Z. Parasitenk. 47, 1--9 (1975) © by Springer-Verlag 1975

Interactions between Encephalitozoon cuniculi and Macrophages Parasitophorous Vacuole Growth and the Absence of Lysosomal Fusion* Earl Weidner Department of Zoology and Physiology, Louisiana Stare University, Baron l~ouge l%eceived February 14, 1975

Summary. Encephalitozoon cuniculi grow within ever-increasing parasitophorous vacuoles (PV) in peritoneal macrophages. The PV boundary membrane conforms to a rich arrangement of blebs; similar, but free vesicles were observed within the PV space. An iron dextran-concanavalin A marker was used to express visually clustered distributions of Con A receptors on the PV boundary blebs and free vesicles; no marker was observed on other membrane surfaces within the PV. These results, combined with the observation that the PV grows while the host cytoplasm decreases in mass, implieate the PV boundary blebs of interiorizing into vesicles by a pinocytic mechanism. Phagocytic vacuoles, secondary lysosomes a r d pinocytic vesieles were labeled by incubating infected macrophages in minimum essential medium with ferritin. Ferritin readily aceumulated in secondary lysosomes and phagocytic vacuoles; however, ferritin was excluded from parasitophorous vacuoles containing E. cuniculi. Acid phosphatase cytochemical reaction product was observed in lysosomes and phagocytic vacuoles; however, parasitophorous vaeuoles with vegetative E. cunicuti were always negativs.

Introduction I n t e r a c t i o n s occurring between two c o m p e t i t i v e systems are exemplified b y t h e i n t r a c e l l u l a r p a r a s i t i s m of m a c r o p h a g e s w i t h Encephalitozoon cuniculi. This small p a r a s i t i c e u c a r y o t e (classified as Microsporida, Protozoa) produces chronic, orten latent, infections in a v a r i e t y of m a m m a ] s , including m a n ( S h a d d u e k , 1971). E. cuniculi e h a r a c t e r i s t i c a l l y grows within a p a r a s i t o p h o r o u s vacuole (PV). T h e size of t h e vacuole can be considerable, as in r a b b i t choroid plexus cells (V£vra et al., 1972). I n mouse peritonea] macrophages, m a t u r i n g p a r a s i t e colonies grow w i t h i n a P V which m a y measure 15-20 ~zm in d i a m e t e r (Nelson, 1962). I n f e c t e d m a c r o p h a g e s c o n t i n u a l l y lose c y t o p l a s m i c mass during P V growth. A n i r o n - d e x t r a n - c o n c a n a v a l i n A m a r k i n g p r o c e d u r e was used to gain some insight into t h e g r o w t h m e c h a n i s m of t h e PV. Also of interest was t h e knowledge t h a t m a c r o p h a g e s h a v e a well d e v e l o p e d ]ysosomal complex. Since E. cuniculi r e a d i l y grows w i t h i n a P V s u r r o u n d e d b y this lysosomal network, we t h o u g h t it would be of some i n t e r e s t to a n a l y s e w h e t h e r t h e r e is an absence of ]ysosomal fusion w i t h t h e P V as in Toxoplasma gondii in mouse m a c r o p h a g e s (Jones a n d Hirsch, 1972). * This work was supported in part by ~ational Science Foundation GA-36198 and DES 75-03 359.

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Materials and Methods Encephalitozoon cuniculi was passaged in young female Tex(S) Swiss mice (Texas Inbred Mice Co., Houston) by injecting parasite-rich peritoneal ascites (0.25 tal) at 12-14 day intervals. Experiments were run on macrophages from 14-day infected mice. A simple two-step procedure, deve]oped by Martin and Spicer (1974), was fotlowed for visualizing concanavalin A (Con A) receptors on or within parasitophorous vacuoles. Infected ceIls were fixed in 2 % glutaraldehyde, washed with bnffer, and exposed to a shearing aetion of a glass homogenizer (Pyrex No. 7727) in order to open parasitophorous vacuoles (PV) for substrate penetration. Vegetative parasites retained attachment to the PV boundary and were useful in subsequent recognition of the PV envelope. Prepared cells were incubated in I mg/tal solution of Con A in phosphate buffered saline (PBS) at pH 7.4 for 20 min. After five washes with PBS at pH 7.4, cells were exposed to 3 ml PBS containing 50 mg iron dextran (Imferon, Lakeside Laboratories, Inc.) for 30 min. Following 5 washes in PBS, cells were prepared in a standard manner for electron microscopy. Controls were run by omitting Con A in PBS prior to the iron dextran step. For ferritin labeling of seeondary lysosomes, host cells were exposed to 5 mg of ferritin/ml in vitro or in vivo following the procedure of Jones and Hirsch (1972). In vivo incubation results were more satisfactory since in vitro cell transfers tended to stimulate intra-PV spore hatching which unfortunately caused PV collapse. After 2 and 18 hr incubations with ferritin, the cells were collectcd by slow centrifugation and fixed in glutaraldehyde, post-fixed in osmium and processed by standard procedure for electron microscopy. For visualizing primary and secondary lysosomes, eells were prepared for acid phosphatase cytochemistry using a modification of the Gomori technique as developed for electron microscopy by Bainton and Farquhar (1968). Cells were fixed in 2% glutaraldehyde in cacodylate buffer and washed several hours in buffer. The incubation medium was made up of 20 ml of 0.125% substrate (beta-glyeerophosphate) and 20 ml of 0.2 M Tris-maleate pH 5.0 (adjusted with 1.0 N HCI); to this was added 1.5 ml 2% lead nitrate (added dropwise) and 5 ml oB 0.1 ~¢IMgCl~. After incubation of celIs in this medium for 20 min at 37°C, cells were rinsed in 5 % acetic acid for 5 min, rinsed in buffer, post-fixed in osmium and processed by standard procedures for electron microscopy. Controls followed the same procedures but without betaglycerophosphate as substrate.

Results P r e s u m p t i v e host p h a g o c y t i c m e m b r a n e closely s u r r o u n d s t h e p l a s m a memb r a n e of E. cuniculi after e n t r y of t h e p a r a s i t e into m a c r o p h a g e c y t o p l a s m . S u b s e q u e n t d i s p r o p o r t i o n a t e g r o w t h of this p r e s u m e d m a c r o p h a g e - d e r i v e d memb r a n e p r o v i d e s t h e beginnings of t h e p a r a s i t o p h o r o u s vacuole (PV) (see Fig. 1). F r e e surfaces of m u l t i p l y i n g p a r a s i t e s e x t e n d into t h e ever-increasing P V space while v e g e t a t i v e p a r a s i t e s r e t a i n localized p l a s m a m e m b r a n e a t t a c h m e n t to t h e P V b o u n d a r y (Fig. 2). N o surface modifications were d e t e c t e d a t t h e m e m b r a n e suffaces a t p o i n t s of t i g h t - j u n c t i o n - l i k e a t t a c h m e n t b e t w e e n t h e p l a s m a m e m b r a n e of t h e p a r a s i t e a n d t h e P V m e m b r a n e (Figs. 2-4). I n f e c t e d m a c r o p h a g e s c o n t i n u a l l y lost cy~oplasmic m a s s while t h e P V grew; moreover, t h e size of t h e P V was p r o p o r t i o n a l t o t h e n u m b e r of v e g e t a t i v e p a r a s i t e s inside. T h e P V m e m b r a n e b o u n d a r y , free of a t t a c h e d v e g e t a t i v e parasites, c o n f o r m e d t o a c a n o p y of blebs w i t h single or m u l t i p l e c l a v a t e profiles (Figs. 3-5). S i m i l a r u n a t t a c h e d vesicles, orten in aggregates, were freely dispersed t h r o u g h o u t t h e P V space; t h e incidence of these vesicles was higher a t p o i n t s where v e g e t a t i v e p a r a s i t e s were a t t a c h e d to t h e P V b o u n d a r y (see Figs. 4, 5).

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Fig. 1. Early developmental stage of vegetative E. cuniculi in mouse peritoneal macrophage. Arrows indicate areas where presumptive host phagocytic membrane is separated from parasite and provides the beginning of parasitophorous vacuole space. × 25000 Fig. 2. Electron micrograph of E. cuniculi in large PV. Vegetative parasites are attached to the boundary of the PV. Note the uniform canopy of small blebs (see arrows) at the PV boundary. × 18000

Concanavalin A (Con A ) Receptor Sites on P V Membrane Glutaraldehyde-fixed parasitophorous vacuoles i n macrophages were rendered accessible to a Con A-iron d e x t r a n m a r k i n g technique for visualizing Con A receptor

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Figs. 3--5. Electron micrographs of PV boundary blebs and vesicles (see arrows) extending into PV spaee. Note vegetative parasites in tight-junction-like attachment to PV boundary. × 45000

sites. I r o n d e x t r a n particles a c c u m u l a t e d in discrete p a t c h e s on t h e P V b o u n d a r y blebs a n d on u n a t t a c h e d vesicles w i t h i n t h e P V space (Figs. 6, 7). F e w or no iron particles wëre o b s e r v e d a t t h e s m o o t h surface of v e g e t a t i v e a n d spore-forming

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Fig. 6. Electron micrograph showing Con A-iron dextran marker on Con A receptor sites within PV (see arrows). Note the uniform distribution of iron particles at macrophage plasma membrane surface (at top of figure). × 25000 Fig. 7. Electron micrograph blow up of a portion of PV boundary showing discrete patches of iron dextran on 1)V blebs (see arrows) and on free vesicles. × 50000

parasites or on other profiles within the PV. The macrophage plasma membrane had a uniform distribution of iron particles at the surface. Control experiments were negative for iron particles at all membrane surfaces.

Absence o/ Lysosomal Activity in Parasitophorous Vacnoles Ferritin-laheling of pinocytic vesicles, secondary lysosomes and phagocytic vacuoles was performed by using a modification of the Jones and Hirsch procedure (1972). After 2 and 18 hr incubations, ferritin was located in secondary lysosomes, phagocytic vacuoles and pinocytic vesicles (Figs. 8-10). Ferritin readily accumulated in secondary lysosomes and to phagocytic vacuoles containing hatched E. cuniculi spores; however, ferritin was always excluded from the PV.

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Fig. 8. Macrophage exposed for 2 hours to ~erritin ~t 37°C. Note ferritin in presumptive second~ry lysosomes (~rrows). Ferritin w~s never observed within int~ct purasitophorous v~cuoles containing vi~ble E. cuniculi. Note the numerous PV blebs and free vesicles in the PV. × 25000 Fig. 9. Higher magnification of ferritin-{illed seeondary lysosomes and PV with attached veget~tive p~rasite (VP). × 40000

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Fig. 10. Electron micrograph of infected cell exposed for 2 hours to ferritin at 37°C. Note abundance of lysosomes loaded with ~erritin surrounding the p~rasitophorous vacuole (PV). Also obvious: vegetative parasite (VP)in tight-junetion-like attaehment to PV boundary, caveolae, and free vesicles, x 31500

Acid Phosphatase Activity in In/ected Macrophage8 This modified Gomori procedure was used to show acid phosphatase activity in primary and secondary lysosomes or within other vacuoles within the macrophage. Although infected macrophages readily showed acid phosphatase activity in lysosomes and phagocytic vacuoles, such activity was never observed within the PV (see Figs. 11-13). Discussion

A number of observations support the idea that the parasitophorous vacuole (PV) boundary blebs are transient membrane configurations interiorizing as vesicles into the PV. First, parasitophorous vacuoles always grow in size while host cytoplasm diminishes; second, the vesicles free within the PV space have a structural similarity to PV boundary blebs; and finally, the Con A-iron dextran label for Con A receptors is a useful marker since the iron particles attach on PV boundary blebs and on the free vesicles. The degree of b]ebbing of the PV boundary depends sornewhat on the host cell parasitized by E. cuniculi. For example, parasitophorous vacuoles in plasma cells are small and display few or no free vesicles or boundary blebs; curiously, only a few parasites develop in such a PV and growth is abbreviated before induction of spore formation (Weidner, unpublished observations). On the other hand, macrophages harboring large colonies of rapidly growing vegetative parasites,

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f PV

"

PV

Fig. 11. Electron micrograph of infected cell exposed to acid phosphatase staining reaction. Reaction product is evident near phagocytic envelope surrounding hatched E. cuniculi spore (HS) and in presumptive lysosomes. × 30000 Figs. 12, 13. Electron micrographs of infected cells exposed to acid phosphatase staining reaction. Reaction product is found in a phagocytic vacuole containing hatched spores (HS) and in lysosomes. Lead reaction product for acid phosphatase activity was not observed with parasitophorous vacuoles (PV). × 25000

i n v a r i a b l y d i s p l a y p a r a s i t o p h o r o u s vacuoles w i t h n u m e r o u s vesicles a n d b o u n d a r y blebs. T h e results of t h e ferritin a n d acid p h o s p h a t a s e e x p e r i m e n t s i n d i c a t e pinocytic vesicles a n d lysosomes fail to fuse w i t h t h e p a r a s i t o p h o r o u s vacuole. This corresponds to t h e Toxoplasma gondii condition in m a c r o p h a g e s (Jones a n d Hirsch, 1972).

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T h e differentia,1 m o v e m e n t of c o n s t i t u e n t s across t h e P V b o u n d a r y is i l l u s t r a t e d b y t h e a p p a r e n t interiorizing of t h e P V b o u n d a r y at t h e same t i m e t h a t lysosomes fail to coalesce w i t h t h e PV. This condition is similar to t h e o b s e r v a t i o n s of E d e l s o n a n d Cohn (1974) who showed t h a t Con A, once b o u n d to t h e m a c r o p h a g e p l a s m a m e m b r a n e , is r a p i d l y interiorized b y classic p i n o c y t i c mechanisms. Once t h e Con A - c o a t e d vesieles were formed, t h e y would fuse with o t h e r Con A vesicles a n d e v e n t u a l l y develop a large Con A - c o a t e d pinosome. This Con A - c o a t e d pinosome d i s p l a y e d a blebbing condition as long as Con A was a t t h e b o u n d a r y surface. A n interesting f e a t u r e oB this s t u d y was t h u t m a c r o p h a g e lysosomes would fail to coalesce w i t h t h e Con A - e o a t e d vesicles or pinosomes. I f t h e a p p a r c n t p i n o c y t i c a c t i v i t y d i s p l a y e d b y p a r a s i t o p h o r o u s vacuolcs h a r b o r i n g E. cuniculi is similar to t h e Con A - c o a t e d pinosome condition, one w o u l d a n t i c i p a t e t h e presence o2 a molecular c o m p o n e n t which, like Con A, induces t h e p i n o c y t i c m e c h a n i s m a n d s i m u l t a n e o u s l y blocks ]ysosomal fusion.

Acknowledgements. The author wishes to aeknowledge the exeellent teehnical assistance of Mrs. Beeky Demler during these studies.

Referenees Bainton, D. F., Farquhar, M. G.: Differenees in enzyme content of azurophil and speei/ic granules of polymorphonuelear leukocytes. J. Cell Biol. 119, 299 (1968) Bennett, H. S.: Cell surface: eomponents and eonfigurations. In: A. Lima-de-Faria (ed.), Handbook of moleeular eytology. New York: Ameriean Elsevier Pub. Co., Inc. 1969 Edelson, P. J., Cohn, Z. A. : Effeets of eoncanavalin A on mouse peritoneal maerophages. I. Stimulation of endocytic activity and inhibition of phago-lysosome formation. J. exp. Med. 140, 1346 (1974) Edelson, P. J., Cohn, Z. A. : Effects of concanavalin A on mouse peritoneal macrophages. II. Metabolism of endoeytized proteins and reversibility of the effeets by mannose. J. ex10. Med. 140, 1387 (1974) Jones, T. C., Hirsch, J. G.: The interaction between Toxo191asma gondii and mammalian cells. II. The absenee of lysosomal fusion with phagocytic vacuoles containing living parasites. J. exp. Med. 186, 1173 (1972) Martin, B. J., Spicer, S. S. : Concanavalin A-iron dextran technique for staining eell surface mucosubstances. J. Histochem. Cytoehem. 22, 206 (1974) Nelson, J. B. : An intracellular parasite resembling a microsporidian associated with ascites in Swiss miee. Proe. Soe. exp. Biol. 109, 714 (1962) Shadduck, J . A . : Eneephalitozoonosis (Nosematosis) and toxoplasmosis. Amer. J. Path. 64, 657 (1971) Vävra, J., Bedrnik, P., Cinatl, J. : Isolation and in vitro eultivation of the mammalian microsporidian Encephalitozoon cuniculi. Fo]ia parasit. (Praha) 19, 349 (1972) Dr. Earl Weidner Dept. of Zoology and Physiology Louisiana State Univ. Baton Rouge Louisiana 70803, USA