CC BY
21УДК 57.086.3:582.282.23
ЦИТОЛОГИЧЕСКИЕ ОСОБЕННОСТИ ДРОЖЖЕВЫХ КЛЕТОК МУЛЬТИРЕЗИСТЕНТНОГО ПАТОГЕНА CANDIDA AURIS
'Васильева Н.В. (директор института, зав. кафедрой), 2Круглов А.Н. (зав. лаб.), Степанова A.A. (зав. лаб.)*, Чилина Г.А. (зав. лаб.), 1Босак И.А. (с.н.с.), Богомолова Т.С. (зав. лаб.)
1НИИ медицинской микологии им. П.Н. Кашкина, СевероЗападный государственный медицинский университет им. И.И. Мечникова, Санкт-Петербург; Национальное агентство клинической фармакологии и фармации, Москва, Россия
©Коллектив авторов, 2018
В работе представлены данные по микроморфологии и ультраструктуре выращенных in vitro дрожжевых клеток Candida auris (РКП^-1821). Отличие мультирезистентного штамма C. auris от других видов рода Candida, в том числе резистентных, состоит в: 1) мелких размерах клеток в сочетании с толстой клеточной стенкой и небольшим диаметром рубчика(ов); 2) наличии несколько рубчиков в составе клеточной стенки; 3) присутствии в зрелых дрожжевых клетках внеклеточного липидного «гало». Описан новый способ вторичного использования запасных липидов зрелой дрожжевой клеткой, происходящий с участием вакуолей.
Ключевые слова: in vitro, Candida auris, культура, микроморфология, мультирезистентность, ультраструктура
СУТОШСЮАЬ FEATURES OF MULTI-RESISTANCE CANDIDA AURIS YEAST CELLS
1Vasilyeva N.V. (director of the institute, head of the department), 2Kruglov A.N. (head of the laboratory), 1Stepanova A.A. (head of the laboratory), 1Chilina G.A. (head of the laboratory), 1Bosak I.A. (senior scientific collaborator), 1Bogomolova T.S. (head of the laboratory)
1Kashkin Research Institute of Medical Mycology, NorthWestern State Medical University named after I.I. Mechnikov, St. Petersburg, 2National Agency of Clinical Pharmacology and Pharmacy, Moscow, Russia
©Collective of authors, 2018
In the present paper the data about the morphology and ultrastructure of C. auris (РКП^-1821) yeast cells growing in vitro were presented. Differences of multi-resitant strain Candida auris from another species of genus Candida, including resistant, consist in: 1) small cell sizes in combination with thick cell wall and small diameter of scar(s); 2) presence of several scars in the composition of cell wall; 3) presence of the extracellular lipids «halo» in the mature yeast cells. The new way of secondary using of storage lipids of mature yeast cells carried out with the participation of vacuoles was described.
Key words: in vitro, Candida auris, culture, morphology, multiresistance, ultrastructure
Контактное лицо: Степанова Амалия Аркадьевна, e-mail: amaliya.stepanova@szgmu.ru
INTRODUCTION
Candida auris is an emerging multidrug-resistant fungal pathogen causing nosocomial and invasive infections associated with high mortality rate [1]. The first C. auris was isolated from the external ear canal of a patient in Japan in 2009 [2]. It was an emerging multidrug-resistant pathogen that was difficult to identify using traditional biochemical methods. C. auris has been recovered in samples from blood, catheter tips, cerebrospinal fluid, bone, ear discharge, pancreatic fluid, pericardial fluid, peritoneal fluid, pleural fluid, respiratory secretions (including sputum and bronchoalveolar lavage), skin and soft tissue samples (both tissue and swab cultures), urine, and vaginal secretions. Clinically, it has been implicated as a causative agent in fungemia, ventriculitis, osteomyelitis, malignant otitis (including otomastoiditis), complicated intra-abdominal infections, pericarditis, complicated pleural effusions, and vulvovaginitis [3]. The data of the morphology of C. auris yeast cells are very important and may shed light on the nature of its multidrug resistance. Thus, the aim of the present paper was to investigation the morphology of this species in vitro growing cultures of C. auris by using light- and transmission electron microscopy (TEM).
MATERIALS AND METHODS
The Candida auris Satoh & Makimura strain PKnrY-1821 (Russian collection of pathogenic fungi) was isolated for the first time in Russia from the blood of patient in 2017. C. auris was identified by MALDI-TOF mass spectrometry and DNA sequencing [4]. The strain was cultivated for 10 days in Sabouraud's medium and wort agar at 37 °C. The morphology of cultures was investigated on stereoscopic microscope Zeiss Stem 2000. The yeast cells structure was observed on Leica DMLB and Axio-images. Z1 (Carl Zeiss) light microscopes with Nomarski optics. For transmission electron microscope (TEM), different part of fungal colonies were fixed in 3% glutaraldehyde for 3 h and post-fixed in 1% osmium tetroxide for 10 h. according to the technique described earlier [5].
The ultrathin sections were cut with a glass knife and stained with uranyl acetate and lead citrate. Finally, the sections were observed in JEM-100 SX TEM (JEOL, Tokyo, Japan).
RESULTS AND DISCUSSION
Colony characteristic. The average diameter of fungal colony after 10 days of growth at 37 °C on the Sabouraud's medium was 3,5 cm (Fig. 1 a), and on the wort agar - 3,5 x 4,0 cm (Fig. 1 b). Colonies were cream-colored, smooth, opaque, soft. On the periphery, the thin rim was present. The colony edge was fair.
Microscopy. During the using routine microscopy (Fig. 1 c) and with Nomarkski optics (Fig. 1 d-g) the yeast cells were ovoid (3,0 x 4,0 ^m), ellipsoidal (2,0-3,3 x 2,4-5,0 ^m) to elongated and colorless. Yeast cells were single, in pair or in small groups. Budding cells were abundant (Fig. 1 d-g). Pseudomycelium was absent. Germ tubes were not formed.
Ultrastructure of the yeast cells. The sizes of C. auris mature yeast cell were in 2-3 times smaller in comparison with C. albicans [5, 6]. The fixation of different cells types of Candida spp. was very difficult that we explain this fact by the presence of pigments in its cytosol which absorb fixatives [5, 7, 8, et al.]. In this investigation, we also
Ï--
Fig. 1. C. auris colonies after 10 days cultivation on Sabouraud's medium (a) and wort agar (b). Light microscopy of C. aurisyeast cells: on wort agar (c) and Sabouraud's medium (d - g). Explanation for this and another figures: Bd - bud, BYC - budding yeast cells, CW - cell wall, Hp - hyphae, LI - lipid inclusion, MC - mother cell, N - nucleus, P - plasma membrane, T - tonoplast, FL -flexible lipids, FLl - flexible part of lipid inclusion, V - vacuole; YC - yeast cell(s).
Fig. 2. Ultrastructure of in vitro growing yeast cells of C. auris.
obtain the medium quality fixation of C. auris yeast cells but we were lucky that independent of this situation may study the mechanism of extracellular lipids origination. The presence of ring from extracellular lipid (Fig. 2 a, b, arrows) the imped the shooting the ultrathin sections so that they destroyed under TEM electronic beam. Thus, we must produce the sections shooting under TEM practically in dark field of vision and that use special conditions for negatives manifestation.
In the central part of the growing yeast cell, one nucleus (1,0 x 1,2 (m) with highly level of chromatization was present (Fig. 2 a, c). It is known that for fungi typical interphase nucleus were with lower level of chromatization and lower level of condensed chromatin which correlated with lower number of chromosome [9]. According the data Finn T. with co-authors. [10] for C. auris was typical the haploid genome. The higher level of interphase nucleus chromatization in yeast cells of C. auris can be caused by the large number of chromosomes in comparison with another candida species. However, for decision of this question needs further investigation.
In the mature (after completion of growth) yeast cells, the main volume was occupied by one large storage lipid inclusion (Fig. 2 d). Large vacuole was present in growing cell (Fig. 2 c), but several small vacuoles were present in mature ones (Fig. 2 d, h, i-m, o, q). The vacuolar content was filled with uniformly distributed fibrillar-granular materials, whose presence gives them a specific appearance.
The cell wall of C. auris mature yeast cells was in twice more thick (0,5-0,6 (m, fig. 2 e, f) than in C albicans (0,3 (m) [5] and consist of two layers: outer thin dark layer (0,05-0,07 (m) and inner thick layer (0,45-0,53 (m) with moderate electron density. There are variable numbers of scars (from 1 to 3) on ultrathin sections in composition of yeast cell wall. We revealed several topography of scar localization (Fig. 3): apical (a), lateral (near apex) (b), apical-lateral (c) and apical-double lateral (d). For the yeast cells of another medically important Candida species were typical presence of one apically localized scar [4, fig. 2 g]. Sometimes we observed the simultaneous development 2-3 buds and daughter cells by one mother yeast cell. These morphological peculiarities give our species «carte blanche» in sense to win the space during the surface colonization and subsequent invasion in tissue.
The mean scar diameter in the C. auris mature yeast cells was (Fig. 2 f) two times small (0,12 (m) than in C. albicans (0,24 (m, fig. 2 g). It may be explained by small sizes of C auris yeast cells. The scar in yeast cells of both species separated from cell walls with special «ring» which were 0,10 (m wide in C auris (Fig. 2 f, arrows) and 1,15 (m - in C. albicans (Fig. 2 g, arrows). In this area of cell wall microfibrills were localized more loosely and this part of cell wall were more plastic so that the development of bud becomes possible. In C. albicans this «ring» was more light (Fig. 2 g, arrows), but it was moderate electron density in C auris (Fig. 2 h, arrows). In our opinion, the scar «ring» was very vulnerable part of yeast cells wall for penetration of antifungals. More small area of scar «ring» in the C auris yeast cells and thick wall made them more resistant in comparison with C. albicans.
The specific peculiarity of the structure of mature yeast cells of C. auris was the presence of extracellular light lipids «halo» (Fig. 2 a, b, arrows) or capsule with flat outer surface and thickness form 0,9 to 1,5 (m, which is 3,0-3,5 times
thicker than cell wall.
Fig. 3. Diagram showing the scar(s) topography in C. auris yeast cells. a - apical, b - lateral, c - apical-lateral, d - apical-double lateral.
Fig. 4. Diagram showing the mechanism of formation of external «halo» from the secondary using of storage lipids of mature yeast cells C. auris.
We revealed the fine mechanism of the extracellular lipids origination in C. auris yeast cells. In mature yeast cells, the main volume occupied the large lipid inclusion (Fig. 2 d, 4 a). In this stage the volume of vacuole was significantly reduced. Several flat vacuole(s) localized between the large lipid inclusion and cell wall (Fig. 2 d, 4 a). Vacuole(s) «envelope» the lipid inclusion (Fig. 2 d, 4 a) and were in tight contact with them. Over time, the solid lipid inclusion passes into more flexible form (Fig. 2 h-m, q, arrowheads, 4 b, black coloring). This process of «soffening» may pass in periphery or inside lipid inclusion (Fig. 2 h-m, q, 4 b). Part of vacuole(s) cover the flexible (more light «gold like» on ultrathin sections) part of lipid inclusion (Fig. 2 h-j, 4 b, c) and by way which similar with autophagy absorbed the
part of lipid inclusion (Fig. 2 k, head of arrow, 4 d). Then by way of the exocytosis part of flexible lipid from vacuole appear in periplasmic space (Fig. 2 n, p, q, arrows, 4 e) and migrate through cell wall (Fig. 2 q, 4 f) on its surface (Fig. 2 a, b, arrows, 4 g). Soon the separate lipid globules on the yeast cell wall surfaces flow together and form unique light «halo» which is in early stage of development may be asymmetrical in thickness (Fig. 2 a) so that the process of migration of flexible lipids from internal part of cell to the surfaces of yeast cell wall was asynchronous.
Thus, in C. auris yeast cells we revealed the unique mechanism of secondary using the internal cellular storage lipid inclusion for external lipid «halo» production, which presence may be very important for its possible protection. The presence of extracellular light lipid «halo» also: 1) increased the adherent properties of our objects during the biofilm formation and in a whole biofilm resistance; 2) will create a problem for host cells of immune system. Contrary with external polysaccharide capsule in yeast cells of Cryptococcus neoformans [11, 12] extracellular light lipid «halo» in C. auris developed only in mature cells. According to the difference in the chemical composition this two cytological external cells attributes typical for some species of pathogenic fungi it was evident that lipid capsule was more resistant in comparison with polysaccharide one.
It was important that in filamentous fungi [S. apiospermum: 13; S. aurantiacum: 14] in the synthesis of external lipids was involved the tubular smooth tubules
of endoplasmic reticulum. Before we expect [13, 14] that the external lipids in this last species may contain pigments which determinate the colony coloring. Perhaps external lipids in this species demonstrated their high level of resistance also. As a rule, during fungal morphogenesis the storage lipids inside cells were used during germ tube or mycelium development, conidia and spores germination. But as a rule, they disintegrated and disappeared during fungal cells senescence. Thus, we revealed very «economical» type the secondary using of storage lipids which pass by way new type of «secretion» with participation of vacuoles. Perhaps, this last one was typical for another yeast cells, especially pathogenic.
CONCLUSION
The data of present work allow considering that C. auris yeast cells multi-drug resistance may be caused by the complex of its features: 1) small sizes in combination with thick cell wall and small diameter of scar(s); 2) several scars in the composition of cell wall; 3) presence of the extracellular light lipids «halo» in mature yeast cells. But in our opinion, the last characteristic was the main in the understanding of its higher level of multiresistance. Perhaps the presence of external «halo» from lipids represents the essential barrier for penetration of antifungals and set problem of elaboration of destroying the drug. However, it was unknown what following cellular mechanism of resistance may «use» yeast cell.
REFERENCES
1. Chowdhary A., Sharma C., Meis J.F. Candida auris: a rapidly emerging cause of hospital-acquired multidrug-resistant fungal infections globally. PLoS Pathog. 2017; 13 (5): e1006290.
2. Satoh K., Makimura K., Hasumi Y., et al. Candida auris sp. nov., a novel ascomycetous yeast isolated from the external ear canal of an inpatient in a Japanese hospital. Microbiol. Immunol. 2009; 53 (1): 41-44.
3. Sears D., Schwartz B.S. Candida auris: an emerging multidrug-resistant pathogen. Intern. J. Infect. Dis. 2017; 63: 95-98.
4. Vasilyeva N., Kruglov A., Pchelin I.M., et al. The first Russian case of candidaemia due to Candida auris. Abstr. 28 ECCMID, Madrid, Spain, 21-24 April, PO311.
5. Степанова А.А., Шевяков М.А., Авдеенко Ю.Л. и др. Изучение слизистой оболочки пищевода при кандидозе у ВИЧ-инфицированного больного. Проблемы медицинской микологии. 2011; 13 (1): 54-61. [Stepanova A.A., Shevyakov M.A., Avdeenko Yu.L. i dr. Izuchenie slizistoj obolochki pishchevoda pri kandidoze u VICH-inficirovannogo bol'nogo. Problemy medicinskoj mikologii. 2011; 13 (1): 54-61 (In Russ)].
6. Елинов Н.П., Васильева Н.В., Степанова А.А. и др. Краткий атлас медицински значимых микромицетов рода Candida / Под. ред. з.д.н. РФ проф. Н.П. Елинова. СПб, 2013:73 с. [Elinov N.P., Vasil'eva N.V., Stepanova A.A. i dr. Kratkij atlas medicinski znachimyh mikromicetov roda Candida / Pod. red. z.d.n. RF prof. N.P. Elinova. SPb, 2013:73 s. (In Russ)].
7. Staniszewska M., Bondaryk M., Siennicka K., Kurzatkowski W. Ultrastructure of Candida albicans pleomorphic forms: phase-contrast microscopy, scanning and transmission electron microscopy. Polish J. Microbiol. 2012; 61 (2): 129-135.
8. Li H., Gong H., Qi Y., et al. In vitro and in vivo antifungal activities and mechanism of heteropolytungstates against Candida species. Scientific Rep. 2017; 7. Article number: 16942.
9. Степанова А.А. Морфогенез грибов порядка Agaricales s.l. (сравнительный ультраструктурный анализ): Автореф. дисс... д.б.н. / АН СССР Ботан. ин-т им. В.Л. Комарова. Л., 1990. [Stepanova A.A. Morfogenez gribov poryadka Agaricales s.l. (sravnitel'nyj uTtrastrukturnyj analiz): Avtoref. diss... d.b.n. / AN SSSR. Botan. in-t im. V.L. Komarova. L., 1990 (In Russ)].
10. Finn T., Novikov A., Bash E., Ben-Ami R. Epidemiology and clinical features of Candida haemulonii and Candida auris isolates in tertiary medical centre in Tel Aviv. EV9033, Abstract.
11. Stepanova A.A., Vasilyeva N.V., Yamaguchi M., et al. Ultrastructural investigations of the yeast^hyphal cell switching of the Cryptococcus neoformans var. grubii in murine brain. Problems in medical mycology. 2017; 19 (2): 19-24.
12. Васильева Н.В., Степанова А.А. Электронно-микроскопическое изучение клеточной стенки и капсулы зрелых клеток слабо и сильновирулентных штаммов Cryptococcus neoformans var. neoformans в ходе in vitro^in vivo трансформации. Проблемы медицинской микологии. 2018; 20 (1):18-24. [Vasil'eva N.V., Stepanova A.A. Elektronno-mikroskopicheskoe izuchenie kletochnoj stenki i kapsuly zrelyh kletok slabo i sil'novirulentnyh shtammov Cryptococcus neoformans var. neoformans v hode in vitro^in vivo transformacii. Problemy medicinskoj mikologii. 2018; 20 (1):18-24 (In Russ)].
13. Stepanova A.A., Vasilyeva N.V., Bogomolova T.S., Chilina G.A. Cytological study of the in vitro growing cells of vegetative mycelium of Scedosporium apiospermum. Problems in medical mycology. 2017; 19 (1): 24-30.
14. Stepanova A.A., Yamaguchi M.M., Chibana H., et al. Comparative ultrastructural analysis of the in vitro growing hyphal cells of Scedosporium aurantiacum. Problems in medical mycology. 2017; 19(3): 18-25.
Поступила в редакцию журнала: 20.09.2018
Рецензент: M. Yamaguchi
