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ЭЛЕКТРОННО-МИКРОСКОПИЧЕСКОЕ ИЗУЧЕНИЕ ИНВАЗИВНОГО АСПЕРГИЛЛЕЗА, ОБУСЛОВЛЕННОГО ASPERGILLUS FUMIGATUS
''Степанова А.А. (зав. лаб.)*, ''Васильева Н.В. (директор института, зав. кафедрой), 2Чжан ф. (директор института, зав. кафедрой), 2Тонг Д. (доцент кафедры), 'Авдеенко Ю.Л. (с.н.с.), 3Богданов А.Н. (профессор кафедры), 3Семелев В.Н. (нач. отд.), 'Шадривова О.В. (ассистент кафедры), 'Климко Н.Н. (зав. кафедрой) 1 НИИ медицинской микологии им. П.Н. Кашкина, СевероЗападный государственный медицинский университет им. И.И. Мечникова, Санкт-Петербург, Россия; 2 Харбинский Медицинский Университет, Харбин, Китай; 3 Военно-медицинская академия им. С.М. Кирова, Санкт-Петербург, Россия
© Коллектив авторов, 2015
Изучены особенности ультраструктурной организации гиф мицелия A. fumigatus в легких больного острым лейкозом и инвазивным аспергиллезом, возникшим после цитостатической терапии. Установлено, что морфогенез клеток мицелия A. fumigatus сопровождался усилением уровня вакуолизации, аккумуляцией небольшого числа запасных липидов, формированием снаружи клеточных стенок толстого темного внеклеточного матрикса. Зрелые клетки гиф мицелия не различались между собой по ультраструктуре интерфазных ядер, митохондрий и компонентов эндомембранной системы, а также по количеству и качеству аккумулируемых запасных веществ. Способность гиф гриба формировать одну гигантскую митохондрию, покоящиеся клетки гиф мицелия, а также хорошо развитый внеклеточный матрикс можно отнести к цитологическим критериям его высокой вирулентности.
Ключевые слова: Aspergillus fumigatus, инвазивный аспергиллез, компоненты клеток, легкие человека, электронная микроскопия
ELECTRON-MICROSCOPIC INVESTIGATIONS OF INVASIVE ASPERGILLOSIS CAUSED wITH ASPERGILLUS FUMIGATUS
'Stepanova А.А. (head of laboratory), Vasilyeva N.V. (director of the institute, head of the chair), 2Zhang F. (director of the institute, head of the chair), 2Tong D. (associate professor), 'Avdeenko Y.L. (senior scientific collaborator), 3Bogdanov А.П (professor of chair), 3Semelev V.N. (head of department), 1 Shadrivova O.V. (assistant of the chair), 'Klimko N.N. (head of the chair)
1 Kashkin Research Institute of Medical Mycology, I.I. Mechnikov North-Western State Medical University, Saint Petersburg, Russia; 2 Harbin Medical University, Harbin, China; 3 Military Medical Academy, Saint Petersburg, Russia
© Collective of authors, 2015
Ultrastructural peculiarities of the organization of A. fumigatus hyphal vegetative cells in lungs of patient with invasive aspergillosis and
* Контактное лицо: Степанова Амалия Аркадьевна, тел.: (812) 303-51-40
acute leukemia have been studied. It was revealed, that the morphogenesis of hyphal cells of A. fumigatus was accompanied by increasing the level of vacuolization, accumulation of a small number of storage lipids, formation outside of the cell walls a wide dark extracellular matrix. The mature hyphal cells didn't differ among themselves according the ultrastructure of interphase nucleus, mitochondria and components of endomembrane system, quantity and quality of accumulated storage substances. Ability of the studied tissular vegetative hyphae to develop one giant mitochondrion, hyphal resting cells and well developed extracellular matrix can be considered as cytological criteria of its virulence.
Key words: Aspergillus fumigatus, cell components, electron microscopy, invasive aspergillosis, lung
INTRODUCTION
Aspergillus fumigatus Fresen. is a major pathogen of invasive aspergillosis in immunocompromised patients [Latge J.-P. // Clin. Microbiol. Rev. - 1999. - Vol. 12, №2]. The literature data about the fine organization of the tissular forms of A. fumigatus is limited. Thus, ultrastructural peculiarity of the integral part of the cell wall - so called «extracellular matrix» of A. fumigatus, damaging of the mouse and human lung tissue, was studied in the certain work [1-5]. Fine structure of the A. fumigatus hyphal cells was investigated in the mouse lung tissue [4] and in the lungs of the patient in association with A. flavus [5]. The aim of this study was ultrastructural investigations of the A. fumigatus hyphal cells, which cause the invasive aspergil-losis in a patient with acute leukemia.
MATERIALS AND METHODS
We investigate the lungs of patient K. (41 y.o.) with invasive aspergillosis and mixed phenotype acute leukemia. Invasive aspergillosis was developed after cytostatic chemotherapy (1-st course induction according the «RACOP» protocol). The diagnosis of invasive aspergillosis was made according EORTC/MSG 2008 criteria (risk factors, clinical and CT scan features, and positive galactomannan test in serum) [6]. Despite antifungal therapy with voriconazole progressed neurological symptoms with the development of coma, and increased respiratory and cardiovascular failure. After 10 days from the start of antifungal therapy the death of patient was verify. According to the results of my-cological examinations of the autopsy material was diagnosed disseminated invasive aspergillosis with lungs, brain and thyroid involvement, caused by Aspergillus fumigatus.
The lung pieces were fixed for light microscopy in 10% formalin solution and then treated according the standard methods. The paraffin sections were stained with hematoxylin-eosin (H-E) and by the Gomori-Grocott (GG) methods. For transmission electron microscopy (TEM) the lung pieces were fixed 3 hours in 3% solution of the glutaraldehyde, and then post-fixed during the 9 hours in 1% solution of osmium tetroxide, dehydrated in series of ethanol and acetone, after what were embedded in the epoxy medium epon-araldite according the previously described method [7]. From epoxy blocks on Pyramitome 11800 LKB we obtain semi-thin sections (3,0-5,0 ^m), which were stained with toluidine blue and investigated with light microscope for revealing the fungal aggregation and subsequent aimed trimming and cutting. Ultrathin sections were cut with glass knifes and stained with uranyl acetate and lead citrate and then observed with transmission electron microscope Jem-100 SX (Jeol, Japan).
RESULTS AND DISCUSSION
During the light microscopic examinations after of the H-E staining we observed in the patient lungs the vast areas
of necrosis with scanty inflammatory infiltration, presented with rare neutrophiles and macrophages. In lung tissue we revealed the fibrinous exudate and areas of aspergillus pneumonia. After the staining with G-G methods in areas of necrosis we identified the variable in size (from 15,0 to 25,0 ^m) aggregations of the radially oriented, branching (Fig. 1 a) and septate vegetative hyphae. The diameter of the hyphae was variable from 3 to 4 ^m.
Under TEM the vegetative cells of fungi may be solitary or form dense aggregations (Fig. 1 b, 2 c, h). The hyphal cells were differed in the presence or absence of vacuoles, and the degree of vacuolization. The apical fungal segments were rich with cytosol and practically without vacuoles (Fig. 1 b). Following the hyphal cells in the peripheral marginal area, were poorly vacuolized (Fig. 1 c-f). Vacuoles were small, with light content, uniformly distributed on the median sections of cells. Interphase nuclei single or in pairs, as a rule, occupied the lumen of the hyphae. They were spherical (1,2 ^m) or slightly ellipsoid (0,8x1,2 ^m) in shape, with lower level of uniformly distributed condensed chromatin (Fig. 1 e, f), contained one large spherical (from 0,4 to 0,5 ^m) excentric nucleolus with a higher contrast and predominance of granular component. Notice, that the lower level of chromatization of interphase nuclei was typical for in vitro growing cells of this [8] and another species of the Aspergillus [9-12].
cells varied from 5 to 7. They were single or often localized in small groups, polymorphic, variable in size (from 0,3 to 0,5 ^m), with a dark matrix and a often light cristae. The dark matrix of these organelles made them easily distinguishable from the moderately electron dense cytosol.
After the poorly vacuolated cells of hyphae followed (towards to the center of the fungal colony) cells with rare light medium size (Fig. 1 g) vacuoles or several large (Fig. 2 a, g), which occupies the main area in the cell. In these actively growing cells of the hyphae, the number of mitochondria was increased to 20-25 on the median section of cell. They are polymorphic. We identified the profiles of the mitochondria considerable in length (about 0,6-0,8 ^m, fig. 1 h) and often annular in shape. Storage substances in the cytosol were observed rarely, mostly it was a few small, rounded and of moderate electron density lipids inclusions, which are localized single or in small groups in the cytosol or near the cell wall (Fig. 2 d). Notice, that in vitro condition the mature hyphal cells of the A. fumigatus synthesized more variable types of storage substances [8] and the degree of last were higher, which can be explained by more nutritional value of the medium, as well as by the necessity to produce the numerous conidiogenous apparatus. Another explanation for the low content of storage substances in mature hyphal cells of the analyzed in present work case is still short time of infection current in the lung tissue of the patient.
Fig. 1. Light- (a) and transimission (b-h) electron microscopic
images of the A. fumigatus hyphal cells in patient lungs parenchyma. Abbreviations used in this and Fig. 2: V - vacuole, FH - fungal hyphae, EM - extracellular matrix, FC - fungal cell, CW - cell wall, LI - lipids inclusions, M - mitochondrion(ia), P - plug, PCW - primary cell wall, S - septum(ae), SCW -secondary cell wall, WB - Woronin body(ies), N - nucleus. Scale bars: a - 20,0 mkm, b - h - 1,5 |m
Mitochondria are uniformly distributed on the cells sections. They number on the median sections of growing
Fig. 2. Ultrastructure of the A. fumigatus vegetative cells in patient lungs parenchyma. Scale bars: a - 1,0 |m, b, j - 0,5 |m, c - j, k - 1,0 |m
Components of the endomembranouse systems were not good developed. They are presented in the form of short slightly curved cisterns of endoplasmic reticulum and small light vesicles, which localized near the cell wall. Single cisterns of the Golgi were not detected. The cytosol
with moderate electron density contains numerous free ri-bosomes. Microbodies were not detected.
The plasma membrane of intact hyphal cells was three-layered, asymmetric. Periplasmic space is absent. The lateral cell walls thin (from 0,15 to 0,20 ^m), light, homogeneous and with lower contrasting randomly oriented microfibrils (Fig. 1 b-f, h, 2 a, b, d - f, h, i, j). Notice that the hyphal cells of A. fumigatus in vitro [8] and in vivo [4, 5] possess with the cell wall similar in thickness and ultrastructure. Cell walls outside were covered with good developed dark granular-fibrilar layer, so-called «extracellular matrix» (Fig. 1 b-e, h, 2 c, h, i). In the mature hyphal cells the thickness of this layer was in 2-6 times (up to 1,0-1,2 ^m) larger in comparison with the cell wall.
During the growth and development of the hyphal cells we not observed significant increasing in the cell wall, what cannot be typical about of the extracellular matrix (Fig. 1 b, c, e, h, 2 c, h). It should be noted, that in the hyphal extracellular matrix of the tissular forms of A. fumigatus [1-5] was thicker and electron dense in comparison with the same in vitro [8], what clearly indicate about its protective function. As a rule, in the more mature part of the fungal aggregations the extracellular matrix of neighboring hyphae come into tight contact (Fig. 2 c, h), so that became the cause for the formation of specific hyphal clusters in the form of biofilm. In the cultural condition the extracellular matrix was revealed in another species, such as A. niger [9], A. flavus [10], A. versicolor [11] and A. terreus [12], but the level of its development in the same condition in A. fumiga-tus was more higher [8].
Rarely we mentioned the resting hyphal cells, which formed the thick (from 0,4 to 0,5 ^m) secondary cell wall (Fig. 2 f), similar in structure with the primary one. The another type of resting cells posses with very thick (from 0,6 to 0,8 ^m) primary cell wall (Fig. 2 d, e, i). In the first case, between the primary and secondary cell walls we detected the thin irregular in thickness dark homogeneous layer (Fig. 2 f, arrow), and the outside of last - a thin extracellular matrix. In the contents of the described both cells types we observed electron-dense cytosol, which often masking their intercellular components (Fig. 2 e, f, h).
It was obvious, that the presence of a thick walls and dark content of such cells indicate that some of the cells of the hyphae of the investigated tissular form of the fungus transit into a resting condition, which at the time of the favorable situation may cause secondary mycotic infection. Similar morphological peculiarity in the structure of hyphal cell walls was revealed on the example of tissular forms of another strain of A. fumigatus, which infected mouse lung [4]. Is still open the following question: are the identified peculiarity was typical for the individual cells of the hyphae, or it occurs within the one hyphae.
It is interesting to note, that during the transition of some hyphal cells in the resting stage its extracellular matrix was locally undergo lysis (Fig. 2 i, arrows) and reduce in thickness until complete disappearance. There is in fact, that the fungal cell wall and its integral part - extracellular matrix, present a dynamic «alliance» in the course of which the regular changes in their thickness and ultrastructure provides strong protection to the fungal pathogen from the cells of the human immune system. It was obviously the domination of the protective functions of the cell wall, but not extracellular matrix, during the transition of hyphal cells in the resting stage.
Intact hyphal cells were separated from each other by transverse wedge-shaped moderate electron density sep-tae (Fig. 2 a, b). The thickness of last was on the average was equal 0,20 ^m near the lateral cell and 0,12 ^m - in the middle part of septum. The ultrastructure of the septal pore apparatus was similar for the cells of the hyphal cells of tissular [4] and cultural forms [8] of A. fumigatus. Septum posses with central pore near which we revealed the Woro-nin bodies and dark variable in morphology plugs (Fig. 2 a, b). The diameter of septal pore was on the average 0,12 ^m. Woronin bodies were small, spherical (0,15 ^m), dark, homogenous, have a three-layered limiting membrane. The number of last components varied from 2 to 4 on median sections of septum. They were located at the same distance from each other and septal pore. Plugs were localized in the lumen of the septal pore. In the growing hyphal cells in the composition of the septal pore apparatus we identified only Woronin bodies, whereas in mature - Woronin bodies and plugs.
Thus, we not found the differences in the structure of septae and components of the septal pore apparatus in the hyphal cells of the tissular [4] and cultural forms [8] of A. fumigatus. The ultrastructure of the pore septal pore apparatus of the filamentous fungi was the rather conservative characteristic which recently attracted the attention of researchers for decision the questions in field of the fungal systematic and phylogeny. The analyses of own data [9-12] and in literature [Robinson P.M., et al.// Trans. Brit. My-col. Soc. - 1969. - Vol. 52, №3; Bojovic-Cvetic D., Vujcic R. // Protoplasma. - 1976. - Vol. 88; Momamy M., et al. // Mycology. - 2002. - Vol. 94, №2; etc.] shows that within the genus Aspergillus significant differences in the structure of the septal pore apparatus between the different species absent. However, according of these cytological characteristics, both as in vitro and in vivo conditions, reliably able provide the fungal identification on the genus level. The homogeneity in the structure of the septal pore apparatus of the members of the genus Aspergillus were the evidence of its naturalness and homogeneity.
The central part of the radially oriented fungal aggregations consists from senescent and dead cells. The contents of the predominant number of the cells was without cytosol (Fig. 2 j), and sometimes - destructive organelles. It is interesting note, that after the complete loosing the hyphal cells contents and the fully destruction of their cell walls, in lung tissue was presented the zones of accumulation of dark extracellular matrix with characteristic spherical light «holes» of locations of once intact fungal cells (Fig. 2 k, arrows).
In the senescent hyphal cells the nuclei lost their group localization, the enlightenment of the cytosol and nucleoplasm were revealed (Fig. 2 j); significantly decreased the number of organelles and storage substances. In a whole, the general pattern of the ultrastructural changes were identical with previously described for senescent growing in vitro cells of this and another species from genus Aspergillus [13].
The investigated in the patient lungs parenchyma the A. fumigatus vegetative cells were formed in mature condition the one giant mitochondrion that according the cytological criterion was the indicator of their high functional activity. Earlier [4], on the example of another strain of this species, which we investigated in mouse lung tissue, we did not reveal such morphological form of this cell component. We believed that the ability of the tissular form of the in-
vestigated in the patient lungs parenchyma vegetative cells of the A. fumigatus to develop a one giant mitochondrion was cytological indicator of its higher virulence, such as it was revealed for Cryptococcus neoformans in mouse brain during its transformation from cultural to tissular form [7].
Another specific features of the studied in present work tissular forms of A. fumigatus was the ability to developed the resting vegetative hyphal cells with dark cytoplasm, thick cell walls and the good developed extracellular matrix. Of course, that the ability to developed the resting cells, which can be considered analogues of infectious propagules with vegetative origin, shows a high level of functional activity of the fungus, and the frequency of their occurrence - about the duration of the infectious process. In our opinion, the presence of such type of the cells in the lung tissue may cause a secondary mycotic infectious process. It was interesting to note, that at the first time the numerous resting hyphal cells with the similar structures were revealed for tissular forms of another strain of the A. fumigatus in the lungs of mouse after 5 days after beginning of infection [4].
Thus, according the ultrastructural data in the studied patients lung tissue was dominated the intact and active hyphal cells of the A. fumigatus. Identified with a light microscope rare cells of immune system in the TEM
practically were not revealed. In a whole, the absence of the pronounced condition of the cells of the host immune system, may explains the intact and active condition of the fungal pathogen in the investigated patients lung tissue.
CONCLUSIONS
1. During the light-microscopic studies of lungs sections of patient with invasive aspergillosis we revealed the radially oriented variable in size aggregations of the A. fumigatus hyphae.
2. Morphogenesis of the A. fumigatus hyphal cells in the lungs parenchyma of the patient with invasive aspergillosis was accompanied with the increasing the level of vacuolization, mitochondrial proliferation, development of one giant mitochondrion, accumulation of a small number of storage substances in the form of lipids inclusions, thickening of the lateral cell wall, formation outside of last the well developed extracellular matrix. Mature hyphal cells did not differ among themselves by the number and fine structure of interphase nuclei, mitochondria, on the quantity and quality of accumulated storage substances and components endomembranous system.
3. The ability of the studied mature vegetative hyphae of the A. fumigatus to developed one giant mitochondria, resting hyphal cells and wide outer extracellular matrix can be consider as cytological factors of its higher virulence.
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Поступила в редакцию журнала 17.06.2015
Рецензент: М.А. Шевяков
