Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections in immunocompromised individuals. It is surrounded by three concentric structures that separate the cell from the extracellular space: the plasma membrane, the cell wall and the polysaccharide (PS) capsule. Although several studies have revealed the chemical composition of these structures, little is known about their ultrastructural organization and remodeling during C. neoformans budding events. Here, by combining the latest and most accurate light and electron microscopy techniques, we describe the morphological remodeling that occurs among the capsule, cell wall and plasma membrane during budding in C. neoformans. Our results show that the cell wall deforms to generate a specialized region at one of the cell’s poles. This region subsequently begins to break into layers that are slightly separated from each other and with thick tips. We also observe a reorganization of the capsular PS around the specialized regions. While daughter cells present their PS fibers aligned in the direction of budding, mother cells show a similar pattern but in the opposite direction. Also, daughter cells form multilamellar membrane structures covering the continuous opening between both cells. Together, our findings provide compelling ultrastructural evidence for C. neoformans surface remodeling during budding, which may have important implications for future studies exploring these remodeled specialized regions as drug-targets against cryptococcosis.
To perform in-vitro studies with Paracoccidioides brasiliensis yeast cells it is necessary to avoid the presence of clumps of cells while maintaining their integrity. Because of the multiple budding type of growth, the bud cells are always attached to the mother cell and the yeast cells keep growing, resulting in the formation of large clumps. In order to obtain free cells, the cultures are usually sonicated. The present study shows that sonication induces lesions in a significant number of cells, as evaluated by labelling of the cells with acridine orange and Janus green vital dyes. In some cases labelling was initially observed in only one cell of the clump; however, the other cells also became labelled after a few minutes. These observations were confirmed by scanning and transmission electron microscopy of treated cells. Colony forming units (c.f.u.) on BHI plates also confirmed the decrease in cell viability following sonication.
12 13 † These authors share the senior position 14 15 Susana Frases +55 (21) 3938-6593 susanafrases@biof.ufrj.br 16 Bruno Pontes +55 (21) 3938-6465 bpontes@icb.ufrj.br 17 18 Abstract 21 22Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections in 23 immunocompromised individuals. It is surrounded by three concentric structures that 24 separate the cell from the extracellular space: the plasma membrane, the cell wall and the 25 polysaccharide capsule. Although several studies have revealed the chemical composition 26 of these structures, little is known about their ultrastructural organization and remodeling 27 during C. neoformans budding event. Here, by combining the state-of-the-art in light and 28 electron microscopy techniques we describe the morphological remodeling that occurs 29 synergistically among the capsule, cell wall and plasma membrane during budding in C. 30 neoformans. Our results show that the cell wall deforms to generate a specialized budding 31 region at one of the cell's poles. This region subsequently begins to break into layers that 32 are slightly separated from each other and with thick tips. We also observe a reduction in 33 density of the capsular polysaccharide around these specialized regions. Daughter cells 34 present a distinct spatial organization, with polysaccharide fibers aligned in the direction 35 of budding. In addition, to control the continuous openings between mother and daughter 36 cells, the latter developed a strategy to shield themselves by forming multilamellar 37 membrane structures in conjunction with their capsules. Together, our findings provide 38 compelling ultrastructural evidence for a dynamic C. neoformans surface remodeling 39 during budding and may have important implications for future studies exploring these 40 remodeled specialized regions as drug-targets against cryptococcosis. 41 65The success of the infection is based on the ability of the fungus to evade the 66 host's immune system. During its evolution, Cryptococcus spp. developed several 67 adaptation mechanisms, known as virulence factors. Some examples are: (I) melanin 68 production and cell wall remodeling (resistance to cell-mediated death and 69 immunomodulation) (Doering et al.
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