CC BY
14УДК 57.012.4:57.086.3:576.3:582.282.23
ultrastructural observation of
cell components
during budding in
yeast MALASSEZIA PACHYDERMATIS
1Yamaguchi M. (associate professor), 1Shimizu K. (assistant professor), Kawamoto S. (professor), 2Stepanova A.A.* (head of the laboratory), 2Vasilyeva N.V. (director of the institute, head of the chair)
1 Medical Mycology Research Center, Chiba University, Chiba, Japan; 2 North-West State Medical University named after I.I. Mechnikov: Kashkin Research Institute of Medical Mycology and Chair of Medical Microbiology, St. Petersburg, Russia
© Collective of authors, 2014
The phase contrast, scanning and transmission electron microscopy were used for observation of the cell components in exponential phase of growth in M. pachydermatis. Lower level of vacuolization, small number of mitochondria, presence of single cisterns of endoplasmic reticulum (ER), absence of secretory vesicles and presence of large lipid inclusion opposite budding scar was typical for mother cells. It was revealed the uniformity in bud formation: the number of mitochondria, storage inclusion, and cisterns of ER were not increased compared with cytosol volume and number of free ribosomes. The increase in nucleus sizes and level of chromatisation were observed in mother cell before budding. The mother cell after septum formation, differs from daughter cell in larger volume of cytosol and presence of thicker cell wall. In contrast, for daughter cell was typical in smaller volume of cytosol and presence of large lipid inclusion. A diagram of organelles transition in M. pachydermatis bud-ding was represented.
Key words: budding, exponential phase of growth, freeze-substitution, Malassezia pachydermatis, in vitro, scanning and transmission electron microscopy, ultrastructure
ультраструктурное исследование
клеточных
компонентов в
ходе почкования
MALASSEZIA PACHYDERMATIS
i Ямагучи М. (адъюнкт-профессор), 1Шимицу K. (ассистент профессора), 1Кавамото С. (профессор), Степанова А.А. (зав. лаб.), Васильева Н.В. (директор института, зав. кафедрой)
* Контактное лицо: Степанова Амалия Аркадьевна, тел.: (812) 303-51-40
i Центр исследований по медицинской микологии, Университет г. Чиба, Япония; 2 Северо-Западный государственный медицинский университет им. И.И. Мечникова: НИИ медицинской микологии им. П.Н. Кашкина и кафедра медицинской микробиологии, Санкт-Петербург, Россия
© Коллектив авторов, 2014
Для изучения перемещения клеточных компонентов в экспоненциальной стадии роста у M. pachydermatis использовали методы фазового контраста, сканирующей и трансмиссионной электронной микроскопии. Для материнской клетки был характерен низкий уровень вакуолизации, небольшое число митохондрий, присутствие одиночных цистерн эндоплазматического ретикулума (ЭР), обилие секреторных пузырьков и присутствие крупного липидного включения напротив рубчика. Выявили единообразие в формировании почки: число митохондрий, запасных включений и цистерн ЭР не увеличивалось по сравнению с объемом цитозоля и числом свободных рибосом. В материнской клетке до начала почкования наблюдали увеличение размеров ядра и уровня его хроматизации. Материнская клетка после формирования септы отличалась от дочерней большим объемом цитозоля и присутствием толстой клеточной стенки. Напротив, для дочерней клетки был типичен меньший объем цитозоля и присутствие крупного липидного включения. Представлена схема, иллюстрирующая перемещение органелл в ходе почкования M. pachydermatis.
Ключевые слова: замораживание-замещение, Malassezia pachydermatis, почкование, сканирующая и трансмиссионная электронная микроскопия, in vitro, ультраструктура, экспоненциальная фаза роста
INTRODUCTION
Malassezia pachydermatis is basidiomycetous, non obligatory lipophilic, skin normal biota yeast of other warm-blooded animals [1 and Marcon M.J., Powell D.A. // Clin. Microbiol. Rev. - 1992. - Vol. 5; Gueho E., et al. // Med Mycol. - 1998. - Vol. 36]. Only these species are zoophilic among members of Malassezia genus. It was the part of the normal cutaneous microbiota of dogs [Bond R., et al. // J. Small Anim. Pract. - 1996. - Vol. 37], cats [2], birds [Breuer-Strosberg R., et al. // Mycoses. -1990. - Vol. 33, №5] and many other mammals and associated with superficial infections of the skin and associated structures, including pityriasis versicolor and follic-ulitis, dessiminated infections in IV-neonates [1]. Also it was reported as agents of more invasive human diseases including deep-line catheter-associated sepsis [Marcon M.J., Powell D.A. // Clin. Microbiol. Rev. - 1992. - Vol. 5]. Previously, some aspects of budding process [Bond R., et al. // J. Small Anim. Pract. - 1996. - Vol. 37] and cell structure [3-6 and GabalM.A., Fagerland J.A. //My-cosen. - 1979. - Vol. 22; Nishima K., et al. // Med. Mycology. - 1991. - Vol. 29, №6; Winiarczyk S. //Arch. Vet. Pol. - 1992. - Vol. 32] were investigated using scanning, transmission and freeze-fracture methods of electron microscopy. The aim of this work was investigation of the cell components changes during in vitro growing in yeast M. pachydermatis using modern methods of light, scanning and transmission electron microscopy and compare this data with previous literature.
MATERIALS AND METHODS
The cultures of two strains of M. pachydermatis (strain PKnTY-1195 from Russian collection of pathogenic fungi and strain 40199 from Culture Collection of the Research Center of Pathogenic Fungi, Chiba University, Japan). The first strain was cultivated 10 days on solid wort agar at 28°. For scanning electron microscopy the part of fungal colonies with nutrient medium were fixed in 3% glutaraldehyde (in 0,1 M cacodylate buffer) for 3 hour, postfixed overnight in 1% osmium tetroxide in the same buffer, dehydrated by ethanol series (30% to 70%), critical-point dried (HCP-2) for 15 min, coated with gold and observed in JSM 35 (JEOL, Tokyo, Japan). The second strain was cultivated for 24 hour in YPD medium (1% (w/v) yeast extract, 2% (w/v) bactopepton and 2% (w/v) glucose on shaker at 30 °C. For morphological characteristics of cultures, we made the temporary preparation of living cells and observed them under the phase-contrast microscope (Olympus BH-2RFCA). For transmission electron microscopy (TEM) the cells were collected by centrifugation and sandwiched between two copper grids. Then samples were then freeze-substituted in 2% osmium tetroxide/aceton at - 80 °C for 48 hour and embedded in epoxy resin according to the method described before [7]. Ultrathin sections (70 nm thick) were cut with diamond knife and stained with uranyl acetate and lead citrate and covered with Super Support Films (Nisshin EM, Tokyo, Japan) observed with JEM-1400EX transmission electron microscope (JEOL, Tokyo, Japan).
RESULT AND DISCUSSION
Scanning electron microscopy. Yeast cells were subspherical to ovoidal in form, 3,0-6,0-2,0-2,5 |m. Buds were as wide as the mother cells [1]. For mature mother cells, M. pachydermatis were typical percurrent [1], monopolar blastic budding [Nishima K., et al. // Med. Mycology. - 1991. - Vol. 29, №6] through broad scar (Fig. 1 a-e), which was border with prominent collarette (Fig. 1c-e). The diameter of the budding scar varied from 1 to 1.5 |m which correlated with data of another authors [3 and Nishima K., et al. // Med. Mycology. - 1991. - Vol. 29, №6]. Under the lower magnification it was visible the adhesiveness feature of cells which situated on different developmental stages (Fig. 1 a). This peculiarity was accordant with observation about capability of cultures of this species to biofilm formation [8]. The cell wall surface structure was smoother in developing bud, but in mother and daughter cells they showed thin-granular texture (Fig. 1 b-e). Exactly after separation, the mother and daughter cells, as a rule, were also localized on tight lateral contact (Fig. 1 e). The cause of this lateral contact may be stipulated by not simultaneous medium (light) part septum disintegration - but in direction from one part to another.
Phase-contrast microscopy. Peculiarity of yeast cells walls (Fig. 1 f) was obvious, including the stage of budding (Fig. 1 g)._
Transmission electron microscopy. Mother cells
before budding. At the median cells section, it was obvious that nucleus occupied the main volume and localized in central part near cell wall (Fig. 1 i, h, 3 a). Nucleus was spherical (on the average 1,0 |m), with lower level of condensed chromatin which uniformly distributed in nucleoplasm. One big (on the average
0.6 x 0,8 | m) lenticular dark nucleolus was situated near nucleus envelope (Fig. 1 j). The low number of dark irregularly distributed ribosomes was observed on outer nucleus membranes. The number of vacuoles at median section was varied from 2 to 3. They were small in size, spherical, as a rule, closely localized and have identical content with tightly packed fibrillar material, which give them specific morphological features (Fig. 1
1, h). We revealed small differences in ultrastructure of vacuolar contents in M. pachydermatis in comparison with Cryptococcus albidus cells [9]. As a rule, the group of vacuoles were localized between the nucleus and lipid inclusion (Fig. 1 i) or under nucleus (Fig. 1 h). The mitochondrial number at median section varied from 2 to 5. They were localized near cell wall, as a rule spherical in form (from 0,5 to 0,7 | m). Large (on the average I,0 |m) irregular in form and median electron density lipid inclusion (Fig. 1 i, h) was visible in the upper part of cells opposite the budding scar. From components of endomembrane system, it was possible to recognized only single (on median section) short straight granular cistern on endoplasmic reticulum (ER) in cell periphery near cell wall (Fig. 1 k). The dark ribosomes were compactly distributed on the surfaces of ER cisterns. The secretory vesicles and single Golgi cisterns on this and other budding stages were absent. For comparison, numerous secretory vesicles were present in C. albidus [9] growing in same condition.
Fig. 1. Scanning (a-e), phase-contrast (f, g) and transmission electron microscopy (i-n) of budding M. pachydermatis cells. Explanation for this and another figures: B - bud, BC - budding cells; C - colarette, CW - cell wall, DC - daughter cell, ER - endoplasmic reticulum, LI - lipid inclusion, M - mitochondrium, MC - mother cell, Mt - microtubule, N - nucleus, Nu - nucleolus, R - ribosomes, S - septum, Sc - scar, Sp - septum, V - vacuole, Vs - vesicles. Scale: a = 15 |m; b, c = 2 |m; d, e, g, i = 0,5 |m; f = 10 |m;
k, l , j = 0,3 |m; h, m, n - 1 |m
Cytosol had a higher electron density, rich with free ribosomes in form of mono- or polysome. Plasma membrane formed the small light narrow (on median section) and more lengthy on skate paradermal sections (Fig. 1 l, arrows) «edge-like» symmetrically localized ring-like swellings which previously detected in typical for M.
pachydermatis and another species from genus Malasse-zia [3, 4 and Nishima K., et al. // Med. Mycology. - 1991. - Vol. 29, №6]. The presence of this cell walls thickening provide its rigidity and protective features. Periplasmic space was absent, because that plasma membrane situated in close contact with cell wall. Cell wall was thick
(on the average 0,3 |m), light, with two distinct layers: of cell adhesion) provide the cell wall surface structure
lower thick (Fig. 2 b, 1) identical in thickness (on aver- and ensure its adhesive peculiarity. Another authors [3,
age 0,2 |m) and upper thin (on average 0,1 |m) and dis- 4] also revealed the presence the thick cell wall in cell of continuous (Fig. 2 b, 2). The presence of upper layer, as this species. it was obvious in fig. 1 h (arrow demonstrate the place
Fig. 2. General view (a, c, e, g) and fragments (b, d, f, h, i) of budding cells M. pachydermatis under transmission electron
microscope. Scale: a, c, d, e, g, = 1 |m; b, f, h, i = 0.5 |m
Budding. At the early stage of bud formation, the cytosol with free ribosomes was revealed in its content (Fig. 1 n). During budding, the density of free ribosomes distribution regarding the visual evaluation was similar in mother and bud compartments. Nucleus in mother cells was translocaled under isthmus (Fig. 1n). Several short microtubules were revealed in central part of mother cell (Fig. 1 n) and inside isthmus. Cytoplasmic microtubules were also observed during budding of M. pachydermatis cells using fluorescence microscopy [4].
Later several small vacuoles (Fig. 1 m, 3 b, c), single mitochondria (from 1 to 2 on median section) migrate from mother cells into bud (Fig. 2 c). Thus, the specific peculiarities of budding of investigated M. pachydermatis were the lower level of mitochondria, which is different from C. albidus [9].
Similar presence of several small vacuoles and small number of single mitochondria were visible during budding (Fig. 1), during (Fig. 3 e) and after septum formation (Fig. 2, 3 f) of the cells of same fungal species according David M. et al. [4]. In the apex of the growing M. pachydermatis bud David M. with co-authors [3] revealed small vesicles.
Notice, that in early (Fig. 1 m, n, 2 a) and final stages (Fig. 2 e, d, g) of bud formation the isthmus was more apparent than in intermedian ones (Fig. 2 c). As a rule, the large lipid inclusion was apparent in bud before nucleus migration (Fig. 2 a) in this part of the system
Fig. 3. Diagram showing the organelles transition during M. pachydermatis budding: a - mother cell before budding, b-d -mother cell during budding, e - mother and bud compartments during septum formation, f - mother and bud compartments with formed septum, g - mother cell after separation from daughter cell, h - daughter cell after separation from mother cell
from mother^ to developing bud. The level of nucleus chromatisation were increased (Fig. 2 c), when the bud was practically fully developed. The nucleus sizes in this period larger than in mother cell before budding (on the average 1,4 |m). Similar changes in nucleus were described for budding cells of Cryptococcus albidus [9]. Direct process of nucleus division was not observed in the present study. According to David M. et al. [4], this process was took places directly during nucleus moving through isthmus and as a result one nucleus revealed in mother and another in bud. Last authors, using fluorescent microscopy, revealed in dividing nucleus the spindle and astral microtubules.
The mother and daughter cells during and after cytokinesis. During cytokinesis the sizes of mother cells varied from 2,0-2,6 to 3-3,5 |m and the bud from 1,8-2,0 |m to 2,0-2,2 |m. Inside isthmus on the level of collar (Fig. 2 e, 3 e) were formed electron-transparent wedge-shaped centripetal growing (Fig. 2 d) septum. At this time, small (on average 1,0 |m) single nucleus revealed in the mother half (Fig. 1 e) and bud ones (Fig. 2 d). In both part of the system of mother^ to bud, the nucleus was localized in more distal (in cell periphery) from isthmus (Fig. 2 e, d). From last they «separated» with several small vacuoles. Eventually the continuous straight (on average 0,18 |m) light septum was formed (Fig. 2g, 3 f). The structure of nucleus in mother and bud half was similar: lower level of chromatin condensation and presence of large (0,6 x 0,8 |m) prominent lenticular
nucleolus (Fig. 2 d, e) were observed. Probably the cell wall material from which composed collarette partially remains around bud scar (Fig. 1 e, arrow).
In this period the volume of bud was smaller in comparison with mother but the number and structure of organelles, components of endomembrane system and free ribosomes were similar. Exclusion compose the presence of large lipid inclusion which may immigrate into bud content not only before septum formation (Fig. 2 a), but also during this process (Fig. 2 e). The presence of storage lipids in daughter cells (Fig. 2 I, 3 h) gives them advantage for rapid and valuable growth and differentiation. In our opinion, the presence in C. albidus [8] mother cell large vacuole and respectively in M. pachydermatis large lipid inclusion between budding scar and interphase nucleus in mother cell temporary block process of budding which allow last situated in «rest» condition. Investigations of another yeast species in same condition and stage let the universality of this finding.
As it was revealed in fig. 2-g, the main pattern of cell walls structure in mother and daughter cell formation was similar; differences in wall thickness were obvious. For daughter cell, thin wall (on average 0,2 | m) in comparison with mother (on average 0,3 |m) one was typical. The growth of daughter cell occurs with participation of vacuoles whose sizes in this period considerably increased (Fig. 2 h). In content of mother cell, the synthesis of storage lipid inclusion (Fig. 3 g-a) later took places.
RESUME
The scanning electron microscopy showed that for M. pachydermatis mother and daughter cells were typical the cell wall with thin-granular texture. The cell surface structure was smooth in developing bud.
In exponential phase mother cells it was typical the lower level of vacuolization, small number of mitochondria, presence of single cistern of ER, absence of secretory vesicles and presence of big lipid inclusion opposite budding scar. The uniformity in vacuolar content structure in this and all subsequent developmental stages was revealed. For interphase nucleus, central-lateral topography and lower level of condensed chromatin was typical.
During bud formation the number of mitochondria, storage inclusion and cisterns of ER were not increased contrary to the volume of cytosol and numbering of free ribosomes. Before budding, the increase in nucleus sizes and level of chromatisation were revealed in mother cell. The mother cell after septum formation differs from daughter cell in the larger volume of cytosol and the presence of thick cell wall. Contrary to mother cell, daughter cell showed small volume of cytosol, free ribosomes and presence of large storage lipid inclusion.
ACKNOWLEDGEMENTS
The authors were grateful to head of Russian Collection of Pathogenic Fungi Chilina G.A. for assign of fungal cultures.
REFERENCES
1. de Hooge G.S., et al. Atlas of clinical fungi (a recent electronic version 3.1). - 2011.
2. Volk A.V., Belyavin C.E., Varjonen K., et al. Malassezia pachydermatis and M. nana predominate amongst the cataneous my-cobiota of Sphynx cats // J. Feline Med. Surg. - 2010. - Vol. 12, №12. - P. 917-922.
3. David M., Gabriel M., Kopecka M. Unusual ultrastructural characteristics of the yeast Malassezia pachydermatis // Scripta medica (Brno). - 2003. - Vol. 76, № 3. - P. 173-186.
4. David M., Gabriel M., Kopecka M. Microtubular and actin cytoskeletons and ultrastructural characteristics of the potentially pathogenic basidiomycetous yeast Malassezia pachydermatis // Cell Biology International. - 2007. - Vol. 31. - P. 16-23. doi: 10.1016/j.cellbi. 2006.09.001.
5. Gueho-Kellerman E., Boekhout T., Begerow. Biodiversity, phylogeny and ultrastructure. In: Malassezia and the skin // Springer Link. - 2010. - P. 17-63.
6. Kurtzman C.P., Fell J.W., Boekhout T. The yeast a taxonomic study. 5th ed. - 2012. - 1500 p.
7. Yamaguchi M., Okada H., Namiki Y. Smart specimen preparation for freeze substitution and serial ultrathin sectioning of yeast cells // J. Electron Microsc. - 2009. - Vol. 58. - P. 261-266.
8. Figueredo L.A., Cafarchia C., Desantis S., Otranto D. Biofilm formation of Malassezia pachydermatis from dog // Vet. Microbiol. - 2012. - Vol. 9, №160. - P. 126-131.
9. Yamaguchi M., Shimizu K., Kawamoto S., et al. Dynamics of cell components during budding of Cryptococcus albidus yeast cells // J. Problems of Med. Mycol. - 2014. - Vol.16, №1. - P. 29-35.
Поступила в редакцию журнала 24.10.2014
Рецензент: Л.Е. Сергеева
