα1-(2Manα1-) 22Man, instead of the previously reported linear side chains. The branched side chains are oversynthesized under acidic conditions.

Characterization of beta-1,2-mannosyltransferase in Candida guilliermondii and its utilization in the synthesis of novel oligosaccharides.

A particulate insoluble enzyme fraction containing mannosyltransferases from Candida guilliermondii IFO 10279 strain cells was obtained as the residue after extracting a 105,000 x g pellet of cell homogenate with 1% Triton X-100. Incubation of this fraction with a mannopentaose, Manalpha1-->3(Manalpha1-->6)Manalpha1-->2Manalpha1+ ++-->2Man, in the presence of GDP-mannose and Mn2+ ion at pH 6.0 gave a third type of beta-1,2 linkage-containing mannohexaose, Manbeta1-->2Manalpha1-->3(Manalpha1-->6)Manalpha1++ +-->2Manalpha1-->2Man , the structure of which was identified by means of a sequential NMR assignment. The results of a substrate specificity study indicated that the beta-1,2-mannosyltransferase requires a mannobiosyl unit, Manalpha1--> 3Manalpha1-->, at the nonreducing terminal site. We synthesized novel oligosaccharides using substrates possessing a nonreducing terminal alpha-1,3-linked mannose unit prepared from various yeast mannans. Further incubation of the enzymatically synthesized oligosaccharide with the enzyme fraction gave the following structure, Manbeta1-->2Manbeta1-->2Manalpha1-->3(Manalpha1- ->6)Manalpha1--> 2Manalpha1-->2Man, which has been found to correspond to antigenic factor 9. Incubation of Candida albicans serotype B mannan with the enzyme fraction gave significantly transformed mannan, which contains the third type of beta-1,2-linked mannose units.

Candida albicans serotype analysis by flow cytometry

Candida albicans strains can be assigned to either of two major serogroups, A or B. Antigenic surface determinants present only in serotype A strains allow such a distinction, which has epidemiologic relevance. Reports have established that the relative distributions of the two serotypes can vary depending on the geographic origin of the isolates. A prevalence of susceptibility to an antifungal agent, flucytosine, was also observed with isolates of serotype A. More recently, it was suggested that the occurrence of serotype B isolates in various clinical forms of candidiasis is increasing. However, this latest finding remains controversial since serotyping results vary widely from one laboratory to another because of the lack of standardized methodologies. Difficulty in interpretation of results, which may lead to erroneous serotype identification, is the major setback associated with current methods. For this study, we thus devised a procedure that relies on flow cytometry and that may eliminate ambiguities in serotype determination. The validation of results was achieved with two types of serotype A-specific antisera, Iatron Factor 6 antiserum and an anti-C. albicans antiserum adsorbed on serotype B yeast cells. Agreement between results obtained with these two reagents was 100% with a wide array of Candida strains. These results confirmed the potential of the flow cytometric procedure as a reliable and reproducible method to establish the serotypes of C. albicans strains. Furthermore, some applications of this procedure to the epidemiological study of this human pathogen are presented.

A new method of antibiotyping yeasts for subspecies discrimination and distribution in human clinical specimens.

A study of the antibiotypes of 764 isolates of the genera Candida and Torulopsis from different clinical specimens is reported. The typing method was based on the susceptibility results obtained by the standardized and partially automated kit ATB-Fungus (API-bioMérieux), giving to each strain a code of six figures, according to these criteria: susceptibility to 5-fluorocytosine, amphotericin B, nystatin, miconazole, econazole, and ketoconazole. Candida albicans serotypes were determined by the Candida Check test (Iatron, Japan). Twenty-six antibiotypes were found in C. albicans (482 isolates), 21 types in serotype A, and 15 in serotype B strains. Candida parapsilosis (115 isolates) was divided into 11 antibiotypes, Torulopsis glabrata (53 isolates) into five, Candida guilliermondii (36 isolates) into 10 and Candida tropicalis (31 isolates) into eight. Depending on the sample origin, 000000 (susceptibility to all the antifungals tested) was the predominant C. albicans antibiotype (92.9% of blood isolates, 41.2% of vaginal isolates, 33.3% of respiratory isolates, isolates, 31.01% or oral and digestive tract isolates, and 25.0% of nail and skin isolates). No predominant antibiotypes were found in strains from respiratory tract, skin ad nails. A reproducibility close to 99% was found with the test. Simplicity and standardization could make this method useful for typing Candida and Torulopsis isolates.