Host cell surface sialic acid residues are involved on the process of penetration of Toxoplasma gondii into mammalian cells

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1 FEMS Microbiology Letters 164 (1998) 323^327 Host cell surface sialic acid residues are involved on the process of penetration of Toxoplasma gondii into mammalian cells V.G. Monteiro a, C.P. Soares a, W. de Souza a;b; * a Laboratoèrio de Biologia Celular e Tecidual, Centro de Biocieências e Biotecnologia, Universidade Estadual do Norte Fluminense, Av. Alberto Lamego 2000, , Campos dos Goytacazes, RJ, Brazil b Laboratoèrio de Ultraestrutura Celular Hertha Meyer, Instituto de Biof èsica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Abstract Ilha do Fundaìo, 21941, Rio de Janeiro, Brazil Received 7 December 1997; revised 19 May 1998; accepted 23 May 1998 Tachyzoites of Toxoplasma gondii are able to infect several cell types tested (wild-type chinese hamster ovary (CHO) cells and glycosylation mutants, Vero and LLCMK2 cells). However, the extent of infection varied. Mutant cells which present few or no surface-exposed sialic acid residues were infected to a lower extent. Similar results were obtained if sialic acid residues were removed by previous neuraminidase treatment. Addition of sialic acid residues to surface-exposed glycoconjugates using fetuin as a sialic acid donor and the trans-sialidase of Trypanosoma cruzi rendered the cells more easily infected by Toxoplasma gondii. These observations indicate that surface-exposed carbohydrate residues of the host cell are involved on the process of Toxoplasma gondii-host cell recognition. z 1998 Published by Elsevier Science B.V. All rights reserved. Keywords: Apicomplexa; Toxoplasma gondii; Parasite-host cell interaction; Neuraminic acid 1. Introduction Some pathogenic protozoa are able to interact with the surface of vertebrate cells triggering a process which leads to their into the cells. For some protozoa, such as those of the genus Leishmania, it has been shown that there is a special tropism of the protozoa for macrophages and that parasite interiorization takes place through a typical phagocytic process. Other protozoa, as Trypanosoma cruzi, penetrate di erent types of cell using both a phagocytic process and another mechanism where * Corresponding author. Tel./Fax: +55 (247) ; wsouza@uenf.br the host cell also plays an important role on the interaction process (reviewed in [1]). Still other protozoa, as is the case of Toxoplasma gondii, have the capacity to invade a broad range of vertebrate cells using several mechanisms including one in which components of the cytoskeleton play a fundamental role [2^5]. Studies carried out with several microorganisms have shown that surface-exposed carbohydrate-containing macromolecules present on the interacting cells play an important role on the early recognition events. For instance, several glycoconjugates of T. cruzi and Leishmania have been implicated in the parasite-host cell interaction process. In the case of T. gondii no surface-exposed carbohydrate / 98 / $19.00 ß 1998 Published by Elsevier Science B.V. All rights reserved. PII: S (98)

2 324 V.G. Monteiro et al. / FEMS Microbiology Letters 164 (1998) 323^327 residues have been detected using lectins [6]. On the other hand, evidence exists that carbohydrate residues exposed on the host cell surface are involved in the interaction with T. cruzi [7]. In the case of T. gondii, however, no such data are available. Some preliminary studies showed that addition of monosaccharides to the interaction medium did not interfere with the interaction process [8]. In contrast, with Eimeria tenella, it has been shown that addition of D- galactose to the interaction medium inhibited sporozoite penetration into chicken kidney cells [9]. Addition of N-acetyl glucosamine also inhibited interaction of Plasmodium falciparum with erythrocytes [10]. In recent years a large number of data have shown that sialic acid residues present on the surface of host cells play some role in the protozoa-host cell interactions. It has been shown that merozoites of P. falciparum recognize N-acetyl neuraminic acid residues on the erythrocyte cell surface [11] and that trypomastigotes of T. cruzi penetrate better cells which express sialic acids on the surface [12,13]. In the present study using a panel of glycosylation mutants of chinese hamster ovary cells, we present evidence that sialic acid residues exposed on the host cell surface are involved on the process of interaction of tachyzoites of T. gondii with the host cell. 2. Materials and methods 2.1. Parasites Tachyzoites from the virulent RH strain of T. gondii were maintained by intraperitoneal passages in Swiss mice and were collected in Ringer's solution at ph 7.2, 48^72 h after infection. The ascite uid obtained from infected mice was centrifuged at 200Ug for 7^10 min at room temperature to remove host cells and debris. The supernatant, which contained the parasites, was collected and centrifuged at 1000Ug for 7^10 min. The pellet obtained was washed two times with phosphate-bu ered saline solution (PBS), ph 7.2, and resuspended to a density of 10 6 parasites/ml in 199 medium. The parasites were used 30^40 min after removal from the mouse peritoneal cavity, and the viability was tested using the Trypan blue test Host cells Cell viability was evaluated using a dye-exclusion test with Trypan blue. We used a panel of chinese hamster ovary (CHO) cells (Table 1) kindly provided by Dr. P. Stanley (Department of Cell Biology, Albert Einstein College of Medicine, New York, USA), including a W5 Gat 32 parental line and Lec 1, IdlD lec 1, lec 2 and lec 8 glycosylation mutants [14]. They were grown in MEM alpha medium (Gibco) supplemented with 10% fetal bovine serum (FBS). For some experiments we also used Vero and LLCMK2 cells maintained in Dulbecco's modi ed Eagle's medium Host cell-parasite interaction Parasites suspended in 199 medium were incubated for 2 or 24 h in the presence of cells using a 10:1 parasite-host cell ratio. In some experiments, after incubation the cultures were washed three times with 199 medium and allowed to interact for 24 h at 37³C in the presence of medium with FBS. After incubation the cells were xed in Bouin's xative solution and stained with Giemsa. The percentage of cells with internalized parasites and the mean number of internalized parasites per cell were determined by randomly counting a minimum of 200 cells in each of the duplicate preparations. The was calculated by multiplication of these two parameters. The value obtained for the control cells was considered as 100 in order to determine the relative es. Experiments were repeated at least three times. Statistical analysis was done using the F student's t-test. We considered as signi cant those di erences above 15% (P ). In some experiments the cells were incubated for 60 min in the presence of U/ml of neuraminidase from Vibrium cholerae (Sigma Chemical Company) dissolved in Hank's solution, ph 5.5, washed with Hank's solution and allowed to interact with T. gondii as described above. In other experiments using W5 and Lec 2 cells, attempts were done to sialylate the cells using T. cruzi trans-sialidase. In this case the lec 2 cell line was incubated for 30 min at 37³C in alpha medium containing 500 Wg/ml fetuin and a 1:1 dilution of the supernatant of a heavily infected culture containing recem-liberated trypomastigotes of

3 V.G. Monteiro et al. / FEMS Microbiology Letters 164 (1998) 323^ Table 1 Carbohydrate structures found in CHO cell lines used Symbols: [ ], sialic acid; b, Gal, GalNAc; F, GlcNAc; a, man and S/T, serine or treonine. T. cruzi which were centrifuged at 2000Ug for 10 min, as previously described [12]. After incubation the cultures were washed with Hank's solution allowed to interact with the parasites as described above. 3. Results and discussion Incubation of the three cell lines tested for 2 h in the presence of tachyzoites of T. gondii using a parasite-cell ratio of 10:1 showed that all cell lines were infected. However, di erences were observed in the indices which were of about 67, 58 and 49 for the parental CHO, LLCMK2 and Vero cells, respectively. Analysis of the infected cells after 24 h showed that the parasites were viable, as assayed by their ability to divide within a cytoplasmic vacuole. Table 2 summarizes the data obtained in some experiments on the penetration of the tachyzoites into di erent CHO glycosylation mutants. All cells could be infected. However, the extent of infection varied according to the cell line. The parental strain showed the highest, while Lec 2 Table 2 Internalization of tachyzoites of Toxoplasma gondii into CHO glycosylation mutants a Cell line cells showed the lowest. Intermediate values were observed for Lec 1, Lec 8 and Id1D lec 1 cells. Table 1 shows the carbohydrate structures found in the CHO cell lines used. The analysis of the data clearly indicates that the highest was observed with cells which present surface-exposed terminal sialic acid residues associated to N- glycosylated glycoconjugates. Cells presenting sialic acid residues associated to O-linked carbohydrate structures showed a much less. Based on the non-signi cant di erences observed among the other cell lines, we can suggest that the presence of residues of galactose, N-acetyl glucosamine or mannose on the host cell surface do not facilitate the process of T. gondii-host cell interaction. Previous studies carried out with P. falciparum and E. tenella suggest that N-acetyl glucosamine and galactose, respectively, play some role on the protozoan-host cell interaction process [9,10]. The observation that sialylated cells are more easily infected with tachyzoites of T. gondii is an additional evi- Table 3 Internalization of tachyzoites of Toxoplasma gondii into control and neuraminidase-treated cells a Cell type Treatment Mean CHO-W5 no 54.3 þ CHO-W5 yes 42.1 þ b Vero no 49.0 þ Vero yes 18.6 þ b LLCMK2 no 57.7 þ LLCMK2 yes 36.2 þ b a Incubation time of 2 h. b P Mean W þ Lec þ b Lec þ b Lec þ b IdlD lec þ a Incubation time of 2 h. b P

4 326 V.G. Monteiro et al. / FEMS Microbiology Letters 164 (1998) 323^327 Table 4 E ect of sialylation of Lec 2 cells on the of tachyzoites of Toxoplasma gondii a Cell type Mean W þ Lec þ c Lec 2+fetuin+TS b 47.5 þ c a Interaction time of 2 h. b Trans-sialidase obtained from Trypanosoma cruzi as described in Section 2. c P dence that this negatively charged carbohydrate residue is involved on the interaction process, as previously reported for the P. falciparum-erythrocyte [10] and the T. cruzi-host cell (reviewed in [1]) interaction processes. In order to con rm the role played by sialic acid residues two approaches were used. First, the parental CHO cells, as well as Vero and LLCMK2 cell lines, were incubated in the presence of neuraminidase under conditions where most of the sialic residues were liberated. As shown in Table 3, such treatment signi cantly reduced the penetration of T. gondii into the cells. The neuraminidase e ect varied according to the cell line and it was more evident with Vero cells. In the case of CHO cells, es similar to those observed with the other cell lines were observed. The second approach consisted in the resialylation of neuraminidasetreated parental Gat cells or the sialylation of Lec 2 cells by incubation of the cells in a medium containing fetuin as a donor of sialic acid and a source of trans-sialidase of T. cruzi. After sialylation of the Lec 2 cells they were more easily infected with tachyzoites (Table 4). Similar results were obtained in experiments of resialylation of neuraminidase-treated W5 cells (not shown). Previous studies have shown that the trans-sialidase of T. cruzi is able to transfer sialic acid from a donor to an acceptor (reviewed in [15]). In conclusion our present observations show for the rst time that sialic acid residues exposed on the surface of the host cell are involved on the process of interaction of tachyzoites of T. gondii with host cells. In view of the fact that di erent cells present di erent density of sialic acid residues, our present results may explain the signi cant di erence observed in the infection of various cell types by T. gondii. Even the parental Gat 2 cells used presented exposed galactose residues as evaluated by binding of lectins which recognize this monosaccharide (data not shown) and by the e ect observed when they were incubated in the presence of trans-sialidase and fetuin, a condition in which a signi cant increase of parasite penetration was observed. Further studies are necessary to determine if the exposed sialic acid residues play apart in the interaction due to a charge e ect or if a parasite sialic acid binding protein is involved on the process. It is relevant that no signi cant di erences in es were observed when T. gondii was allowed to interact with CHO cells in charged glycoconjugates such as heparan sulfate and chondroitin [16]. Acknowledgments This work has been supported by Conselho Nacional de Desenvolvimento Cient è co e Tecnoloègico (CNPq), Financiadora de Estudos e Projetos (FI- NEP), Fundac aìo de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), Programa de Nuécleos de Exceleência (PRONEX) and Fundac aìo Estadual Norte Fluminense (FENORTE). References [1] Araujo-Jorge, T.C. (1989) The biology of Trypanosoma cruzimacrophage interaction. Mem. Inst. Oswaldo Cruz 84, 441^ 462. [2] Dobrowolski, J.M. and Sibley, L.D. (1996) Toxoplasma gondii invasion of mammalian cells is powered by the actin cytoskeleton of the parasite. Cell 84, 933^939. [3] Kasper, L.H. and Mineo, J.R. (1994) Attachment and invasion of host cells by Toxoplasma gondii. Parasitol. Today 10, 184^188. [4] Morisaki, J.H., Heuser, J.E. and Sibley, L.D. (1995) Invasion of Toxoplasma gondii occurs by active penetration of the host cell. J. Cell Sci. 108, 2457^2464. [5] Silva, S.R.L., Meirelles, S.S.L. and De Souza, W. (1982) Mechanism of entry of Toxoplasma gondii into vertebrate cells. J. Submicrosc. Cytol. 14, 471^482. [6] Cintra, W.M., Silva-Filho, F.C. and De Souza, W. (1986) The surface charge of Toxoplasma gondii: a cytochemical study. J. Submicrosc. Cytol. 18, 773^781.

5 V.G. Monteiro et al. / FEMS Microbiology Letters 164 (1998) 323^ [7] Araujo-Jorge, T.C. and De Souza, W. (1984) Interaction of Trypanosoma cruzi with macrophages: e ect of previous incubation of the parasites or the host cells with lectin. Z. Parasitenkd. 72, 153^171. [8] Crane, M.J. and Dvorak, J.A. (1982) In uence of monosaccharides on the infection of vertebrate cells by Trypanosoma cruzi and Toxoplasma gondii. Mol. Biochem. Parasitol. 3, 333^ 341. [9] Baba, E., Uno, H., Sadano, N., Fukata, T., Sasai, K. and Arakawa, A. (1996) Eimeria tenella: Role of carbohydrates on sporozoite at the penetration into cultured cells. Exp. Parasitol. 83, 67^72. [10] Jungery, M., Pasvol, G., Newbold, C.I. and Weatherall, D.J. (1983) A lectin-like receptor is involved in invasion of erythrocytes by Plasmodium falciparum. Proc. Natl. Acad. Sci. USA 80, 1018^1022. [11] Friedman, M.J., Blankenberg, T., Sensabaugh, G. and Tenforde, T.S. (1984) Recognition and invasion of human erythrocytes by malarial parasites: contribution of sialoglycoproteins to attachment and host speci city. J. Cell Biol. 98, 1672^ [12] Ciavaglia, M.C., Carvalho, T.U. and De Souza, W. (1993) Interaction of Trypanosoma cruzi with cells with altered glycosylation patterns. Biochem. Biophys. Res. Commun. 193, 718^721. [13] Rocilda, P., Schenckman, F., Vanderchove, F. and Schenckman, S. (1993) Mammalian cell sialic acid enhances invasion by Trypanosoma cruzi. Infect. Immun. 61, 898^902. [14] Stanley, P. (1984) Glycosylation mutants of animal cells. Annu. Rev. Genet. 18, 525^552. [15] Schenckman, S., Eichinger, D., Pereira, M.E. and Nussenzweig, V. (1991) Structural and function properties of Trypanosoma trans-sialidase. Annu. Rev. Microbiol. 48, 499^ 523. [16] Mack, D., Kasper, L. and Mcleod, R. (1994) Alterations in cell surface glycosylation, heparin, and chondroitin sulfate do not modify invasion of CHO cells by the Ptg B strain of Toxoplasma gondii. J. Eukaryot. Microbiol. 41, 14S.