Soichi Yoshida

Environmentally controlled dimorphic cycle in a fission yeast

The fission yeast Schizosaccharomyces pombe shows bipolar growth and is a convenient model for studying cell polarity and polar growth. This paper shows that the related Schiz. japonicus var. japonicus can switch to unipolar growth and can exist in both yeast and mycelial phases. On solid media, the yeast phase is unstable and prone to switch to the mycelial form, which shows unipolar growth by tip elongation. The hyphae can colonize the body of the substrate (true mycelium) or just its surface (pseudo-mycelium). The yeast-to-mycelium transition and the growth of the mycelium are regulated by a nutritional gradient and are associated with extensive vacuolation. The mycelium can convert into arthroconidia or return to the yeast phase in response to environmental changes. These environmentally controlled morphological transitions make Schiz. japonicus var. japonicus an attractive model for the investigation of cell polarity and morphogenesis.

Cell-surface hydrophobicity and lipolysis as essential factors in human tinea nigra

Summary. Hydrophobic adhesion of cells of the black yeast Hortaea werneckii (Horta) Nishimura & Miyaji, causative agent of human tinea nigra, was established by microbial adhesion to hexadecane (MATH) and adhesion to polystyrene, and compared with adhesion of other species of black yeasts. Additional ecophysiological tests were performed. Hortaea werneckii cells proved to have a high degree of hydrophobicity (98.5% MATH). The species is unable to degrade keratin, but shows significant lipolytic activity. It is concluded that H. werneckii is a commensal, that shows lipophilic adhesion to human skin and survives by the assimilation of excretion products.

Microtubules and actin cytoskeleton in Cryptococcus neoformans compared with ascomycetous budding and fission yeasts.

Actin cytoskeleton and microtubules were studied in a human fungal pathogen, the basidiomycetous yeast Cryptococcus neoformans (haploid phase of Filobasidiella neoformans), during its asexual reproduction by budding using fluorescence and electron microscopy. Staining with rhodamine-conjugated phalloidin revealed an F-actin cytoskeleton consisting of cortical patches, cables and cytokinetic ring. F-actin patches accumulated at the regions of cell wall growth, i. e. in sterigma, bud and septum. In mother cells evenly distributed F-actin patches were joined to F-actin cables, which were directed to the growing sterigma and bud. Some F-actin cables were associated with the cell nucleus. The F-actin cytokinetic ring was located in the bud neck, where the septum originated. Antitubulin TAT1 antibody revealed a microtubular cytoskeleton consisting of cytoplasmic and spindle microtubules. In interphase cells cytoplasmic microtubules pointed to the growing sterigma and bud. As the nucleus was translocated to the bud for mitosis, the cytoplasmic microtubules disassembled and were replaced by a short intranuclear spindle. Astral microtubules then emanated from the spindle poles. Elongation of the mitotic spindle from bud to mother cell preceded nuclear division, followed by cytokinesis (septum formation in the bud neck). Electron microscopy of ultrathin sections of chemically fixed and freeze-substituted cells revealed filamentous bundles directed to the cell cortex. The bundles corresponded in width to the actin microfilament cables. At the bud neck numerous ribosomes accumulated before septum synthesis. We conclude: (i) the topology of F-actin patches, cables and rings in C. neoformans resembles ascomycetous budding yeast Saccharomyces, while the arrangement of interphase and mitotic microtubules resembles ascomycetous fission yeast Schizosaccharomyces. The organization of the cytoskeleton of the mitotic nucleus, however, is characteristic of basidiomycetous yeasts. (ii) A specific feature of C. neoformans was the formation of a cylindrical sterigma, characterized by invasion of F-actin cables and microtubules, followed by accumulation of F-actin patches around its terminal region resulting in development of an isodiametrical bud.

Killing of Histoplasma capsulatum by γ-interferon-activated human monocyte-derived macrophages : evidence for a superoxide anion-dependent mechanism

The interaction of human macrophages with the yeast form of the thermally dimorphic fungal pathogen, Histoplasma capsulatum, was studied. Macrophages derived from monocytes by culture in vitro for 3 days ingested H. capsulatum, but were neither fungicidal or fungistatic. In contrast, when monocytes were exposed to human recombinant gamma-interferon (gamma-IFN) during their differentiation into macrophages, those macrophages were able to reduce the number of ingested or adherent cfu of H. capsulatum by 44-75% in 2 h. Activation of macrophages for fungicidal activity by gamma-IFN was dose dependent and 500-1000 units ml were optimal. Antibody to gamma-IFN abrogated the gamma-IFN activation process. Killing of H. capsulatum by activated macrophages in 2-h assays could be inhibited by superoxide dismutase but not by sodium azide.

Antifungal activity of murine polymorphonuclear neutrophils against Histoplasma capsulatum

Murine polymorphonuclear neutrophils (PMN) were examined for fungicidal and fungistatic activity against yeast cells of Histoplasma capsulatum. In a 16-h limiting dilution assay (LDA) (constant number of PMN versus decreasing numbers of yeast cells) where PMN to yeast cell ratios were high (0.4-1.5 x 10(5):1), murine PMN exhibited a limited fungicidal effect on H. capsulatum as measured by sterilization of cultures containing one to four yeast cells. On the other hand, inoculum colony forming units (c.f.u.) of H. capsulatum were not reduced by PMN in short-term (2 h) assays (PMN to yeast cell ratio, 500:1) even though H. capsulatum stimulated a brisk oxidative burst in PMN. In the same assays 84% of Candida albicans cells were killed. These findings indicate that H. capsulatum was resistant to killing by products of the oxidative burst generated by PMN and that it was slightly sensitive to PMN in 16-h LDA assays where a different microbicidal mechanism may be operative. In long-term co-culture assays, where the PMN to yeast cell ratio was 500:1, PMN consistently inhibited the replication of H. capsulatum after 24, 48, or 72 h of incubation. Inoculum c.f.u. failed to increase significantly in co-cultures, whereas in culture medium alone c.f.u. increased several-fold in 72 h. Based on these novel findings, we speculate that PMN might play a role in resistance to H. capsulatum due to their strong fungistatic activity and to a lesser extent their limited fungicidal action.

The cytoskeleton in the unique cell reproduction by conidiogenesis of the long-neck yeast Fellomyces ( Sterigmatomyces ) fuzhouensis

Summary.The morphology of conidiogenesis and associated changes in microtubules, actin distribution and ultrastructure were studied in the basidiomycetous yeast Fellomyces fuzhouensis by phase-contrast, fluorescence, and electron microscopy. The interphase cell showed a central nucleus with randomly distributed bundles of microtubules and actin, and actin patches in the cortex. The conidiogenous mother cell developed a slender projection, or stalk, that contained cytoplasmic microtubules and actin cables stretched parallel to the longitudinal axis and actin patches accumulated in the tip. The conidium was produced on this stalk. It contained dispersed cytoplasmic microtubules, actin cables, and patches concentrated in the cortex. Before mitosis, the nucleus migrated through the stalk into the conidium and cytoplasmic microtubules were replaced by a spindle. Mitosis started in the conidium, and one daughter nucleus then returned to the mother via an eccentrically elongated spindle. The cytoplasmic microtubules reappeared after mitosis. A strong fluorescence indicating accumulated actin appeared at the base of the conidium, where the cytoplasm cleaved eccentrically. Actin patches then moved from the stalk together with the retracting cytoplasm to the mother and conidium. No septum was detected in the long neck by electron microscopy, only a small amount of fine “wall material” between the conidium and mother cell. Both cells developed a new wall layer, separating them from the empty neck. The mature conidium disconnected from the empty neck at the end-break, which remained on the mother as a tubular outgrowth. Asexual reproduction by conidiogenesis in the long-neck yeast F. fuzhouensis has unique features distinguishing it from known asexual forms of reproduction in the budding and fission yeasts. Fellomyces fuzhouensis develops a unique long and narrow neck during conidiogenesis, through which the nucleus must migrate into the conidium for eccentric mitosis. This is followed by eccentric cytokinesis. We found neither an actin cytokinetic ring nor a septum in the long neck, from which cytoplasm retracted back to mother cell after cytokinesis. Both the conidium and mother were separated from the empty neck by the development of a new lateral wall (initiated as a wall plug). The cytoskeleton is clearly involved in all these processes.

Histopathological and electron microscopical studies on experimental Penicillium marneffei infection in mice

Experimental Penicillium marneffei infection in mice was investigated histopathologically and by electron microscopy. Viable conidia (5 x 10(6) cells) of P. marneffei were inoculated into each mouse of group A (BALB/c mice) and group B (BALB/c-nu/nu-SIc mice) through the tail vein. All the mice were sacrificed at intervals and the livers were examined. In group A, the conidia were phagocytosed by Kupffer cells soon after inoculation, and proliferated by fission in the cytoplasm. Marked proliferation of yeast cells was observed 7 and 14 days after inoculation. With proliferation of the fungus, the number of lysosomes in Kupffer cells increased, and numerous granulomas were formed in the liver. These granulomas consisted mainly of macrophages with yeast cells, together with a few polymorphonuclear leukocytes, lymphocytes and giant cells. From 28 days on yeast cells were gradually cleared from the granulomas, and 56 days after inoculation almost all the granulomas disappeared. In group B, at an early stage of infection, similar pathological changes to those seen in mice of group A were observed. However, as the infection progressed, the number of granulomas continued to increase and yeast cells continued to proliferate although lymphocytes did not infiltrate these granulomas. With proliferation of yeast cells the liver tissue was replaced with both yeast cells engulfed by macrophages and extracellular yeasts, and dissemination occurred.

Early death at medium acidification and survival after low pH adaptation in Cryptococcus neoformans

WhenCryptococcus neoformans was grown in yeast nitrogen base (YNB) supplemented with 0.5% glucose, the medium was acidified to below pH 3 during the exponential growth phase, which caused early growth-phase death in susceptible strains. Even in resistant strains, 30–70% cells died if incubated for 2 d in YNB supplemented with 1.5% glucose, whereas the remaining cells survived long. Two types of fatal alterations have been observed in dead cells. In the first type, release of cytoplasm occurred through weakened parts of the cell wall; structures attached to cell walls of dead cells were shown to be rich in proteins by FITC staining, indicating their cytoplasmic origin. In the second type, cells shrank distinctly with no sign of wall rupture. The shrinkage may be due to dysfunction of the plasma membrane at low pH. The mechanism of cell survival in medium below pH 3 was also examined. Aniline blue alone, or calcofluor together with methylene blue, allowed cell wall glucan or chitin and dead cell cytoplasm to be stained simultaneously. In the later stages of incubation, cells showing bright staining for cell wall glucan and chitin emerged. These changes in cell wall synthesis could be considered as an adaptation mechanism to acidification of the medium, because such cells survived longer than cells showing no change in the cell wall staining pattern.

Intra-strain variability of Cryptococcus neoformans can be detected on Phloxin B medium

A method was devised for easy detection of intra‐strain variability of the human pathogenic yeast Cryptococcus neoformans. Cultivation of strains on a medium containing Phloxin B resulted in different coloured colonies. Generally, colonies were either pink or red; however there were also several colony‐colour segregant in which both colours could be observed. A number of these segregants were isolated and analysed. Virulence factors such as the cell and capsule sizes were measured; further temperature sensitivity, growth rates, mating‐types and melanin production were also studied. Segregants were examined by random amplified polymorphic DNA (RAPD) fingerprinting and electrophoretic karyotyping by pulsed‐field gel electrophoresis (CHEF). They showed both phenotypic and genotypic differences. The main differences appeared in phenotypic characters and RAPD patterns; while the chromosomal patterns remained unchanged. Reversion frequency analysis revealed that the reason for this segregation could be due to phenotypic switching. The physiological reason for the colour changes was also investigated and was attributed to the differential ability of the cells to accumulate Phloxin B either into their capsules or into their cells. The method described here is potentially applicable for the detection of strain heterogeneity in both basic and clinical microbiology laboratories.

A basis for resistance of blastomyces dermatidis killing by human neutrophils: inefficient generation of myeloperoxidase system products

The mechanism by which the yeast form of Blastomyces dermatitidis resists killing by human peripheral blood polymorphonuclear neutrophils (PMN) was investigated. The metabolic products of the oxidative burst generated during the interaction of PMN and B. dermatitidis or Candida albicans were detected by lucigenin- or luminol-enhanced chemiluminescence (CL). Interaction of PMN and C. albicans resulted in luminol-enhanced CL 100-fold greater than that generated by PMN and B. dermatitidis. This correlated with killing of C. albicans and resistance of B. dermatitidis. Since B. dermatitidis and PMN interactions resulted in significant lucigenin-enhanced CL, deficient luminol CL was not due to a lack of products from the NADPH oxidase system. Killed B. dermatitidis cells at 37 degrees C were more efficient than live cells in stimulating PMN for luminol-enhanced CL; however, only fragmented B. dermatitidis cells elicited luminol-enhanced CL equivalent to that of C. albicans. Since lysates of PMN were active in a cell-free hydrogen peroxide-peroxidase-halide system, resistance of B. dermatitidis to PMN was not due to a defect in PMN peroxidase. Taken together, these findings indicate that resistance of B. dermatitidis to killing by PMN results from inefficient generation of products from the peroxidase-dependent PMN microbicidal system.