Novel strategy in Trypanosoma cruzi cell invasion: Implication of cholesterol and host cell microdomains

International Journal for Parasitology 37 (2007) 1431–1441 www.elsevier.com/locate/ijpara

Novel strategy in Trypanosoma cruzi cell invasion: Implication of cholesterol and host cell microdomains Maria Cecı´lia Fernandes a, Mauro Cortez a, Kelly Aparecida Geraldo Yoneyama b, Anita Hilda Straus b, Nobuko Yoshida a, Renato Arruda Mortara a,* a

b

Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Sa˜o Paulo - Escola Paulista de Medicina, Rua Botucatu, 862, 6o andar, Sa˜o Paulo, SP 04023-062, Brazil Departamento de Bioquı´mica, Universidade Federal de Sa˜o Paulo - Escola Paulista de Medicina, Rua Botucatu, 862 Sa˜o Paulo, SP 04023-062, Brazil Received 2 April 2007; accepted 24 April 2007

Abstract Trypanosoma cruzi, the etiological agent of Chagas’ disease, is an obligatory intracellular parasite in the mammalian host. In order to invade a wide variety of mammalian cells, T. cruzi engages parasite components that are differentially expressed among strains and infective forms. Because the identification of putative protein receptors has been particularly challenging, we investigated whether cholesterol and membrane rafts, sterol- and sphingolipid-enriched membrane domains, could be general host surface components involved in invasion of metacyclic trypomastigotes and extracellular amastigotes of two parasite strains with distinct infectivities. HeLa or Vero cells treated with methyl-b-cyclodextrin (MbCD) are less susceptible to invasion by both infective forms, and the effect was dose-dependent for trypomastigote but not amastigote invasion. Moreover, treatment of parasites with MbCD only inhibited trypomastigote invasion. Filipin labeling confirmed that host cell cholesterol concentrated at the invasion sites. Binding of a cholera toxin B subunit (CTX-B) to ganglioside GM1, a marker of membrane rafts, inhibited parasite infection. Cell labeling with CTX-B conjugated to fluorescein isothiocyanate revealed that not only cholesterol but also GM1 is implicated in parasite entry. These findings thus indicate that microdomains present in mammalian cell membranes, that are enriched in cholesterol and GM1, are involved in invasion by T. cruzi infective forms.  2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Trypanosoma cruzi; Trypomastigote; Amastigote; Membrane rafts; Cholesterol; Cell invasion

1. Introduction The involvement of plasma membrane microdomains in the entry process and intracellular replication of microbial pathogens is becoming increasingly evident. Cellular invasion by viruses (Pelkmans, 2005) and the uptake of a great number of intracellular bacteria into professional or nonprofessional phagocytic cells (Gulbins et al., 2004; Lafont and van der Goot, 2005) has been shown to require a specialized host membrane microenvironment, organized into membrane rafts. These microdomains are small (10–100 nm), heterogeneous, highly dynamic, sterol- and

*

Corresponding author. Tel.: +55 11 5579 8306; fax: +55 11 5571 1095. E-mail address: [email protected] (R.A. Mortara).

(glyco)sphingolipid-enriched domains that compartmentalize cellular processes (Simons and Ehehalt, 2002; Pike, 2006). Membrane rafts were originally proposed to be involved in the sorting of proteins to the apical surface of polarized epithelial cells, with which specific proteins interact (Simons and Ikonen, 1997). Cholesterol is thought to serve as a spacer between the hydrocarbon chains of sphingolipids and to function as a dynamic adhesive that keeps the raft assembly together (Simons and Toomre, 2000). The role of cholesterol and membrane rafts in the uptake of larger pathogens like protozoa is beginning to be addressed. Host cell cholesterol is recruited during Toxoplasma gondii invasion (Coppens and Joiner, 2003) and the closely related apicomplexan parasite Plasmodium falciparum targets sphingomyelin and cholesterol to the parasitophorous vacuole (Lauer et al., 2000). Recently, it has also been shown

0020-7519/$30.00  2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2007.04.025

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that Leishmania donovani uptake into macrophages also depends on host cell cholesterol, since its depletion with nystatin or methyl-b-cyclodextrin (MbCD) inhibits the process (Pucadyil et al., 2004; Tewary et al., 2006). The implication of host cell cholesterol or membrane microdomains in the entry process of Trypanosoma cruzi has not yet been addressed. Trypanosoma cruzi is the etiological agent of Chagas’ disease and it is known that the parasite is able to infect a wide variety of mammalian host cells (Brener, 1973; De Souza, 2002). However, this ability to enter non-phagocytic mammalian cells may vary widely between strains and infective forms (Ruiz et al., 1998; Neira et al., 2002; Fernandes and Mortara, 2004; Silva et al., 2006; Yoshida, 2006). Numerous studies have been performed in order to understand the molecular mechanisms that underlie the rather complex process of parasite entry into mammalian host cells. Unfortunately, some of these have proposed mechanisms that turned out not to be as general as initially supposed (Ortega-Barria and Pereira, 1991; Tardieux et al., 1992; Ming et al., 1995) and until now attempts to completely abolish host cell colonization have not succeeded. The variety of invasion strategies engaged by T. cruzi metacyclic trypomastigotes as well as extracellular amastigotes is consistent with the complex repertories of surface molecules that both infective forms of the parasite have evolved to ensure infection (reviewed in Mortara et al., 2005; Yoshida, 2006). The search for specific protein host cell receptors involved in T. cruzi invasion has turned out to be a laborious and challenging task (Yoshida, 2006), so we decided to examine the possibility that lipids, abundant and widespread in host cell membranes, could play a role in this process. In the present work we have evaluated the ability of the parasite to invade cholesterol-depleted cells as well as the ability of steroldepleted parasites to invade untreated cells. We demonstrate, for the first time, the involvement of cholesterol in the T. cruzi entry process. Host cell cholesterol was shown to be recruited to the invasion site of both infective forms studied and pre-treatment of HeLa and Vero cells with cholesterol sequestering agent, MbCD, inhibited parasite invasion. In addition, binding of a cholera toxin B subunit (CTX-B) to GM1, a marker of membrane rafts, significantly decreased parasite infectivity, indicating that not only cholesterol but also membrane rafts participate in this process. 2. Materials and methods 2.1. Cells and parasites Vero and HeLa cell lines obtained from Instituto Adolpho Lutz (Sa˜o Paulo, SP, Brazil) were cultivated in Dulbecco’s modified Eagles medium (DMEM) (Cultilab, Campinas, SP, Brazil) with 10% FCS (Cultilab) in a humid atmosphere of 5% CO2 at 37 C. Cell-derived trypomastigotes from G (Yoshida, 1983) and CL (Brener and Chiari,

1963) strains were obtained after infection of semi-confluent Vero cell lines. Cells grown in 75 cm2 flasks were infected with recently released cell-derived trypomastigotes (108 parasites/ml). Infection proceeded overnight at 37 C in DMEM supplemented with 10% FCS. The supernatant was then replaced by DMEM 2% FCS and cells were kept at 37 C. Tissue culture-derived trypomastigotes emerge from Vero cells after approximately 6 days of infection. Extracellular amastigotes were obtained from the differentiation of cell-derived trypomastigotes isolated in liver infusion tryptose (LIT) – define medium. Vero cell-derived trypomastigotes were isolated from culture supernatants of infected cells by centrifugation at 2500g for 5 min. The pellet was resuspended in LIT medium, pH 5.8, and incubated for 24–48 h at 37 C, and at least 95% pure extracellular amastigotes were obtained (Ley et al., 1988; Mortara, 1991). Metacyclic trypomastigotes from both strains were obtained from the axenic differentiation of stationaryphase hemocultures of mice previously infected with tissue-culture trypomastigotes, in liver infusion tryptose (LIT, Camargo, 1964) containing 5% FCS and 0.2% glucose, or Grace’s medium (CL strain) at 28 C and purified by ionic exchange chromatography (Yoshida, 1983). 2.2. Cell invasion assays Semiconfluent Vero or HeLa cells were infected with metacyclic trypomastigotes or amastigotes at a proportion of 10:1 parasites/cell (CL strain trypomastigotes and G strain amastigotes) and 25:1 parasites/cell (G strain trypomastigotes and CL strain amastigotes). After 1 h, DMEM supplemented with 5% FCS was removed and cells were washed five times with PBS. Cells were then fixed with either 3.5% formaldehyde in PBS for filipin labeling, or with methanol for Giemsa staining. The number of intracellular parasites were counted in 500 cells in duplicate coverslips. The percentage of infectivity was calculated according to the formula: number of internalized parasites · 100/number of intracellular parasites in control cells. 2.3. Cholesterol depletion and repletion For cholesterol depletion, cells were washed twice with PBS and incubated at 37 C for 45 min with MbCD (Sigma Chemical Co., St. Louis, USA) at the indicated concentration in DMEM (without serum). Cell viability after MbCD treatment was confirmed by 2-(4,5-dimethyl2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide (MTT) reaction (Mosmann, 1983). For cholesterol repletion, Vero cells were incubated with the indicated concentration of water soluble cholesterol (Sigma Chemical Co., St. Louis, USA) in DMEM without serum for 30 min, or 90 min incubation with DMEM with 10% or 20% FCS. After the procedure, cells were washed twice with PBS before adding the parasites. Infection was performed as described above.

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2.4. Lipid extraction and high performance thin layer chromatography (HPTLC) analysis

made with SigmaStat (Version 1.0, Jandel Scientific), employing Student’s t test for significance and SD.

After MbCD treatment, parasites and cells were pelleted by centrifugation and lipids present in the supernatant were recovered directly by partition with the same volume of 1butanol (Yoneyama et al., 2006). The butanol fraction was dried under a stream of N2, resuspended in chloroform/ methanol (2:1, v/v) and analyzed by high-performance thin layer chromatography (HPTLC) on Silica Gel 60 plates (E. Merck, Darmstadt, Germany). For sterol extraction of the pellet content, treated cells and parasites were resuspended three times with 10 vol of isopropanol/hexane/water (55:25:20, v/v/v) and twice with chloroform/methanol (2:1, v/v). Extracts were pooled and dried as described above, resuspended in chloroform/methanol (2:1, v/v) (Straus et al., 1993). HPTLC was developed in chloroform/ethyl ether/acetic acid (97:2.3:0.5, v/v/v) and sterols were visualized as dark spots using copper acetate reagent (10% CuSO4Æ5H2O in 3% H3PO4) (Fewster et al., 1969). Ergosterol and cholesterol (Sigma) were used as standards for the quantification of HPTLC by Scion densitometric analysis.

3. Results

2.5. Competition assays with cholera toxin-B Vero cells on coverslips were incubated with indicated concentrations of cholera toxin subunit B (CTX-B, Molecular Probes, Eugene, OR, USA) at 4 C for 20 min, according to the manufacturer’s instructions, rinsed three times with PBS, and cell invasion was carried out as described above. 2.6. Filipin and GM1 labeling After aldehyde fixation, cells were washed with PBS and then permeabilized with PGN solution (PBS, 0.15% gelatin, 0.1% sodium azide) containing 0.1% saponin for 15 min. Samples were then incubated 1 h with filipin III (Sigma) 100 lg/ml diluted in PGN. For GM1 labeling, fixed cells were incubated with a 1 lg/ml cold solution of CTX-B – Alexa Fluor 488 (Molecular Probes) for 30 min. Coverslips were then incubated with 10 lM 4 0 ,6diamidino-2-phenylindole (DAPI) in PGN. Slides were mounted in Vectashield (Vector) and images were acquired on a Nikon E600 fluorescence microscope attached to a Nikon DXM1200 digital camera using ACT-1 software. Image J (http://rsb.info.nih.gov/ij/) was used to analyze the fluorescence intensity distribution, to enhance contrast and brightness of acquired images, and for combining images. 2.7. Quantitation of experiments and statistical calculations All experiments were performed with duplicate coverslips and repeated at least three times. Five hundred cells per coverslip were analyzed. Statistical calculations were

3.1. Host cell cholesterol extraction reduces Trypanosoma cruzi infection In order to determine whether host cell domains enriched in cholesterol play a role in T. cruzi cell invasion, Vero and HeLa cells were exposed to MbCD and then infected with metacyclic trypomastigotes or extracellular amastigotes of two parasite strains that display distinct infectivities (Fernandes et al., 2006; Neira et al., 2002) and belong to the two major phylogenetic groups: T. cruzi I (G) and T. cruzi II (CL) (Briones et al., 1999; Brisse et al., 2000; Souto et al., 1996). MbCD extracts cholesterol molecules from the plasma membrane and this lipid is trapped in the internal hydrophobic core of the MbCD molecule (Yancey et al., 1996). Removal of cholesterol from both host cell lines significantly decreased the invasion index of metacyclic trypomastigotes and extracellular amastigotes of G and CL strains, indicating that host cell cholesterol and/or membrane organization is important for the entry process of both infective forms (Fig. 1A). Surprisingly, the pre-treatment of HeLa cells with 10 mM MbCD did not have the same inhibitory effect on G strain metacyclic trypomastigote invasion. Although to a lesser extent, MbCD inhibited approximately 30% of the infectivity of these trypomastigotes. In addition, we observed that the percentage of inhibition was higher for metacyclic trypomastigotes compared with extracellular amastigotes (Fig. 1A and B). At high concentrations (30 mM) MbCD inhibited metacyclic trypomastigote entry by 80% whereas the extracellular amastigote invasion index decreased 45–55% with all concentrations tested. Only metacyclic trypomastigote invasion appeared to be a MbCD dose-dependent process, indicating that cellular invasion by these infective forms are more reliant on host cell cholesterol compared with extracellular amastigote entry. 3.2. T. cruzi infectivity is reestablished in cells replenished with cholesterol To ensure that inhibition of T. cruzi invasion (Fig. 1A) was specifically due to removal of cholesterol and not to an alteration of the association of other molecules that control signaling pathways and/or parasite internalization after MbCD treatment, cells were replenished with cholesterol by incubation with medium containing different concentrations of water soluble cholesterol or FCS. After this treatment, cells were submitted to invasion assays with metacyclic trypomastigotes and extracellular amastigotes of both strains. Our results indicate that after cholesterol repletion, the invasion index of CL strain metacyclic trypomastigotes (Fig. 2A) and G strain extracellular amastigotes (Fig. 2B) was almost fully recovered. Interestingly, the

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Fig. 1. Host cell cholesterol depletion inhibits Trypanosoma cruzi invasion. (A) G and CL strain metacyclic trypomastigotes (MT) (left panel) and extracellular amastigotes (EA) (right panel) were allowed to invade Vero or HeLa cells pretreated with 10 mM methyl-b-cyclodextrin (MbCD). (B) Vero cells were incubated with indicated concentrations of MbCD and invasion assays were performed with CL strain metacyclic trypomastigotes and G strain extracellular amastigotes. The same results were obtained with infective forms of distinct strains (data not shown). Percentage (%) of infection was calculated as described in Section 2. Values are means ± SD of three experiments performed in duplicate. *Statistically significant differences (P < 0.01) between treated and untreated cells.

increase in cellular cholesterol content led to a concomitant increase in the ability of the parasites to invade Vero cells (Fig. 2). When MbCD-treated cells where incubated with 0.05 mM of water soluble cholesterol, the percentage of infection for both metacyclic trypomastigotes (Fig. 2A) and extracellular amastigotes (Fig. 2B) increased significantly (approximately 60%) compared with Vero cells that had not been cholesterol replenished. Invasion recovery was also observed when cells were incubated for 90 min, after cholesterol depletion with MbCD, with medium containing 20% FCS (estimated final cholesterol concentration of 0.6 mM). Similar results were obtained with G strain metacyclic trypomastigotes and CL strain extracellular amastigotes (data not shown). Altogether these results therefore confirm that the reduced T. cruzi invasion index in cholesterol-depleted cells (Fig. 1A) is a specific effect, not strain dependent, and can be reversed by cholesterol replacement.

astigotes and extracellular amastigotes were treated with MbCD (10 mM) for 45 min, washed and allowed to invade HeLa or Vero cells. Interestingly, depletion of metacyclic trypomastigote sterol from either G or CL strains decreased their infectivity by approximately 30% (Fig. 3A). By contrast, pre-treatment of extracellular amastigotes of both strains did not affect their efficiency in entering non-phagocytic cells (Fig. 3B). This is a strong indication that extracellular amastigotes do not explore sterols and possibly parasite membrane microdomains. Considering the different repertoire of surface molecules engaged in the invasion process of each infective form, this result is not surprising and corroborates recent findings that metacyclic trypomastigotes and extracellular amastigotes employ different strategies to invade host cells (Neira et al., 2002; Mortara et al., 2005; Fernandes et al., 2006; Yoshida, 2006). 3.4. MbCD efficiently extracts cholesterol and ergosterol

3.3. Depletion of parasite sterol only decreases the infectivity of metacyclic trypomastigotes We also investigated whether depletion of parasite sterol affected its ability to enter host cells. Metacyclic trypom-

Although the efficiency of MbCD in extracting sterols other than cholesterol has been demonstrated elsewhere (Ohvo-Rekila et al., 2000) the possibility that ineffective sterol extraction occurred was investigated. To determine

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Fig. 2. Cholesterol replenishment after methyl-b-cyclodextrin (MbCD) treatment restores Trypanosoma cruzi infectivity. Vero cells pretreated with 10 mM MbCD were incubated with either water soluble cholesterol for 30 min or culture medium supplemented with FCS for 90 min prior to CL strain metacyclic trypomastigote (A) or G strain extracellular amastigote (B) invasion. The same results were obtained with infective forms of distinct strains (data not shown). Percentage (%) of infection was calculated as described in Section 2. Values are means ± SD of three experiments performed in duplicate. *Statistically significant differences (P < 0.01) between cells replenished and not replenished ().

whether host cell cholesterol and T. cruzi ergosterol had been efficiently depleted upon MbCD treatment, whole cells (Fig. 4A) and MbCD-containing supernatants (Fig. 4B) were subjected to lipid extraction and HPTLC analysis (Fig. 4A and B). As determined by densitometry, MbCD significantly decreased sterol content of treated cells and parasites in a dose-dependent manner (Fig. 4A). The total amount of host cell cholesterol decreased by approximately 60% (Fig. 4A, first panel, lane 3) after treatment with 10 mM MbCD, the concentration used throughout our experiments. HPTLC also confirmed that parasite sterol was efficiently sequestered and treatment of metacyclic trypomastigotes with the same MbCD concentration removed comparable amounts of sterol. Similar results were obtained after treatment of extracellular amastigotes, confirming that depletion of equivalent sterol amounts had no effect on the infectivity of extracellular amastigotes, although it reduced host cell invasion by metacyclic trypomastigotes (Fig. 3A and B). These data corroborate recent findings that demonstrate diminished Leishmania (Viannia) braziliensis infectivity after treatment with MbCD (Yoneyama et al., 2006). In addition, supernatant analysis confirmed that sterols were also efficiently sequestered from the cells and were present in medium after MbCD treatment (Fig. 4B). The same results were obtained after lipid extraction of both G strain metacyclic

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Fig. 3. Pre-treatment of metacyclic trypomastigotes with methyl-b-cyclodextrin (MbCD) decreases their infectivity. (A) G and CL strain metacyclic trypomastigotes and (B) extracellular amastigotes were pretreated with 10 mM MbCD and incubated with Vero or HeLa cells for 60 min. Percentage (%) of infection was calculated as described in Section 2. Values are the means ± SD of three experiments performed in duplicate. *Statistically significant differences (P < 0.01) between treated and untreated parasites.

trypomastigotes, and G as well as CL strain extracellular amastigotes (data not shown). MbCD efficiently extracts a considerable percentage of parasite ergosterol, confirming that the observed reduction of metacyclic trypomastigote infectivity or the lack of effect on extracellular amastigotes upon MbCD treatment are not due to poor ergosterol sequestering, but rather to distinct characteristics of these infective forms. These results thus indicate that although metacyclic trypomastigotes rely both on host cell and parasite sterol, extracellular amastigotes appear to preferentially engage host cell cholesterol to accomplish invasion (Figs. 1 and 3). 3.5. Cholesterol accumulates at the entry site of metacyclic trypomastigotes and extracellular amastigotes Accumulation of cholesterol during cell invasion of T. cruzi has not yet been examined. In order to confirm that host cell cholesterol could be recruited to the site of parasite attachment, Vero cells were incubated with either metacyclic trypomastigotes or extracellular amastigotes for 30 min. After aldehyde fixation, coverslips were labeled with filipin, a fluorescent polyene antifungal agent that upon binding to cholesterol can be imaged by fluorescence microscopy (Kruth and Vaughan, 1980). Cholesterol

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assembly at the site of invasion of metacyclic trypomastigotes was evident (Fig. 5). Extracellular amastigotes also seemed to recruit cholesterol at the attachment site, but as expected, not all parasites were found to concentrate intense labeling (Fig. 5). Cholesterol-enriched domains were also recruited at the entry site of both T. cruzi infective forms in HeLa cells (data not shown). 3.6. T. cruzi invasion is decreased upon host cell incubation with cholera toxin Membrane rafts are enriched in cholesterol and GM1 (Simons and Toomre, 2000). In order to investigate whether not only cholesterol but also membrane rafts participate in parasite internalization, we performed competition assays with CTX-B that specifically binds to plasma membrane GM1 (Lencer and Tsai, 2003). To determine whether this alternative course of blocking interaction with membrane rafts would alter T. cruzi infectivity, Vero cells were pretreated for 30 min with different concentrations of CTX-B and then infected with metacyclic trypomastigotes or extracellular amastigotes. The infectivity of both infective forms was significantly reduced when cells were pretreated with 1 and 2 lg/ml CTX-B (Fig. 6A and B) suggesting that host cell GM1-rich membrane rafts are also exploited by T. cruzi during invasion. Although cholera toxin consistently decreased the invasion index of metacyclic trypomastigotes and extracellular amastigotes, the percentage of inhibition was lower than observed when cells were pretreated with MbCD (Fig. 1A). Given the biochemical differences between MbCD and CTX-B and their distinct modes of action, this result is not unexpected. Unlike MbCD which temporarily disassembles membrane rafts, the inhibition of metacyclic trypomastigotes and extracellular amastigotes invasion by CTX-B could occur by steric hindrance or temporary internalization of a subset of membrane rafts. The remaining proportion of parasites that invade host cells in the presence of CTX-B therefore achieve invasion through effective competition for intact membrane rafts. 3.7. Raft marker is recruited during T. cruzi invasion To further demonstrate the implication of membrane rafts during T. cruzi entry we performed GM1 labeling

using fluorescently labeled CTX-B in fixed Vero cells recently infected with metacyclic or extracellular amastigotes. Our results show that parasites of both infective forms recruited the plasma membrane raft marker GM1 (Fig. 7A and B). The parasitophorous vacuoles of both metacyclic trypomastigotes and extracellular amastigotes were enriched in this specific glycosphingolipid, indicating that not only cholesterol but also specialized membrane rafts enriched in GM1 are implicated in T. cruzi invasion of distinct infective forms and distinct phylogenetic lineages. 4. Discussion The role of membrane rafts is becoming increasingly recognized in signal transduction and entry of pathogens in host cells (Incardona and Eaton, 2000; Lafont and van der Goot, 2005; Manes et al., 2003). Altogether, we believe our results show for the first time that cholesterol and specialized membrane rafts enriched in GM1 are involved in T. cruzi cell entry. The use of cyclodextrins for extraction of host cell cholesterol to determine its importance during invasion by different pathogens is a well-established procedure (Coppens and Joiner, 2003; Pucadyil et al., 2004; Seveau et al., 2004; Yancey et al., 1996; Yoneyama et al., 2006). However, this treatment may affect not only raftassociated proteins (Ilangumaran and Hoessli, 1998) but also non-raft proteins as a result of the presence of cholesterol in non-raft domains (Abrami and Der Goot, 1999). Through experiments of depletion followed by replenishment of host cell cholesterol, we demonstrated that cholesterol is directly involved in T. cruzi cell entry. The effect of cholesterol depletion on T. cruzi invasion did not appear to be cell type dependent. However, the extent of the inhibitory effect was found to be cell- and strain-dependent, since treatment of HeLa cells decreased the infectivity of G strain metacyclic trypomastigotes by only 20–30%. It has been shown that cholesterol depletion impairs actin polymerization and membrane ruffling required for Listeria monocytogenes cellular invasion (Seveau et al., 2004). The involvement of the target cell actin microfilament system in T. cruzi cell invasion studies with different infective forms and target cells have revealed interesting differences (Mortara, 1991; Mortara et al., 2005; Proco´pio et al., 1999). It was recently shown that HeLa cell invasion by metacyclic trypomastigotes of G and CL strains

c Fig. 4. Methyl-b-cyclodextrin (MbCD) treatment efficiently removes both cholesterol and ergosterol. Vero cells and CL strain metacyclic trypomastigotes were incubated with or without MbCD for 1 h and pelleted by centrifugation. Pellet (A) and supernatant (B) of Vero cells (upper panels) and metacyclic trypomastigotes (MT) (lower panels) were collected and subjected to lipid extraction, and the sterol content analyzed by high-performance thin layer chromatography developed in chloroform/ethyl ether/acetic acid (97:2.3:0.5, v/v/v) as described in Section 2. Note the decrease in the total sterol amount upon MbCD treatment (A) and the consequent increase in cholesterol (upper panel) and ergosterol (lower panel) in the supernatant (B). The amount of sterol (in lg) in each band was quantitated by densitometry. Similar results were obtained after MbCD treatment of either extracellular amastigotes (G or CL strain) or G strain metacyclic trypomastigotes (not shown). Chol, standard cholesterol; Erg, standard ergosterol. Fig. 5. Cholesterol accumulates at the Trypanosoma cruzi invasion site. Invasion assays were performed in Vero cells with CL strain metacyclic trypomastigotes (A,B,C) or G strain extracellular amastigotes (D,E,F) as described in Section 2. Coverslips were fixed and labeled with filipin. (A,D) Phase contrast images; (B,E) filipin fluorescence; (C,F) same images as in (B) and (E), loaded with a hi (red)–low (blue) intensity color lookup table (Image J). Arrows indicate sites of parasite invasion and filipin/cholesterol concentration. Bar = 10 lm.

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involves, respectively, the disruption and recruitment of cell actin microfilaments (Ferreira et al., 2006), whereas amastigote invasion requires functional actin meshwork

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(Proco´pio et al., 1999). MbCD inhibited HeLa cell invasion by highly infective CL trypomastigotes to a greater extent than G strain parasites. The reason for such differences is

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Fig. 6. Cholera toxin-B (CTX-B) inhibits Trypanosoma cruzi invasion. Vero cells were incubated with the indicated concentrations of CTX-B 30 min before infection with CL strain metacyclic trypomastigotes (A) or G strain extracellular amastigotes (B). The same results were obtained with infective forms of distinct strains (data not shown). Values are means ± SD of three experiments performed in duplicate. *Statistically significant differences (P < 0.01) between CTX-B treated and untreated cells.

unknown and a possible explanation could be that the expected effect of cholesterol depletion on host cell actin reorganization required for parasite entry might involve other, as yet uncharacterized components. Regarding extracellular amastigote invasion, cholesterol depletion had a comparable inhibitory effect on parasites of either the G or CL strain (Fig. 1). Nevertheless, the observations described here are consistent with the notion that each parasite-host cell pair mobilizes specific interacting components (Mortara et al., 2005) and clearly indicate that host cell cholesterol is also required for cellular invasion of both T. cruzi infective forms. The expression of distinct repertories of parasite surface molecules that can take part in the invasion process may also be related to the differences observed. Metacyclic trypomastigotes trigger invasion through an 82 kDa glycoprotein or a 35/50 kDa mucin-like component, which are Ca2+ signaling inducing molecules (Ramirez et al., 1993; Ruiz et al., 1993, 1998; Yoshida et al., 1989; Yoshida, 2006). Extracellular amastigotes express a major glycoprotein designated Ssp-4 (Andrews et al., 1987) which contains carbohydrate epitopes that are possibly involved in cell invasion (Barros et al., 1997; Silva et al., 2006). These parasite surface glycoproteins bind to yet undefined host

cell receptor(s) and molecular partitioning at the host cell membrane, followed by assembly of membrane rafts, are processes likely to occur. In all instances, host cell cholesterol depletion only resulted in partial inhibition of T. cruzi invasion. This indicates that cholesterol-rich membrane microdomains are not an exclusive portal of entry for T. cruzi, but are rather well exploited routes that guarantees the parasite success in entering a wide range of non-phagocytic cells. Phosphatidylinositol-3-kinase, which at least in some cell types partitions into membrane (Li et al., 2004), appears to be involved in the initial invasion steps by T. cruzi infective forms (Wilkowsky et al., 2001; Woolsey et al., 2003) and could provide the necessary regulatory element that controls the involvement of membrane components. Metacyclic trypomastigote gp82 is a glycoprotein containing N-linked oligosaccharides (Ramirez et al., 1993) that is anchored to the parasite membrane by a glycophosphatidylinositol (GPI) anchor (Cardoso de Almeida and Heise, 1993). Binding of gp82 triggers a transient increase in intracellular Ca2+ concentration in the host cell (Ruiz et al., 1998). However, a signaling cascade is also initiated at the parasite cell surface and proceeds downstream through a sequence involving PTK (protein tyrosine kinase), PLC (phospholipase C) and IP3 (inositol 1,4,5-triphosphate), leading to Ca2+ mobilization. These functions are widely mediated by proteins that specifically partition to the membrane raft, such as GPI-anchored proteins (Li et al., 2003). It still remains unclear whether microdomains on the parasite membrane act as platforms for these signaling events or what the role of sterol in the gp82 signaling cascade would be. Conversely, G strain metacyclic trypomastigotes that are less infective preferentially engage another GPI-anchored molecule gp35/50 (Schenkman et al., 1993) to invade host cells and the signaling cascade triggered in the parasite is distinct from that induced by gp82. PTK and PLC are not implicated and instead, cyclic AMP is speculated to be involved (Neira et al., 2002; Ferreira et al., 2006; Yoshida, 2006). Regardless of the molecule implied on invasion and the signaling cascade triggered in the parasite, our results demonstrate that cell invasion by metacyclic trypomastigotes of both strains is hindered upon sterol depletion of the parasite, indicating that microdomains present in these infective forms are also involved in cellular invasion. Clearly, cholesterol depletion of host cells had a more significant inhibitory effect on the invasion by metacyclic trypomastigotes, leading us to conclude that host cell cholesterol is preferentially mobilized in this pathway. The invasion of host cells by T. cruzi extracellular amastigotes is a much less well-understood process and the components involved are only beginning to emerge. Signaling pathways involved in cell invasion by extracellular amastigotes of G and CL strains diverge, primarily as regards the activation of a tyrosine kinase activity, predominant in the more infective G strain (Fernandes et al.,

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Fig. 7. Recruitment of raft marker GM1 during Trypanosoma cruzi entry. Invasion assays were performed as described in Section 2. After fixation, coverslips with invading metacyclic trypomastigotes (A,B,C) or extracellular amastigotes (D,E,F) were labeled with fluorescent subunit of cholera toxin (B,E). (A,D) Phase contrast images; (B,E) CTX-B fluorescence. (C,F) Merged image CTX-B fluorescence (cyan) and DAPI (red). Magnification bars: 5 lm.

2006). The 84 kDa membrane glycoprotein carrying the epitope defined as Ssp-4 (Andrews et al., 1987) was also originally shown to be GPI-anchored to the parasite membrane (Andrews et al., 1988). Moreover, the expression of Ssp-4 related carbohydrates epitopes is also distinct among extracellular parasites of divergent strains and it is believed to be involved in extracellular amastigote invasion (Silva et al., 2006). The nature of the carbohydrate(s) is still unknown and their role in T. cruzi biology is not clear. Recent studies revealed that expression of the epitope defined by mAb 1D9 was not sufficient to confer higher infectivity as originally imagined (Mortara et al., 1999) and there could be other factors that modulate the infectivity of extracellular amastigotes from different isolates (Silva et al., 2006). However, our results demonstrate that unlike metacyclic trypomastigotes, extracellular amastigote sterol, independent of the parasite strain, had no significant role in

the invasion process and possible microdomains in the parasite membrane do not appear to be mobilized during host cell infection. As a final point, we have demonstrated that not only cholesterol but host cell membrane rafts are involved in T. cruzi cell invasion process. GM1 has been used extensively as a membrane raft marker (Simons and Ikonen, 1997; Pang et al., 2004; Seveau et al., 2004; Rodriguez et al., 2006). Fluorescence studies showed that some newly formed parasitophorous vacuoles of both infective forms were enriched in membrane rafts. In addition, competition assays with CTX-B revealed that both infective forms of T. cruzi distinct strains displayed reduced infectivity upon prior incubation of Vero cells with CTX-B. Overall these results imply similarity, at least in part, between the uptake of cholera toxin and the T. cruzi invasion pathway, leading us to conclude that cholesterol and GM1 present in

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membrane rafts are cellular components mobilized by T. cruzi to achieve internalization. In this work we demonstrated a new T. cruzi invasion strategy that targets specialized membrane microdomains, designated membrane rafts, as a portal to enter host cells. What remains to be established is whether/how rafts are responsible for providing a suitable environment for the association of the diverse membrane glycoproteins implied in T. cruzi infectivity and whether rafts are directly involved in the discrimination between signaling machineries engaged by different strains. Also, since only partial inhibition was achieved, the precise mechanisms involving lipid raft mobilization among distinct cell lines and parasite forms and strains, as well as a possible role in cytoskeleton reorganization, deserves further investigation. Acknowledgements This work was part of Maria Cecı´lia Di Ciero Fernandes’s PhD thesis and was supported by a fellowship from Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo, FAPESP. We also thank our colleague Helio K. Takahashi for suggestions and criticisms. Financial support from the Brazilian agencies, FAPESP, CNPq and CAPES through grants and fellowships is also acknowledged. References Abrami, L., Der Goot, F.G., 1999. Plasma membrane microdomains act as concentration platforms to facilitate intoxication by aerolysin. J. Cell Biol. 147, 175–184. Andrews, N.W., Hong, K.-S., Robbins, E.S., Nussenzweig, V., 1987. Stage-specific surface antigens expressed during the morphogenesis of vertebrate forms of Trypanosoma cruzi. Exp. Parasitol. 64, 474–484. Andrews, N.W., Robbins, E.S., Ley, V., Hong, K.S., Nussenzweig, V., 1988. Developmentally regulated, phospholipase C-mediated release of the major surface glycoprotein of amastigotes of Trypanosoma cruzi. J. Exp. Med. 167, 300–314. Barros, H.C., Verbisck, N.V., Silva, S., Araguth, M.F., Mortara, R.A., 1997. Distribution of epitopes of Trypanosoma cruzi amastigotes during the intracellular life cycle within mammalian cells. J. Eukaryot. Microbiol. 44, 332–344. Brener, Z., 1973. Biology of Trypanosoma cruzi. Annu. Rev. Microbiol. 27, 347–382. Brener, Z., Chiari, E., 1963. Variac¸o˜es morfolo´gicas observadas em diferentes amostras de Trypanosoma cruzi. Rev. Inst. Med. Trop. Sa˜o Paulo 5, 220–224. Briones, M.R., Souto, R.P., Stolf, B.S., Zingales, B., 1999. The evolution of two Trypanosoma cruzi subgroups inferred from rRNA genes can be correlated with the interchange of American mammalian faunas in the Cenozoic and has implications to pathogenicity and host specificity. Mol. Biochem. Parasitol. 104, 219–232. Brisse, S., Dujardin, J.C., Tibayrenc, M., 2000. Identification of six Trypanosoma cruzi lineages by sequence-characterised amplified region markers. Mol. Biochem. Parasitol. 111, 95–105. Camargo, E.P., 1964. Growth and differentiation in Trypanosoma cruzi: origin of metacyclic trypomastigotes in liquid media. Rev. Inst. Med. Trop. Sa˜o Paulo 6, 93–100. Cardoso de Almeida, M.L., Heise, N., 1993. Proteins anchored via glycosylphosphatidylinositol and solubilizing phospholipases in Trypanosoma cruzi. Biol. Res. 26, 285–312.

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