Our basic cell biology research was aimed at investigating the effect on eukaryotic cells of the sudden loss of the F-actin cytoskeleton. Cells treated with latrunculin A (LA) in yeast extract peptone dextrose (YEPD) medium were examined using phase-contrast and fluorescent microscopy, freeze-substitution, transmission and scanning electron microscopy, counted using a Bürker chamber and their absorbance measured. The cells responded to the presence of LA, an F-actin inhibitor, with the disappearance of actin patches, actin cables and actin rings. This resulted in the formation of larger spherical cells with irregular morphology in the cell walls and ultrastructural disorder of the cell organelles and secretory vesicles. Instead of buds, LA-inhibited cells formed only 'table-mountain-like' wide flattened swellings without apical growth with a thinner glucan cell-wall layer containing β-1,3-glucan microfibrils. The LA-inhibited cells lysed. Actin cables and patches were required for bud formation and bud growth. In addition, actin patches were required for the formation of β-1,3-glucan microfibrils in the bud cell wall. LA has fungistatic, fungicidal and fungilytic effects on the budding yeast Saccharomyces cerevisiae.
- MeSH
- aktiny antagonisté a inhibitory MeSH
- antifungální látky farmakologie MeSH
- bicyklické sloučeniny heterocyklické farmakologie MeSH
- mikrobiální viabilita účinky léků MeSH
- mikroskopie MeSH
- počet mikrobiálních kolonií MeSH
- Saccharomyces cerevisiae cytologie účinky léků fyziologie MeSH
- Saccharomycetales cytologie účinky léků fyziologie MeSH
- thiazolidiny farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
AIMS: Limited aeration has been demonstrated to cause slowdown in proliferation and delayed budding, resulting eventually in a unique unbudded G2-arrest in the obligate aerobic pathogenic yeast Cryptococcus neoformans. Also, the ability to adapt to decreased oxygen levels during pathogenesis has been identified as a virulence factor in C. neoformans. The aim of this study was to identify and characterize genes that are necessary for the proliferation slowdown and G2-arrest caused by limited aeration. METHODS: Random mutants were prepared and screened for lack of typical slowdown of proliferation under limited aeration. The CNAG_00156.2 gene coding for a zinc-finger transcription factor was identified in mutants showing most distinctive phenotype. Targeted deletion strain and reconstituted strain were prepared to characterize and confirm the gene functions. This gene was also identified in a parallel studies as homologous both to calcineurin responsive (Crz1) and PKC1-dependent (SP1-like) transcription factors. RESULTS: We have confirmed the role of the cryptococcal homologue of CRZ1/SP1-like transcription factor in cell integrity, and newly demonstrated its role in slowdown of proliferation and survival under reduced aeration, in biofilm formation and in susceptibility to fluconazole. CONCLUSIONS: Our data demonstrate a tight molecular link between slowdown of proliferation during hypoxic adaptation and maintenance of cell integrity in C. neoformans and present a new role for the CRZ1 family of transcription factors in fungi. The exact positioning of this protein in cryptococcal signalling cascades remains to be clarified.
- MeSH
- anaerobióza MeSH
- antifungální látky farmakologie MeSH
- biofilmy růst a vývoj MeSH
- Cryptococcus neoformans účinky léků genetika růst a vývoj MeSH
- delece genu MeSH
- flukonazol farmakologie MeSH
- kontrolní body buněčného cyklu genetika MeSH
- mikrobiální viabilita MeSH
- proteiny Caenorhabditis elegans genetika MeSH
- transkripční faktory genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: This basic research aimed to investigate the effects of the actin inhibitor latrunculin A (LA) on the human pathogen Cryptococcus neoformans, by freeze-substitution (FS) and electron microscopy (EM), to determine whether the actin cytoskeleton can become a new antifungal target for inhibition of cell division. METHODS: Cells treated with LA for 20 h in yeast-extract peptone dextrose medium were investigated by phase-contrast and fluorescent microscopy, FS and transmission EM, counted in a Bürker chamber and the absorbance was then measured. RESULTS: The disappearance of actin patches, actin cables and actin rings demonstrated the response of the cells of C. neoformans to the presence of the actin inhibitor LA. The removal of actin cables and patches arrested proliferation and led to the production of cells that had ultrastructural disorder, irregular morphology of the mitochondria and thick aberrant cell walls. Budding cells lysed in the buds and septa. CONCLUSION: LA exerts fungistatic, fungicidal and fungilytic effects on the human pathogenic yeast C. neoformans.
- MeSH
- aktiny antagonisté a inhibitory MeSH
- antifungální látky farmakologie MeSH
- bicyklické sloučeniny heterocyklické farmakologie MeSH
- buněčná stěna účinky léků metabolismus MeSH
- buněčné dělení účinky léků MeSH
- Cryptococcus neoformans účinky léků metabolismus MeSH
- elektronová mikroskopie metody MeSH
- fluorescenční mikroskopie metody MeSH
- kryptokokóza farmakoterapie metabolismus mikrobiologie MeSH
- lidé MeSH
- mikrofilamenta účinky léků metabolismus MeSH
- proliferace buněk účinky léků MeSH
- thiazolidiny farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The F-actin cytoskeleton of Cryptococcus neoformans is known to comprise actin cables, cortical patches and cytokinetic ring. Here, we describe a new F-actin structure in fungi, a perinuclear F-actin collar ring around the cell nucleus, by fluorescent microscopic imaging of rhodamine phalloidin-stained F-actin. Perinuclear F-actin rings form in Cryptococcus neoformans treated with the microtubule inhibitor Nocodazole or with the drug solvent dimethyl sulfoxide (DMSO) or grown in yeast extract peptone dextrose (YEPD) medium, but they are absent in cells treated with Latrunculin A. Perinuclear F-actin rings may function as 'funicular cabin' for the cell nucleus, and actin cables as intracellular 'funicular' suspending nucleus in the central position in the cell and moving nucleus along the polarity axis along actin cables.
- MeSH
- aktiny analýza MeSH
- bicyklické sloučeniny heterocyklické farmakologie MeSH
- buněčné jádro ultrastruktura MeSH
- Cryptococcus neoformans účinky léků fyziologie ultrastruktura MeSH
- dimethylsulfoxid farmakologie MeSH
- elektronová mikroskopie MeSH
- faloidin analogy a deriváty MeSH
- fluorescenční mikroskopie MeSH
- mikrofilamenta ultrastruktura MeSH
- mikrotubuly účinky léků MeSH
- modulátory tubulinu farmakologie MeSH
- mořské toxiny farmakologie MeSH
- nokodazol farmakologie MeSH
- rhodaminy MeSH
- scavengery volných radikálů farmakologie MeSH
- thiazolidiny farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Cryptococcus neoformans is an obligate aerobic pathogenic yeast causing lung infection typically followed by spread to the central nervous system. During pathogenesis, it relies on well-established virulence factors. This review focuses on the emerging role of cryptococcal adaptation to hypoxia in pathogenesis. METHODS AND RESULTS: We examined the MedLine database for information on the cryptococcal hypoxia response. While several recent papers describe components of two presumable hypoxia-sensing pathways including description of their target genes, a link of this system to the hypoxic tuning of proliferation is still missing. In addition, an interpretation of this knowledge in respect to the general picture of microbial pathogenesis is lacking. CONCLUSIONS: There seems to be a striking parallel between biofilm formation in bacteria, which results in chronic dormant infection with the potential for acute outbreaks, and the dormant state of primary infection followed by secondary outbreaks in C. neoformans. We propose a hypothesis that cryptococcal response to hypoxia might be the driving force for developing a state of dormant infection which is characterized by slowed proliferation and extensive changes in transcriptome and phenotype. This state enables C. neoformans to survive in host and possibly develop life-threatening acute outbreaks later. Hence, conventional well-aerated planktonic culture is not a good in vitro model for studying the pathogenesis of infection and we advocate the development of a more adequate model. Our further conclusion is that the ability of the immune system and antifungal agents to cope with hypoxia-adapted cells is crucial for the successful eradication of cryptococcal infection.
