Dynamin inhibitor impairsToxoplasmagondiiinvasion

The protozoan parasite Toxoplasma gondii infects its host cells through an active mechanism. In this work, we obtained evidence that host cells also play a fundamental role during the infection process. We found that previous incubation of the host cells, but not the parasites, with Dynasore, a small molecule that inhibits dynamin GTPase activity, markedly reduced the penetration of T. gondii tachyzoites into LLC-MK2 cells. In contrast, parasite adhesion to the host cell surface increased, as observed both by light and electron microscopy. Intriguingly, the few parasites internalized by Dynasore-treated cells remained in vacuoles located at the periphery of the cell, in contrast to the perinuclear localization seen in the control.


Introduction
Some pathogenic protozoa have developed the ability to interact and be recognized by components of the host cell surface.In the case of phagocytic cells, these protozoa can be internalized through typical phagocytic processes.In the case of nonprofessional phagocytic cells, it has been shown that some protozoa can trigger signaling events that lead to their internalization (Sibley, 2004;Boothroyd & Dubremetz, 2008).Toxoplasma gondii, the agent of toxoplasmosis, a disease with a high prevalence throughout the world and a leading cause of death in immunocompromised patients, is able to infect virtually all nucleated cells of warm-blooded animals (Joiner & Dubremetz, 1993).Despite the large number of studies analyzing the interaction process of T. gondii and the host cell, the mechanisms used by the protozoan to infect the cells remain controversial.Some studies have suggested that the process of infection depends primarily on the ability of the parasite to move by a typical gliding process, and the secretion of a large set of proteins from highly specialized organelles such as the micronemes and rhoptries located at the anterior portion of the proto-zoan.Other studies have postulated the coparticipation of the host cell cytoskeleton (Morisaki et al., 1995;Dobrowolski & Sibley, 1996;De Souza et al., 1998;MacLaren & De Souza, 2002) and protein kinases (Ferreira et al., 2003;MacLaren et al., 2004;Da Silva et al., 2009) during the interaction process.Following invasion, the parasite resides inside a special endocytic vacuole, known as the parasitophorous vacuole (PV), which does not fuse with host cell endosomes and lysosomes.Following exponential growth inside the PV, tachyzoites leave the host cell by a mechanism triggered by an increase in the cytoplasmic calcium concentration within the host cell (Mordue et al., 1999;Hoff & Carruthers, 2002;Sibley, 2004).
Dynamin is a multidomain GTPase, which plays a crucial role in clathrin-dependent endocytosis, vesicle formation at the trans-Golgi network and plasma membrane caveola fission to form transport vesicles and phagosomes.It is a key element that regulates membrane tubulation and the fission of budding vesiculotubular structures, and is essential for the formation of phagosomes in macrophages, especially at the stage of membrane extension around the particle (Gold et al., 1999).The GTPase activity of dynamin can be inhibited by Dynasore, a small molecule that was identified from a library of 16 000 compounds (Kirchhausen et al., 2008).In this study, we used Dynasore, which has been shown to prevent vesicle budding from the plasma membrane, as well as the release of these vesicles (Macia et al., 2006), to investigate the role of dynamin in the process of invasion of host cells by T. gondii tachyzoites.
Our observations clearly show the participation of the host cell during the process of parasite invasion into host cells.

Chemicals
Dynasore was kindly provided by Dr Tomas Kirchhausen (Department of Cell Biology, Harvard University, Boston, MA and IDI Research Institute, Boston, MA).It was dissolved in dimethyl sulfoxide so that a stock concentration of 200 mM was obtained.Aliquots were stored at À 40 1C and diluted to final concentrations in the culture medium just before use.All other reagents were purchased from Sigma Chemical Company (St. Louis, MO) and IDI Research Institute (Boston, MA).

Parasites and host cell culture
Toxoplasma gondii tachyzoites of the RH wild-type strain were obtained by peritoneal washes of 2-3-day-infected mice.The cell suspension was centrifuged at 1000 g for 10 min to remove cell debris and peritoneal leukocytes, and the number of parasites in the supernatant was determined by counting in a Neubauer chamber.The parasites were resuspended in Dulbecco's modified Eagle's medium (DMEM).Swiss mice were bred at the Universidade Estadual do Norte Fluminense animal facility.
The experimental protocol was approved by the Instituto de Biofísica Carlos Chagas Filho (Universidade Federal do Rio de Janeiro) Ethics Committee for animal experimentation.Macaca mulatta monkey kidney cells (LLC-MK2) were maintained in vitro in DMEM supplemented with 10% fetal bovine serum at 37 1C in a 5% CO 2 atmosphere.Cellular viability was determined using neutral red, and absorbance values were determined using an enzyme-linked immunosorbent assay reader.For light microscopy, cultures were fixed and stained using the Panotic Solution Kit s .

In vitro infections
Confluent monolayers of LLC-MK2 cells were infected with five parasites per cell for light microscopy or 10 parasites per cell for electron microscopy assays.Dynasore, at concentrations varying from 20 to 100 mM, was tested for its effect on parasite attachment and internalization into the host cells.
Based on the results obtained, a concentration of 80 mm was selected for the experiments involving microscopy.Treatments were performed for 20 min.For the reversibility assay, samples were briefly washed three times.Images were taken after 40 min of host cell-parasite interaction.For control, neither the parasites nor the host cells were incubated with Dynasore before protozoan-host cell interaction.In other experiments, parasites were pretreated or Dynasore was added to medium containing parasites for interaction with host cells.Interaction was carried out at 37 1C in a 5% CO 2 atmosphere.After interaction, the supernatant containing parasites that had not attached or entered into host cells was aspirated and host cell monolayers were fixed for further observation by light or electron microscopy.All experiments included nontreated infected monolayers of LLC-MK2 cells as control.Experiments were performed in triplicate in three independent experiments.For each experiment, 10 loci were counted in each glass coverslip.In order to determine the influence of Dynasore treatment on the intracellular localization of the few parasites that invaded the host cell, measurement of parasite-host cell nucleus distance was made using a version 3.0 SEMAPHORE s processing system.

Statistical analysis
The statistical analysis was conducted using ANOVA with the Bonferroni test.The results were considered significant when P o 0.05.

Light microscopy
After incubation in the presence of the parasite, the LLC-MK2 cells were fixed and stained with the Panotic Solution Kit s .Observations were carried out on a Zeiss-Axioplan light microscope equipped with a Nomarski differential interferential contrast system.

Electron microscopy
For field emission scanning electron microscopy, the host cells were cultivated on round sterile coverslips in 24-well plates.After Dynasore treatment, followed by interaction with parasites, the cell monolayers were washed and fixed in a solution containing 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, postfixed for 1 h with 1% OsO 4 in 0.1 M cacodylate buffer, pH 7.2, plus 0.8% potassium ferrocyanide, dehydrated in ethanol, critical point dried with CO 2 , sputtered with gold and observed in a Jeol 6340 field emission scanning electron microscope operated at 5.0 kV and 12 mA.Occasionally, some samples were dry-cleaved with a carbon adhesive tape, to expose the inner portion of the cell.
For transmission electron microscopy, the cells were allowed to grow on 60-mm 2 Petri dishes or in 25-cm 2 tissue culture flasks.After the experimental procedure, cell monolayers were fixed as described above, dehydrated in ethanol and flat embedded in Polybed (Polysciences s ).En face sectioning avoided the removal of the cells from the substratum, which could disrupt and disorient their architecture.Ultrathin sections were stained with uranyl acetate and lead citrate and observed on a Zeiss 900 transmission electron microscope.

Dose--response curve analysis
In order to investigate whether Dynasore, a dynamin inhibitor, had any effect on the attachment and invasion of tachyzoites to host cells, LLC-MK2 cells were pretreated with 20, 40, 60, 80 and 100 mM of the drug before the addition of parasites.Figure 1a shows that Dynasore had a dosedependent increase effect on inhibition and blocked 60% of T. gondii invasion when used at a concentration of 80 mM.In contrast, it markedly increased the adhesion of the parasites to the host cell surface (Fig. 1b), indicating that the parasites seen outside failed to invade.
Significant inhibition was also obtained when Dynasore was added to the medium during the interaction.Indeed, under this experimental condition, parasite invasion was inhibited by 80% at 100 mM (Fig. 1c).
To see whether the effect of Dynasore was reversible, the cells were initially incubated in the presence of the drug and washed before interaction with the parasites.As shown in Fig. 1d, Dynasore inhibition was significantly lower in this condition, indicating the reversibility of its effect.It is important to point out that Dynasore did not interfere with host cell morphology, as seen by light microscopy, or with cell viability, as evaluated using the neutral red assay (data not shown).Previous incubation of parasites with Dynasore did not interfere with parasite attachment and invasion (data not shown).
One fundamental step in the life cycle of some intracellular parasites such as T. gondii is the mechanism used by them to infect host cells.Evidence obtained by several groups has established that T. gondii approaches and binds Fig. 1.Graphs of Dynasore effect over Toxoplasma gondii invasion of LLC-MK2 cells.(a) The inhibitory effect on T. gondii invasion after pretreatment of host cells with Dynasore was dose-dependent, reaching 60% at a concentration of 80 mM; (b) Pretreated samples showed higher rates of adhesion, reaching 70% as compared with the control samples.% of adhesion was based on the total parasites counted on the coverslip: inside (intracellular) and outside (adhered to host cell surface); (c) When Dynasore was added to the interaction medium together with parasites, a greater inhibition of invasion was observed, reaching 70% and 80% at concentrations of 80 and 100 mM, respectively; (d) Reversibility of the effect of Dynasore was successfully tested after washing pretreated samples, resulting in the recovery of 70% of invasion when compared with nontreated cells; (e) Measurement of host cell nucleus and intracellular parasite distance in nontreated and Dynasore-treated cells.
to the host cell surface through gliding motility, a process in which parasitic actin plays an important role (Sibley, 2004;Soldati & Meissner, 2004).Subsequently, sequential triggering of microneme and rhoptry secretion is responsible for parasite invasion and assembly of the PV.The participation of the host cell in this process is usually underestimated.
Indeed, there is little information about the participation of host cell surface receptors during parasite invasion (Sibley, 2004).Previous experiments, in which host cells were treated with cytochalasin, which disrupts the actin cytoskeleton, suggested the participation of microfilaments in the interaction process (Silva et al., 1982;De Souza et al., 1998).However, because parasitic actin was also affected by cytochalasin, it remained unclear whether this effect was due to interference with parasite actin (Dobrowolski & Sibley, 1996).
Our present observations that previous treatment of the host cell, but not of T. gondii tachyzoites, with Dynasore significantly inhibited parasite internalization strongly support the idea that the host cell cytoskeletal machinery plays a fundamental role in the process of parasite invasion.It has been shown that Dynasore impairs the pinching off of the plasma membrane required for PV formation, a process that requires the GTPase activity of dynamin, by interfering both with initial vesicle formation and with vesicle release (Macia et al., 2006).It is important to note that even at a concentration of 100 mM, Dynasore did not interfere with the binding and the internalization of parasites.The effect was observed only when host cells were in contact with Dynasore.The effect was even more evident when the drug was present throughout the parasite-host cell interaction process.Indeed, under this condition, inhibition of cell infectivity reached up to 80%.This is the highest inhibitory effect reported for invasion of host cells by T. gondii.Our present observations are in close agreement with previous studies in other biological systems, such as infection by papillomaviruses (Abban et al., 2008) and phagocytosis by Sertoli cells (Otsuka et al., 2009), in which Dynasore has a marked inhibitory effect.As a consequence of inhibition of protozoa internalization, there was a marked increase in the number of parasites attached to the host cell surface.

Microscopy analysis
Light, scanning and transmission electron microscopy observations of the interaction of T. gondii with Dynasoretreated cells showed that most of the parasites were spread over the cell surface (Figs 2a and 3a), with the anterior portion of the cell body extended and some conoids extruded toward the host cell surface (Fig. 3b-c).Several parasites were observed to be attached to the host cell surface and partially internalized (Figs 2b and 3d-e), differing from control samples (Fig. 2c).In some cases it was possible to observe filopodia-like structures from the host cell over the protozoan surface (Fig. 3f).Tachyzoites that were internalized by Dynasore-treated cells remained at the cell periphery.In control preparations, PVs containing internalized parasites were always close to the nucleus of the host cell (Fig. 2c).The same features described in the host cell pretreatment assay were observed by light microscopy after a brief parasite interaction, followed by Dynasore treatment (data not shown).
The apparent delay in infection was also observed by transmission electron microscopy (data not shown), and Fig. 4 shows abundant host cell filopodia in contact with tachyzoite, as well as the accumulation of endocytic vesicles at the point of interaction.
Our morphological observations using both light and electron microscopy show that Dynasore does not inhibit the initial process of cell invasion, because images showing attached tachyzoites with the most proximal apical region partially internalized were observed.A similar effect was observed previously when host cells were treated with Wortmanin, an inhibitor of PI3-kinase, a protein involved in the complete sealing of surface projections involved in endocytic processes (MacLaren et al., 2004).Indeed, recent observations point to a certain partnership between PI(4,5)P2-bound dynamin 2 and actin reorganization, resulting in the assembly of lamellipodia and ruffles (Otsuka et al., 2009).Even when Dynasore treatment blocked tachyzoite internalization, we observed, both by scanning and transmission electron microscopy, the presence of surface projections from the host cell touching the parasites.
We also observed that the few parasites that entered into Dynasore-treated cells remained at the cell periphery and did not move to the perinuclear region of the host cell as occurs in untreated cells.This is evidenced in Fig. 1e, which shows the average distance between host cell nucleus and intracellular parasites.We do not have a clear explanation for this fact; however, it is possible that inhibition of the dynamin system in some way also interferes with the host cell cytoskeleton required for the transport of the initial PV from the cell periphery to its most central portion.On the other hand, inhibition by Dynasore could be avoided by washing it from the medium before host cell infection, which is in agreement with the recovery of transferring endocytosis reported by Macia et al. (2006).Coppens et al. (2006) reported the participation of GRA7, a dynamin-like protein, in the process of acquisition of material coming from the host mammalian cell via the exploitation of the host endo-lysosomal system.At this point, we have not tested whether Dynasore would inhibit this process, thus impairing parasite survival or multiplication, but it remains as an issue for further investigation.
In conclusion, the use of Dynasore allowed us to show clearly that the host cell plays an active and important role during T. gondii invasion, somehow contradicting the dogma that the host cell is passively invaded by the parasite.The high efficiency of Dynasore in blocking cell infection by  T. gondii also suggests that blocking the entry of tachyzoites into host cells may be a strategy in the design of new drugs for the treatment of toxoplasmosis.

Fig. 2 .
Fig. 2. Light microscopy of the Toxoplasma gondii invasion assay.(a, b) Pretreatment of host cells with Dynasore.Arrows point to parasites adhered to the host cell surface, indicating the blockage of the invasion process.(c) Control showing the aspect of a typical infection, with parasites internalized in PVs established at perinuclear sites of a host cell.Scale bars = 6 mm.

Fig. 3 .
Fig. 3. Scanning electron microscopy of the Toxoplasma gondii invasion assay after treatment of LLC-MK2 cells with Dynasore.(a) Adhesion of tachyzoites to the host cell plasma membrane.Parasites are seen adhered to the apical portion toward the host cell membrane.(b) Single tachyzoite adhered to the LLC-MK2 plasma membrane with the conoid extruded, as evidenced in the inset.(c) Tachyzoite over host cell plasma membrane.(d-e) Impairment of invasion.Parasites with cell bodies half-way inside the host cell.The hourglass constriction at the point of contact between the parasite and the host cell, typical of a moving junction, is seen in both micrographs.Host cell membrane filopodia (arrow) can be clearly seen in the inset of (e).(f) Host cell membrane extension making lateral contact with the tachyzoite.Scale bars: (a) 1 mm; (b) 1 mm, inset 250 nm; (c, d) 1 mm; (e) 1 mm, inset 500 nm; and (f) 1 mm.

Fig. 4 .
Fig. 4. Transmission electron microscopy of Toxoplasma gondii interaction with the host cell after Dynasore treatment.Host cell filopodia are observed around the tachyzoites (arrows).At the site of contact between the host cell and the parasite, a large flow of vesicles (arrowheads) is seen, possibly due to some impairment caused by Dynasore treatment.Scale bar = 2 mm.