Growth strategy of the pathogenic yeast Cryptococcus neoformans submerged culture under different cultivation formats.

Growth patterns of Cryptococcus neoformans submerged culture in different culture volumes, intensity of agitation and types of sealing were evaluated to better understand the physiological role of hypoxia response in this yeast. When low intensity agitation was set at high culture volumes and air exchange between the cultivation vessel and external environment was not abolished completely, the cells proliferated slowly but steadily. On the other hand, when the intensity of agitation was high but the vessel was withheld from fresh air supply, the cells first proliferated rapidly, then arrested completely and finally died. Therefore, the central strategy of C. neoformans here seems to lie in its proliferation-rate adjustment to the available oxygen levels and not in its capacity to survive under anoxia. The data support the opinion that the cultures grown under limited aeration (even though not completely withheld from fresh air supply) are much closer to the real cryptococcal life in human tissues than conventional well-aerated exponential cultures.

Peculiar clusters of daughter cells observed in Cryptococcus neoformans grown in sealed microtiter plates.

Cryptococcus neoformans was grown in 96-well microtiter plates sealed by foil which is less than 0.01 % permeable to oxygen. On day 14 of the cultivation, we observed peculiar clusters of small droplike daughter cells arranged around < or = 4 % of mother cells. The fact that most of the other cells had died indicates that few cells had been able to survive hypoxic conditions and escape the cell-cycle arrest. However, their daughters were unable to separate from them and to continue their proliferation under such conditions.

Prevalence of genes encoding extracellular virulence factors among meticillin-resistant Staphylococcus aureus isolates from the University Hospital, Olomouc, Czech Republic.

A rather fast and complicated progression of an infection caused by some strains of Staphylococcus aureus could be associated with the expression and co-action of virulence factor complexes in these strains. This study screened the antibiotic susceptibility and prevalence of virulence markers in isolates of meticillin-resistant S. aureus (MRSA) obtained from patients hospitalized at the University Hospital in Olomouc, Czech Republic. A total of 100 isolates was screened for 13 genes encoding extracellular virulence determinants (tst, pvl, eta, etb, sea, seb, sec, sed, see, seg, seh, sei and sej) and for their distribution in sample types. Eighty-nine isolates were positive for at least one of the genes. Genes for etb, pvl, see and seh were not detected in any of the MRSA isolates. No statistically significant differences in the occurrence of the determinants studied among sample types were found.

Secreted aspartate proteinases, a virulence factor of Candida spp.: occurrence among clinical isolates.

Production of secreted aspartate proteinases was determined in a set of 646 isolates of Candida and non-Candida yeast species collected from 465 patients of the University Hospital in Olomouc (Czechia) in the period 1995-2002, and Candida samples obtained from 64 healthy volunteers using solid media developed for this purpose. Using random amplified polymorphic DNA analysis (RAPD) 79 Candida isolates from blood were analyzed to show potential relationships between clustering of the fingerprints and extracellular proteolytic activity of these strains. C. albicans, C. tropicalis and C. parapsilosis possess always proteolytic activity while non-Candida species did not display any proteolysis. A tight relationship between fingerprints and extracellular proteolysis in the Candida isolates was not shown. A remarkable consistency between fingerprint clusters and proteolysis occurred in a subset of C. parapsilosis samples. Suboptimal pH of the growth medium was shown to facilitate the investigation of potential co-incidence of genotypic and phenotypic traits.

Rylux BSU stimulates spore germination in Trichophyton mentagrophytes and Aspergillus fumigatus and increases the survival rate after UV-irradiation.

Calcofluor-allied optical brightener Rylux BSU stimulated spore germination rate in Trichophyton mentagrophytes and Aspergillus fumigatus both if supplemented into Sabouraud glucose agar and if used for pretreatment of spore suspension prior to inoculation at low concentrations. Maximum stimulation of germination was obtained if 0.2% Rylux BSU was used for pretreatment in aqueous solution for 1 d prior to inoculation (130% in T. mentagrophytes and 150% in A. fumigatus, respectively). Pretreatment with 1% Rylux BSU provided strong protection against UV-irradiation and resulted in increased yields of cultural variants after UV-irradiation.

Deficit in oxygen causes G(2) budding and unbudded G(2) arrest in Cryptococcus neoformans.

Cryptococcus neoformans exhibited diphasic growth when grown under limited aeration. First, it grew exponentially, but at OD 1, the concentration of dissolved oxygen in culture decreased to 1 mg l(-1) and a second phase of slow growth was started. This phase was characterized by a shift of budding from S to G(2), a sharp decrease in budding index and a sharp increase in the proportion of unbudded G(2) cells to 80%. Thus, a deficit in oxygen was demonstrated to delay the timing of budding, prolong the G(2) phase and cause accumulation of cells after DNA synthesis, but before commitment to budding.

Diversity among wild type and vaccination strains of Trichophyton verrucosum investigated using random amplified polymorphic DNA analysis.

We initially tested 20 primers for their ability to amplify genomic DNA of Trichophyton verrucosum using RAPD. Six of these were selected for further study aimed at discrimination of wild type and vaccination strains of T. verrucosum. The results indicate that RAPD successfully distinguished all strains included in the study. In addition, results of corrected cluster analysis were consistent with the fact that the avirulent vaccination strains (T. verrucosum TV-M9 and T. verrucosum TV-M-130) were prepared by ultraviolet (UV) light induced mutagenesis of the standard wild type strain T. verrucosum Strádznice. No marker for a/virulence was detected. These outcomes suggest new possibilities for epidemiological analyses, for discrimination among different vaccination strains and studies of fungal population in vaccinated/infected hosts.

Nikkomycin Z counteracts Rylux BSU and Congo red inhibition of Saccharomyces cerevisiae growth but does not prevent formation of aberrant cell walls.

Rylux BSU and congo red bind to chitin, interfere with proper cell-wall assembly, and stimulate chitin synthesis by increasing, most probably, chitin synthase 3 (ChS3) levels in Saccharomyces cerevisiae. On the other hand, the antibiotic nikkomycin Z inhibits chitin synthesis competitively. As ChS3 is the critical target of nikkomycin Z, its effect was tested in cells inhibited in growth by Rylux BSU or Congo red. Nikkomycin Z counteracted this inhibition but did not counteract aberrant cell-wall formation. These results indicate that chitin synthesis stimulation is the key step in Rylux BSU and congo red inhibition and support the idea that increase in chitin synthesis represents a compensatory response to damaged cell-wall structure. As Rylux BSU and congo red bind to newly synthesized chitin, further damage is caused in the wall and the response works in this case contrariwise. Nikkomycin Z breaks this vicious circle by counteracting the chitin synthesis stimulation.

Comparison of chitin content in the apical and distal parts of fungal hyphae in Basidiobolus ranarum, Neurospora crassa and Coprinus sterquilinus.

Primary cell wall is synthesized in the growth zone of hyphal apex in fungi and rigidified during maturation along the newly formed hypha. Cross-linking of cell-wall components and self-assembly of individual polysaccharide chains into microfibrils are supposed to be involved in the rigidification process. We determined the relative chitin content in the cell wall of hyphal tips and distal walls of three fungal species and demonstrated a general increase in relative chitin content in mature cell walls. Thus, this increase can be supposed to raise cell-wall rigidity as the principal role of chitin in the determination of cell-wall rigidity is beyond doubt.

Signalling towards cell wall synthesis in budding yeast.

The budding yeast Saccharomyces cerevisiae has long proved to be a very useful model in cell biology. Its cell morphology is established and maintained at least in part by the cell wall, a rigid but dynamic structure that affords mechanical protection. Although fungal cell walls represent an unique phenomenon, recent progress in research has shown striking parallels between yeast and mammalian cells in the area of cell morphogenesis and proliferation. Further studies promise to shed common light on the processes of cell morphogenesis including the intersections with proliferation control. This review focuses on the recent progress in this promising area in the yeast Saccharomyces cerevisiae. The process of cell wall synthesis in Saccharomyces cerevisiae was reviewed by several authors recently. Briefly, the cell wall represents a complex structure of cross-linked chitin, beta-(1,6)-d-glucan, beta-(1,3)-D-glucan and mannoproteins. Chitin and beta-(1,3)-D-glucan are synthesized by enzymatic complexes at the cell membrane and extruded into the periplasmic space, mannoproteins are synthesized along the yeast secretory pathway, and the site of beta-(1,6)-D-glucan synthesis is still unknown. The principal motif which interconnects individual cell wall constituents was recently identified by Kollár et al. The mechanisms of cross-linking of the polymers in the wall remain unknown, however. Recently, nevertheless, substantial progress has been achieved in understanding the signalling pathways which target the cell wall construction.

Effect of the fluorescent brightener Rylux BSU on the cell wall chitin content in Basidiobolus ranarum.

In Basidiobolus ranarum an artificial cell dimorphism was found if cultivated in presence of Rylux BSU previously. We have found an increase of glucosamine content in purified cell walls of Basidiobolus ranarum grown in presence of Rylux BSU in SGA. The relative increase in glucosamine content did correspond with the increase of Rylux BSU present in SGA. The results are discussed with the conclusion that not only the chitin synthesis but also the mechanisms of polarized growth are influenced if Basidiobolus ranarum is cultivated in presence of Rylux BSU.

The fluorescence brightener Rylux BSU induces dimorphism in Basidiobolus ranarum.

The fluorescence brightener Rylux BSU (RBSU) showed an affinity for polysaccharide components of cell walls and accumulated in the extension zones of hyphal apices in Basidiobolus ranarum. It inhibited the polarized growth of mycelial hyphae and induced isotropic growth resulting in spherical thick-walled cells up to 456 microm in diameter. On the inner cell wall surface, massive protuberances were formed. The cell wall and protuberances were positive in PAS and the Grocott method and stained with fluorochromes Blankophor BA, Calcofluor, Uvitex 2B, Rylux BSU and FITC-labeled WGA- and ConA-lectins. The WGA-FITC fluorescence intensity of the wall's outermost layer, if not connected with neighbouring cells, and the fluorescence intensity of the innermost layer and of some protuberances mainly in their apical parts were on the average twice higher than the fluorescence intensity of the remaining wall material. RBSU binding to the cell wall material was stable. The process of converting from polarized to isotropic growth was reversible, depending upon contact with RBSU-containing medium. Repeated transfers of cells from RBSU-containing medium to an RBSU-free medium resulted in the development of apical swollen dumbbell-shaped cells.

[Quantitative determination of yeast in sputum--a direct microscopy method]. / Kvantitativní stanovení kvasinek ve sputu--prímá mikroskopická metoda.

The authors describes a fluorescence microscopic method using fluorochrome Blankophor (Bayer) which binds to chitin of the cell walls of yeasts and filamentous fungi. The authors processed, using this method, 50 specimens of sputum and compared the results with those of cultivation examinations. In five instances, where cultivation was negative by the microscopic method large numbers of fungi were detected. The method is suitable for quantitative assessment of yeasts and other fungi in sputum and other clinical specimens (urine, irrigation of cavities etc.). The author discusses the interpretation of microscopic findings.