Cytoplasmic antigens unique to the mycelial or yeast phase of Candida albicans.

Crossed immunoelectrophoresis with absorption in situ was used to distinguish the cytoplasmic antigens unique to the mycelial or yeast phase of Candida albicans from cytoplasmic antigens shared by both phases. The soluble cytoplasmic extracts of each growth phase had at least six distinct antigenic constituents not shared by the other phase. This technique is recommended for the analysis of closely related antigenic complexes.     

Use of immunoblotting to identify antigenic differences between the yeast and mycelial phases of Candida albicans.

Western blotting was applied to the analysis of Candida albicans in the yeast and mycelial phases in an attempt to recognise mycelial specific antigens which might be of serodiagnostic value. The antisera were prepared in rabbits by immunising them with pressates of C albicans type A NCTC 3153 in the yeast phase or the mycelial phase. These were blotted against C albicans in the yeast and mycelial phases and the yeast phase of C parapsilosis, C krusei, C tropicalis, and Torulopsis glabrata. Cross reactivity was greatest against C parapsilosis. One yeast specific mannoprotein was identified with a molecular weight of 49 000. No mycelial specific antigens could be identified.     

Characterization of cell wall proteins of yeast and hydrophobic mycelial cells of Candida albicans.

Cell surface hydrophobicity (CSH) of blastoconidia and blastoconidia bearing germ tubes of Candida albicans ATCC 26555 was monitored by assessing attachment of polystyrene microspheres to the cell surface, and we found that mature hyphae were significantly hydrophobic. Treatment of intact cells with low concentrations of beta-glucanase (Zymolyase 20T) or proteases abolished or significantly reduced attachment of latex beads to hyphae. This effect paralleled an obvious reduction in CSH of the entire cell population, as measured by an aqueous-hydrocarbon biphasic partitioning assay. Analysis of the cell wall material released by Zymolyase and adsorbed on polystyrene microspheres indicated that germ tube-specific cell wall proteins and mannoproteins with apparent molecular masses of 20 to 67 kDa may be responsible for the hydrophobicity of hyphae. Zymolyase released from blastoconidia cell walls a different set of proteins and mannoproteins that were able to adsorb to polystyrene microbeads. Such molecular species might in turn be responsible for the CSH exhibited by blastoconidium populations as determined by the biphasic partitioning assay, although these probably hydrophobic components can be masked on the surface of blastoconidia, as the latter had no or very few latex microspheres attached to their surfaces. Treatment of cells of both C. albicans morphologies with 2-mercaptoethanol released qualitatively distinct species of polystyrene-adsorbed proteins and mannoproteins from yeast and mycelial cells. These observations suggested that hydrophobic proteins and mannoproteins that could be associated with CSH are bound to the cell wall structure through diverse types of linkages.     

Antifungal activity of Lavandula angustifolia essential oil against Candida albicans yeast and mycelial form.

The antifungal activity of the essential oil of Lavandula angustifolia Mill. (lavender oil) and its main components, linalool and linalyl acetate, was investigated against 50 clinical isolates of Candida albicans (28 oropharyngeal strains, 22 vaginal strains) and C. albicans ATCC 3153. Growth inhibition, killing time and inhibition of germ tube formation were evaluated. The chemical composition of the essential oil was determined by gas chromatography and mass spectrometry. Lavender oil inhibited C. albicans growth: mean minimum inhibitory concentration (MIC) of 0.69% (vol./vol.) (vaginal strains) and 1.04% (oropharyngeal strains); mean MFC of 1.1% (vaginal strains) and 1.8% (oropharyngeal strains). Linalool was more effective than essential oil: mean MIC of 0.09% (vaginal strains) and 0.29% (oropharyngeal strains); mean MFC of 0.1% (vaginal strains) and 0.3% (oropharyngeal strains). Linalyl acetate was almost ineffective. Lavender oil (2%) killed 100% of the C. albicans ATCC 3153 cells within 15 min; linalool (0.5%) killed 100% of the cells within 30 s. The essential oil inhibited germ tube formation (mean MIC of 0.09%), as did the main components (MIC of 0.11% for linalool and 0.08% for linalyl acetate). Both the essential oil and its main components inhibited hyphal elongation of C. albicans ATCC 3153 (about 50% inhibition at 0.016% with each substance). Lavender oil shows both fungistatic and fungicidal activity against C. albicans strains. At lower concentrations, it inhibits germ tube formation and hyphal elongation, indicating that it is effective against C. albicans dimorphism and may thus reduce fungal progression and the spread of infection in host tissues.     

Analysis of the enzymatic activity of mycelial and yeast phases of Penicillium marneffei.

The cell-associated and extracellular enzymatic activities were examined in a total of 10 Penicillium marneffei isolates. Both mycelia and yeast expressed alkaline phosphatase, acid phosphatase and naphthol-AS-BI-phosphohydrolase activities, whereas a variety of other enzyme activities, including trypsin, chymotrypsin and alpha-fucosidase were absent. There was some inter-isolate variation in both mycelia and yeast in the activities of other enzymes such as esterases and galactosidases. Enzyme activities did not change significantly over the course of culturing in three representative isolates.     

Optimisation of Candida albicans typing by pulsed-field gel electrophoresis.

Six strains of Candida albicans were subjected to pulsed-field gel electrophoresis (PFGE) using the CHEF-DRIII system (BioRad). Hansenula mingei YB-4662-VIA and Saccharomyces cerevisiae YNN 295 (BioRad) were used as size markers (1.05-3.13 and 0.22-2.2 megabase pairs [Mbp] respectively) for comparison of DNA molecules. The DNAs were resolved by a three-block protocol with pulse times of 120 s for 24 h, 240 s for 36 h and 300 s for 17 h. The voltage was set at 4.5 V/cm for the first two blocks and 4.0 V/cm for the final block. PFGE was carried out under these conditions using different agarose concentrations, types and concentrations of buffer, temperatures, and sizes of agarose gel plug. The resolution and mobility of DNAs were affected by some of these variables. Separation of C. albicans by PFGE was optimal at 12 degrees C with 1.0 x Tris-borate-EDTA (TBE) buffer using 1.2% agarose. Resolution of banding patterns was dependent on size of DNA plug used.     

Epidemiological analysis of Candida albicans strains by multilocus enzyme electrophoresis.

Genotypic diversity in a collection of 98 isolates of Candida albicans was assessed by multilocus enzyme electrophoresis. Four of the 10 enzyme loci studied were polymorphic. The electrophoretic patterns observed were compatible with those expected for a diploid organism. The 98 isolates were assigned to 14 electrophoretic types, each of which was represented by from 1 to 21 isolates. Samples from various clinical sites of seven bone marrow transplant patients treated in the same unit within a 13-month period were obtained repeatedly. Three patients were found to be colonized with more than one strain. In one patient, flucytosine-resistant strains were isolated after systemic antifungal treatment was started. These isolates had electrophoretic types different from those of the strains that colonized the patient before treatment. There was no evidence that cross-infection between these patients occurred in the hospital. The population structure of C. albicans is discussed in regard to the multilocus genotype data.     

Characterization of cell wall proteins from yeast and mycelial cells of Candida albicans by labelling with biotin: comparison with other techniques.

Candida albicans ATCC 26555 blastoconidia and blastoconidia bearing germ tubes were metabolically labelled by incubating the cells with 14C-labelled protein hydrolysate and were subsequently tagged with biotin. Double-labelled (radioactive and biotinylated) cell wall proteins and glycoproteins were extracted from intact cells of both growth forms by treatment with 2-mercaptoethanol (beta ME) and with beta-glucanases (Zymolyase) after treatment with beta ME. The beta ME- and Zymolyase-extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotted (immunoblotted) to nitrocellulose paper. Polyacrylamide gels were stained with Coomassie blue and processed for fluorography. Western blot analysis was performed either with peroxidase conjugated-concanavalin A (ConA) or Extravidin. Blotted proteins were also reacted with polyclonal antibodies and monoclonal antibodies against mannoprotein components from mycelial cell walls of the ATCC 26555 strain. Labelling with biotin allowed identification of a complex array of cell wall protein and glycoprotein components within a very wide molecular mass range (from 650 to 13 kDa). These appeared to be genuine cell wall components. Biotinylated high-molecular-mass glycoproteins that were not stained with Coomassie blue or that appeared as poorly resolved polydisperse bands by indirect ConA-peroxidase staining of Western blots were detected as sharply defined bands following reaction with the Extravidin-peroxidase conjugate. Biotinylated molecules retained unaltered reactivities against ConA, polyclonal antibodies, and monoclonal antibodies.     

A proteomic view of Candida albicans yeast cell metabolism in exponential and stationary growth phases

The facultative pathogenic fungus Candida albicans has to come up with dynamic metabolic adaptation programs in order to be able to survive within a variety of niches in the human host, each of which has its different nutrient availability. Using a large-scale two-dimensional (2-D) protein gel electrophoresis approach, we analyzed the adaptation mechanisms to nutrient limitation in a batch culture in complex medium with glucose as carbon source. To this end, we constructed a 2-D reference map of cytoplasmic proteins and quantitatively compared protein accumulation of growing yeast cells with those from the stationary phase. This yielded characteristic proteome signatures for each physiological state. During exponential growth, proteins required for the synthesis of RNA, DNA, and proteins, including components of purine and pyrimidine synthesis pathways and ribosomal proteins, were over-represented. The stationary-phase signature revealed a complex reprogramming of metabolic networks: Up-regulation of glyoxylate cycle, gluconeogenesis, and glutamate degradation signaled a switch to the utilization of alternative carbon sources instead of the exhausted glucose. Induction of proteins involved in defense against oxidative and heat stress indicates a change in redox balance and reactive oxygen species concentrations.     

Candida albicans group A-specific soluble antigens demonstrated by quantitative immunoelectrophoresis.

Soluble cytoplasmic extracts of Candida albicans groups A and B were prepared and compared by quantitative immunoelectrophoresis experiments performed with a commercial anti-C. albicans group A immune serum. Although crossed immunoelectrophoresis, tandem crossed immunoelectrophoresis, and line immunoelectrophoresis revealed many cross-reactions between the two groups, some components seemed to be specific to group A. However, the complexity of the extracts studied did not allow us to demonstrate specific constituents with these methods. Crossed-line immunoelectrophoresis with and without absorption of antibodies in situ was then used, and four specific antigens unique to group A cytoplasmic extract were demonstrated, one of which appeared to be quantitatively important. The value of various quantitative immunoelectrophoretic methods applied to complex antigenic preparations is discussed.     

Rapid killing of monocytes in vitro by Candida albicans yeast cells.

To study the interaction between Candida albicans blastoconidia and human phagocytes, we incubated peripheral leukocytes with fungi for 1 h at 37 degrees C and stained the cells with fluorescent vital stains ethidium bromide (EB) and fluorescein diacetate. Fungi that had been phagocytosed showed little staining; however, some leukocytes containing blastoconidia exhibited nuclear staining with EB, even though their cell membranes showed no signs of penetration by fungi. The number of EB-positive leukocytes was related to viability of the yeast cells and the temperature at which they were maintained before use. Because efforts to quantitate EB-positive leukocytes microscopically were frustrated by cell aggregation, we labeled the leukocytes with 51Cr and measured isotope release. We determined that leukocytes incubated with viable fungi released significantly more isotope than cells incubated alone or with killed blastoconidia. Furthermore, 51Cr release correlated directly with concentration of fungi in the assay, time of incubation, and temperature at which fungi were maintained before use. Using a number of isolates of C. albicans and several other species of Candida, we found that all exhibited cytotoxic activity against leukocytes, but the level of activity varied among organisms. Finally, we depleted or enriched peripheral leukocytes for specific cell populations and determined that only monocytes released more 51Cr after incubation with viable blastoconidia. Blastoconidia can lyse phagocytic cells through germination and penetration of cell membranes within 1 to 2 h, but the cytotoxic phenomenon we describe occurs within 15 to 30 min after yeast cells have been phagocytosed. Therefore, this capacity may represent a more immediate response by blastoconidia against phagocytosis and killing by monocytes.     

Purification and characterization of a major cytoplasmic antigen of Candida albicans.

In previous work (Jones, Infect, Immun. 30:78-89, 1980) a major cytoplasmic antigen of Candida albicans was identified. In both humans and experimental animals, this antigen is released from C. albicans during the course of an invasive C. albicans infection and elicits a specific antibody response. In this study, we used diethylaminoethyl cellulose chromatography and concanavalin A-Sepharose chromatography to obtain purified preparations of the major cytoplasmic antigen from crude cytoplasmic extracts of C. albicans. Column chromatography yielded a purified preparation of the major cytoplasmic antigen, which produced a single line in polyacrylamide gel electrophoresis. Using crossed immunoelectrophoresis, we detected small concentrations of contaminating antigens in the purified preparations. We found that the major antigen was a single polypeptide chain containing about 435 amino acid residues and had a molecular weight of 54,300. This antigen did not possess any of 19 common activities. Enzyme linked immunosorbent assays are being developed to detect this antigen in serum and to detect antibody against the antigen.     

Identification of wall-specific antigens synthesized during germ tube formation by Candida albicans.

Walls of the two cellular forms (blastoconidia and mycelia) of Candida albicans ATCC 26555 were obtained from cells metabolically labeled (6-h pulse) with 14C-protein hydrolysate and [3H]threonine. Walls were purified by thorough washings with buffered and sodium dodecyl sulfate solutions and digested with Zymolyase 20T. The enzymatic treatment released four major high-molecular-weight mannoproteins (HMWM), with apparent molecular masses of 650, 500, 340, and 200 kilodaltons (HMWM-650, HMWM-500, HMWM-340, and HMWM-200, respectively), from yeast cells, whereas two high-molecular-mass mannoproteins (HMWM-260 and HMWM-180) were solubilized from mycelial cells. Some additional minor low-molecular-weight species were also detected in the enzymatic digests of walls from both types of cell. Single and dual pulse-chase experiments indicated that the HMWM-260 and HMWM-180 species reflect de novo synthesis of new proteins specific for the mycelia and do not represent a topological rearrangement of blastoconidium wall components. Monoclonal antibodies were raised against the HMWM-260 species (quantitatively the predominant component in the mycelial walls), and polyclonal rabbit antibodies were obtained against yeast or mycelial cell walls. Anti-mycelial cell wall polyclonal antibodies were adsorbed to whole killed blastoconidia to remove antibodies against common blastoconidium and mycelial wall antigens. Titration by enzyme-linked immunosorbent assay revealed that the monoclonal antibodies could recognize an epitope of the protein moiety of the HMWM-260 mannoprotein. Immunoblotting and immunofluorescence techniques using these monoclonal and polyclonal antibodies confirmed that the HMWM-260 and HMWM-180 species are specific components of the envelope of the mycelial cell walls.     

Analysis of cell wall extracts of Candida albicans by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot techniques.

Cell walls of intact yeast- and mycelial-phase Candida albicans B311 were extracted with different compounds: dithiothreitol, dithiothreitol with protease, dithiothreitol with lyticase, and dithiothreitol with protease followed by beta-glucuronidase with chitinase. Extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot techniques. Dithiothreitol extracts contained the most satisfactory array of components for study. Analysis of these extracts demonstrated that the outer cell wall layers of Candida blastoconidia and germ tubes contained a complex array of polysaccharides, glycoproteins, and proteins. The proteins contributed to a latticework stabilized by covalent bonds that was important in determining the porosity of the outer cell wall layers. When equivalent weights were analyzed, mycelial-phase extract contained a more varied array of proteins than did yeast-phase extract. Only a portion of proteins in mycelial-phase extract elicited antibody responses in hyperimmunized rabbits or infected humans. A polysaccharide-rich, high-molecular-weight component (migrating at a position that would correspond to proteins having molecular weights of 235,000 to 250,000) and a protein component (molecular weight, 19,000) were readily demonstrable in the mycelial-phase extract but could not be identified in the yeast-phase extract.     

Identification of two germ-tube-specific cell wall antigens of Candida albicans.

Outer cell wall layers of intact yeast- and mycelial-phase Candida albicans B311 were extracted with dithiothreitol. Antisera against mycelial-phase organisms were absorbed with yeast-phase organisms or yeast-phase extract and used to stain Western blots of sodium dodecyl sulfate-polyacrylamide gels loaded with yeast- and mycelial-phase extracts. Autoradiography of gels loaded with extracts from organisms surface labeled with 125I was used to detect surface antigens containing proteins. Antigen bands of interest identified in Western blots were cut from the blots and used to immunize rabbits. Two antigens were identified in the mycelial-phase extract which were not present in the yeast-phase extract. The first was a 19-kilodalton protein that was present in the cell walls of germ tubes but was not expressed on their surfaces. The second was a polysaccharide-rich high-molecular-weight antigen which was expressed on the surface of the germ tube. Treatment of mycelial-phase extract with protease and endo-beta-N-acetylglucosaminidase H demonstrated that this antigen was composed of polysaccharides linked through di-N-acetylchitobiose groups to proteins.     

beta-Glucosidase in Candida albicans and its application in yeast identification.

In this report we attempt to explain the discrepancy between beta-glucosidase (EC 3.2.1.21) activity in Candida albicans as measured by commercial kits and that found in an experimental assay. beta-Glucosidase activity in American and Israeli isolates of C. albicans was evaluated with the API ZYM and YeastIdent systems (Analytab Products) and with experimental biochemical assays. Activity was found with whole cells and cell extracts of isolates from both sources. The greatest beta-glucosidase activity was found at pH 5.0 and with p-nitrophenyl-beta-glucopyranoside (PNP-BDG) as the substrate. In assays with beta-naphthyl-beta-D-glucopyranoside and 6-bromo-2-naphthyl-beta-D-glucopyranoside (6-Br-2-naphthyl-BDG), no enzyme activity was detected in whole cells and only limited activity was found in cell extracts of isolates from both sources. In studies with PNP-BDG at pH 5.0 and 7.5, 29 to 38% less activity was found at both pHs with American whole cells, and minor activity (20%) was found at pH 7.5 with isolates from both sources. Because assays with PNP-BDG in cell extracts of isolates from both sources showed no significant differences in activity, the more limited beta-glucosidase activity in American whole cells was most likely due to less efficient transport. Because the API ZYM system uses 6-Br-2-naphthyl-BDG as the substrate and because the substrate is buffered at pH 7.5 in the API YeastIdent kit, both systems appear to be of limited value for the detection of beta-glucosidase activity in C. albicans.     

Characterization of Candida albicans cell wall antigens with monoclonal antibodies.

The antigenic composition of Candida albicans is very complex. In order to study the antigenic relationship between blastoconidia and germ tubes of C. albicans, we produced several monoclonal antibodies and analyzed their reactivity against cell wall antigens either in intact cells or in cells treated with dithiothreitol. Overall, four types of reactivity were found. Monoclonal antibodies 3D9 and 15C9 stained the germ tubes only when tested by indirect immunofluorescence. However, they showed a different reactivity by immunoblotting. Monoclonal antibody 3D9 reacted with antigens with molecular masses of > 200 and 180 kDa specifically expressed in the germ tube. Monoclonal antibody 15C9 reacted with antigens of 87, 50, and 34 kDa present in the germ tube extract and with antigens of 92, 50, 34, and 32 kDa present in the blastoconidium extract. The reactivity of blastoconidia treated for different times with dithiothreitol with these monoclonal antibodies was also studied by enzyme-linked immunosorbent assay. The reactivity of monoclonal antibody 3D9 did not significantly change during the cell wall extraction. However, the reactivity of monoclonal antibody 15C9 was increased for blastoconidia extracted for 60 min and decreased markedly for blastocondia extracted for 120 min. Monoclonal antibody G3B was nonreactive by indirect immunofluoresence but reacted with antigens of 47 and 38 kDa present in the germ tube extract and with an antigen of 47 kDa present in the blastoconidium extract. Monoclonal antibody B9E stained both morphological phases by indirect immunofluorescence. By immunoblotting, it reacted with antigens of > 70 kDa present in the germ tube extract and with antigens of > 63, 56, 47, and 38 kDa present in the blastoconidium extract. Based on the results presented in this study, four types of antigens are described. Type I antigens are expressed on the outermost layers of the germ tube cell wall only. Type II antigens are expressed both on the germ tube cell wall surface and within the blastoconidium cell wall. Type III antigens are found within the cell wall of both blastoconidia and germ tubes. Type IV antigens are expressed on both the blastoconidium and germ tube surface. Two types more can be hypothesized for antigens expressed on the blastoconidium cell surface and within the germ tube cell wall (type V) and for those expressed on the blastoconidium surface only (type VI).     

Candida albicans cell wall antigens for serological diagnosis of candidemia.

Serological tests for diagnosis of disseminated fungal infections in the immunocompromised host are used with varying results. In the present study, the relative ability of antibodies to specifically recognize Candida albicans cell wall components was evaluated in order to find antigenic markers for serological diagnosis of candidemia. Native C. albicans cell wall fragments (CW), periodate- (CWIO4) and proteinase-K- (CWP) treated CW, a mildly extracted phosphopeptidomannan (PPM), and beta(1-3)(1-6)-glucan were used as antigens in ELISA with sera from rabbits immunized with C. albicans (n = 10), patients with culture proven candidemia (n = 8) and healthy individuals (n = 8). The antibody response in rabbits consisted predominantly of anti-PPM antibodies, a finding that was substantiated by inhibition-ELISA. Consistently, periodate treatment (CW104) destroyed a major proportion of the antigenic epitopes. Low rabbit antibody levels were found against glucan, the major Candida cell wall component. These results supported the conclusion that glucan is localized mainly in the inner part of the C. albicans cell wall. In contrast to rabbits' serum IgG antibody response against PPM, which was at least tenfold higher than that raised against CW, patients with candidemia had similar IgG antibody levels against both antigens. These levels were significantly higher than those seen in healthy controls (CW, P = 0.0005 and PPM, P < 0.0001). Although the human anti-glucan and anti-CWIO4 IgG antibody levels were low overall, they were nonetheless significantly increased in the patient group (P = 0.0159 for antiglucan and P = 0.0491 for anti-CWIO4). In addition, a correlation was noticed between levels of these antibodies. No significant differences were found between patients and controls for IgM antibodies when CW, CWIO4, PPM and Glu were used as antigens. In conclusion, IgG antibodies to PPM and native cell wall fragments (CW) were highly discriminatory for recognition of candidemia and these antigens are thus promising candidates for use in serodiagnosis.     

Multiple LTR-retrotransposon families in the asexual yeast Candida albicans

We have begun a characterization of the long terminal repeat (LTR) retrotransposons in the asexual yeast Candida albicans. A database of assembled C. albicans genomic sequence at Stanford University, which represents 14.9 Mb of the 16-Mb haploid genome, was screened and >350 distinct retrotransposon insertions were identified. The majority of these insertions represent previously unrecognized retrotransposons. The various elements were classified into 34 distinct families, each family being similar, in terms of the range of sequences that it represents, to a typical Ty element family of the related yeast Saccharomyces cerevisiae. These C. albicans retrotransposon families are generally of low copy number and vary widely in coding capacity. For only three families, was a full-length and apparently intact retrotransposon identified. For many families, only solo LTRs and LTR fragments remain. Several families of highly degenerate elements appear to be still capable of transposition, presumably via trans-activation. The overall structure of the retrotransposon population in C. albicans differs considerably from that of S. cerevisiae. In that species, retrotransposon insertions can be assigned to just five families. Most of these families still retain functional examples, and they generally appear at higher copy numbers than the C. albicans families. The possibility that these differences between the two species are attributable to the nonstandard genetic code of C. albicans or the asexual nature of its genome is discussed. A region rich in retrotransposon fragments, that lies adjacent to many of the CARE-2/Rel-2 sub-telomeric repeats, and which appears to have arisen through multiple rounds of duplication and recombination, is also described.     

Isolation and characterization of an avirulent Candida albicans yeast monomorphic mutant.

Mutagenesis of Candida albicans strain ATCC 26555 with N-methyl-nitro-N-nitrosoguanidine followed by plating on solid yeast nitrogen base-N-acetylglucosamine medium at 37 degrees C yielded colony morphology variants that were characterized as forming smooth colonies, in contrast to the rough colonies formed by the parental strain. One yeast monomorphic mutant, CAL4, was studied in detail. Strain CAL4 is defective in filamentous growth, unable to form hyphae or pseudohyphae in vivo and in vitro. These filamentous structures are not elicited by commonly used external stimuli such as serum. The mutant had no obvious alterations in its mannan, glucan or chitin content. The total quantity of non-covalently linked wall proteins was reduced in the mutant strain, but the electrophoretic pattern shown by these proteins was identical to that of proteins from the parental strain. CAL4 showed major differences from the parental strain in its formation of covalently linked wall proteins. An important aspect of these differences lay in the practical absence of proteins recognized by two monoclonal antibodies, 1B12 and 3H8, which are considered valuable tools in the diagnosis of candidiasis in part because they normally react strongly with all strains. The C. albicans mutant, blocked in yeast-mycelium transition, was avirulent in a mouse model, although it was able to grow in animal tissues.