CC BY
16УДК 57.012.4:582.282.23
УЛЬТРАСТРУКТУРНОЕ ИССЛЕДОВАНИЕ РАННИХ СТАДИЙ ТРАНСФОРМАЦИИ ДРОЖЖЕВОЙ ФОРМЫ В ГИФАЛЬНУЮ У CRYPTOCOCCUS NEOFORMANS VAR. GRUBII IN VIVO
''Степанова A.A. (зав. лаб.)*, ''Васильева Н.В. (директор института, зав. кафедрой), 2Ямагучи M. (адъюнкт-профессор), 2Чабана Х. (ассистент профессора), 'Босак И.А. (с.н.с.)
1 НИИ медицинской микологии им. П.Н. Кашкина, СевероЗападный государственный медицинский университет им. И.И. Мечникова, Санкт-Петербург, Россия; 2Центр исследований по медицинской микологии, Университет г. Чиба, Япония
© Коллектив авторов, 2017
Проведены ультраструктурные исследования трансформации дрожжевых клеток C. neoformans var. grubii в монокариотическое ги-фальное состояние в мозге мышей через 7 дней от начала эксперимента. Выявлено 2 стадии в трансформации материнской дрожжевой клетки в гифальную. На первой стадии из содержимого материнской клетки в содержимое гифального выроста переходит цитозоль со свободными рибосомами, пузырьки, мелкие вакуоли, митохондрии и липидные включения. Во вторую стадию дифференциации гиф (после появления, последующего деления и увеличения числа ядер), синтез цитозоля, свободных рибосом, секреторных пузырьков, пролиферация митохондрий с формированием гигантской органеллы, синтез запасных липидов, клеточной стенки и полисахаридной капсулы, имели место. Одиночные интерфазные ядра равномерно расположены в монокариотической гифе. Впервые обнаружили, что клетки гиф монокариотического мицелия, как и материнские дрожжевые клетки, покрыты широкой полисахаридной капсулой идентичной структуры.
Ключевые слова: Cryptococcus neoformans var. grubii, мозг мышей, монокариотический мицелий, ультраструктура
ULTRASTRUCTRAL INVESTIGATIONS OF EARLY STAGES OF TRANSFORMATION OF YEAST FORM TO HYPHAL IN CRYPTOCOCCUS NEOFORMANS VAR. GRUBII IN VIVO
'Stepanova A.A. (head of the laboratory), 'Vasilyeva N.V. (director of the institute, head of the chair), 2yamaguchi M. (grand-fellow), 2Chibana H. (assistant professor), 'Bosak I. A. (senior scientific collaborator)
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, Japan
© Collective of authors, 2017
Контактное лицо: Степанова Амалия Аркадьевна, тел.: (812) 303-51-40
The ultrastructural investigations on the switching from yeast to monokaryotic hyphal state in mouse brain at 7 day experiments in C. neoformans var. grubii were carried out. It was found that 2 stages were present in switching of mother yeast cell to hyphal one. In the first stage, cytosol with free ribosomes, vesicles, small vacuoles, mitochondria and lipid inclusions passed from mother cells to hyphal cells. In the second stage of hyphal differentiation (after appearance, subsequent divisions and increasing the number of nuclei), the synthesis of cytosol, free ribosomes, secretory vesicles, proliferation of mitochondria with formation of giant organelle, synthesis of storage lipids, cell wall and polysaccharide capsule, apparently, took place. The single interphase nuclei regularly present in the monokaryotic hyphae. For the first time, we found that the hyphal monokaryotic cells to be coated with polysaccharide capsule with identical structure to that of the mother cells, but more thicker.
Key words: Cryptococcus neoformans var. grubii, monokaryotic mycelium, murine brain, ultrastructure
INTRODUCTION
Cryptococcus neoformans var. grubii is the opportunistic world-wide distributed human pathogen. More than 90% of infections worldwide are due to haploid strains of C. neoformans var. grubii, which possess the serotype A capsular epitope [1]. This species was isolated from soil, wood, rotting vegetables, fruits, fruit juices, dairy products, and pigeon excreta [2 and etc.]. It was a common cause of meningoencephalitis among the AIDS patients [3]. We previously investigated the pattern of ultrastructural changes of yeast cells of the strain PKOTY-1165 during in vitro ^ in vivo transition [4]. Light microscopic investigations of three day culture in vitro and 7 day experiments revealed formation of hyphal elements by yeast cells of last species in murine brain tissue [5]. Sexual cycle of C. neoformans var. grubii was investigated by Nielsen K. with coauthors in detail [Nielsen K., et al. // Infect. Immun. - 2003. -Vol. 71]. Since there were no reports about the cardinal process of transformation of yeast to hyphal form on the ultrastructural level in Cryptococcus species, the present work was undertaken to investigate this process.
MATERIALS AND METHODS
The strain (PKnTY-1165, Russian collection of pathogenic fungi) of Cryptococcus neoformans var. grubii Franzot, Salkin & Casadevall was isolated from spleen of HIV-infected patient and then was used in modeling of experimental cryptococcosis.
This strain was cultured in vitro on Sabouraud's agar for 3 days at 37° C. Virulence of strains was determined previously [5] (LD-50 after intravenous injection varied from 8T02 to M07 cells/mouse). The strain PKnrY-1165 characterized with LD50 < 106 cells/mouse, was considered as low virulent [5].
For modeling of experimental cryptococcosis we used mouse male with mass of body 18 - 20 g. The fungal suspension was made in sterile 0,9 % NaCl solution and then used for intravenously infection in mouse caudal vein with using 0,5 ml of this suspension.
For light and transmission electron microscopy (TEM), the pieces of mouse brain at 7 days of experiments were fixed with glutaraldehyde-osmium in 0,1 M cacodylate buffer according to the methods we published previously [Nielsen K., et al. // Infect. Immun. - 2003. - Vol. 71]. For light microscopy semi-thin sections (3-5 ^m) from epoxy blocks were cut on the Pyramitome 1180 (LKB), colored with toluidine blue and investigated under light microscope Leica LB2 (Leica Microsystem Inc, Germany). Ultrathin sections were cut on Ultratome LKB V, stained with uranyl acetate (10 minute) and lead citrate (5 minute)
nPQBflEMbl MEflHUHHCKOI/l MHKOflOrHH. 2017, T.19, №2
and examined under JEM 100 SX (JEOL, Tokyo, Japan).
RESULTS AND DISCUSSION
At 7 day of experiments in mouse brain we revealed compact aggregation of yeast cells spherical in form and variable in size (Fig. 1 a, arrow) which situated in different stages of development. The average sizes of mature yeast cells were 6,0 x 8,0 ^m. In the peripheral part of yeast cell aggregation the yeast cell forming hyphae can be sometimes recognized (Fig. 1 b). The form of the hyphae is tubular with variable diameter (from 1,4 to 2 ^m) and length (from 2,0 to 4,0 ^m). It was interesting that for another species from genus Cryptococcus - C. laurentii also developed monokaryotic mycelium at 7-10 days on malt extract agar [Kurtzman C.P. //Mycologia. - 1973. - Vol. 65]. According to the data in literature [WangP., et al. // Mol. Cell. Biol. -2000. - Vol. 20] sexual reproduction has been defined for all C. neoformans variation.
In the basal part of mother cell before starting filamentation, we revealed the interphase nucleus (Fig. 2 a) and in apical part opposite budding scar, with mean diameter 1,0 ^m, one large and several median in size vacuoles with light content and curved tonoplast. Previously, similar nucleolar and vacuolar topography we described for in vitro growing mother yeast cells of Cryptococcus albidus [6] before budding. Nucleus and vacuoles occupied the main cell volume. The ellipsoidal (1,5 x 2,0 ^m) nucleus contains moderate level of randomly distributed condensed chromatin and one large (0,5 ^m) spherical nucleolus, composed of similar amount of granular and fibrillar components. On the outer nuclear membrane the rare ribosomes were revealed. The cell wall was thin (0,10-0,12 ^m), moderate electron density, one-
layered and with tightly localized microfibrills. The average thickness of polysaccharide capsule was 3,2 ^m. The structure of capsule under TEM was very simple: dark (Fig. 1 j) moderate density microfibrills with variable length were localized in light capsular matrix.
Nucleus in the mother yeast cell during formation of hyphal projection change its position with vacuoles and now localized in apical part of cell (Fig. 1 c). At this time, in region of budding scar the short cylindrical in form hyphal projection was formed in which dark cytosol and numerous free ribosomes were revealed.
Mitochondria were localized in the periphery of cell; its number varied from 4 to 6 on median cell section. They were spherical (0,6 ^m) or ellipsoidal (0,4 x 0,6 ^m) and had moderate electron density matrix, which was similar to the electron density of cytosol. The storage substances were in the form of small (0,4-0,6 ^m) median electron density having single or in small group lipid inclusions, which localized near cell wall (Fig. 1 c).
Cytosol was rich with free ribosomes. Moderate number (from 5 to 20 on median cell section) single or in small groups small (60-70 nm) light secretory vesicles were distributed in cytosol of mother cell in this stage (Fig. 1 e). Cisterns of endoplasmic reticulum, single cisterns of Golgi and microbodies were not found in this and another stages, as in mother cell, since in forming hypha. Plasma membrane was in close contact with the electron-transparent thin (0,10 ^m), one-layered, moderate electron density cell wall with tightly oriented microfibrills. The mother yeast cells, which formed hyphal elements, covered with polysaccharide capsule (Fig. 1 f) with the same thickness and structure as in mother cells before filamentation.
Fig. 1. Light (a, b) and transmission electron microscopy (c - m) of growing C. neoformans var. grubii cells in murine brain. Explanation for this and another figures: BS - budding scar; BT - brain tissue; CW - cell wall, H - hypha; HG - hyphal germ; HP -hyphal projection; LI - lipid inclusion; M - mitochondrium(a), Mf - microfibrills; Mt - microtubule, N - nucleus, Nu - nucleolus, PC - polysaccharide capsule; S - septum; V - vacuole, Vs - vesicles; YC - yeast cell. Scale: a - 100 |m; b - 4 |m; c, f, h - 1 |m; d - 0,5
|m; I - k - 1,5 |m; e, g, l, m - 2 |m.
During subsequent grows, the hypha fill with cytosol, numerous free ribosomes (Fig. 1 e), several vesicles with light content and dark granule (Fig. 1 j), single mitochondrium (Fig. 1 h, j, k), small vacuoles (Fig. 1 h, i) and single small lipid inclusions (Fig. 1 g).The forming hypha, as a rule, more wide that budding scar (Fig. 1 e, f, h-j, l), but sometimes identical with last in diameter (Fig. 1 c, g, j, k). Hyphal cell wall in 2 time more thin (0,5 цт), that the same of mother cells. Apical part of developing hypha may be dome-shaped hemi-spherical (Fig. 1 c, e, f, h - i, l); more rare with irregular contour (Fig. 1 g, k, arrow) or it was covered with structure similar to budding scar which was typical for mother yeast cells (Fig. 1 m), but not typical for developing bud (Fig. 2 b). Soon the nucleus migrated in the part near isthmus (Fig. 1 h).
Mf^ LI
PC
b
Fig. 2. Ultrastructure of growing mother cells of the C. neoformans var. grubii before formation of hyphae (a) and during budding (b) in murine brain. Scale: a - 1 |m; b - 2 |m.
In the content of monokaryotic hypha single ellipsoidal (1,5 x 2,0 ^m) interphase nuclei were visible (Fig. 3 a, c-e), which localized on large distance one from another. The nucleus occupied main hyphal lumen, with moderate level of randomly distributed condensed chromatin and large ellipsoidal (0,6 x 0,7 ^m) nucleolus, which localized near nuclear membrane and composed of similar amount of granular and fibrillar components. The active functional condition of nucleolus correlated with large number of free ribosomes in cytosol. The density of organelles distribution in content of monokaryotic hypha was very high. Between the nuclei were visible large light or with dark fibrillar inclusions (Fig. 3 a) small or median size vacuoles. Mitochondria were numerous, polymorphic, concentrated near nuclei (Fig. 3 e). Often possible observed the long profiles of this component in hyphal content (Fig. 3 a). The analysis of serial sections demonstrated the presents of giant mitochondrium in content of hyphal cell. For comparison, during the development in vitro and in vivo (mouse brain, 7 days of infection) mother cells of used in this work strain PKnTY-1165 giant mitochondrium not formed. For comparison, the giant mitochondrium was also formed during the conidial growth in Aspergillus fumigatus [7].
From components of endomembrane system we revealed only secretory vesicles. They were in moderate number, localized in small groups near cell walls (Fig. 3 b) and with light content. Sometimes its profile greatly expanded (Fig. 3 b), what, perhaps, may be indicator of its transformation in vacuole. In apical part of hypha secretory vesicles were practically absent (Fig. 1 l, m).
The number and sizes of storage lipids was similar to that of mother cells, which formed the hyphal elements. Several long microtubules were found near outer nuclear membrane (Fig. 3 d). The cytosol of monokaryotic hyphae was dense and rich with free ribosomes.
Plasma membrane was in tight contact with the thin (from 0,10 to 0,12 ^m) moderate electron density one-layered cell wall. It was important, that outside the forming monokaryotic hypha present good developed polysaccharide capsule (Fig. 1 f, j, m, 2 a), which structure step-by-step change simultaneously with the same of mature yeast cells [WangP., et al. // Mol. Cell. Biol. - 2000. - Vol. 20] and form more complex structure (Fig. 1 m), additional layers were formed. The average thickness of polysaccharide capsule was 6,0 ^m which was wider than that of mature mother cells.
The first septum was formed in the region of isthmus (Fig. 3 f) when mother cell connect with monokaryotic hypha. This septa was right, with average thickness 0,15 ^m, electon-transparent, solid (without septal pore) and without clamp connection. In this period, the structure of mother cell, in a whole, was similar to the structures before starting of hyphal elements development (Fig. 3 h). Differences were revealed in absence of storage lipids in content of mother cell, which, evidently were used during hyphal formation, what was typical for germinating conidia [Wang P., et al. // Mol. Cell. Biol. - 2000. - Vol. 20]. Then the middle layer of septum was dissolved (Fig. 3 h, arrow) and, thus, mother cell separated from monokaryotic hyphae (Fig. 3 h). After this period, the mother cell was living (intact), but not fully destroyed, as it was typical for germination conidia [WangP., et al. // Mol. Cell. Biol. - 2000. - Vol. 20]. It was important and obvious, that monokaryotic hyphae lose its connection with mother cell and subsequent stages of formation of dikaryotic hyphae pass independent of this cell type. Thus, arise very important question about pattern of morphogenetical transformation: it may be possible that one yeast mother cell may successively formed bud and hypha or strongly in fungal population some mother cells developed only yeast and another one only hyphal elements? It was possible suggest, that one mother cells of analyzed C. neoformans var. grubii strains may developed repeatedly as bud, since hyphal element.
Fig. 3. Transmission electron microscopy (a-j) of growing C. neoformans var. grubii cells in mouse brain. Numbers in Fig. j shows the layers in composition of polysaccharide capsule. Scale: a, c, f-i - 2 |m; b, e - 1 |m; d, j - 0,5 |m.
We revealed only early stages of septal development (Fig. 3 g, arrow) in another part of hyphae, which pass not synchronous.
Very rare we observed the hyphal fragments with fully developed polysaccharide capsule, which typical for mature yeast mother cell, which consisted of 3 layers [Wang P., et al. // Mol. Cell. Biol. - 2000. - Vol. 20]. We described this structural capsular pattern as fifth type. Thus, the development specific for analyzed species of Cryptococcus type of polysaccharide capsule determinate genetically and so that was same for yeast and monokaryotic hyphae.
According to our data, the mother cells of C. neoformans var. grubii at 7 day experiments in mouse brain may developed, as bud, since the cells of monokaryotic hyphal elements. The complex mother yeast cell ^ hypha was present very dynamic asymmetrical unipolar growth alliance, which developed in two stages. In the first stage of the formation of hypha, cytosol with free ribosomes, vesicles, small vacuoles, mitochondria and lipid inclusions passed in hyphal projection from yeast mother cell. Later one of the nucleus after division also immigrate in developing hyphae. We did not observe the place and pattern of first nucleus division, but it was very interesting comparison with the place of division during budding. In the second stage of hyphal differentiation (after appearance and subsequent divisions and increasing the number of nuclei), the synthesis of cytosol, free ribosomes, secretory vesicles, proliferation of mitochondria with formation of giant organelle, synthesis of storage lipids, cell wall and capsule, apparently, occur without direct participation of yeast mother cell. So that, on second stage and after formation of
first septum between the mother cell and developed hypha in the region of isthmus, the morphogenesis of mycelium passed separately from mother cell. According to our data, the hyphal cell wall coated with thicker polysaccharide capsule, in comparison with mother yeast cells, whose structure was identical to the same of last one. The presence of capsule protect hyphal cells and determinate the sliding among the fungal and host cells. On some sections of hyphal profiles we observed that its apex «covered» with the «budding scar», which, perhaps, protect this very important for invasion part of hypha. Also possible suggest, that this structure very important for «recognition» of fungal partners and adhesion during possible hyphal fusion and formation of dikaryotic mycelium.
It was obvious, that according to the ultrastructural data, activity of the cells of monokaryotic hypha in complex yeast mother cell ^ hypha may be higher. Another indicator of great activity of hyphal form in comparison with yeast one was the formation of a thicker polysaccharide capsules. So that, the level of «aggressivity» in fungal population, which was composed of yeasts and hyphae was not identical, what provide some «confusion» for host immune system. According to Lin X. [8], the transition of unicellular yeast cells to the multi-cellular filamentous might be essential for the survival of the C. neoformans in mammalian host. In our opinion, the first signal for transition to hyphal formation was nucleus division in hyphal protrusion. It is obvious, that the presence of more active hyphal state of pathogenic cryptococcus promote the more quickly distribution of fungal infection process in host tissue.
REFERENCES
1. Chayakulkeeree M., Perfect J.R. Cryptococcosis// Infect. Dis. Clin. North Am. - 2006. - Vol. 20. - P. 507-544.
2. Litvintseva A.P., Carbone I., Rossouw J., et al. Evidence that the human pathogenic fungus Cryptococcus neoformans var. grubii may have evolved in Africa. - 2011. PLOS, May 11, DOI: 10.1371/journal.pone.0019688.
3. Idnurm A., Bahn Y.S., Nielsen K., et al. Deciphering the model pathogenic fungus Cryptococcus neoformans // Nat. Rev. Microbiol. - 2005. - Vol. 3. - P. 753 -764.
4. Vasilyeva N. V., Stepanova A.A., Sinitskaya I.A. Peculiarities of Cryptococcus neoformans cell morphogenesis of in dependence on strain's virulence // Problems in Medical Mycology. - 2009. - Vol. 9. - 23-30.
5. Vasilyeva N.V. The factors of Cryptococcus neoformans virulence and its role in pathogenesis of cryptococcosis: Diss. Doct. Biol. Sc., 2005 - 340 pp.
6. Yamaguchi M., Shimizu K., Kawamoto S., et al. Dynamics of cell components during budding of Cryptococcus albidus yeast cells // Problems in Medical Mycology. - 2014. - Vol. 16. - P. 29-35.
7. Stepanova A.A., Sinitskaya I.A. Cytological investigations of Aspergillus fumigatus Fres. germinating conidia // Problems in Medical Mycology. - 2012. - Vol. 14. - P. 43-53.
8. Lin X. Cryptococcus neoformans: morphogenesis, infection, and evolution // Infect. Genet. Evol. - 2009. - Vol. 9. - P. 401-416.
Поступила в редакцию журнала 10.02.2017 Рецензент: Т.С. Богомолова
