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ЭЛЕКТРОННО-
МИКРОСКОПИЧЕСКОЕ
ИЗУЧЕНИЕ
PSEUDALLESCHERIA BOYDII, P. ELLIPSOIDEA И P. ANGUSTA
Степанова A.A. (зав. лаб.)*, Васильева Н.В. (директор института, зав. кафедрой)
Северо-Западный государственный медицинский университет им. И.И. Мечникова, НИИ медицинской микологии им. П.Н. Кашкина, Санкт-Петербург, Россия
Представлены данные световой и электронной микроскопии по цитологической организации выращенных in vitro клеток гиф Pseudallescheria boydii, P. ellipsoidea и P. angusta. На полутонких эпоксидных срезах колоний изученных штаммов P. boydii, P. ellipsoidea и P. angusta особенности анатомического строения были идентичными; выявленные различия в толщине воздушного и субстратного мицелия, а также плотности расположения зрелых конидий различных штаммов P. boydii демонстрируют межштаммовые различия.
Клетки мицелия изученных видов содержали одно ядро с низким содержанием конденсированного хроматина. Морфогенез клеток воздушного и погруженного мицелия сопровождался усилением вакуолизации, увеличением числа митохондрий с формированием одной гигантской митохондрии в клетках субстратного мицелия, синте-зомразнообразных запасных веществ, компонентов эндомембранной системы и разных типов внеклеточных метаболитов.
Ключевые слова: сравнительный анализ, клетки гиф, in vitro, Pseudallescheria boydii, P. ellipsoidea, P. angusta, штаммы, ультраструктура
ELECTRON-MICROSCOPIC INVESTIGATIONS OF PSEUDALLESCHERIA BOYDII, P. ELLIPSOIDEA AND P. ANGUSTA
Stepanova A.A. (head of the laboratory), Vasilyeva N.V. (director of the institute, head of the department)
North-Western State Medical University named after I.I. Mechnikov, Kashkin Research Institute of Medical Mycology, St. Petersburg, Russia
The light- and electron microscopic data about the cytological organization of in vitro growing hyphal cells of Pseudallescheria boydii, P. ellipsoidea and P. angusta were presented. Peculiarities of anatomical structure on semi-thin epoxy sections between the investigated P. boydii, P. ellipsoidea and P. angusta strains colonies were identical; differences in thickness of aerial and substrate mycelium; the density of mature conidia distribution in the different strains cultures in P. boydii demonstrated the inter-strains differences.
Cells of the mycelium of investigated species contained one nucleus with the low level of condensed chromatin. Morphogenesis of aerial and submerged mycelium cells included increasing of vacuolization, development of the one giant mitochondrion (in the submerged mycelial cells), synthesis of various types of storage substances, components of endomembrane system and different types of extracellular metabolites.
Key words: comparative analysis, hyphal cells, in vitro, Pseudallescheria boydii, P. ellipsoidea and P angusta, strains, ultrastructure
Контактное лицо: Степанова Амалия Аркадьевна, e-mail: amaliya.stepanova@szgmu.ru
INTRODUCTION
The genus Pseudallescheria (Ascomycota, Euascomycetes, Microascales, Microascaceae) include 10 species. Pseudallescheria is the teleomorphic state of the anamorph - Scedosporium. These fungus are ubiquitous, saprophytic and can be isolated from agricultural soil and polluted water [1]. These fungi cause cutaneous infection and disseminated invasive infections in immunocompromised patient which are potentially fatal [1-3]. The application of septal ultrastructure to ascomycete systematics has been limited [4]. In literature the data on the ultrastructural patterns of hyphal cells morphogenesis of Pseudallescheria species is absent. This knowledgeis is very important for understanding, firstly, the degree of the relationships between the different Pseudallescheria species and, secondly, with its anamorphs. Thus, the aim of the present work was comparative analysis of the in vitro growing hyphal cell morphogenesis on the example of different strains of P. boydii and another species - P. ellipsoidea and P. angusta. It was important to compare this data with the similar obtained for its anamorphs - Scedosporium apiospermum [5] and S. aurantiacum [6].
MATERIALS AND METHODS
Four strains of P. boydii (CBS 117410, 117432, 120157, 301.79), one of P. ellipsoidea (CBS 301.709) and P. angusta (CBS 254.72) were cultured in vitro on potato dextrose agar (20 days incubation at 28° C) and were verified by rDNA ITS sequencing.
For scanning electron microscopy (SEM) the part of fungal colonies with nutrient medium after 7 and 20 days of sowing was transported in 3% glutaraldehyde (on 0,1 M cacodylate buffer) for 3 hour, post-fixed overnight in 1% osmium tetroxide in the same buffer, dehydrated by ethanol series (30°^70°), critical-point dried (HCP-2) for 15 min, coated with gold and observed in JSM 35 (JEOL, Tokyo, Japan).
For transmission electron microscopy (TEM) the pieces of medium with different part of fungal colonies after 7 and 20 days of cultivation were fixed for 3 h in 3% glutaraldehyde and post-fixed in 1% osmium tetroxide during 10 h. Samples were dehydrated through series of ethanol and acetone and embedded in epon-araldite epoxy resin. Prior to TEM observation, light microscopic investigations of semi-thin epoxy sections (3-5 ^m) were performed on Pyramitome 1180 (LKB, Bromma, Sweden) by using glass knives. Epoxy sections were stained with toluidine blue and investigated under a light microscope Leica DM 4000 (Leica Microsystem Inc, US). Ultrathin sections were cut with an Ultratome V 2088 (LKB, Bromma, Sweden), stai-ned with uranyl acetate and lead citrate and examined under a JEM-100 CX II transmission electron microscope (Jeol, Tokyo, Japan).
RESULTS AND DISCUSSION
Semi-thin longitudinal epoxy sections through the 20-days old mature part of strains colony demonstrated the presence of 3-ree distinct layers (Fig. 1 a-e, layers 1-3). The arrows on this figure demonstrate the nutrient medium surfaces. The 1-st outer layer demonstrated the aerial mycelium and 2-nd and 3-rd - submerged. All 3-ree layers were composed from dead or senescent hyphal cells. In one strain of P. boydii (CSB 117410) the thickness of aerial and submerged mycelium was equal (Fig. 1 a). In another P.
boydii strain (CBS 117432, fig. 1 b) the aerial mycelium was 2 times thinner than the submerged and in the strain CBS 120157 - 7 times (Fig. 1 c). In P. ellipsoidea (CBS 301.709) cultures the thickness of aerial mycelium in 6 times thinner than submerged (Fig. 1 d) and in cultures of P. angusta (CBS 254.72) the aerial mycelium was in 3 times thinner than the submerged (Fig. 1 e). In cultures of all analyzed species mature conidia were observed in the 3 lower submerged layers, but their concentration in the upper part (layers 2) of submerged mycelium was maximal (Fig. 1 a-e). As it was obvious on the semi-thin sections that the most higher level of the mature conidial concentration were typical for the P boydii strain CBS 117432 (Fig. 2 b, layer 2). Hence, the common patterns of anatomical structure between the investigated P. boydii, P. ellipsoidea and P. angusta strains cultures were identical. Differences in the thickness of aerial and submerged mycelium and the density of mature conidia between the different strains cultures of P boydii show the inter-strains differences.
Under SEM (Fig. 2) in the surfaces of central mature parts of 20 days old cultures of all investigated P. boydii, P. ellipsoidea and P. angusta strains we revealed the single small or variable in sizes clusters of bodies (Fig. 2 a-g). In cultures of the P boydii (strains CBS 117410, fig. 2 a), CBS 117432 (Fig. 2 b), CBS 120157 (Fig. 2 c) we observed the spherical sclerotia (Table) with crystalline texture of surfaces, but according data in literature [3] for this species were typical cleistothecia (Table). In mature cultures of P. ellipsoidea (strain CBS 301.79, fig. 2 d, e, table) and P. angusta (CBS 254.72) we revealed sclerotia (Fig. 2 d, f) and cleistothecia (Fig. 2 e, g). But in last species sclerotia were ellipsoidal. Sclerotia were also present in mature cultures of S. aurantiacum [6], but absent in S. apiospermum (Table) [5].
The diameter of the mature hyphal cells of aerial and submerged mycelium of the investigated cultures varied from 3,5 to 4,5 ^m. The hyphal cells of all investigated P boydii strains (Table, fig. 3 a, f, q) contain one ellipsoidal interphase nucleus with irregular envelope. Contrary the hyphal cells of P ellipsoidea (Fig. 3e, c, table) and P angusta (Fig. 3 b, d) contain from 1 to 2 nuclei with the similar ultrastructure. For comparison hyphal cells of S. apiospermum [5] and S. aurantiacum [6] also posses of one interphase nucleus, but vegetative cells of first species has more large interphase nucleus in comparison with P. boydii, P. ellipsoidea and P. angusta (Table). The interphase nucleus localized in the central part of hyphal cell and often occupied its lumens, contain the moderate level of randomly distributed condensed chromatin. Nucleolus localized near nuclear envelope (Fig. 3 j), spherical (0,5 ^m), contain granular and fibrillar components.
The morphogenesis of hyphal cells in all investigated strains cultures pass on 3-ree main stages: 1) growth, 2) maturation and 3) senescence. The first (Fig. 3 e, 5 a) and third (Fig. 3 d, q, 5 e) stages pass uniformly, but in second stage (Fig. 5 b-d) significant differences were revealed between mature hyphal cells of P. boydii (CBS 117410, 117432, 129157, fig. 5 b), P ellipsoidea (CBS 301.79, fig. 5 c) and P angusta (CBS 254.72, fig. 5 e).
Young hyphal cells contained uniformly localized small vacuoles (Fig. 3 e, 5 a). They may be light or contain thin-fibrillar material (Fig. 3 e). Mitochondria (from 6 to 8 on median cell section) distributed on the periphery of cells near the cell wall. They were single or in small groups, spherical (0,6 ^m) or ellipsoidal (0,5 x 0,6 ^m). Mitochondrial matrix was dark in comparison with cytosol.
During the growth of hyphal cells of aerial and submerged mycelium the number of mitochondria
Table
The main cytological characteristics of in vitro growing hyphal cells of S. apiospermum, S. aurantiacum, P. boydii, P. ellipsoidea and P. angusta
Strains Size of mature sclerotia/ cleistothecia, pm Number of Interphase nuclei Diameter ofthe Interphase nucleus, pm Level of chroma-tlzatlon Contour ofthe nuclear envelope Presence of mitochondrial reticulum In aerial and submerged mycelium Types ofstorage substances In mature hyphal cells Presence ofRER andSER Presence of mlcrobodles In the hyphal cells of aerial and submerged mycelium Presence ofthe extracellular slime Presence of extracellular llpophyllc substances
Scedosporium apiospermum
RCPFF 1491/1057 -/- 1 2,0x1,2 Low Irregular +/+ LI, a, GV, PG +/+ +/+ - -
RCPFF 490/712 -/- 1 2,0x1,2 Low Irregular +/+ LI, a, GV, PG +/+ +/+ - -
Scedosporium auranthiacum
CBS 116910 100-120/- 1 1,78x1,42 Low Regular -/- LI, a +/- +/+ + -
CBS 136047 80-110/- 1 1,80x1,6 Low Regular -/- LI, a +/- +/+ + -
CBS 136049 90-100/- 1 1,70x1,4 Low Regular -/- LI, a +/- +/+ + -
CBS 136.046 90-100/- 1 2,5x2,0 Low Irregular +/+ LI, a, LIV, GV +/+ +/+ + +
Pseudaiiescheria boydii
CBS 117410 50/-* 1 1,2x1,5 Low Irregular -/+ LI, a, ß +/- +/+ + -
CBS 117432 70/-* 1 1,5x3,5 Low Irregular -/+ LI, a, ß +/- +/+ + -
CBS 120157 50/-* 1 1,2x2,0 Low Irregular -/+ LI, a, ß +/- +/+ + -
Pseudaiiescheria ellipsoidea
CBS 301.79 50/70 1-2 1,3x2,0 Low Irregular -/+ LI,a,ß,GV, FB, PG +/- +/+ - -
Pseudaiiescheria angusta
CBS 254.72
40x60/ 80x100
1-2
1,2x2,0
Low
Irregular
-/+
LI,a,ß,GV, FB, PG
Notice: -* - we not revealed cleistothecia, but according Hoog - flbroslnous bodies; GER - granular endoplasmic reticulum; GV -endoplasmic reticulum, PG - polyphosphate granule In vacuole.
G.S. de, et al. (2011) they were typical for this species; a - alpha-glycogen; IS- betta-glycogen; FB protein globule In vacuole; LI - lipid Inclusions; LIV - lipophilic substances In vacuole; RER - rough
was increased (10-17 on median section). They were polymorphic (0,2-0,4 ^m), straight or slightly curved and situated in near cell wall. Often on the sections of cells of submerged mycelium of the all investigated in present work species we revealed long (3,5-4,3 ^m) profiles of the one giant organelle, so called «mitochondrial reticulum» (Fig. 3 f, q, table).
In the investigated strains of P. boydii and P. ellipsoidea mitochondrial reticulum was revealed only for hyphal
cells of submerged mycelium, but in P. angusta and S. apiospermum - in aerial and submerged (Table). In hyphal cells of S. aurantiacum (Table) mitochondrial reticulum was absent. The presence of mitochondrial reticulum in hyphal cells of aerial and submerged reticulum of S. apiospermum, S. aurantiacum (CBS 136.046) and P. angusta demonstrated its high metabolic activity. In all analyzed species and strains this component present in cells of submerged mycelium.
Fig. 1. Light microscopy of longitudinal semi-thin epoxy sections of the mature part of colony P. boydii (a-d) and P. angusta (e). a - CBS 117410, b - CBS 117432, c - CBS 120157, d - 301.79, e - CBS 254.72. Magnification: a-c - x 400.
Fig. 2. Ultrastructure of the sclerotia (a-d, f ) and cleistotecia (e, g) in cultures of P. boydii (a-e) and P. angusta (f, g). a - CBS 117410, b - CBS 117432, c - CBS 120157, d, e - 301.79, f, g - CBS 254.72. Scale bars: a, c, d, e, - 10 |m, b, f - 50 |m, g - 20 |m.
Fig. 3. Ultrastructure of the hyphal cells of P. boydii (a, e, f, h, q), P. angusta (b, d, j, m-o) and P. ellipsoidea (c, g, i, k, l, p). Scale bars: a, d, e, f, l, g, q, h, m - 1 |m, b, c - 1, 5 |m, i, j, k - 0,2 |m, n-p - 0,1 |m. Abbreviations used: EM - extracellular matrix, ER - endoplasmic reticulum, FB - fibrosinous body, GV - protein granule in vacuole, LI - lipid inclusion(s), M - mitochondrium, N - nucleus, Nu - nucleolus, PG - polyphosphate granules, V - vacuole.
Fig. 4. The structure of the external metabolites of the hyphal cells of aerial mycelium: a - P. boydii (CBS 117432), b-d - P. angusta (CBS 254.72). Scale bars: a, d - 2 |m, b - 0,5 |m, c - 1, 5 |m.
Fig. 5. Diagrams showing the peculiarity of development the hyphal cells of P. boydii (a, b, e), P. ellipsoidea (a, c, e) and P. angusta (a, d, e). Abbreviations used: a - alpha-glycogen, R - betta-glycogen, CW - cell wall, FB - fibrosinous body, ER - rough endoplasmic reticulum, GV - protein globule in vacuole, LI - lipid inclusions, M - mitochondrium, N - nucleus, Nu - nucleolus, S -septum. Polyphosphate granules in vacuoles of P. ellipsoidea and P. angusta for senescence hyphae (e) not demonstrated.
The components of the endomembranous system were not so good developed. Only moderate number of long straight or slightly curved cisterns of rough endoplasmic reticulum were revealed (Fig. 3 h). Small number (from 3 to 6 on median cell section) single or in small groups light secretory vesicles (60-70 nm) with thin-fibrillar content were distributed in cytosol. The rare (from 1 to 2 on median section) of large (0,3-0,4 ^m) microbodies with thin-fibrillar content revealed near the cell wall (Fig. 3 i, f, table) of hyphal cells in P boydii (CBS 120157), P. ellipsoidea (CBS 301.79) and P. angusta (CBS 254.72).
Cytosol was moderately electron dense and rich with free ribosomes. Plasma membrane was three-layered, closely contact with the electron-trans-parent thin (0,05 ^m) light cell wall (Fig. 3 e, arrow) which in hyphal cells of submerged mycelium covered with dark extracellular matrix (Fig. 3 e).
During the hyphal cell growth the size of vacuoles was increased. When hyphal cells finished growth the stage of maturation start. During this process synthesis of storage substances began. In cells of aerial and submerged mycelium of P boydii, P. ellipsoidea and P. angusta the three types of storage substances were revealed: lipid inclusions (Fig. 3 d, l, table), rosettes of a- (Fig. 3 q, j, l, arrows) and granules of 6-glycogen (Fig. 3 a, b, k, arrows). In the mature hyphal cells of P. ellipsoidea (CBS 301.79, fig. 3 k) and P angusta (CBS 254.72, fig. 3 l) we observed the globule (0,20,6 ^m) of protein in vacuoles and fibrosinous bodies with variable size and form in cytosol (Table, fig. 3 b, g, n-p). Lipid inclusions dominate, variable in size (0,3-0,6 ^m). As a rule, they were surrounded with rosettes of a-glycogen (Fig. 3 l). The rosettes of a- and granules of 6- glycogen were numerous and tightly localized. Dark single variable in size (0,2-0,6 ^m) protein globules localized in vacuolar content near the tonoplast (Fig. 3 k, m). In fungi fibrosinous bodies are rare type of fungal storage substances. They were revealed in the yeast cells of Cryptococcus neoformans [7, 8] and vegetative mycelium of Aspergillus versicolor [9] and A. candidus [10].
The mature cells of aerial mycelium in P boydii (Table, fig. 4 a, arrow) produced the moderate amount of external thin-fibrillar slime. External secondary metabolites were more abundant and variable in morphology (Fig. 4 b-d) in P angusta. The external metabolites outside the hyphal cells walls of aerial mycelium of P. ellipsoidea were absent, what distinguished this species also from investigated species of S. apiospermum and S. aurantiacum (Table). For hyphal cells of aerial mycelium of S. apiospermum [5] and one strain from four investigated S. aurantiacum [6] the synthesis and secretion of abundant external lipophilic substances were typical. In our opinion the rare type of smooth endoplasmic reticulum, which we revealed in the cells of aerial mycelium of this species, participate in the synthesis of this lipophilic substance. It may be pigment, which presence of in extracellular space of the hyphal cells of aerial mycelium determinate its yellow color [5, 6].
The presence of different types of extracellular metabolites outside the hyphal cells of investigated in vitro growing strains S. apiospermum, S. aurantiacum, P. boydii and P angusta demonstrated its high metabolic activity, what sharply distinguished this species from P. ellipsoidea (Table). For comparison, for the cells of aerial mycelium of another species of pathogenic fungi such peculiarity was not typical [11-14]. In our opinion the external substances
play the adherent, protective and nutrition functions.
In the stage of senescence, large central vacuole was developed in the content of hyphal cells (Fig. 3 d, 5 e). The volume of cytosol, the amount of the storage substances and cell organelles significantly decrease. The volume of the protein globules in vacuoles, lipid inclusions and amount of glycogen in cytosol gradually decreased. Numerous small (0,1 - 0,2 ^m) dark polyphosphate granules (Fig. 3 d, table) appeared in the vacuolar content of hyphal cells of P. ellipsoidea and P angusta. The process of senescence passes according the way which was typical for S. apiospermum [5] and S. aurantiacum [6].
CONCLUSION
The longitudinal epoxy semi-thin sections through the 20-days old mature part of strains colony of investigated species of Scedosporium (S. apiospermum, S. aurantiacum) and Pseudallescheria (P ellipsoidea, P. angusta) demonstrated the presence of three distinct layers: one layer typical for aerial mycelium and two for submerged one. The mature conidia were revealed in all this layers. But strains of this species differ according the thickness of this layers and concentration of mature conidia.
In cultures of S. apiospermum, contrary with S. aurantiacum (Table), sclerotia and cleistothecia were absent. Cultures of P. boydii differ from its same its anamorph S. apiospermum by the presence of sclerotia (Table) and cleistothecia [3]. Cultures of P ellipsoidea and P angusta also produced sclerotia and cleistothecia, but differed according its form (spherical in P. ellipsoidea and ellipsoidal in P. angusta).
The hyphal cells in P ellipsoidea and P angusta differ from the same of P boydii, S. apiospermum [5] and S. aurantiacum [6] by the presence of variable number (from 1 to 2) of interphase nuclei (Table). Becourse S. apiospermum and its teleomorph P boydii have the same numbers of nuclei was possible to expect that for anamorphs of P. ellipsoidea and P angusta were typical presence of 1-2 nucleus. The large interphase nuclei were typical for hyphal strains of S. apiospermum and one strain (CBS 136.046) S. aurantiacum (Table). Small nuclei were revealed in hyphal cells of P boydii, P. ellipsoidea and P angusta. The hyphal cells of tree strains (CBS 116910, 136047, 136049) of S. aurantiacum according to this characteristic occupied the intermediate position.
Hyphal cells of investigated species differ according the presence of mitochondrial reticulum which presence shows the higher level of their metabolic activity. In S. aurantiacum hyphal cells (for exclusion CBS 136.046) this component was absent. In hyphal cells of both strains cultures of S. apiospermum and one strain S. aurantiacum (CBS 136.046) mitochondrial reticulum was typical for aerial and submerged mycelium. It was possible to notice that the last strain of S. aurantiacum in comparison with another produced more numerous types of storage substances (Table) [6]. Rough endoplasmic reticulum was revealed in hyphal cells of the mycelium in all species (Table), but smooth - in S. apiospermum and S. auratiacum (CBS 136.046). Microbodies were typical for all investigated cells of aerial and submerged mycelium (Table).
In general the conducted investigations demonstrated that early and final stages of morphogenesis in investigated fungi pass uniformly and differences in mature hyphal ultrastructure were present between analysed species
and sometimes strains. S. apiospermum differ from S. aurantiacum and P. boydii, P. ellipsoidea and P. angista by the absence of sclerotium (Table).
Inter-strain differences between the mature hyphal cells of S. apiospermum, S. auranticum (for exclusion of strain CBS 136.046) and P. boydii were absent (Table). Cells of vegetative mycelium in cultures of P. ellipsoidea and P. angista synthesize the greatest types of the same storage substances. Only the mature cells of analyzed teleomorphic species (P boydii, P. ellipsoidea and P. angista) may synthesize in cytosol dark granules of 6-gly-cogen (Table). The distinctive structural feature of P. ellipsoidea and P. angista mature hyphal cells were ability to synthesize the
fibrosinous bodies. Mature hyphal cells of S. apiospermum contrary with the same of P. boydii synthesized more numerous types of storage substances.
Ultrastructural data about mature hyphal structure provide us more deep information about the morphological and physiological similarity between the strains of one species and different Scedosporium/Pseudallescheria species.
ACKNOWLEDGEMENT
We thank to Prof. G. Sybren de Hoog from the Centraalbureau voor Schimmelcultures (CBS) for providing cultures of P boydii, P. ellipsoidea and P. angusta strains used in this study.
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Поступила в редакцию журнала:22.02.2019
Рецензент: Т.С. Богомолова
