Different ultrastructural morphology of Pneumocystis carinii derived ...

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morphology of Pneurnocysfis carinii derived from mice, rats, and rabbits. APMIS ... Key words: Pneumocystis carinii; electron microscopy; host-species-linked ...

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Different ultrastructural morphology of Pneumocystis carinii derived from mice, rats, and rabbits M. H. NIELSEN,’ 0. F? SETTNES,’ E. M. ALIOUAT,’ J.-C. CAILLIEZ’ and E. DEI-CAS’ ‘Institute of Pathological Anatomy, *Institute of Medical Microbiology and Immunology, University of Copenhagen, Copenhagen, Denmark and ’U42 INSERM, Villeneuve d’Ascq, Lille, France

Nielsen, M. H., Settnes, 0. F?, Aliouat, E. M., Cailliez, J.-C. & Dei-Cas, E. Different ultrastructural morphology of Pneurnocysfis carinii derived from mice, rats, and rabbits. APMIS 106: 771-779, 1998. Pneurnocysfis carinii (PC) is a fungus present in the lungs of many mammal species. Even though studies of the genome, the isoenzymes, and the antigens have proved some host-species-linked heterogeneity, the existence of distinct Pneumocystis species or subspecies has still not been accepted. Comparative studies of the ultrastructural morphology of pneumocysts derived from several host species may support evidence of host-species-linked heterogeneity. We have compared the ultrastructural morphology of pneumocysts derived from mice, rats, and rabbits. The density of membrane-limited electron-dense cytoplasmic granules was found to be higher in mouse-derived pneumocysts than in rabbitderived pneumocysts, and furthermore the average diameter of the granules from mouse pneumocysts was larger than that of granules from rabbit-derived pneumocysts. The average diameter of the filopodia of mouse-derived pneumocysts was smaller than that of filopodia from rat-derived pneumocysts, which was smaller than that of filopodia from rabbit-derived pneumocysts. Globular electrondense bulbous dilatations at the tip of the filopodia were described for the first time and they were only found on filopodia of mouse-derived pneumocysts. These distinct host-species-linked morphological differences of pneumocysts from mouse, rat, and rabbit may support previous biochemical data indicating the existence of different Pneumocystis species or subspecies.

Key words: Pneumocystis carinii; electron microscopy; host-species-linked heterogeneity; membranelimited cytoplasmic granules; rat; mouse; rabbit.

0. F? Settnes, Institute of Medical Microbiology and Immunology, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen N, Denmark.

Pneurnocystis carinii (PC) is present in the lungs of many mammals. It is a harmless microorganism in immunocompetent individuals, but may cause severe and fatal pneumonia in immunocompromised or strongly immunosuppressed hosts, as now evidenced in human and veterinary medicine. The ultrastructure of PC has been described in many reports (Rusfolo 1994). Evidence of the life cycle of this organism has also been Received September 3, 1997. Accepted February 3, 1998.

provided (Vuvra & Kucera 1970; Campbell 1972; Vossen et al. 1978; Yoshida et al. 1984; Matsurnoto & Yoshida 1986; Haque et al. 1987; Itatani & Marshall 1988; Bedrossian 1989; Nielsen & Settnes 1991; Cushion et al. 1991; Palluault et al. 1992; Goheen et al. 1992, Itatani 1996). The basic biology of PC is still largely unknown, and it was only recently, through similarities between the 16s-rRNA-coding regions, rendered probable that PC had a closer phylogenetic relationship to the higher fungi than to protozoa (Edman et al. 1988; Kwon-Chung 1994; 77 1

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Stringer et al. 1992; Stringer 1996; Cailliez et al. 1996). Studies of the PC genome (Wakefield et al. 1990; Sinclair et al. 1991;Edlind et al. 1992; Gigliotti et al. 1993; Lee et al. 1993; Stringer et al. 1993; Keely et al. 1994; Peters et al. 1994a, b; Weinberg et al. 1994; Hunter & Wakefield 1996; Wakefield et al. 1997), of isoenzymes (Dei-Cas et al. 1994; Mazars et al. 1995, 1997), and of surface carbohydrates (Graves et al. 1986; Walzer & Linke 1987; Kovacs et al. 1989; Gigliotti et al. 1988; Gigliotti 1992; Smulian & Walzer 1992; Bauer et al. 1993; Christensen et al, 1996) indicate a host-species-linked heterogeneity of PC. The existence of distinct species or subspecies of Pneumocystis has been suggested, but not yet accepted (Bartlett et al. 1996). Except for the results of a few studies (Aliouat et al. 1993; Gigliotti et al. 1993a; Dei-Cas et al. 1994) it is not clear to what extent PC isolates from certain species may infect other species. There are few comparative studies on the ultrastructure of pneumocysts obtained from different hosts (Palluault et al. 1992; Dei-Cas et al. 1994). However, these studies indicate the existence of host-related morphologic differences with regard to the pneumocyst filopodia. Extended knowledge of these differences may be used together with other criteria to separate possible PC species or subspecies in the future. In the present study a comparison was made between the ultrastructure of PC in lung tissues from mice, rats and rabbits, and various ultrastructural differences are reported, some of which have not previously been described.

MATERIALS AND METHODS

Animals Two female SCID mice (Iffacredo, Lyon, France), aged 4 to 6 weeks, were nasally instilled with 40X lo6 pneumocysts 11 weeks before they were killed. Four female outbred mice aged 4 to 6 weeks were treated with prednisolone (40 mg/l in their drinking water) for 3, 4, 4 and 6 months, respectively, before they were killed. Two female Wistar rats (Iffacredo, Lyon, France), 5 and 6 weeks of age, were treated with dexamethasone (2 mg/l in their drinking water) for 2 and 3 months, respectively, before they were killed. Two male rabbits (Vasseur, Prouzel, France), 28 days of age, one untreated and one treated for

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one week with prednisolone (25 ml/l in their drinking water) were also included.

Preparation for electron microscopy All animals were anesthetized with ethyl ether. The thorax was opened and an incision was made in the left atrium wall of the heart. The lungs were then perfusion-fixed by injection into the right ventricle of 2% glutaraldehyde in phosphate buffer [pH 7.21 with 6% sucrose added (650 mOsmol). After 20 min the lung tissue was cut into l X l X l mm blocks, which were kept in the same fixative for an additional 4 h. The blocks were then fixed for 4 h - some for 18 h in 1% osmium tetroxide in 0.1 M cacodylate buffer [pH 7.21, dehydrated, and embedded in Epon.

Morphometry The density of cytoplasmic granules was obtained using a single lattice grid with a distance of 0.5 pm between test lines on electron micrographs at X66,OOO magnification. The number of granules per area of cytoplasm was=N: (PX0.52), where N is the total number of granules counted and P the number of test line intersections over the same cells. Granules and filopodia for diameter measurements were sampled at random using electron micrographs with a magnification of X66,OOO or X82,500. Only structures strictly cross-cut were measured.

RESULTS The lungs of dexamethasone-treated and control rabbits had a fairly normal histological appearance, whereas the lungs of corticosteroidtreated mice and rats showed increased cellularity of the lung tissue, including the alveolar walls. Large clusters of pneumocysts were noted in many alveoli. By electron microscopy it was found that the pneumocysts were not evenly distributed. In some areas they completely filled the alveoli, in other areas they were either abTABLE 1. Cytoplasmic densities and mean diameters of cytoplasmic granules in pneumocysts derived from SCID mice, outbred mice, and rabbits Cytoplasmic Mean SD N density diameter (eranules/um2) (nm) SCID mice 5.18* 91 9.6 50 Outbred mice 87 10.3 50 Rabbits 1.61 68 8.8 37 * includes measurements of pneumocysts from outbred mice as well. SD=standard deviation. N=number of granules measured.

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sent or only a few scattered pneumocysts could be found attached to the alveolar wall. As judged from the overall density of pneumocysts, there was no marked difference in the intensity of the PC infection in SCID mice and outbred mice or between animals of the same species treated with corticosteroids for different periods of time. The ultramicroscopic structure of pneumocysts derived from mice, rats and rabbits was in principle similar, and with few exceptions the study confirmed previously published findings (Matsumoto & Yoshida 1986; Haque et al. 1987; Itatani & Marshall 1988; Nielsen & Settnes 1991; Palluault ef al. 1992; Goheen et al. 1992). How-

ever, consistent differences were seen regarding the morphology of the cytoplasmic granules and the filopodia. Most significant was the presence of electrondense membrane-limited granules in the cytoplasm of trophozoites, but not in precysts or cysts of pneumocysts derived from mice and rabbits (Figs. 1 , 2, 3, 7). These granules were never seen in the cytoplasm of rat-derived pneumocysts. They were about three times as numerous in mice-derived pneumocysts as in rabbitderived pneumocysts, and furthermore they had a larger average diameter in mice-derived pneumocysts, i.e. 89 nm vs 68 nm in the rabbit-derived pneumocysts (Table 1). The granules were

Fig. 1. P. carinii at the alveolar wall of a SCID mouse. N denotes nucleus, M mitochondrium, MG membranelimited cytoplasmic granules, and E erythrocyte in lung capillary. Magnification: X 19,000. Fig. 2. Part o f a P. carinii cell from a SCID mouse. MG denotes membrane-limited cytoplasmic granules, CM cell membrane, and CW cell wall. Arrow denotes electron-dense material between granule and cell membrane. Magnification: X 128,000.

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Figs. 3 & 5. P. carinii from a steroid-treated rabbit. F denotes filopodia, N nucleus, NN nucleolus, M mitochondrium, MG membrane-limited cytoplasmic granules. Glycogen granules (G) may surround a clear area (CL). Magnification: Fig. 3= X 19,000, Fig. 5= X63.000. Fig. 4. P. carinii from an untreated rabbit. C denotes cyst with intracystic bodies, N trophozoite nucleus, CL clear area surrounded by glycogen granules (G), F filopodia. Magnification: X25,OOO. Fig. 6. P. carinii from a steroid-treated Wistar rat. Cross-sectioned filopodia (F) with occasional glycogen granule (G).Magnification: X63,OOO.

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TABLE 2. Diameters of cytoplasmic core ofjilopodia from pneumocysts derived f r o m mice, rats, and rabbits Mean diameter SD* N (nm) 14.5 50 Mice 48 25.0 50 Rats 52 21.6 50 Rabbits 104 * SD=standard deviation. N=number of filopodia measured.

dispersed in the cytoplasm, but most were located in the peripheral part of the cytoplasm where some of them were seen “attached” to the cell membrane by electron-dense material (Fig. 2, arrow). A fusion of the granules with the cell membrane was never observed. The content of the granules was electron dense, homogeneously punctate. Filopodia were mostly filiform extensions of the pneumocyst cell enclosing the cell wall as well as the cell membrane and a core of cytoplasm. The filopodia were often branching

(Figs. 5, 7 , 9). In some lung areas, pneumocysts had many filopodia; in other areas, they had few or none. The presence or absence of filopodia was apparently not related to the local density of pneumocysts or to the extent of pneumocysthost cell surface contact. As regards shape and size, the filopodia showed great variations. However, when filopodia from the three host animal species were compared, consistent host-related differences were found. In general, rabbit-derived pneumocysts had filopodia which were wide, almost spherical and short-stalked (Figs. 3, 4, 5 ) with occasional small cytoplasmic inclusions such as glycogen granules (Fig. 5). The mean diameter of the electron-lucent cytoplasmic core with cell membrane was 104 nm in rabbit-derived pneumocysts (Table 2). In pneumocysts from mice (Figs. 7 , 8, 9) and rats (Fig. 6) the filopodia had the shape of slender, often branching filiform cell extensions with an electron-lucent core. The mean diam-

Fig. 7. P. carinii from a steroid-treated outbred mouse. Filopodia (F), some of which are branching, and some with electron-dense spherical dilatations (H). Membrane-limited cytoplasmic granules are marked MG. Magnification: X 16,000. Figs. 8 & 9. P. carinii from steroid-treated outbred mice. Filopodia (F) with electron-dense hyphal body-like dilatations (H). Magnification: X63,OOO.

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eter of the core with the cell membrane of the filopodia of mice-derived pneumocysts was 48 nm and in rat-derived pneumocysts 52 nm. Filopodia of mice-derived pneumocysts occasionally had a 150 nm to 250 nm wide bulbous dilatation at their tip with a homogeneously stained electron-dense content (Figs. 7, 8, 9). As for the fine structure of pneumocysts from the three host species, no additional morphological differences were noted. This accounts for the size and shape of cells, nuclei, mitochondriae with cristae, rough and smooth endoplasmic reticulum, ribosomes, glycogen granules, lipid inclusions and cytoplasmic vesicles. Spherical or more irregularly shaped emptylooking areas were occasionally present in the cytoplasm of pneumocysts from all three host species (Figs. 4, 7). Sometimes these clear areas were surrounded by densely packed glycogen granules (Figs. 3, 4). Smooth endoplasmic reticulum or Golgi cisterns were sparce and distinct Golgi zones (dictyosomes) were not observed. Nuclear associated dense bodies (NAO) were not with certainty found in any of the many hundred pneumocysts examined. The cell wall of trophozoites and cysts had the same fine structure. Typically, the cell membrane is smooth in trophozoites, but wrinkled in maturating or mature cysts. DISCUSSION In the present study we describe for the first time the limiting membrane of the spherical electron-dense cytoplasmic granules of pneumocysts, and furthermore that their size and cytoplasmic density may vary in isolates from different host species. Thus, we found that the cytoplasmic density of the 87-91 nm granules of mice-derived pneumocysts was three times as high as that of the 68 nm granules of rabbitderived pneumocysts, whereas rat-derived pneumocysts had no granules. In a review of the morphology of the pneumocyst the dense granules were described as universal cytoplasmic organelles of pneumocysts (Ruffolo 1994). From the present and a previous study (Nielsen & Settnes 1991) we may modify this statement and add that the granules may be absent from ratderived pneumocysts. However, that this may not be the case in all rat-derived pneumocysts 776

is evidenced from the papers describing or just illustrating the granules in rat-derived pneumocysts (Barton & Campbell 1969; Vavra & Kucera 1970; Bouton et al. 1977; Takeuchi 1980; Matsumoto & Yoshida 1986; Yoshikawa et al. 1987; Sukura 1995; Itatani 1996). In contrast, we always observed the dense cytoplasmic granules in mouse- and rabbit-derived pneumocysts, which confirms previous findings made by others (Tamura et al, 1978; Dei-cas et al. 1989, 1994). The dense granules have been observed in human-derived pneumocysts too by Campbell (1972) and recently by Benjield (personal communication 1996). However, none of the abovementioned studies has compared the cytoplasmic density and the size of the granules in pneumocysts derived from different hosts. We conclude that the spherical membrane-limited granule is a normal cytoplasmic organelle of pneumocysts, which, however, may be absent or at least very scarce in rat-derived organisms. The size of the granules and their cytoplasmic density varied depending on the host species from which the organisms were derived. Nothing is known about the biochemical composition and function of the dense granules. We may state, however, that their presence in trophozoites, and not in precysts or cysts, indicates important functions for the metabolically most active organisms. Granules called “Golgi vesicles” were described in rabbit-derived pneumocysts (Palluault et al. 1992). They were labeled with colloidal gold-conjugated Concanavalin A. In a previous work (Palluault et al. 1990), ultracytochemical evidence was presented for the existence of a Golgi complex in pneumocysts, i.e. thiamine pyrophosphate and beta-glycerophosphate activity in Golgi-like and primary lysosome structures. However, we have not observed distinct Golgi zones (dictyosomes) and this was also mentioned by other authors who have questioned whether pneumocysts have a proper Golgi apparatus (Vuvra & Kucera 1970; Bouton et al. 1977; Haque et al. 1987; Yoshida 1989; Ruffolo et al. 1989; Bartlett et al. 1996). Filopodia are filiform extensions of trophozoite cells. They increase the resorptive cell surface area greatly, but it is not known why they are absent in some clusters of pneumocysts and yet abundant in neighboring clusters. It is known that fungi develop large mycelia in areas

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rich in nutrients and that the mycelium is sparse or nonexistent in areas devoid of nutrients. The obtained nutrients are then shared through a communication network of hyphas. Such a communication network has earlier been suggested for pneumocysts (Murphy et al. 1977). This would be in accordance with the genetic evidence of a close relation of pneumocysts to fungi (Edman et al. 1988; Stringer et al. 1992; Pixley et al. 1991). There are morphological differences among filopodia of pneumocysts isolated from different host species. Thus, it was found (Dei-Cas et al. 1994) that filopodia of rabbit-derived pneumocysts were shorter and wider than filopodia of mice-derived pneumocysts. The present study confirms this observation and demonstrates a similar size difference between the filopodia of rabbit- and rat-derived pneumocysts. However, only filopodia of mice-derived pneumocysts have globular electron-dense bulbous dilatations at their tip. These “hyphal body”-like structures have to our knowledge not previously been described in pneumocysts. The nucleus-associated organelle (NAO) or spindle pole body is the analogue of the centriole of the fungus cell. Its presence was earlier described in rat-derived pneumocysts (Goheen et al. 1992), but not until recently (Ztatani 1996) has it been convincingly demonstrated in ratderived pneumocysts. Why the NAOs were not observed in the present study may be explained by the fixation procedure used. Goheen et al. (1992) showed for the first time that potassium ferrocyanide/osmium tetroxide post-fixation clearly enhances the staining contrast of many structures in the cytoplasm of pneumocysts. This is also the case with the NAOs (Itatani 1996). Failure to demonstrate the NAOs in any of the pneumocyst cells in this study may thus be caused by the omission of the potassium ferrocyanide/osmium tetroxide post-fixation. As mentioned in the introduction to this paper, studies of the PC genome, of PC isoenzymes, and of PC surface antigens have led to the suggestion of a host-species-linked heterogeneity of pneumocysts. The present study has extended and confirmed previous knowledge respecting the differences in ultrastructural morphology between pneumocysts derived from different host species (Palluault et al. 1992; DeiCas et al. 1994) and proved the existence of a

host-species-linked morphological heterogeneity of pneumocysts as well. Taken together the morphological findings may thus support the hypothetical existence of distinct Pneumocystis species or subspecies. This research was supported by European Concerted Action, BioMedl. CT94 1118. Part o f the results have been presented at the European Concerted Action BioMed-1 Second Specialized Meeting: In vitro Studies in Pneumocystis Research. Copenhagen, September 5-8, 1996.

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