Molecular Microbiology (1997) 26(1), 163–173
Participation of myosin in gliding motility and host cell invasion by Toxoplasma gondii Janice M. Dobrowolski, Vern B. Carruthers and L. David Sibley* Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Ave., St Louis, MO 63110, USA.
classical phagocytosis and is not accompanied by condensation of host cell actin filaments or phosphorylation of host cell proteins upon parasite entry (Morisaki et al., 1995). Invasion is also strictly dependent on the actin cytoskeleton of the parasite with little contribution from the host cell microfilament network (Dobrowolski and Sibley, 1996). While invasion is an active process on the part of the parasite, the mechanism for generating the force necessary for cell penetration remains unclear. Because T. gondii is an obligate intracellular parasite, its ability to gain entry into the host cell is paramount for survival and relevant to its pathogenesis during both acute and chronic infection. T. gondii belongs to the phylum Apicomplexa that contains a diverse array of medically important parasitic protozoans, including Plasmodium, the causative agent of malaria, and Eimeria and Cryptosporidium, the causative agents of enteric infections in animals and man (Dubey, 1977). Apicomplexan parasites lack organelles, such as cilia or flagella, throughout most of their life cycles and are unable to swim through fluids. Instead, all members of the phylum exhibit an unusual form of gliding motility that is strictly substrate dependent (Russell and Sinden, 1981; King, 1988). During gliding, the parasite moves across the surface without forming protrusions, such as pseudopodia or lamellipodia; this absence of cell shape deformation distinguishes gliding from the crawling motility of amoeboid protozoans or vertebrate cells (Mitchison and Cramer, 1996). Most apicomplexan parasites have a characteristic crescent shape. Consequently, gliding is manifest as either circular spirals or a series of helical turns revolving around the long axis of the body (Russell and Sinden, 1981; King, 1988; Stewart and Vanderberg, 1988). As well as supporting migration across solid substrates, including host cell surfaces, gliding motility likely provides the force for cell entry as both processes are dependent on the parasite’s actin cytoskeleton (Dobrowolski and Sibley, 1996). T. gondii is highly polarized and has a complex cytoskeleton consisting of an apical microtubule organizing centre, known as a conoid, connecting 22 singlet microtubules that extend longitudinally approximately two-thirds the length of the cell (Russell and Sinden, 1982; Nichols and Chiappino, 1987). A second set of posteriorly organized filaments that extends forward and interdigitates between the microtubules has also been described in related apicomplexan parasites, although the composition
Summary
Toxoplasma gondii is an obligate intracellular parasite that actively invades mammalian cells using a unique form of gliding motility that critically depends on actin filaments in the parasite. To determine if parasite motility is driven by a myosin motor, we examined the distribution of myosin and tested the effects of specific inhibitors on gliding and host cell invasion. A single 90 kDa isoform of myosin was detected in parasite lysates using an antisera that recognizes a highly conserved myosin peptide. Myosin was localized in T. gondii beneath the plasma membrane in a circumferential pattern that overlapped with the distribution of actin. The myosin ATPase inhibitor, butanedione monoxime (BDM), reversibly inhibited gliding motility across serum-coated slides. The myosin lightchain kinase inhibitor, KT5926, also blocked parasite motility and greatly reduced host cell attachment; however, these effects were primarily caused by its ability to block the secretion of microneme proteins, which are involved in cell attachment. In contrast, while BDM partially reduced cell attachment, it prevented invasion even under conditions in which microneme secretion was not affected, indicating a potential role for myosin in cell entry. Collectively, these results indicate that myosin(s) probably participate(s) in powering gliding motility, a process that is essential for cell invasion by T. gondii. Introduction
Toxoplasma gondii is proficient at invading all types of nucleated cells within its many hosts, which include virtually all warm-blooded vertebrates. Invasion of vertebrate cells by T. gondii is fundamentally different from phagocytosis or induced endocytic uptake used by most intracellular pathogens. Invasion occurs much faster than Received 27 May, 1997; revised 14 July, 1997; accepted 15 July, 1997. *For correspondence. E-mail
[email protected]; Tel. (314) 362 8873; Fax (314) 362 1232. Q 1997 Blackwell Science Ltd
m
164 J. M. Dobrowolski, V. B. Carruthers and L. D. Sibley of these filaments is unknown (D’Haese et al., 1977). Apicomplexans belong to the Aveolates, which typically have a system of flattened membrane cisternae that lie beneath the plasma membrane. In the Apicomplexa, this structure is known as a pellicle or the inner membrane complex (IMC) (Dubremetz and Torpier, 1978). The IMC overlies the microtubular cytoskeleton and contains protein particles that are aligned parallel to the microtubules and participate in maintaining cell shape and polarity (Morrisette et al., 1997). T. gondii tachyzoites expresses a single cytoplasmic isoform of actin that is localized beneath the plasma membrane and in scattered clusters throughout the cytosol (Dobrowolski et al., 1997). Despite compelling evidence that actin filaments are necessary for motility and invasion, the majority of actin appears in a soluble form in parasite extracts (Dobrowolski et al., 1997). Actin filament assembly, while necessary, is probably insufficient to generate the force for motility. Instead, actin filaments probably act as a scaffold for a conventional motor protein such as myosin. Myosin, previously localized primarily at the apical end of T. gondii using heterologous antisera to cricket muscle myosin (Schwartzman and Pfefferkorn, 1983), has not been extensively studied in apicomplexan parasites. To explore the role of myosin in T. gondii motility and invasion, we have characterized a small myosin recognized by a polyclonal antibody generated against a highly conserved myosin peptide. Myosin was detected in T. gondii beneath the plasma membrane, where it co-localized with actin in a circumferential pattern. Furthermore, the myosin inhibitors, butanedione monoxime (BDM) and KT5926, blocked gliding and cell entry by T. gondii, implying an important role for myosin in motility and invasion.
Results
Fig. 1. A. Western blot analysis of platelet and T. gondii myosin with affinity-purified a-LEAF antibodies. B. Co-sedimentation of actin and myosin from the saponin-soluble fraction of T. gondii lysates. Precipitates formed after the addition of lysis buffer (control), ATP or AMP-PNP were pelleted by centrifugation, separated on SDS–PAGE gels and transferred to nitrocellulose. Left, Western blot of precipitates using rabbit anti-T. gondii actin serum. Right, Western blot of precipitates using a-LEAF antibodies.
Detection of a small myosin in T. gondii To characterize myosins in T. gondii, we used an affinitypurified rabbit antiserum generated against a highly conserved peptide. This antiserum, referred to as a-LEAF recognizes myosin isoforms I, II and VI by Western blot (T. Mitchison, personal communication). As expected, platelet myosin II (a gift from T. Mitchison, UCSF) was readily recognized by a-LEAF antiserum as an intense band of approximately 200 kDa (Fig. 1A). a-LEAF antibodies recognized a predominant band of approximately 90 kDa in RH strain parasite cell lysates (Fig. 1A). A second, slightly smaller band seen in Fig. 1 may arise from partial degradation, as it was not observed using fresh lysates prepared with protease inhibitors (data not shown). Longer exposures of the blot did not reveal any additional bands in T. gondii (not shown). The peptide
sequence recognized by a-LEAF is conserved in all classes of myosin except the highly divergent ninaC (Mooseker and Cheney, 1995), suggesting that the 90 kDa band recognized here is the major myosin isoform expressed by tachyzoites of T. gondii.
Co-sedimentation of actin and myosin in T. gondii We have shown previously that the majority of actin in detergent extracts of T. gondii is globular (Dobrowolski et al., 1997). As expected, only trace amounts of insoluble actin were detected in the pellet of parasite lysates (Fig. 1B). The addition of ATP greatly increased the amount of precipitable actin and resulted in co-precipitation of myosin (Fig. 1B). These results indicate that the Q 1997 Blackwell Science Ltd, Molecular Microbiology, 26, 163–173
Myosin-based motility in Toxoplasma 165 fluorescence (not shown). Staining with affinity-purified a-LEAF antibodies revealed a beaded staining pattern for myosin, which extended around the periphery of the tachyzoite (Fig. 2B). Actin was also localized in a circumferential pattern around the cell periphery and as diffuse cytoplasmic staining, as revealed by co-staining with the mAb C4 (Fig. 2C). Immunoelectron microscopy was also used to examine the distribution of myosin and actin in T. gondii. Myosin was localized beneath the plasma membrane of the cell in a pattern that extended around both ends of the parasite and in scattered clusters throughout the cytoplasm (data not shown). Double staining with mAb C4 (18 nm gold) and a-LEAF (12 nm gold) antibodies revealed that actin and myosin were co-localized beneath the plasma membrane of the parasite (Fig. 3A). In parasites that were incubated in hypotonic medium (25 mM KCl) during fixation, the plasma membrane expanded away from the IMC. Under these conditions, myosin was found along the plasma membrane (arrowheads), while actin remained associated with the IMC (arrow) (Fig. 3B).
Myosin inhibitors block T. gondii motility
Fig. 2. Co-staining of myosin and actin within extracellular tachyzoites. Myosin was detected with a-LEAF (B, FITC staining) and actin with mAb C4 (C, Texas Red staining). Both proteins are concentrated beneath the plasma membrane and scattered throughout the cytosol. Bar ¼ 5 mm.
90 kDa protein recognized by a-LEAF forms an insoluble complex with actin, a hallmark feature of myosins. Surprisingly, actin was not precipitated by the non-hydrolyzable analogue AMP-PNP and, under these conditions, myosin also remained soluble (Fig. 1B).
Co-localization of myosin with actin in T. gondii tachyzoites Indirect immunofluorescence staining of T. gondii was carried out to co-localize myosin and actin within saponinpermeabilized, extracellular parasites. Parasites stained with non-immune rabbit serum showed minimal background Q 1997 Blackwell Science Ltd, Molecular Microbiology, 26, 163–173
To determine if myosin plays a role in parasite motility, we tested the effects of two inhibitors, BDM and KT5926, on parasite gliding. BDM is a low-affinity inhibitor of myosin ATPase that blocks the actions of a variety of myosins but has no effect on actin filaments or kinesin (Cramer and Mitchison, 1997). KT5926 is a myosin light-chain kinase (MLCK) inhibitor that blocks both Ca2þ /calmodulin-dependent and -independent smooth muscle MLCKs (Nakanishi et al., 1990). We monitored the motility of T. gondii tachyzoites by immunofluorescence staining with antibodies specific to SAG1, the major surface protein of T. gondii, to detect the trails left by gliding parasites (Fig. 4A). In the presence of 0.1 mM Cyt D, parasite gliding was partially inhibited (Fig. 4A’) and 0.2 mM Cyt D completely inhibited gliding (Fig. 4A’’). Both myosin inhibitors also significantly blocked the gliding motility of T. gondii. Gliding was partially inhibited at a concentration of 20 mM BDM (Fig. 4B’) and nearly completely blocked at 40 mM (Fig. 4B’’). While these are relatively high concentrations, they are entirely consistent with the effects of this inhibitor on myosinspecific functions in higher eukaryotic cells (Cramer and Mitchison, 1995; 1997). Gliding was partially inhibited by KT5926 at a concentration of 1 mM (Fig. 4C’), and complete inhibition was seen at 5 mM (Fig. 4C’’). The concentrations of KT5926 that inhibited parasite gliding are somewhat higher than those expected for a direct effect on MLCK (20 nM IC50 for mammalian MLCK) (Nakanishi et al., 1990). Since KT5926 may inhibit other protein kinases besides MLCK, we tested the ability of calphostin C to block gliding.
166 J. M. Dobrowolski, V. B. Carruthers and L. D. Sibley Fig. 3. Co-localization of actin and myosin in extracellular T. gondii tachyzoites by immunoelectron microscopy. A. Co-staining with mAb C4 (18 nm gold particles) and a-LEAF (12 nm gold particles) showing clusters of actin and myosin beneath the parasite plasma membrane. B. In osmotically swollen cells, the plasma membrane separates from the inner membrane complex revealing that myosin is associated with the plasma membrane (arrowheads) and actin remains along the inner membrane complex (arrow). Bars ¼ 200 nm.
Calphostin C is a specific inhibitor of protein kinase C (IC50 ¼ 0.05 mM) that inhibits PKC purified from Toxoplasma (C. Beckers, personal communication) and has little effect on mammalian MLCK (IC50 for MLCK $ 5 mM) (Kobayashi et al., 1989). Calphostin C did not inhibit the gliding of T. gondii tachyzoites at 0.1 mM (Fig. 4D’) or at 1 mM (Fig. 4D’’), indicating that the action of KT5926 in blocking parasite motility is not caused by the inhibition of protein kinase C. To quantify the effects of inhibitors on gliding, we monitored the average trail length for individual parasites stuck to serum-coated glass compared with the parasite cell length (approximately 7 mm). During the incubation period used here, parasites deposited a trail of 3–4 body lengths on average, or approximately 20–30 mm. Treatment with the drugs Cyt D, KT5926 and BDM each inhibited the average trail length by 70–90% when compared with medium or dimethyl sulphoxide (DMSO) alone controltreated cells (Fig. 5). In parasites that were treated with inhibitors, then washed and allowed to recover, trail length returned to approximately normal levels (Fig. 5). The reversibility of inhibitors is expected from their modes of action and verifies that their effects were not the result of non-specific decreases in viability.
Myosin inhibitors block T. gondii invasion into mammalian cells To determine whether myosin also contributes to invasion by T. gondii, we tested the ability of BDM and KT5926 to block entry into host cells. Parasite numbers were determined by a quantitative assay using the exogenous reporter enzyme b-Gal expressed by a stable transformant harbouring the lacZ gene (Seeber and Boothroyd,
1996). Following synchronous infection, parasites were allowed to grow overnight before quantification of cell numbers by b-Gal content. Neither BDM or KT5926 significantly altered the division rate of parasites during the 24 h culture period (data not shown). Thus, the differences shown in Fig. 6 reflect inhibition of parasite invasion. As shown previously, Cyt D blocked the invasion of T. gondii into host cells when compared with untreated parasites (Fig. 6) or those treated with DMSO alone (not shown). The myosin inhibitors, BDM and KT5926, both reduced the number of intracellular parasites in a dosedependent manner, although the latter drug was considerably more potent (Fig. 6). We have shown previously that Cyt D blocks invasion without affecting parasite binding to the host cell (Dobrowolski and Sibley, 1996); thus, the reduced b-Gal activity observed here is directly attributable to reduced entry. However, the effects of KT5926 and BDM on host cell binding by T. gondii are unknown. To establish if the myosin inhibitors were blocking the attachment or invasion of host cells, we examined the number of parasites that remained extracellular yet adherent to the host cells against those that successfully invaded during a short infectious pulse. Extracellular parasites were distinguished by immunofluorescence staining with a rabbit antibody to the surface protein SAG1 before the addition of detergent to permeabilize the monolayer, while both extracellular and intracellular parasites were revealed by a mouse mAb to SAG1 that was added after detergent permeabilization. Treatment with KT5926 dramatically reduced the attachment of parasites to the host cell monolayer, while the effect of BDM treatment on cell attachment was less pronounced (Fig. 7A). Separate from their effects on attachment, KT5926 and BDM also prevented cell entry, Q 1997 Blackwell Science Ltd, Molecular Microbiology, 26, 163–173
Myosin-based motility in Toxoplasma 167
Fig. 4. Effect of cytoskeletal inhibitors on gliding motility of T. gondii. Tachyzoites were pretreated with drugs and allowed to glide on serum-coated slides. Trails were visualized by staining with anti-SAG1 antibodies followed by a fluorescent secondary antibody. A. DMSO control. Inhibition of gliding by Cyt D occurs at both low (0.1 mM) (A’) and high (0.2 mM) (A’’) concentrations. B. Medium control. BDM partially inhibited gliding at 20 mM (B’) (low) and completely inhibited gliding at 40 mM (B’’) (high). C. DMSO control. KT5926 partially inhibited gliding at 1 mM (low) (C’) and completely inhibited gliding at 5 mM (high) (C’’). D. DMSO control. Calphostin C did not significantly inhibit gliding at 0.1 mM (low) (D’) or 1.0 mM (high) (D’’) concentrations.
as seen by the decrease in the number of parasites that successfully entered the host cell relative to the total number of cell-associated T. gondii (Fig. 7A). The effects of BDM in blocking cell invasion were more apparent using the two-colour immunofluorescent assay (Fig. 7A) vs. the b-Gal assay (Fig. 6), a result that presumably reflects differences in incubation times and the washing efficiencies between the two assays. Despite these differences in relative potency, both experiments indicate that BDM inhibits cell invasion by T. gondii, primarily by reducing cell penetration.
Effects of myosin inhibitors on secretion of the parasite adhesin MIC2 We have recently characterized a multifunctional adhesin Q 1997 Blackwell Science Ltd, Molecular Microbiology, 26, 163–173
of T. gondii, called MIC2, which is released from micronemes during cell attachment (Wan et al., 1996; Carruthers and Sibley, 1997). The substantial reduction in cell binding resulting from treatment with myosin inhibitors suggested they may act by preventing the release of MIC2, which is not normally found on the cell surface until contact with the host cell is established (Carruthers and Sibley, 1997). We examined the release of MIC2 by extracellular parasites, which occurs spontaneously at a low rate, after treatment with medium alone, DMSO, BDM or KT5926. A dramatic reduction in the release of MIC2 was observed even at the lowest doses of KT5926 used (88% inhibited at 1.0 mM) (Fig. 7B). In contrast, no reduction in the release of the dense granule protein, GRA1, was observed (data not shown). Although treatment with 20 mM BDM modestly reduced the secretion of MIC2
168 J. M. Dobrowolski, V. B. Carruthers and L. D. Sibley
Fig. 5. Gliding motility of T. gondii is reversibly inhibited by cytoskeletal agents. Treatments with Cyt D (0.2 mM), BDM (40 mM) or KT5926 (3 mM) all significantly decreased average trail lengths left by parasites gliding on serum-coated slides (þ). These effects were reversible in parasites that were treated then washed before use (¹).
(approximately 35% inhibition), treatment with 40 mM or 60 mM had no effect on its release (# 5% inhibition) (Fig. 7B and data not shown). Because these doses of BDM are relatively high, we evaluated the possibility that its effects were the result of non-specific lysis. Release of cytoplasmic b-Gal from treated cells indicated minimal cell lysis (0–1%) at 20 mM or 40 mM and slightly higher levels at 60 mM BDM (approximately 10%). Discussion We have provided new evidence that T. gondii contains a
Fig. 6. Effects of cytoskeletal inhibitors on host cell invasion by T. gondii tachyzoites. A. Both BDM and KT5926 significantly reduced tachyzoite invasion into host cells compared with untreated controls. Cyt D, a known inhibitor of parasite invasion, was included as a positive control for inhibition. Parasite numbers were estimated from a standard curve relating cell numbers to b-Gal enzyme activity.
small myosin that is located in both the anterior and the posterior ends of the parasite, where it is concentrated beneath the plasma membrane. A single, predominant isoform of myosin of approximately 90 kDa was detected using an anti-myosin antibody to probe Western blots of T. gondii. The 90 kDa T. gondii myosin co-precipitated with filamentous actin in detergent-permeabilized parasite lysates in an ATP-dependent manner. Finally, parasite motility and host cell invasion were blocked by the myosin inhibitors, BDM and KT5926, which appear to have separate effects in blocking the release of an important cell adhesin (primarily effected by KT5926) and in directly inhibiting cell entry by T. gondii (primarily effected by BDM). Collectively, these studies support a role for myosin in motility and cell invasion by Toxoplasma. To investigate myosins in T. gondii, we used a polyclonal antisera generated against a conserved peptide found in virtually all myosins described to date (the exception being the highly divergent ninaC of Drosophila) (Mooseker and Cheney, 1995). As myosins have not been characterized extensively in T. gondii, this anti-peptide antisera allowed us broadly to examine the diversity of isoforms expressed by this parasite. Surprisingly, Western blotting of T. gondii with a-LEAF antibodies revealed a single isoform of approximately 90 kDa, which is somewhat smaller than previously described unconventional myosins (Mooseker and Cheney, 1995). This antisera recognizes myosin II from a variety of organisms, and the failure to detect large myosins in T. gondii suggests that they are either not very abundant or highly divergent. We have shown previously that actin in T. gondii is primarily in a globular form even under conditions that normally favour the assembly or stabilization of filaments in other organisms (Dobrowolski et al., 1997). The reasons for this instability are unknown. However, in the present study, we report that the addition of ATP induced the formation of a complex that precipitated actin together with myosin. Formation of this precipitate was dependent on ATP hydrolysis, similarly to the previous finding that the addition of ATP but not AMP-PNP induces actin precipitation in extracts of Eimeria parasites (Preston and King, 1992). The requirement for ATP is unexpected, as hydrolysis is typically not required for actin filament polymerization in eukaryotic cells. Nevertheless, this assay should allow more complete characterization of the conditions and proteins that regulate actin filament polymerization and their interaction with myosin in force generation in T. gondii. To evaluate the diversity of myosin genes expressed in Toxoplasma, we examined the EST database, which contains over 8000 partial cDNA sequences generated from tachyzoite mRNAs (Washington University – Merck Toxoplasma EST Project). Several of these T. gondii EST sequences resemble myosin I isoforms (GenBank N82503 Q 1997 Blackwell Science Ltd, Molecular Microbiology, 26, 163–173
Myosin-based motility in Toxoplasma 169
Fig. 7. Myosin inhibitors block both attachment and invasion of host cells by T. gondii. A. Attached (open bars) vs. invaded (closed bars) parasites were distinguished by two-colour immunofluorescent staining (see Experimental procedures ). Treatment with KT5926 significantly decreased cell attachment, while BDM showed only a modest reduction. Both drugs also blocked invasion, reflected in a reduction in the percentage of intracellular parasites. Control is the average of medium alone and DMSO alone. B. Secretion of MIC2 as detected by Western blotting with mAb 6D10. The inhibitor BDM had a moderate effect on MIC2 release at 20 mM but no effect at 40 or 60 mM. In contrast, the inhibitor KT5926 greatly decreased MIC2 release at all doses examined. Left control is medium alone, right control is DMSO alone.
and W00066) and correspond to a small myosin gene in T. gondii that has recently been sequenced in its entirety and named TgM-A: it predicts a protein with a molecular mass of 93 kDa and contains the peptide sequence recognized by a-LEAF (Heintzelman and Schwartzman, 1997). Based on these similarities, it is likely this gene encodes the 90 kDa myosin described here. Small, unconventional myosins, typified by myosin I, exist as monomers, have relatively short tail regions and function as mechanochemical enzymes that hydrolyze ATP (Mooseker and Cheney, 1995). They contribute to pseudopod extension, membrane ruffling, phagocytosis, cell motility and organelle movements in both protozoal and vertebrate cells (Adams and Pollard, 1989; Jung et al., 1989; Pollard et al., 1991; Wessels et al., 1991; Titus et al., 1993). T. gondii does not exhibit most of these behaviours, and its major form of locomotion is gliding motility, suggesting that this process may be mediated by the 90 kDa myosin Q 1997 Blackwell Science Ltd, Molecular Microbiology, 26, 163–173
described here. Two other myosin alleles have also been described from T. gondii cDNAs, TgM-B and TgM-C (Heintzelman and Schwartzman, 1997); the reason for the failure of the anti-LEAF antibody to recognize these isoforms in tachyzoites is presently unclear but could result from low expression. To establish if myosin was involved in parasite motility and host cell invasion, we tested the effects of several specific inhibitors on T. gondii. Both BDM and KT5926 inhibited the gliding of parasites on serum-coated surfaces, while the protein kinase C inhibitor, calphostin C, had no effect. BDM and KT5926 also partially blocked the attachment of T. gondii to host cells and prevented cell entry. Interpretation of these findings is complicated by the fact that these drugs may also disrupt host processes required for the engulfment of microorganisms. However, this is unlikely to account for the reduction in T. gondii invasion, as we have previously shown that the host cell cytoskeleton plays little role in T. gondii entry (Morisaki et al., 1995; Dobrowolski and Sibley, 1996). Moreover, the action of these inhibitors in blocking parasite gliding motility, which occurs in the absence of host cells, supports the interpretation that they act directly on a target(s) within the parasite. While KT5926 clearly inhibited both motility and invasion by T. gondii, the IC50 (approximately 0.5–1.0 mM) is considerably higher than that reported for mammalian MLCK (IC50 20 nM) (Nakanishi et al., 1990). This result may reflect a difference in enzyme sensitivity for T. gondii MLCK that has not been characterized previously. Alternatively, it is possible that T. gondii myosin is regulated instead by heavy-chain phosphorylation, as is typical of other protozoan myosin-I isoforms (Mooseker and Cheney, 1995), and that the observed inhibition results from action on another kinase, such as calmodulin kinase, which is also highly sensitive to KT5926 (IC50 0.004 mM) (Calbiochem). Rather than acting on MLCK, the effect of KT5926 in reducing cell adhesion is probably caused by the dramatic reduction in the secretion of MIC2, a multispecific adhesin involved in cell attachment by T. gondii (V. B. Carruthers and L. D. Sibley, in preparation). MIC2 is stored within small secretory vesicles, known as micronemes, which are released on contact with the host cell (Carruthers and Sibley, 1997). The inhibition of MIC2 release may indicate a role for myosin in secretion in T. gondii, as is the case for the small myosin, myoA, in Aspergillis (McGoldrick et al., 1995). The inhibition of secretion is specific to microneme release, as KT5926 treatment had no effect on the secretion of dense granules (data not shown). Reduced microneme release may also account for the observed inhibition of motility by KT5926, as MIC2 is also present in the trails and may play a role in gliding (S. Ha˚kansson and L. D. Sibley, unpublished results). Treatment with BDM also partially reduced parasite
170 J. M. Dobrowolski, V. B. Carruthers and L. D. Sibley attachment to host cells, although this effect was less dramatic and not directly related to MIC2 secretion. The more dramatic effect of BDM was to reduce parasite invasion into host cells. BDM has been described as a lowaffinity, yet specific, inhibitor of myosin ATPases that blocks myosin II-based cell motility in PtK2 cells and myosin Ic-based contractile vacuole function in Acanthamoeba (Cramer and Mitchison, 1995). BDM also prevents the backward flow of lamellipodia in neuronal growth cones, a process probably mediated by myosin V (Lin et al., 1996). In each of these examples, myosins act as molecular motors to generate force by moving along actin filaments. In an analogous manner, the inhibitory effects of BDM treatment on parasite motility suggest that myosin plays a critical role in generating the force necessary for both gliding and cell penetration by T. gondii. In neuromuscular cells, BDM has also been shown to affect calcium release from the sarcoplasmic reticulum (Phillips and Altschuld, 1996) and to affect calcium currents, possibly because of a phosphatase-like activity (Coulombe et al., 1990). Determining whether BDM inhibits motility in T. gondii by direct inhibition of the myosin ATPase or by a secondary mechanism awaits further biochemical studies. Support for a model in which myosin participates in gliding motility was also provided by the localization of myosin within T. gondii. Immunofluorescence localization of the 90 kDa myosin in T. gondii using the a-LEAF antisera revealed peripheral staining that extended around the circumference of the parasite with some diffuse cytoplasmic labelling. Previous studies using an antisera raised against cricket muscle myosin reported staining that was restricted to the apical pole of the parasite (Schwartzman and Pfefferkorn, 1983). The T. gondii protein(s) recognized by this heterologous polyclonal serum was not characterized but may represent a subclass of myosin that is apically localized. In contrast, the circumferential pattern of the 90 kDa myosin co-localized with actin as shown by immunoelectron microscopy. In cells that were osmotically swollen, myosin remained attached to the plasma membrane, while actin was associated with the IMC. This arrangement suggests that myosin interacts with phospholipids or a membrane protein, while actin is associated with the cell cortex. Such a model is consistent with the idea that the IMC performs a structural role in maintaining cell shape: association of actin filaments with the IMC would thereby ensure a stable, polarized substrate for myosin to move along. Apicomplexan parasites travel across the substrate at 1–10 mm s¹1 during gliding (King, 1988). In the case of T. gondii, which moves at about 1–2 mm s¹1, this rate closely matches the rate of cell entry (Morisaki et al., 1995). The formation of a trail containing plasma membrane proteins that are shed during gliding indicates that
forward motion arises from an anterior to posterior membrane flow (King, 1988). To date, all of the cell surface proteins of T. gondii have been found to lodge in the membrane via glycosyl-phosphotidylinositol anchors, including SAG1 (Tomavo et al., 1989), and are, therefore, unlikely to participate directly in gliding, as they do not span the plasma membrane. Instead, parasite proteins that traverse the membrane and interact with the substrate to transfer the mechanical force generated by actino-myosin across the cell membrane are likely to be critical components of motility. Recent evidence indicates that the transmembrane protein called TRAP is essential for motility in malarial sporozoites (Sultan et al., 1997). The protein MIC2 is a functional homologue of TRAP, indicating that it may play an analogous role in the motility of T. gondii. T. gondii provides a model system for examining the role of myosin in gliding motility because of its wide host cell range, ease of growth, efficient invasion process and simple in vitro assay for motility. In addition to providing an insight into the mechanochemical force necessary to drive cell locomotion, gliding motility is presumably essential for the survival of apicomplexans, the majority of which are obligate intracellular parasites. We have previously shown that Cyt D specifically blocks parasite motility and invasion and that both processes are critically dependent on actin filaments in the parasite (Dobrowolski and Sibley, 1996). The present studies indicate that parasite motility is probably powered by a myosin motor that acts on actin filaments. The distribution of the 90 kDa myosin, which lies beneath the plasma membrane, places it in an ideal position to transmit mechanical energy into forward motion and thus propel gliding and cell invasion.
Experimental procedures
Growth and isolation of parasites Human foreskin fibroblasts (HFFs) were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum, 2 mM glutamine and 20 mg ml¹1 gentamycin. RH strain parasites were grown in monolayers of HFF cells and harvested as described previously (Morisaki et al., 1995). For some experiments, the following transgenic lines of T. gondii expressing b-galactosidase (b-Gal) were used to quantify parasite numbers: the 2F clone of the RH strain containing GRA1/lacZ (Dobrowolski and Sibley, 1996) or a clone of the PLK strain containing pTUB-b-Gal (Seeber and Boothroyd, 1996) (kindly provided by J. Boothroyd, Stanford University, USA). Parasites were washed and resuspended in Hank’s balanced salt solution containing 10 mM HEPES and 1 mM EGTA (HHE).
Precipitation of actin–myosin complexes in vitro Freshly isolated parasites were resuspended in actin lysis buffer containing 90 mM KCl, 10 mM HEPES, 0.5 mM EGTA, Q 1997 Blackwell Science Ltd, Molecular Microbiology, 26, 163–173
Myosin-based motility in Toxoplasma 171 5 mM MgCl2 and 0.1% saponin and incubated on ice for 30 min. Samples were centrifuged at 100 000 g for 30 min, and the high-speed supernatant containing soluble actin was saved. A 30 ml aliquot of supernatant was mixed with 10 ml of lysis buffer alone or supplemented to 25 mM ATP or 25 mM AMP-PNP and incubated at room temperature for 5 min. Samples were centrifuged at 16 000 g for 30 min at 48C, and pellets were analysed by SDS–PAGE and Western blotting.
Antibodies Actin was detected with the mAb C4 (Boehringer Mannheim), which reacts to all known isoforms of actin (Lessard, 1988), or a rabbit polyclonal anti-T. gondii actin serum described previously (Dobrowolski et al., 1997). Myosin was detected using an affinity-purified polyclonal antibody (referred to as a-LEAF) generated against a 25 amino acid sequence (CNPILEAFGNAKTIKNNNSSRFGKY; a gift from T. Mitchison, UCSF, USA). Secondary antisera consisting of goat anti-mouse IgG or goat anti-rabbit IgG conjugated to fluorescein isothiocyanate (FITC) or Texas Red were purchased from Jackson ImmunoResearch Laboratories.
Western blotting Freshly isolated parasites were lysed in sample buffer, separated on SDS–polyacrylamide gels under reducing conditions (Laemmli, 1970) and blotted to nitrocellulose by semidry electrophoretic transfer. Blots were blocked in phosphate-buffered saline (PBS) with 5% dry milk, 5% goat serum and incubated with primary antibodies followed by goat anti-rabbit IgG or goat anti-mouse IgG conjugated to peroxidase (Kirkegaard & Perry), developed with Luma-Phos reagent (Pierce) and exposed to Kodak XAR film.
Immunofluorescence (IF) localization Freshly isolated parasites were fixed with 2.5% formalin, 0.05% glutaraldehyde, 0.02% saponin in actin stabilization buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl2 and 125 mM KCl). For the localization of actin and myosin, fixed slides where then extracted in acetone at ¹208C. Slides were blocked for 30 min in PBS-10% FBS, then incubated with primary antibodies diluted in PBS-1% FBS, followed by fluorescently conjugated secondary antibodies. Slides were mounted in Pro-Long anti-fade media (Molecular Probes), examined and photographed on a Zeiss Axioscope microscope.
or goat anti-mouse IgG coupled to 18 nm gold (Jackson ImmunoResearch Laboratories). Grids were counterstained with 0.15 M oxalic acid/2% uranyl acetate and supported with a 1:2 mixture of methylcellulose and 4% uranyl acetate. Sections were examined and photographed on a Zeiss EM 109 microscope.
Gliding assays Freshly isolated parasites were resuspended in HHE alone or HHE containing DMSO, BDM dissolved in water (Sigma Chemical), KT5926 dissolved in DMSO (Calbiochem-NovaBiochem), calphostin C dissolved in DMSO (CalbiochemNovaBiochem) or cytochalasin D dissolved in DMSO (Cyt D) (Sigma) and incubated at 378C for 15 min. Pretreated parasites were then placed into serum-coated chamber slides (Nunc) and allowed to glide for 15 min at 378C followed by fixation in 2.5% formalin/PBS. Trails left by gliding parasites were detected by incubation with mAb DG52 (a gift from J. Boothroyd, Stanford University, USA), which reacts with the SAG1 surface protein of T. gondii, and stained with FITClabelled goat anti-mouse IgG antibodies. Gliding was quantified by measuring the approximate trail length as a multiple of the parasite body length (approximately 7 mm) from 10 separate 1000× microscopic fields (approximately 50 cell trails). For reversibility experiments, parasites were treated with inhibitors for 15 min at 378C and washed by centrifugation at 600 g, then resuspended in HHE three times before use in gliding assays. Experiments were repeated two or more times, and data are expressed as mean 6 SD from a representative experiment.
Invasion and growth assays Freshly isolated b-Gal-expressing PLK strain parasites were resuspended in modified Eagle medium (MEM) plus 3% FBS alone or containing DMSO, BDM, KT5926 or Cyt D for 15 min at 378C. Parasites were added in the presence of drug to confluent HFF cells seeded in a 24-well plate and allowed to invade for 1 h at 378C. Cells were washed three times with PBS and treated with 50 mg ml¹1 phleomycin for 2 h to kill any remaining extracellular parasites. Plates were incubated for 24 h in DMEM-10% FBS at 378C, lysed in PBS containing 1% Triton X-100, and b-galactosidase activity was quantified in microtitre plates using the substrate chlorophenol red b-Dgalactopyranoside (Eustice et al., 1991). To determine the effects of inhibitors on growth, infected monolayers grown on chamber slides were incubated overnight in DMEM-10% FBS at 378C, and the mean number of parasites per vacuole was determined. Experiments were repeated three or more times, and data are expressed as mean 6 SD from a representative experiment.
Immunoelectron microscopy Freshly isolated parasites were washed in PBS and fixed as above, except using 0.5% glutaraldehyde for 1 h at 48C. After embedding in 10% gelatin, samples were infiltrated with 0.3 M sucrose and frozen in liquid N2 . Ultrathin cryosections were blocked in 20 mM glycine followed by PBS-10% FBS. Cryosections were incubated with primary antibodies followed by goat anti-rabbit IgG antibodies coupled to 12 nm colloidal gold Q 1997 Blackwell Science Ltd, Molecular Microbiology, 26, 163–173
Parasite attachment vs. invasion To distinguish attachment from invasion, the following twocolour immunofluorescence assay was used to identify parasites that remained extracellular from those that had entered the host cell. Freshly isolated b-Gal-expressing RH strain parasites were resuspended in MEM-3% FBS alone or
172 J. M. Dobrowolski, V. B. Carruthers and L. D. Sibley supplemented with DMSO, BDM, KT5926 or Cyt D and incubated at room temperature for 15 min. Parasites were inoculated onto chamber slides containing HFF monolayers and allowed to invade at 378C for 10 min in the presence of drugs. Slides were rinsed with PBS, fixed as above and stained with polyclonal rabbit anti-SAG1 (a gift from L. Kasper, Dartmouth Medical School, USA), followed by Texas Red–goat anti-rabbit IgG. Monolayers were then permeabilized with 0.01% saponin and incubated with mAb DG52 to SAG1 followed by FITC–goat anti-mouse IgG. Attached (red staining) vs. invaded parasites (green staining) were quantified by replicate counting of at least 50 host cells. Experiments were repeated three times and data are expressed as the mean 6 SD from a representative experiment.
MIC2 secretion assay Freshly isolated b-Gal-expressing RH strain parasites were harvested and treated with drugs as described above and then incubated in polystyrene tubes at 378C for 30 min before cooling to 08C. Parasites were removed by centrifugation twice at 1000 g, and the supernatants were mixed with SDS sample buffer and analysed by Western blot. Secretion was determined by detection of MIC2 (mAb 6D10) (Wan et al., 1996), and inadvertent lysis was monitored by the release of b-Gal (mAb 401-a, kindly provided by J. Sanes, Washington University, USA), as determined by quantitative phosphorimaging analysis and comparison with cell standards.
Acknowledgements We thank Drs John Boothroyd, Lloyd Kasper and Tim Mitchison for generous gifts of antibodies, reagents and helpful advice; Drs Joe Schwartzman and Matt Heintzelman for sharing unpublished data; Marylin Levy for expert technical assistance with electron microscopy; and Drs Sebastian Ha˚kansson and Kathy Miller for a critical review of the manuscript. This work was supported by a grant from the NIH (AI34036). J.M.D. was supported in part by an NIH training grant (AI07172). L.D.S. is a New Investigator in Molecular Parasitology supported by the Burroughs Wellcome Fund.
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