Schistosoma mansoni: Cell-specific expression and secretion of a serine protease during development of cercariae

75,87-98(1992)

EXPERIMENTALPARASITOLOGY

Schistosoma mansoni: Cell-Specific Expression and Secretion Serine Protease during Development of Cercariae ZVI FISHELSON,* MATTHEW *Department Departments

PAYMAN

PETITT,t

of Chemical of fPathology

of a

AMIRI,? DANIEL S. FRIEND,? MOSHE MARIKOVSKY,* GEORGE NEWPORT,$ AND JAMES H. McKERRowt’l

Immunology, The Weizmann Institute of Science, (Box 0506) and #Pharmaceutical Chemistry (Box San Francisco, California 94143, U.S.A.

Rehovot

76100,

Israel;

and

O&6), University of California,

FISHELSON,Z.,AMIRI,P.,FRIEND, D. AND MCKERROW, J. H. 1992. Schistosoma

S., MARIKOVSKY,M.,PETITT,M.,NEWPORT,G., mansoni: Cell-specific expression and secretion of a serine protease during development of cercariae. Experimental Parasitology 75, 8798. Eukaryotic serine proteases are an important family of enzymes whose functions include fertilization, tissue degradation by neutrophils, and host invasion by parasites. To avoid damaging the cells or organisms that produced them, serine proteases must be tightly regulated and sequestered. This study elucidates how the parasitic blood fluke Schistosoma mansoni synthesizes, stores, and releases a serine protease during differentiation of its invasive larvae. In situ hybridization with a cDNA probe localized the protease mRNA to acetabular cells, the first morphologically distinguishable parasite cells that differentiate from the embryonic cell masses present in the intermediate host snail. The acetabular cells contained vimentin but not cytokeratins, consistent with a mesenchymal, not epithelial, origin. Antiprotease antibodies, localized by immunoperoxidase, showed that the protease progressively accumulated in these cells and was packaged in vesicles of three morphologic types. Extension of cytoplasmic processes containing protease vesicles formed “ducts” which reached the anterior end of fully differentiated larvae. During invasion of human skin, groups of intact vesicles were released through the acetabular cytoplasmic processes and ruptured within the host tissue. Ruptured protease vesicles were noted adjacent to degraded epidermal cells and dermal-epidermal basement membrane, as well as along the surface of the penetrating larvae themselves. These observations are consistent with the proposed dual role for the enzyme in facilitating invasion of host skin by larvae and helping to release the larval surface glycocalyx during metamorphosis to the next stage of the parasite. 0 1992 Academic Press, Inc. INDEX DESCRIPTORS AND ABBREVIATIONS: Cercaria; Schistosoma mansoni; Protease; Acetabular gland cells; Differentiation; Trematode; Complementary deoxyribonucleic acid (cDNA); Messenger ribonucleic acid (mRNA); Kilodalton (kDa).

the aquatic environment, where they find their human host. Previous studies identified a 2%kDa serine elastase, released in secretions of cercariae, which degraded a broad spectrum of host dermal and epiderma1 macromolecules (McKerrow ef al. 1985a,b, 1989). Inhibition of this protease by synthetic protease inhibitors prevents larvae from invading skin (Cohen et al. 1991). Concurrent with invasion, the cercaria transforms into a second larval form-the schistosomulum(a). One of the important

INTRODUCTION

Schistosomes initiate infection of the human host with penetration of intact skin by an aquatic larval form, the cercaria(ae) (Stirewalt 1974; Cline 1989). Cercariae develop in an intermediate host snail and, when mature, are released (“shed”) into ’ To whom correspondence should be addressed at Department of Veterans Affairs Medical Center, San Francisco, Anatomic Pathology Service-113B, 4150 Clement Street, San Francisco, CA 94121. Fax: (415) 750-6947.

a7 0014-4894/92 $5.00 Copyright 8 1992 by Academic Press, Inc. All rights of reproduction in any fomt reserved.

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morphologic changes that takes place is the loss of the surface glycocalyx. The glycocalyx provides the cercaria with protection against the osmotically hypotonic environment of fresh water, but also is a potent activator of the host complement system and its cercaricidal activity (Marikovsky et al. 1988b; Samuelson and Caulfield 1985). Therefore, loss of the glycocalyx during host invasion by the cercaria means that the resulting schistosomulum is more refractory to complement damage. The same serine protease that functions to facilitate invasion apparently also helps to remove the glycocalyx (Marikovsky et al. 1988b). Using immunological and molecular probes, we have documented a unique system of cell-specific biosynthesis, storage, and release of the protease during development of cercariae. MATERIALSANDMETHODS Isolation of developing and mature cercariae. Biomphalaria glabrata snails were infected with miracidia of Schistosoma mansoni, Puerto Rican strain, as described previously (Lim and Heyneman 1972). At 4-7 weeks postinfection the snail hepatopancreas was removed and fixed in 10% phosphate-buffered formalin for in situ hybridization, in 2% paraformaldehyde for light microscopic localization by antibody, or in 1.5% glutaraldehyde for electron microscopy as described below. In situ hybridization with protease cDNA. The method of Haas et al. was used as described in detail previously (Newport et al. 1988). The probe was a full-length cDNA (Newport et al. 1988). Immunolocalization of the serine protease at the light microscopic level. Rabbit anti-cercaria protease antisera was prepared as previously described (Marikovsky et al. 1990). Recently shed cercariae (less than 1 hr) or infected snail hepatopancreas tissue (7 weeks postinfection) was fixed in 2% paraformaldehyde, embedded in methacrylate plastic, sectioned, and stained according to the methods of Beckstead (1983). Primary antisera were used at 1: 10,000 and secondary goat anti-rabbit IgG peroxidase-conjugated antisera at 1:200. Monoclonal antibody to cytokeratins (BecktonDickinson, 1:50; plus Boehringer-Mannheim, 1:300) and vimentin (Accurate Chemical, 1:10) were reacted with plastic-embedded sections and developed with

secondary antibody (Vector stain) as per the manufacturer’s recommendations. Ultrastructural studies of acetabular cells and ultrastructural immunolocalization of the serine protease. Isolated cercariae and snail hepatopancreas were fixed in 1.5% glutaraldehyde and 0.1% acrolein in 0.1 M sodium cacodylate buffer containing 1% sucrose at pH 7.4, for 2 hr at 22°C. Tissue and cells were postfixed in 1% 0~0~ in 0.1 M acetate veronal buffer for 1 hr at 4°C and in 2% tarmic acid in 0.05 M sodium cacodylate buffer, pH 7.4, for 1 hr. The tissues were also immersed in Kellenberger’s 0.5% uranyl acetate in a 0.1 M acetate veronal buffer, pH 6, for 1 hr. Dehydration and embedding were routine. Thin sections were stained with lead and uranyl acetate and examined in a JEOL 100 CX electron microscope operating at 80 kV. For immunocytochemistry of frozen thin sections, cells and tissue were fared in 4% phosphate-buffered paraformaldehyde for 2 hr and then frozen in liquid nitrogen. All the procedures followed were those published by Tokuyasu (1980, 1986). Sections were cut on a Reichert ultracut cryotome. The immunogoldlabeled ultrathin frozen sections depicted in this paper were with a 1:50 dilution of the antibody. After ultrathin cryosectioning, the sections were picked up on carbon-coated copper grids. For immunolabeling, the grids were floated on drops of solutions on paralilm. All steps were done at room temperature except the last step of methylcellulose embedding, which was done at 4°C. The procedures followed were those published by Tokuyasu (1980, 1986).

RESULTS

In the intermediate host snail, cercariae develop in sac-like structures derived from the daughter sporocyst form of the parasite (Stirewalt 1974; Schutte 1974). At 7 weeks following infection of snails with miracidia (the ciliated larvae released from eggs in fresh water), cercarial development in sporocysts was found to be asynchronous, and all stages of cercarial development were represented, providing an advantageous setting for observing cell differentiation. Figure 1A shows a section through an infected snail sporocyst with a group of cercariae at various stages of development including primitive embryonic cell masses, early cellular differentiation, and fully mature larvae. Some of the first cells that are morphologically distinguishable within the embryonic cell mass are large cells with

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prominent nuclei and nucleoli. These develop into the “pre- and postacetabular glands,” named for their proximity to the large attachment structure (acetabulum) of the larvae (Stirewalt 1974). At the earliest stages of their differentiation these cells already contain mRNA for the cercarial protease (Fig. 1B). As cercariae develop further, mRNA synthesis becomes restricted to the cytoplasm at the edges of the cell, presumably due to accumulation of protease-containing vesicles (see below). Figure 1E shows immunolocalization of the cercarial protease during acetabular cell differentiation. There is progressive accumulation of the protease within the cyto-

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plasm of the same early differentiating cells which contain the RNA transcripts visualized by in situ hybridization. Figure 2 shows that the enzyme is packaged in vesicle-like organelles that become so numerous within the cytoplasm that other organelles (and mRNA transcripts as noted above) are pushed to the side. By electron microscopy of aldehyde-fixed cells, three morphologic types of vesicles are distinguished. Two are found in the postacetabular glands. The first is the homogeneous dense vesicle (Fig. 2b), and the second is a vesicle with a dark central core (Fig. 2~). The third type is more heterogeneous in appearance, with numerous elec-

FIG. 1. (A) Section of infected intermediate host snail showing cercariae at different stages of development. Dark oval clusters of primitive embryonic cells are seen within sporocyst sacs on the left. In the center, several cercariae that have begun to differentiate are present within a larger sporocyst sac. Arrows point to large acetabular “gland” cells with clear, vesicular cytoplasm and prominent, dark nuclei. Hematoxylin-eosin-stained section; bar, 50 pm. (B) In situ hybridization of tritium-labeled protease cDNA with tissue sections of infected snails. Arrows point to acetabular cells with numerous small, dark, uniform autoradiographic grains indicating hybridization to mRNA. At the upper left, cells are just differentiating from embryonic mass. In the center, a nearly developed cercaria is seen with tail (T) and acetabular gland (G). G marks a differentiating gland cell with compression of cytoplasm containing autoradiographic grains to edge of cell (between arrow and G) by expansion of clear area (G). This clear area corresponds to the region of cytoplasm shown in Fig. 2d. Hematoxylin and eosin stain following autoradiography; bar, 50 pm, (C) Control for in situ hybridization in which tissue section was pretreated with 500 mg/ml of RNase (Sigma) and 50 mg of RNase TUrnI (Sigma) before hybridization (dark-field illumination). (D) In situ hybridization performed as in B but illuminated by dark-field to highlight autoradiographic grains. Note the bright clusters of autoradiographic grains in acetabular cells of four different developing cercariae. At lower left an irregular unreactive area similar to that marked by G in B is seen. (E) Immunoperoxidase localization of protease using monospecific antisera. Immunoperoxidase reaction appears dark. Three developing cercariae are present in this field. On the right, acetabular cells are just beginning to differentiate from embryonic tissue. The distinctive large acetabular cell nuclei appear as clear areas within the cytoplasm, which is darkly stained by immunoperoxidase-localized antisera. In the middle and on the left, cercariae at later stages of development have irregular cytoplasmic evaginations of the acetabular cells (arrows). These evaginations will ultimately extend to the anterior end of the head, forming the “ducts” that transport secretory vesicles as seen in G and Fig. 2a. (F) Control immunoperoxidase reaction using the same secondary immunoperoxidase-linked anti-rabbit antibody, but anti-human platelet antisera (a gift of Dr. Dorothy Bainton) as primary antibody. (G) Cercariae after emergence from snail host. Three cercariae are seen in the field. Two are cut in cross section or tangentially and the third longitudinally. Dark peroxidase localization of antiprotease antisera can be seen in the region of acetabular glands as well as cell processes that now extend to the anterior end of the head. Some secretion can be seen just beginning to leak out from the orifice at the anterior end of the head. (H) Three cercariae (C) invading human skin. The surface of skin is at the left and the dermal extracellular matrix at the right. The cercaria in the middle of the field has created a tunnel in the epidermis and is just beginning to enter the extracellular matrix of the superficial dermis on the right. Immunoperoxidase localization of protease (arrows) shows that it diffuses radially in front of the advancing organism. Also note the dark immunoperoxidase staining along the sides of all three cercariae.

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tron lucent areas, and is found exclusively in the preacetabular glands (Fig. 2~). By frozen thin section after paraformaldehyde fixation, all the vesicles appear more homogeneous, and all vesicles in both pre- and postacetabular cells contain abundantly labeled protease by immunogold cytochemistry (Figs. 2d and 3). In standard electron micrographs, the protease-positive cells do not rest on an obvious basal lamina, nor do they display obvious junctional complexes between adjacent cells. Later in cercarial development, the nuclei of the acetabular cells undergo atrophy, and evaginations of cytoplasm extend toward the anterior end of the organism (Fig. 1E). These cytoplasmic processes become the “ducts” through which secretory vesicles are transported to an anterior orifice (Fig. IG). Because of the accumulation of enzyme within the cells, as these processes reach the anterior end of the organism, they are already tilled with secretory vesicles (Fig. 2a). As noted above, the two groups of cells containing protease vesicles have been called pre- and postacetabular “glands” (Stirewalt 1974; Dorsey and Stirewalt 1971). The term gland was used in its broadest sense to define a cell or organ that elaborates and secretes a substance. As shown in Figs. 2 and 3, the ducts of these unicellular glands are in fact not epitheliallined tubes, as is the case with vertebrate

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exocrine glands like the pancreas, but cytoplasmic processes containing enzyme vesicles as well as other cytoplasmic organelles. This observation, also noted in ultrastructural studies of Dorsey and Stirewalt (1971), led us to examine whether the glands themselves were of epithelial origin. Sections of developing cercariae were reacted with antisera against cytokeratins, an intermediate filament marker of epithelial cells, or vimentin, a marker of mesenchymal cells. No staining was seen with anticytokeratin antibodies, but perinuclear staining was seen with anti-vimentin antibodies in the developing acetabular cells (Fig. 4). Immunolocalization of the protease in cercariae penetrating human skin shows that it is released at the anterior end of the organism and diffuses through the dermal extracellular matrix ahead of the advancing organisms (Fig. 1H). Enzyme can also be seen associated with the surface of the organism as it moves through the tunnel it has created. Figure 5 demonstrates that the organism releases intact vesicles which rupture in the host tissue as well as along the surface of the cercaria. DISCUSSION

Invasion of host tissue by many pathogenic bacteria, fungi, and parasites is facilitated by proteolytic enzymes released by these organisms (McKerrow et al. 1989). In

FIG. 2. (a) A heavily tannic acid-stained cercaria revealing the glycocalyx on the surface of the cell (right side of micrograph) and a tannic acid-impregnated orifice through which protease-containing vesicles exit (marked by an asterisk). The entering orifice on the left (marked by an arrow) is a cytoplasmic process (“duct”) from a postacetabular gland cell that is already ffied with secretory vesicles and other cytoplasmic organelles even before emergence of cercaria from snail. (b) A type of secretory vesicle found predominantly in the postacetabular gland cells. (c) Two other morphologic configurations of secretory vesicles. The section is at the boundary of a postacetabular gland cell on the left and a preacetabular cell on the right. One type with a dense core is present in postacetabular cells and another, to the far right, was a vesicular, looser matrix and is present exclusively in preacetabular gland cells. All vesicles are bound by a unit membrane. (d) Immunocytochemical preparation of a frozen thin section. The immunogold label reacts with the specific protease epitope in these vesicles. In frozen thin sections of sporocysts, the morphology of vesicles is homogeneous. These vesicles are in a postacetabular cell.

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FIG. 3. Immunogold preparation with the anti-cercarial serine protease antibodies showing highly reactive, compressed vesicles in four adjacent preacetabular cell processes. These vesicles would correspond to those seen on the right of Fig. 2C in standard electron micrographs. Note the low background between labeled cells and, near the apices of these four cells, the lack of junctional membrane differentiations.

the case of the schistosome cercaria, prepackaged protease is released in response to the stimulus of host skin lipid. While tracing the biosynthesis of this enzyme in developing larvae, we have observed that the cells which differentiate into the protease-secreting acetabular cells are among the first to differentiate from the embryonic tissue that ultimately gives rise to cercariae. As soon as these cells are morphologically distinguishable, mRNA for the enzyme is being synthesized in them and

translated into protein. The protease is stored in intracellular vesicles which accumulate in the cytoplasm, pushing other cytoplasmic organelles aside. Further differentiation of the cells occurs by evagination of cytoplasm (already filled with secretory vesicles) to form processes that extend to the anterior end of the larva. Synthesis of mRNA has ceased by the time the cercariae are ready to emerge from the snail. There are important parallels as well as differences in the synthesis, storage, and

FIG. 4. (A) Developing cercariae showing absence of immunoperoxidase reaction in acetabular cells (arrows) with antikeratin antisera. Epithelium of surrounding snail hepatopancreas shows positive reaction (brown stain). Immunoperoxidase localization with hematoxylin counterstain; bar, 50 pm. (B) Positive immunolocalization of antisera to vimentin in acetabular gland cytoplasm and perinuclear region (arrows). Note that the surrounding snail epithelium is nonreactive except interdigitating mesenchymal-derived cells at the base of the epithelium. Immunoperoxidase localization with hematoxylin counterstain.

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FIG. 5. Transmission electron micrograph of preacetabular gland secretory vesicles released from cercaria. Some vesicles are still intact while others are beginning to rupture (arrows). Cercaria is in the upper right of the field. Dark knob-like bodies are actin “spikes” on surface of cercaria. One is indenting a vesicle just above the E. Also visible above the E is dissolution of dermal-epidermal basement membrane just below and to the left of the group of vesicles. R marks remnants of degraded epidermaf cell. E is dermal extracellular matrix. Some vesicle packages are intimately associated with the surface of the cercaria (C), as was seen at the light microscopic level (Fig. IF).

secretion of the cercarial serine protease compared to the serine proteases of higher eukaryotes (Neurath 1986). The cercarial enzyme is structurally related to the trypsin family of eukaryotic serine proteases and has greatest structural similarity to rat pancreatic elastase I and II (Newport et al. 1988). As is the case with secreted serine proteases of higher eukaryotes, the expression of the protease gene is cell-specific, and a proenzyme (zymogen) is the initial protein form synthesized (Newport et al. 1988). Like pancreatic, neutrophil, mast cell, or sperm serine proteases (Neurath 1986; Yanagimachi 1988), the cercarial pro-

tease is sequestered in intracellular vesicles prior to secretion. Ultrastructural studies of cell-cell contacts and analysis of intermediate filaments of the acetabular cells suggest they are of mesenchymal, not epithelial, origin, more akin to the vertebrate mast cell than to the pancreatic acinar cell (Neurath 1986). It was formerly thought that only the preacetabular cell vesicles contained the enzyme (Stirewalt 1974), but immunolocalization shows that vesicles, in both the pre- and postacetabular cell, contain the protease (Figs. 2 and 3). This confirms the observations of Marikovsky et al. on vesi-

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cles in mature cercariae. Due to better fixation, the distinct morphology of pre- and postacetabular vesicles is more readily seen in these mature cercariae after their release from the snail (Marikovsky et al. 1990). Whether the differences in vesicle morphology affect protease activation in any way is at present unclear because it is not known where zymogen activation takes place. The preacetabular cell vesicles have been shown to contain high levels of calcium (Davies 1983), and it is possible that ion flux across the vesicle membrane may result in changes in pH or ionic strength that result in zymogen activation. As with other serine proteases, the stability of the protein is probably increased in the presence of calcium and concomitant release of calcium with the enzyme might therefore provide an optimal environment for its action. Alternatively, as Dresden and Edlin (1974) suggested, very high calcium concentrations within the vesicle might be inhibitory to the enzyme, until both enzyme and calcium diffuse away from the ruptured vesicle within the host. Indeed, while lower concentrations of calcium enhance activity of at least one biochemical form of the enzyme (McKerrow et al. 1985b), calcium concentrations higher than 10 mM were found to be inhibitory to the cercarial enzyme (McKerrow et al. 1985b; Dresden and Edlin 1974). While intracellular packaging of the serine protease is similar to that seen in pancreatic zymogen vesicles, release of the vesicles appears to be via apical rupture or dissolution of cytoplasmic extensions of the acetabular cells rather than by membrane exocytosis. Intact vesicles are released during secretion as suggested by Stirewalt and Dorsey (1974) and confirmed by the scanning electron micrographs of Samuelson et al. (1984). A final important observation is that when protease-containing vesicles are released, they are found both in the host tis-

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sue ahead of the cercaria (Fig. 1F) and along the sides of the cercaria as it moves through “tunnels” resulting from host tissue digestion (Fig. 5). This is similar to observations of acrosin release during sperm penetration through the zona pellucida (Yanagimachi 1988) and confirms previous studies which showed that secreted postacetabular cell mucins form a film or overlay around the cercarial bodies (Stirewalt and Walters 1973). Localization of released vesicles is therefore consistent with the proposed dual role of the protease in tissue digestion and glycocalyx removal (Samuelson and Caulfield 1985; Marikovsky et al. 1988a; Fishelson 1989), allowing the cercaria to invade host skin as well as to escape immune damage by the complement system. ACKNOWLEDGMENTS This work was supported by the NIAID J.H.M.), the Edna McConnell Clark (G.N.), the John D. and Catherine T. Foundation (Z.F.), and the Rockefeller (Z.F.).

(AI20452 to Foundation MacArthur Foundation

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