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УЛЬТРАСТРУКТУРА СТАРЕЮЩИХ И ОБЕЗВОЖЕННЫХ КЛЕТОК CRYPTOCOCCUS NEOFORMANS
''Степанова А.А. (зав. лаб.)*, ''Васильева Н.В. (директор института, зав. кафедрой), 2Ямагучи М. (главный специалист), 2Чибана Х. (профессор)
1 НИИ медицинской микологии им. П.Н. Кашкина, СевероЗападный государственный медицинский университет им. И.И. Мечникова, Санкт-Петербург, Россия; 2 Центр исследований по медицинской микологии, Университет г. Чиба, Япония
© Коллектив авторов, 2016
Методами световой, сканирующей и трансмиссионной электронной микроскопии были изучены клеточные и субклеточные аспекты морфогенеза и старения клеток 16 штаммов Cryptococcus neoformans. Показано, что основная модель морфогенеза клеток в пределах культуры протекает по типу «веера». Детально изучен процесс старения дрожжевых клеток на ультраструктурном уровне. Впервые представлены данные об ультраструктуре обезвоженных дрожжевых растущих in vitro клеток культур C. neoformans.
Ключевые слова: Cryptococcus neoformans, морфогенез, обезвоженные и стареющие дрожжевые клетки, in vitro, ультраструктура
ULTRASTRUCTURE OF SENESCENT AND DESICCATED CELLS OF CRYPTOCOCCUS NEOFORMANS
'Stepanova A.A. (head of the laboratory), 'Vasilyeva N.V. (director of the institute, head of the chair), 2yamaguchi M. (grand-fellow), 2Chibana H. (associated professor)
i Kashkin Research Institute of Medical Mycology of Northwestern State Medical University named after I.I. Mechnikov, St. Petersburg, Russia; 2 Medical Mycology Research Center, Chiba University, Chiba, Japan
© Collective of authors, 2016
Using light, scanning and transmission microscopy, the cellular and sub-cellular aspects of pattern of morphogenesis and senescence of the 16 strains cells of C. neoformans were investigated. It was shown that the general pattern of cell morphogenesis inside culture was «fan-like». The process of yeast cell senescence on ultrastructural level was studied in detail. For the first time the data about the ultrastructure of desiccated yeast cells of in vitro growing C. neoformans cultures were presented.
Key words: Cryptococcus neoformans, desiccated and senescent yeast cells, in vitro, morphogenesis, ultrastructure
Контактное лицо: Степанова Амалия Аркадьевна, тел.: (812) 303-51-40
INTRODUCTION
Senescence is the final stage of cell morphogenesis. Before this the cellular and sub-cellular aspects of fungal cell senescence was investigated on several species of fungi imperfecti [Molitoris H.P. // Veroff. Inst. Meeresforsch. Bremerhaven. - 1974. - Vol. 5], discomycetes [Kamaletdinova F.I., Vasilyev A.E. Cytology of Discomycetes, 1982.], basidiomycetes [Stepanova A.A., Vasilyev A.E. Ultrastuctural bases of mushroom morphogenesis, 1994], euromycetes from genus Aspergillus [1] and Trichophyton [2]. Cellular and sub-cellular aspects of this process of C. neoformans were unknown, but their knowledge may be very important because they can be considered as a «control» for future determination in vitro and in v^ experiments on the cellular mechanisms of influence of host cells and drugs on the structure of this fungal cells.
The purpose of the work was to study the cellular pattern in vitro morphogenesis, the ultrastructure of senescent and desiccated C. neoformans cells.
MATERIALS AND METHODS
16 strains (PKnrY-719, 881, 1063, 1067, 1085, 1088, 1091, 1093, 1095, 1105, 1165, 1166, 1175, 1176, 1178, 1180) of C. neoformans (Russian Collection of Pathogenic Fungi, Kashkin Research Institute of Medical Mycology) were isolated from patients and cultured in vitro (Sabouraud's agar, 3 days after sowing, 37 °C). Virulence of strains was determined previously [3] (LD-50 after intravenous injection varied from 8-102 to 1407 cells/mouse). Strains with LD-50 102-104 were considered as strongly virulent, with LD-50 105-106 - moderately virulent and with LD-50 more than 106 - low virulent. The diameter of mature in vitro growing C. neoformans cells was determined under the light-microscope using Indian ink. These measurements were carried out on 100 mature cells.
For light and electron microscopy the pieces of solid nutrient medium with part of fungal colonies was fixed with using glutaraldehyde-osmium in 0,1 M cacodylate buffer according the methods which we published previously [3]. For light microscopy semi-thin sections (3-5 ^m) were cut on the Pyramitome 1180 (LKB) and colored with toluidine blue. Ultrathin sections we cut on Ultratome LKB V, stained with uranyl acetate (10 minute) and lead citrate (5 minute) and examinated under JEM 100 CX. For scanning electron microscopy samples were fixed in 3 % glutaraldehyde (on 0,1 M cacodylate buffer), dehydrated in series of ethanol (30°^70°), critical-point dried for 15 min, and then evaporated with gold and investigate under JSM 35. The measurements of the mean diameter of desiccated cells and its walls thickness under TEM were carried out on 20-25 cells of C. neoformans with using program Statistics v 6,0.
RESULTS
Cellular aspects of senescence. In the light microscope at the cross semi-thin epoxy sections of C. neoformans colonies, colored with toluidine blue, was visible alternation of longitudinal and radial oriented dense (Fig.1 a, 1, 2) and the light areas (Fig.1 a, 3), which organized as a result of differences in the density of concentration of the fungal yeast cells.
Dense zones of two types: 1) a wedge-shaped (expanded to the margin of the colonies (Fig.1 a, 1) and extending
■
from the center of the colony to the marginal part, and 2) more thin (Fig.1 a, 2) young strips with the same thickness of radially oriented aggregation of yeast cells with different lengths, as a rule, localized in the lower half of the colony. Between these dense zones of yeast cells concentration was localized more light wedge-shaped area (Fig.1 a, 3), in which the density of the yeast cells was noticeably lower than that of the above. Alternation of dense (Fig.1 b, 1) and light areas (Fig.1 b, 2) were clearly revealed and on a cross sections of semi-thin epoxy sections.
The study of fungal cultures in the scanning electron microscope (Fig.1 c, d ) also revealed the presence of the above described areas. The dense areas (Fig.1 c) was represent the budding, growing and mature yeast cells, while the light - on different stages of senescence, dead and desiccated (Fig.1 d ) cells. The described cellular pattern of in vitro C. neoformans morphogenesis we term as «fanlike».
Desiccated (dormant) yeast cells. The origin of desiccated yeast cells for in vitro growing C. neoformans may be possible in 2 ways: 1. directly from the daughter cell after its separation from the mother one; 2. from mature yeast cells. Scanning electron microscopy shows evidence of the presence part of polysaccharide capsule (Fig.1 d, e) around the developing desiccated yeast cells. The formation of this type of cells was accompanied with its dehydration, decrease in size and encapsulated after which they transit in the state of dormancy and represent the infection propagules. The sizes of desiccated yeast cells varied from 2.60 to 3.67 ^m depending on the strain. (Table). Desiccated yeast cells we identify in 11 from 16 investigated strains cultures (70 % from the total number of studied strains). From these 11 strains - 4 were strong (PKnrY - 1088, 1105, 1166, 1175), 3 medium (PKnry -1063, 1067, 1176) and 3 low virulent (PKnrY - 719, 881, 1165, 1178).
Table
The mean values of diameter and cell walls (^m) of desiccated
and mature yeast cells of C. neoformans
Strain Diameter of desiccated yeast cells Cell wall thickness of desiccated yeast cells Diameter of mature yeast cells Cell wall thickness of mature yeast cells
719 2,90 0,45 6,32 0,40
881 2,60 0,57 6,00 0,37
1063 3,00 0,60 5,82 0,43
1067 3,50 0,50 7,43 0,18
1088 2,60 0,43 5,10 0,21
1105 3,00 0,39 7,28 0,21
1165 3,67 0,70 6,75 0,12
1166 3,30 0,56 6,65 0,14
1175 2,90 0,47 6,57 0,42
1176 3,00 0,59 5,85 0,21
1178 3,50 0,48 7,64 0,23
Comparison of the mean diameter of desiccated and mature yeast cells of investigated C. neoformans cultures was revealed (Table) that they are in 2 times more less that mature cells of the same strain. The thickness of the cell wall of desiccated and mature yeast cells does not vary (strains 719 and 1175) or in several (from 1,4 to 5,8) times greater for the first one. In fig. 2 we presented the diagram, which illustrated the significant differences in the mean value of cell diameter (a) and wall thickness (b) in mature and desiccated yeast cells of C. neoformans.
As can be seen in the scanning electron microscope, dehydration of such types of cells was accompanied by the separation of their cell wall from their polysaccharide capsules, the upper half of which completely undergo lysis during which the fungal cells became naked (C. neoformans strip-tease) (Fig.1 d, e). The completely developed desiccated yeast cells have a smooth surface (Fig.1 e).
DYC MYC " Five MYC
H »54 0»* Ah
P = 0.0000001 p = 0.0003
Fig. 2. Diagram which illustrated the mean value of cell diameter (a) and wall thickness (b) in mature and desiccated yeast cells of C. neoformans. DYC, desiccated yeast cells, MYC, mature yeast cells.
Thus, it is obvious that desiccated yeasts cells (infectious propagules) could leave the fungal colony and distribute by air flows. There was evidence of presence in senescent parts of colonies the devastated «half» from the once full developed polysaccharide capsules (Fig.1 f ).
The cytosol of mature desiccated yeast cells was so dense that the nucleus and cytoplasm components were identified with difficulty (Fig.1 g, h, k) or not detected (Fig.1 j ). It was possible to identify only storage substances in the form of variable morphology fibrosinous bodies (Fig.1 g, l), lipid inclusions (Fig.1 m) and glycogen granules (Fig.1 j, m). Another distinctive feature of the ultrastructure of these cells was the presence of a thick (Table; Fig.1 g-m, Fig.3 d-f) cell wall. The cell wall could be a single-layer dark homogeneous (Fig.3 d), two-layer dark with a more subtle and loose outer layers (Fig.3 e), and three-layered with a thin dark homogeneous inner (Fig.3 f, 1), less dense thick homogenous middle (Fig.3 f, 2) and thin dark fibrillar outer layers (Fig.3 f, 3). These types of the cell wall ultrastructure correspond to the three types of structure of bud scar (an integral part of the cell wall, Fig.3 a-c, arrow) with 3 different anatomical patterns.
Subcellular aspects of senescence. The senescence processes of the C. neoformans cells occurred after the formation of large light central vacuole (Fig.3 g, m) and were accompanied by a reduction of cytosol volume, an increase in electron density, and the reduction of organelles and free ribosomes numbers. Frequently we observed the local autolysis, during which areas of cytosol with organelles, free ribosomes and storage substances occur into the vacuolar contents by formation of tonoplast invaginations (Fig.3 m, arrows), which then isolated inside vacuoles as vesicles which undergo disruption. Negative contrast frequent was typical for nucleus and membranes of organelles (Fig.3 g). It was important to note that the first senescence changes in some cells occur in the nucleus structure (Fig.3 h, arrow), in other mitochondria (Fig.3 i, arrow), and in some cases in both types of these cells components.
The nucleoplasm was fully clarified (Fig.3 j, arrow). Chromatin was organized in dark homogeneous shapeless dark masses of variable morphology, which localized near the nucleolar envelope (Fig.3 j). Nucleolus decreased in size, granular component disappeared (degranulation), became
Fig. 1. Semi-thin longitudinal (a), transverse (b) sections and scanning electron micrographs of 3-old cultures of C. neoformans. Ultrathin sections (g-m) of desiccated yeast cells of C. neoformans. CW, cell wall; DY, desiccated yeast; FB, fibrosinous bodies; G, glycogen; LI, lipid inclusion; M, mitochondrion; N, nucleus; PC, polysaccharide capsule. The numerations (b) indicate the dark and light zones in fungal colonies. a, b, strain PKnrY-1178; c-f, strain PKnrY-1063; g, strain PKnrY-719; h, strain PKnrY-1063; j, strain PKnrY-1165; k, strain PKnrY-1088; l, strain PKnrY-1178; m, strain PKnrY-1105. Bars: a - 2 mm, b - 1 mm, c, e, f - 3 |m, d - 6 |m,
g - m - 0,6 |m.
Fig. 3. Ultrastructure of bud scar (a-c), cell walls (d-f) of mature desiccated yeast cells and ultrastructure of senescent cells of C. neoformans (g-w). CW, cell wall; G, glycogen; LI, lipid inclusion; M, mitochondrion; N, nucleus; Nu, nucleolus; BS, bud scar; P, periplasmic space; Pl, plasma membrane; T, tonoplast. The numerations (d-f) indicate the cell wall layers. a, d, n, strain PKnrY-1080; b, e, strain PKnrY-1165; c, f, strain PKnrY-1067; g, strain PWTY-719; h, o, strain PKnrY-881; i, k, strain PKnrY-1091; l, m, strain PKnrY-1105; q, r, u, strain PKnrY-1180; p, s, t, strain PKnrY 1176; v, strain PKnrY-1063; w, strain PKnrY-1093. Bars: a, d-f -0,2 |m; b, c - 0,5 |m; g-i, l, n-f, s - 1 |m; j, k, m - 1,5 |m; t, u, v, w - 0,5 |m.
more contrast and irregular in shape. Disappearance of nucleolus takes places after the complete lysis of chromatin (Fig. 3 l ). Periplasmic space was more wide, lighter and irregular (Fig.3 k).
The grouped mitochondrial orientation disappeared. In the content of senescent mitochondria often the large dark irregular in shape zones was revealed (Fig.3 i, arrow). Contents of mitochondrial cristae and between the outer and inner membranes became lighter (Fig.3 j). Then cristae undergo swelling, reduced in size and number. In some mitochondria matrix clarify locally, while in other, they disappeared completely. Then cristae also disappeared. These changes were accompanied by mitochondrial swelling, whose form became irregular. After the complete cristae destruction and internal envelope membranes, these organelles transformed into small, irregular shaped vacuoles (Fig.3 j).
Tonoplast of large and small vacuoles later broke (Fig.3 n, arrows), which resulted in the release of hydrolytic enzymes in the cytoplasm and subsequent autolysis of the cell. Despite the fact that in senescent cells the first changes were observed in the structure of the nucleus and mitochondria, however, they, together with plasma membrane (Fig.3 l) and storage substances (Fig.3 o, p) long time identified in cells without cytosol.
Cisterns of endoplasmic reticulum became more irregular and disintegrated into numerous vesicular elements. Membranes of peroxisomes became more undulate; in its matrix local light zones were revealed. Soon the peroxisome membrane broke and its contents appeared in the cytoplasm.
In some cells, granules of glycogen reduced in number and subsequently undergo hydrolysis. In these parts of cytosol became obvious in the light area, as a rule, imitating the form of glycogen's rosettes. In other cells glycogen was detected in the later stages of senescence - in cells without cytosol and organelles (Fig.3 o).
Lipids inclusions, fibrosinous bodies, polyphosphate granules in vacuoles, cytosol and mitochondria decreased in number and size. Lipid inclusions were often observed in completely dead cells (Fig.3 p).
Plasma membrane remained in the cell until the complete destruction of its contents (Fig.3 p) and obtained irregular shape (Fig.3 n), gradual reduced in length and moved to the central part of the cell (Fig.3 k), where broke up into fragments (Fig.3 l), which undergo subsequent vesiculation. Exactly after its destruction the process of cell death comes to the end.
Typically for the cell wall thinning (Fig.3 o, p, q, r) and contrast reducing led to the fact that it lost its form and became irregular (Fig.3 r, s). These changes began and took place simultaneously with changes in the internal contents of the cells. In the walls of cells, practically without content appear local gaps (Fig.3 s, arrows), leading to its break-up into multiple fragments. The cell walls of kept for long time typical for its size and structure (Fig.3 t).
Destructive changes in ultrastructure of polysaccharide capsules occurred synchronously with those taking place in the structure of the cell wall. At that the microfibrills (Fig.3 u, v, w, arrows) of polysaccharide capsules became rare, short, poorly contrasting, strongly deformed and eventually completely revealed the cell wall.
DISCUSSION
It is known that the infectious propagules for many human fungal pathogens to be spores, but some author [4] hypothesized that in case with C. neoformans it may be desiccated yeast cells also. Another numerous studies [5; Neilson J.B., et al.ll Infect. Immun. - 1977. - Vol. 17; Casadevall A., Perfect J. Cryptococcus neoformans, 1998; et al.] reveal that small, desiccated, encapsulated C. neoformans yeast cold be recovered from soil. In some work specific role of spores in the pathogenesis of this fungi was not addressed [Sukroongreung S., et al. // Med. Mycol. - 1998.
- Vol. 36; Zimmer B.L., et al. // Mycopathologia. - 1984. -Vol. 85]. Ruiz A.R. et al. [Ruiz A., et al. ll Infect. Immun.
- 1981. - Vol. 31] reported that 7.5% of the viable airbone the C. neoformans cells in aerosolized pigeon droppings were from 1.1 to 3.3 ^m in diameter, what intimate with our data about diameter of desiccated yeast of this fungal species. At the same time, as it was known [3; Ruiz A., et al. ll Infect. Immun. - 1981. - Vol. 31], the size of the mature C. neoformans yeast cells was bigger and range from 4 to
20 [!M.
According to our unpublished data [6], some of the studied strains of C. neoformans are capable to develop desiccate yeast cells in the lungs and brain of murine after 7 days of experiments. In our opinion they can be initiated the secondary infectious process in host tissue and ability of strains quickly form large number of «generative population» in vitro and in vivo can be considered as one of the factors of its potential virulence.
We expect that C. neoformans «fan-like» cellular in vitro pattern of morphogenesis may be typical for another species of yeast. The whole described subcellular aspects of C. neoformans senescence was very similar to the case presented for discomycetes [Kamaletdinova F.I., Vasilyev A.E. Cytology of discomycetes, 1982], basidiomycetes [3], species from genus Aspergillus [Stepanova A.A., Vasilyev A.E. Ultrastuctural bases of mushroom morphogenesis, 1994] and Trichophyton [2], what be evidence of some conservatism in this final stage of fungal morphogenesis.
RESUME
1. The described cellular pattern of in vitro C. neoformans morphogenesis we term as «fan-like». In the lower part of colonies new strips composed from developing yeast cells were formed during colony grows.
2. A characteristic ultrastructural feature of senescing C. neoformans cells was the fact that in some in vitro growing cells first destructive processes affect the nucleus, in other
- mitochondria, and in third - nucleus and mitochondria. Subsequent changes to the structure of the cells were quite similar.
3. The completely developed desiccated yeast cells in vitro have a smooth surface and range the size from 2,60 to 3,67 ^m depending on the strain numeration. They have ultrastructure of dormant cells and possess with thick cell walls and dark cytosol.
REFERENCES
1. Stepanova A.A., Sinitskaya I.A. Ultrastructural aspects of cells senescence of some Aspergillus species // Проблемы медицинской микологии. - 2009. - Т. 11, №4. - P. 24-29.
2. Savitskaya T.I., Stepanova A.A., Sinitskaya I.A., Krasnova E.V. Ultrastructural aspects of senescence of the vegetative mycelium cells of some Trichophyton species // Проблемы медицинской микологии. - 2011. - Vol. 13, №2. - P. 106.
3. Vasilyeva N.V. The pathogenic factors of Cryptococcus neoformans and its role in pathogenesis of cryptococcosis: Dissert. of Ph.D. - St. Petersburg, 2005. - 340 p.
4. Velagapudi R., Hsuch Y-P., Geunes-Boyer S., et al. Spores as infection propagules of Cryptococcus neoformans // Infect. Immun. - 2009. - Vol. 77. - P. 4345-4355.
5. Giles S.S., Taylor R.T., Dagenais R.T., et al. Elucidating the pathogenesis of spores from the human fungal pathogen Cryptococcus neoformans // Infect. Immun. - 2009. - Vol. 77. - P. 3491-3500.
6. Stepanova A.A., Vasilyeva N.V., Yamaguchi M., et al. Ultrasrtuctural patterns of the interactions between the murine lung macrophages and the yeast cells of the Cryptococcus neoformans with different virulence // In press.
Поступила в редакцию журнала 07.11.2016
Рецензент: Н.П. Елинов
