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The Korean Journal of Parasitology Vol. 38, No. 4, 257-261, December 2000

The effect of microfilament inhibitor on the infection in vitro

Jae-Ran YU* and Sung-Don CHOI

Department of Parasitology, College of medicine, Konkuk University, Chungju 380-701, Korea

Abstract: This study was focused on the effects of microfilament inhibitor, Cytochalasin D (CD) on the invasiveness of sporozoites of Cryptosporidium spp. into the host cells. MDCK and AGS cell lines were used as host cells for C. parvum and C. muris, respectively. When MDCK cells were pretreated with CD for 1 hr before inoculation of the sporozoites, C. parvum infection was significantly inhibited when compared to the control cells. These inhibitory effects of CD on the rate of infection were dose-dependent. In addition, C. muris infection was hampered when AGS cell lines were pretreated with CD. However, the capability of invasiveness of the sporozoites into the host cells was not greatly influenced by the pretreatment of sporozoites with CD before infection. These results suggest that microfilaments of host cells, rather than parasites, play an important role for the invasion of Cryptosporidium spp. Key words: microfilament, cytochalasin D, , Cryptosporidium muris

human such as C. baileyi and C. muris (Ditrich INTRODUCTION et al., 1991; Katsumata et al., 2000). C. parvum and C. muris have been reported Genus Cryptosporidium is a well known to have a large amount of actin in their coccidian protozoa which has a wide range of cytoplasm and pellicles all through their hosts. Although a lot of different names of the developmental stages (Yu and Chai, 1995; Yu, species have been reported, only six species, 1998) and the gene that encodes actin has such as C. parvum, C. muris, C. baileyi, C. been isolated in C. parvum (Kim et al., 1992). meleagridis, C. serpentis, and C. nasorum, are However, the function of actin in this organism generally accepted as truly valid ones has not been well defined yet. For the (O’Donoghue, 1995). The majority of human successful infection of , invasive zoites has been reported in are the most important developmental stages. immunocompromised patients infected with C. They are suspected to invade host cells via a parvum (Griffiths, 1998) and they usually capping phenomenon in Eimeria spp. and the suffer from a chronic, devastating illness parasite’s contractile system is considered to refractory to all therapeutic modalities. be involved in this process (Russell, 1983). For However, there are two more species other the host cell invasion of Cryptosporidium, there than C. parvum that are known to infect are very little findings known until now. In this study we investigated the role of microfilament �Received 8 September 2000, accepted after during invasion of Cryptosporidium to host revision 3 November 2000. cells using microfilament inhibitors. *Corresponding author (e-mail: jaeran.yu@ mail.kku.ac.kr) ─258─

DMSO concentrations were always under MATERIALS AND METHODS 0.05% (v/v). RPMI 1640 containing the desired concentrations of CD, 0.5, 1.0, 2.5, 4.0 or 5.5 Oocyst preparation µg/ml, were added to the monolayers. Controls Oocysts of C. parvum were collected and were untreated monolayers and monolayers to purified as described by Yu et al. (2000). The which was added medium containing DMSO oocysts of C. muris were isolated from the (0.05%) alone. The experiments were then naturally infected laboratory mice (C57BL/6) divided into two groups. In one group, at the laboratory of Parasitology in Konkuk monolayers were preincubated with various University in 1997, and have been maintained concentrations of CD or DMSO for 1 hr before through in vivo cultivation using the same Cryptosporidium inoculation and the same mouse (SPF) strain. The oocyst collection and concentrations of CD were also included in the purification methods are the same as those of media after infection. In the other group, C. parvum. But mice were not immuno- sporozoites of Cryptosporidium were suppressed for the C. muris oocyst collection preincubated with various concentrations of and the feces were collected from 20 to 40 CD or DMSO for 1 hr, and then challenged days after oral infection. into monolayers which has not been exposed to CD. The media for monolayers did not Host cell culture and sporozoite contain CD after sporozoite inoculation. inoculation As host cell lines, Madin-Darby Canine Infection evaluation kidney cell (MDCK) and human stomach The cover glass was taken out from the well adenocarcinoma (AGS) (Korean Cell Line Bank, after 24 hr of incubation at 37°C in a 6% CO2- Seoul, Korea) were used for C. parvum and C. 94% air humidified incubator, washed in PBS, muris, respectively. The cells were incubated in pH 7.4 and fixed in methanol for 1 min. The RPMI 1640 with L-glutamine (Sigma) cover glass was stained by Hematoxylin and containing 15 mM N-2-hydroxyethylpiperazine Eosin and then mounted on the slide glass. -N’-2-ethanesulfonic acid (HEPES), 14 mM The total number of parasites infected sodium bicarbonate, 100 U of penicillin per monolayers was counted from 25 fields under ml, 100 µg of streptomycin per ml, and 0.25 µg of a light microscope with a resolution of 1,000 of amphotericin B per ml. For a routine cell x (oil) lens (Olympus). The experimental data of passage, 10% fetal bovine serum (FBS) (Gibco each group, repeated four times, were BRL, Gaithersburg, MD) was used, however, compared with those of control group. The after sporozoite inoculation, 1% FBS was used significant difference between the groups was for the MDCK cells and 10% FBS was used for evaluated by Student’s t-test. AGS cells. The host cells for infection were plated onto coverglass 22 mm2 placed in 6 well RESULTS plates and incubated at 37°C in a 6% CO2- 94% air humidified incubator. Oocysts were Cytochalasin D effect on C. parvum washed once with RPMI 1640 medium after infection in vitro excystation by incubating in 1% trypsin in Cytochalasin D at the concentrations tested PBS at 37°C for 1 hr, and inoculated 4 x 106 did not show any toxic effect on the MDCK cell sporozoites into subconfluenced monolayers monolayer within 24 h culture. The sporozoite per well. of C. parvum invasion into MDCK cells was not affected by DMSO treatment (Fig. 1). The Cytochalasin D treatment infected parasite number of C. parvum in A stock solution of cytochalasin D (CD; MDCK cells significantly decreased (P < 0.05) Sigma) was prepared by dissolving in dimethyl compared to that of control group when the sulfoxide (DMSO; Sigma) and adjusting the sporozoites were inoculated to host cells concentration of CD to 100 µg/ml in RPMI preincubated with CD (Fig. 1A). Although low 1640. This was stored at -20°C and final concentrations of cytochalasin D had a little

─260─ with CD (Fig 2B). These results suggest that development of new therapeutic drugs for the microfilament of AGS cells plays an treatment of cryptosporidiosis. essential role for successful infection of C. In the present study, pretreatment of host muris sporozoites. cells with CD inhibited Cryptosporidium species invasion into the host cells. Both C. DISCUSSION parvum and C. muris infection were prevented with the disruption of host cells’ The role of cytoskeletal proteins in coccidian microfilaments using CD. Therefore, the parasite’s invasion has been evaluated using microfilament of the host cells may play an inhibitors against microtubule or important role in Cryptosporidium entry by microfilament. Cytochalasin D is well known making host cells participate actively in the microfilament inhibitor that shows inhibitory entry process, like in T. gondii or Eimeria spp. effect by binding on the subunits of actin and Another possible explanation could be that, in actomyosin (Tannenbaum et al., 1977). mammalian cell lines, the membrane receptors CD was proved not to affect the attachment are known to be anchored by a subtending of but to prevent the entry cytoskeleton and could act as a substratum into peritoneal macrophages and into the for the parasite’s contractile system to work bladder tumor 4934 cells (Ryning and against (Shelterline, 1980). Therefore, the Remington, 1978). In this paper, it was disruption of microfilaments in the host cells suggested that T. gondii acts on the may induce structural changes, causing the nonphagocytic cell in some undefined way, surface receptors to be masked or distorted inducing phagocytosis rather than utilizing the during such process, which would inhibit cell merely as a passive agent during the entry sporozoite to recognize receptors for invasion process. In other words, host cells actively into the cell. The membrane bound host cell participate in the process by which T. gondii receptors related to the cytoskeletal system gains entry to the cells. Additionally, in case of may have a function in the initial process of Eimeria species, it was implied that a Cryptosporidium invasion. microfilamentous system is active during a In this study, C. parvum invasion was host cell invasion because the invasive partially, but significantly, inhibited when the behavior of E. acervulina and E. tenella was sporozoites were pretreated with CD before inhibited by cytochalasin B (Russell, 1983). infection. The effect of CD was known to be The cytoskeletal system is known to regulate reversible within 60 min after removal of this the sporozoite motility as well, thus being an agent (Ryning et al., 1978). However, since important factor for host cell invasion in T. most sporozoites of Cryptosporidium invade gondii and Eimeria species (Schwartzman and host cells within 60 min, the time interval is Pfefferkorn, 1983; Russell and Sinden, 1981). considered to be enough to observe the The fact that the sporozoite motility of Eimeria inhibitory effect of CD on the sporozoites was inhibited by treatment with cytochalasin invasion. The result with sporozoites is B, but not by colchicine, vinblastine sulfate or somewhat different from previous studies other microtubule inhibitor, indicated that all reported by Forney et al. (1998). They aspects of motility are microfilament-based described, although the sporozoite motility contractile system type. decreased with CD treatment, sporozoite’s There are several reports describing the role invasion into host cell was not affected. There of cytoskeletal system in the process of C. also are some key points which could explain parvum invasion into host cells. Wiest et al. the discrepancies between the previous (1993) reported that microtubule inhibitors findings by Forney et al. (1998) and present induced 77% worm number reduction in C. study. One thing is that they did not disrupt parvum infection into human enterocyte cell the microfilament of host cells, so there was no line. This study showed that microtubules description about the role of microfilament of have an important role for the C. parvum host cells. Another one is that different cell invasion and may represent targets for lines, which might have different amounts and ─261─ distribution of microfilaments, were used for Cryptosporidium muris infection in humans. C. parvum infection. In addition, different CD Am J Trop Med Hyg 62: 70-72. concentrations tried for both two studies may Kim K, Gooze L, Petersen C, Gut J, Nelson RG be responsible for different results between (1992) Isolation, sequence and molecular two studies. karyotype analysis of the actin gene of In terms of C. muris infection, we found that, Cryptosporidium parvum. Mol Biochem Parasitol 50: 105-114. whereas the host cell microfilament disruption O’Donoghue PJ (1995) Cryptosporidium and induced lower infection rates, the inhibition of cryptosporidiosis in man and animals. Int J the microfilament of parasite itself did not Parasitol 25: 139-195. inhibit the parasite invasion. Since the effect of Russell DG, Sinden RE (1981) The role of the CD on the motility of C. muris sporozoite was cytoskeleton in the motility of coccidian not observed in this study, it is hard to sporozoites. J Cell Sci 50: 345-359. determine the exact role of the parasite Russell DG (1983) Host cell invasion by motility in the host cell invasion. The reason : an expression of the parasite’s for using AGS cell lines as host cells in C. contractile system? Parasitology 87: 199-209. muris infection is due to the higher Ryning FW, Remington, JS (1978) Effect of susceptibility of the AGS cells than the MDCK cytochalasin D on Toxoplasma gondii cell cells against C. muris infection (data not entry. Infect Immun 20: 739-743. shown). Schwartzman JD, Pfefferkorn ER (1983) In conclusion, this study shows that intact Immuofluorescent localization of myosin at host cell microfilament is required for the the anterior pole of the coccidian, Toxoplasma successful infection of Cryptosporidium gondii. J Protozool 30: 657-661. Shelterline P (1980) Cell surface motility: species. Further investigation with respect to interactions between membrane proteins and the interaction of microfilament between host the cytoskeleton. In Microtubules and cells and Cryptosporidium is needed for the full microtubule inhibitors (ed. M. DeBrabander understanding of host-parasite relationship of and J. DeMey) Amsterdam and New York: this parasite. Elsevier/North Holland. Tannenbaum J, Tannenbaum SW, Godman GC REFERENCES (1977) The binding sites of cytochalasin D. J Cell Physiol 91: 225-237. Ditrich O, Palkovic L, Sterba J, Prokopic J, Wiest PM, Johnson JH, Flanigan TP (1993) Loudova J, Giboda M (1991) The first finding Microtubule inhibitors block Cryptosporidium of Cryptosporidium baileyi in man. Parasitol parvum infection of a human enterocyte cell Res 77: 44-47. line. Infect Immun 61: 4888-4890. Forney JR, Vaughan DK, Yang S, Healey MC Yu JR, Chai JY (1995) Localization of actin and (1998) Actin-dependent motility in myosin in Cryptosporidium parvum using Cryptosporidium parvum sporozoites. J immunogold staining. Korean J Parasitol 33: Parasitol 84: 908-913. 155-164. Griffiths JK (1998) Human cryptosporidiosis: Yu JR (1998) Distribution of actin and Epidemiology, transmission, clinical disease, tropomyosin in Cryptosporidium muris. treatment, and diagnosis. Adv in Parasitol 40: Korean J Parasitol 36: 227-234. 38-87. Yu JR, Choi SD, Kim YW (2000) In vitro infection Katsumata T, Hosea D, Ranuh IG, Uga S, Yanagi of Cryptosporidium parvum to four different T, Kohno S (2000) Short report: possible cell lines. Korean J Parasitol 38: 59-64.