Michiko Tokunaga

Respiration of medically important Candida species and Saccharomyces cerevisiae in relation to glucose effect

Strains of medically important Candida species (C. albicans, C. tropicalis, C. parapsilosis and C. [Torulopsis] glabrata) and Saccharomyces cerevisiae were examined for a glucose effect on respiratory activity. Reduced O2-consuming ability and a relative decrease in cytochrome type c, as determined by polarography and spectrophotometry, respectively, were observed in glucose-grown S. cerevisiae cells in contrast with acetate- or ethanol-grown cells. In glucose-grown cells of C. glabrata, O2 consumption was also reduced without any change in the cytochrome pattern compared to acetate-grown cells, while no such decrease was detected in any of the other strains of Candida species tested. These results suggest that the medically important Candida species, except for C. glabrata, can be categorized as members of the glucose-insensitive yeast type with respect to respiration.

Adansonian study of Candida albicans: intraspecific homogeneity excepting C. stellatoidea strains

Fifty-six strains of Candida albicans (40 fresh human isolates, 10 laboratory strains, and 6 Candida stellatoidea strains), seven strains of other Candida species, and one strain of Saccharomyces cerevisiae were examined for a total of 182 biochemical and physiological characters. As 121 characters proved positive or negative in all of the strains, analysis of similarity values (simple matching coefficients) derived from the remaining 61 characters revealed that C. albicans strains could be discriminated from other species with similarity values of 70% or lower, and that the cluster of C. albicans strains with internal similarity values above 70% could be divided into two subclusters representing classical C. albicans and C. stellatoidea, with intergroup similarity of 70 to 85% and intragroup similarities of 85% or higher. No discernible difference was noted between isolates from pathological specimens and those from healthy individuals.

Initial attachment of Candida albicans cells to buccal epithelial cells. Demonstration of ultrastructure with the rapid-freezing technique

The rapid-freezing technique was applied in association with scanning and transmission electron microscopy to observe the initial attachment (or contact) ofCandida albicans cells to exfoliated human buccal epithelial cells. Low temperature scanning electron microscopy provided detailed three-dimensional morphological features of the yeast-epithelial cell association; adhesion ofC. albicans cells to host cells was primarily owing to an interaction between fibrillar layer of the yeast cell wall and the membrane interdigitations of the epithelial cells. Such a particular interconnection between the two cells was confirmed by the freeze-substitution fixation for transmission electron microscopy. These results clearly demonstrate the outermost fibrillar cell wall layer ofC. albicans responsible for adhesion to host cells.The rapid-freezing technique was applied in association with scanning and transmission electron microscopy to observe the initial attachment (or contact) of Candida albicans cells to exfoliated human buccal epithelial cells. Low temperature scanning electron microscopy provided detailed three-dimensional morphological features of the yeast-epithelial cell association; adhesion of C. albicans cells to host cells was primarily owing to an interaction between fibrillar layer of the yeast cell wall and the membrane interdigitations of the epithelial cells. Such a particular interconnection between the two cells was confirmed by the freeze-substitution fixation for transmission electron microscopy. These results clearly demonstrate the outermost fibrillar cell wall layer of C. albicans responsible for adhesion to host cells.

DNA homology between Candida albicans strains: Evidence to justify the synonymous status of C. stellatoidea

Genetic relatedness between strains of C. albicans and C. stellatoidea was studied by measuring G + C content and overall sequence homology. G + C contents determined by high-performance liquid chromatography were 32.6 to 34.2% for 26 strains of C. albicans and 33.0 to 33.9% for eight strains of C. stellatoidea. DNA-DNA hybridization with two C. albicans and two C. stellatoidea probes revealed that all 34 test strains formed a single cluster in which the extents of hybridization with the heterologous probes ranged between 77.9 and 105.6% of those with the homologous probes. These results give support to the unification of C. albicans and C. stellatoidea into a single species.

Germ tube-forming cells of Candida albicans are more susceptible to clotrimazole-induced killing than yeast cells

Yeast and germ tube-forming cells of Candida albicans were compared with respect to their susceptibility to killing induced by the imidazole antifungal clotrimazole. Cultures consisting largely of germ tube-forming cells or exclusively yeast cells were prepared by incubating cells of a germ tube-proficient strain in a proline-containing phosphate buffer at 37 degrees C or 25 degrees C, respectively. When treated with clotrimazole at 37 degrees C, the cultures of germ tube cells lost colony-forming ability much more rapidly than those of yeast cells. However, this difference was diminished in the cells preincubated at 37 degrees C but prevented from forming germ tubes by 5 mM cysteine, a suppressor of germ tube formation. In another C. albicans isolate showing a very poor capacity to form germ tubes at 37 degrees C, such a difference in killing rate was much smaller than that for the germ tube-proficient strain. Furthermore, when an isogenic pair of strains, one proficient and the other deficient in germ tube formation, were compared with each other, germ tube-forming cultures of the former were found to be more sensitive than yeast cell cultures of the latter. It is inferred from these results that the germ tube-forming cell of C. albicans is more sensitive to clotrimazole-induced killing than the yeast cell.

Surface of the Cell Packets Induced from a Micrococcus lysodeikticus (luteus) Mutant

resolving power. We present a concept that the peripheral wall of M. lysodeikticus consists of at least two layers which are functionally different, although it appears to be one uniform layer in observations of a thin section with a transmission electron microscope (2-4, 7, 8, 10), and that the septum is formed regardless of the outermost layer of the peripheral wall. For scanning electron microscopy, the method of Amako and Umeda (1) was modified. The cell packets were induced from M. lysodeikticus MT by culture in a liquid medium supplemented with trypsin at a final concentration of 5 pgiml as described previously (6). The cultured cells were centrifuged, washed once with deionized water at 4 C, and suspended in 2 % glutaraldehyde in 0.1 M sodiumcacodylate buffer, pH 7.2 (fixative solution). A drop of the cell suspensions was placed on a thin layer of agar prepared on a chip of glass fiber filter (Whatman GF/F). After about 5 min the chip in a small Petri dish was covered with the fixative solution, and after 3 hr standing at room temperature it was washed three times with the cacodylate buffer at intervals of 30 min, dehydrated in a graded series of ethanol, and finally dryed by the critical point drying method. The specimens were coated with gold-palladium (60-40%) of about 5 nm thickness using a JFC1100 Fine Coat Ion Sputter ( JEOL Ltd., Tokyo) and observed with an Akashi ISIDS130 scanning electron microscope (Akashi Seisakusho Ltd., Tokyo) operated at 5 kV. A scanning electron micrograph of the cell packets induced from M. lysodeikticus MT is shown in Fig. 1, which supports the observation of a thin section of the cell packets with a transmission electron microscope (8). The cell packets have an interstice between the unit tetrads which are linked by the bridging structure formed by the outermost layer of the cell wall. They seem to be in rags because the bridging structure is broken here and there, originally or artificially. The photograph also provides another confirmation of the mechanism of the formation of the bridging structure by the outermost layer as described previously (8). The unique structure of the cell packets is formed when the cell wall extension and

Cell Surface of Micrococcus luteus: Chemical Treatment of the Cells and Teichuronic Acids on the Surface

Micrococcus luteus IFO 3333 cells, both treated with chemical reagents and non‐treated, were observed with a scanning electron microscope (SEM). The agglutinability of the cells with antiserum containing anti‐teichuronic acid antibody was examined. The binding of protein A‐gold particles to the cells, mediated with the antiserum, was also observed with SEM. The surface of a M. luteus cell consisted of two or three areas with borders—the rough and the smooth areas, or the rough, the slightly rough, and the smooth areas; fluffy materials were clearly seen in the rough area. Gold particles were observed uniformly and densely on the whole cell surface. However, either mild acid treatment or mild Smith degradation of the cells altered the fluffy rough area to a rough one, and extremely decreased the agglutinability and the binding of protein A‐gold particles. Teichuronic acids appeared to be distributed uniformly on the whole cell surface of M. luteus IFO 3333.

Comparison of Two Kinds of Cell Packets of Micrococcus luteus by Scanning Electron Microscopy: Outermost Layer Maintaining the Packet Structure

Two kinds of cell packets of Micrococcus luteus, one having teichuronic acids (TUA) in the cell wall and the other lacking TUA. have been independently reported by two groups of workers. A comparison by scanning electron microscopy of these packets provided a possibly consistent interpretation for the seemingly conflicting opinions whether TUA were involved in packet induction. It was strongly suggested that the packets having TUA in the wall were rigidly maintained by a bridging structure of the outermost layer of the peripheral wall, while the packets lacking TUA showed low contribution of the outermost layer to the bridging structure probably due to the absence of TUA.

Cell Surface of a Tetrads‐Forming Mutant of Micrococcus luteus: Chemical Treatment of the Cells and Teichuronic Acids on the Surface

Tetrads‐forming mutant MT cells of Micrococcus luteus, both treated with chemical reagents and non‐treated, were observed with a scanning electron microscope (SEM). The agglutinability of the cells with antiserum containing anti‐teichuronic acid antibody was examined. The binding of protein A‐gold particles to the cells, mediated with the antiserum, was also observed with SEM. A tetrad surface, not surface of each of four “unit monococci” constituting a tetrad, consisted of two or three smooth areas with borders. The difference in the surface features between M. luteus wild‐type IFO 3333 (Monodane et al, Microbiol. Immunol. 33: 165–174, 1989) and the mutant MT cells is discussed.