Protist, Vol. 165, 293–304, May 2014 Published online date 24 March 2014


Tritrichomonas foetus Displays Classical Detergent-resistant Membrane Microdomains on its Cell Surface Ivone de Andrade Rosaa,b , Georgia Atellac , and Marlene Benchimola,1 aUniversidade

Santa Úrsula, Rua Jornalista Orlando Dantas 59, Botafogo, CEP 22231-010 Rio de Janeiro, RJ, Brazil bInstituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil cInstituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Brazil Submitted January 14, 2014; Accepted March 19, 2014 Monitoring Editor: Michael L. Ginger

Tritrichomonas foetus is a serious veterinary parasite that causes bovine trichomoniasis, a sexually transmitted disease that results in reproductive failure and considerable economic losses in areas that practice natural breeding. T. foetus is an extracellular parasite, which initially adheres to and infects the urogenital tract using a diverse array of surface glycoconjugates, including adhesins and extracellular matrix-binding molecules. However, the cellular mechanisms by which T. foetus colonizes mucosal surfaces and causes tissue damage are not well defined. Several studies have demonstrated the involvement of pathogen or host lipid rafts in cellular events that occur during pathogenesis, including adhesion, invasion and evasion of the immune response. In this study, we demonstrate that detergentresistant membranes are present in the plasma membrane of T. foetus. We further demonstrate that microdomains are cholesterol-enriched and contain ganglioside GM1-like molecules. In addition, we demonstrate that lipid microdomains do not participate in T. foetus adhesion to host cells. However, the use of agents that disrupt and disorganize the plasma membrane indicated the involvement of the T. foetus lipid microdomains, in cell division and in the formation of endoflagellar forms. Our results suggest that trophozoites and endoflagellar forms present a different plasma membrane organization. © 2014 Elsevier GmbH. All rights reserved. Key words: Tritrichomonas foetus; detergent-resistant membrane microdomains; lipid rafts; methyl-␤cyclodextrin; filipin; ganglioside GM1.

Introduction Tritrichomonas foetus is a serious veterinary pathogen that causes bovine trichomoniasis, a sexually transmitted disease that results in reproductive failure and considerable economic losses 1

Corresponding author; fax +55-21-9853-2754 e-mail [email protected] (M. Benchimol). 1434-4610/© 2014 Elsevier GmbH. All rights reserved.

in areas that practice natural breeding (Alstad et al. 1984; BonDurant 1985; Clark et al. 1983). This disease has distinct clinical presentations in bulls and cows (BonDurant 1997). In bulls, the parasite is detected in the preputial cavity, the urethra and deeper parts of the urogenital tract (Parsonson et al. 1974). Infected bulls can harbor T. foetus throughout their lives without exhibiting clinical symptoms. In contrast, in cows, the effects of the

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disease vary from asymptomatic infection to severe clinical manifestations that include vaginitis, cervicitis, endometritis and pyometra which can result in transient infertility or fetal loss (Anderson et al. 1996; López et al. 2000; Parsonson et al. 1976). T. foetus is an extracellular parasite, which adheres to and infects the urogenital tract (Honigberg 1978). Previous studies have suggested that a diverse array of surface glycoconjugates, including adhesins (Corbeil et al. 1989; Singh et al. 1999; Woudwyk et al. 2013) and extracellular matrix (ECM)-binding molecules (Petrópolis et al. 2008; Silva-Filho and de Souza 1988), play important roles during the interaction of the parasite with its host. However, the cellular mechanisms by which T. foetus colonizes mucosal surfaces and causes tissue damage are not well understood. Evidence suggests that plasma membrane lipids are not distributed homogeneously and that microdomains with specialized functions exist in the plasma membrane (Simons and Toomre 2000). One such domain, the lipid raft, is a highly ordered and tightly packaged membrane domain with low fluidity. Lipid rafts are enriched in cholesterol or other sterols, glycosphingolipids, and phospholipids with a higher degree of saturated fatty acyl chains than the rest of the membrane (Maxfield 2002; Simons and Toomre 2000). The presence of membrane microdomains allows for the inclusion and exclusion of specific membrane proteins based on their attachment to the membrane via lipid anchors or specific protein-lipid interactions. Glycosylphosphatidylinositol (GPI)-anchor, double acylation and transmembrane proteins with the capacity to interact with cholesterol are examples of proteins modified with a hydrophobic attachment that are often found in lipid microdomains (Laughlin et al. 2004). A type of these microdomains is termed detergent-resistant membranes (DRMs) because they are resistant to solubilization in cold nonionic detergents, especially Triton X-100 at 4 ◦ C (Brown and Rose 1992; Goldston et al. 2012). Recent studies concerning the physiological role of lipid rafts have demonstrated that these membrane regions play important roles in a variety of cellular functions, including polarization, signal transduction, endocytosis, secretion and cell-cell and cell-pathogen adhesion (Antal and Newton 2013; Bal et al. 2013; Grimmer et al. 2002; Ha et al. 2003; Harris et al. 2001; Koumangoye et al. ˜ et al. 1999; Pierini et al. 2003; Resnik 2011; Manes et al. 2011; Sharma et al. 2012). The presence of these microdomains has also been demonstrated in parasitic protozoa, such as Trypanosoma

brucei, Leishmania spp., Toxoplasma gondii, Plasmodium spp., Giardia intestinalis and Entamoeba histolytica (Goldston et al. 2012). In addition, some studies have demonstrated the involvement of raftlike membrane domains in the interaction between host cells and pathogenic protozoa (Leishmania spp., Plasmodium falciparum and Giardia lamblia) (Dermine et al. 2005; Goldston et al. 2012; Humen et al. 2011; Karmakar et al. 2011; Koshino and Takakuwa 2009; Murphy et al. 2007; Sen et al. 2011; Yoneyama et al. 2006). However, there are no reports concerning lipid microdomains in T. foetus. Because trichomonad membranes contain cholesterol and sphingolipids (Beach et al. 1990), it is conceivable that lipid domains exist in the plasma membrane of this organism. The aims of this study were to identify and characterize lipid microdomains in T. foetus and determine whether these domains are involved in the interaction of this parasite with host-cells in vitro.

Results Detection of Cholesterol and Lipid Microdomains in T. foetus T. foetus cells that were fixed and stained with filipin exhibited an intense fluorescence in their plasma membranes, in their flagellar membranes and in some organelles (Fig. 1), indicating the presence of cholesterol in these structures. To determine whether the cholesterol molecules were localized in microdomains, fluorescent lipid analogs were used to distinguish raft regions from other membrane domains. T. foetus were disrupted by methyl-␤cyclodextrin (MBCD), a derivate of the family of cyclic oligomers of glucose which have a polar surface and a hydrophobic cavity that can accommodate small hydrophobic molecules (Beseniˇcar et al. 2008), and were stained with DilC16, an order-preferring lipid analog, and FAST-Dil, a nonorder-preferring lipid analog (Fig. 2A-D). Control cells that were stained with DilC16 exhibited fluorescence only in the plasma membrane, whereas control cells stained with FAST-Dil also exhibited fluorescence in intracellular structures. In contrast, DILC16 labeling of MBCD-treated cells was abolished or concentrated in a single region of the plasma membrane (Fig. 2E-H). Similar results were observed for cells treated with filipin (Fig. 2I-L). In addition, in treated cells that were labeled with fluorescent cholera ␤-toxin (CTx␤-Alexa-488), which detects the glycosphingolipid lipid raft marker GM1, fluorescence was absent in the plasma membrane

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Figure 1. Cholesterol detection in T. foetus using fluorescent filipin. T. foetus were observed by both differential interference contrast (DIC) (A) and fluorescence (B) microscopy. Fluorescent filipin labeling was observed on the plasma membrane, the flagellar membrane and in intracellular structures. Scale bar, 2 ␮m.

Figure 2. Fluorescence microscopy of T. foetus stained with the fluorescent lipid analogs DiIC16 and FAST-DiI. Both DiIC16, a lipid raft marker, and FAST-DiI, a marker of non-raft membranes, labeled the plasma membrane of untreated cells (B and D). Treatment with the cholesterol-depleting agent MBCD or the cholesterol-sequestering agent filipin resulted in an altered DiIC16 staining pattern (F and J), but the FAST-DiI staining pattern was unchanged (H and L). Panels A, C, E, G, I, and K show DIC images. Scale bars, 2 ␮m.

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Figure 3. Fluorescence microscopy of T. foetus stained with fluorescent cholera toxin subunit ␤ (CTx␤). Control cells (B) and cells treated with MBCD (D) were stained with fluorescent CTx␤, a lipid raft marker. Panels (A) and (C) are DIC images. Scale bars, (A) 2 ␮m; (C) 4 ␮m.

but present in some intracellular organelles (Fig. 3).

DRM Purification and Characterization of Lipid Microdomains DRM extracts prepared using Triton X-100 were sonicated and subjected to floatation centrifugation over a continuous sucrose gradient. The plasma membrane preparation formed a single prominent band at a density of 10.19 g/ml. The band was collected in fraction 6 (data not shown), which showed an enrichment of cholesterol and a low protein content (Fig. 4). The lipid raft marker CTx␤ was detected strongly in fractions 6 to 8; however, light labeling was also observed in fractions 9 and 10 by slot-blot analysis (Fig. 5). Fractions positive for CTx␤ corresponded to cholesterol-rich fractions 6 and 7 when analyzed using HPTLC (Fig. 6). Lipid analysis was performed to quantify the distribution of lipid classes in the DRM fractions 6 and 7. As expected for lipid raft sub-domains, cholesterol was the major neutral lipid (Fig. 7).

The Role of Lipid Microdomains in T. foetus Adhesion The role of DRMs in T. foetus adhesion was analyzed using scanning electron microscopy (SEM) after treatment with MBCD or filipin. Our results

Figure 4. Quantitative analysis of cholesterol content and protein content in membrane fractions from T. foetus. Membrane preparations from T. foetus were fractionated to isolate cholesterol-enriched DRMs. The cholesterol content of each fraction was measured using a sensitive cholesterol oxidase-based fluorometric assay (bars). The cholesterol content of each fraction was normalized to the total protein content (line), which was measured by the Lowry method.

indicated that there was no difference in cell adhesion when comparing the adherence of control and treated T. foetus to MDCK cells or control T. foetus to MDCK treated (Figs 8, 9A). However, some alterations on morphology of the MDCK cells were observed. MDCK cells that were incubated with untreated T. foetus exhibited a more stretched profile with alteration on confluence than cells that were incubated with treated T. foetus (Fig. 8A-B). These data was confirmed by competition assays, where the number of the parasites adhered to MDCK cells preincubated with the DRM fraction of the T. foetus was similar to the control experiments (Fig. 10A). In addition, alterations were observed in treated T. foetus, including cells with ruptured membranes and cells with duplicated structures, such as flagella (Fig. 8C-D) and axostyles, indicating a division process. Quantitative and statistical analyses demonstrated that the frequency of the reduction of the endoflagellar forms and cells in mitosis were

Figure 5. Detection of GM1 via slot blot of sucrose gradient fractions obtained during isolation of DRMs from T. foetus. Collected fractions (F1-F12) were adsorbed to nitrocellulose membranes, and membranes were probed with a 1/1,000 dilution of horseradish peroxidase-conjugated Cholera-␤-Toxin (HRP-Ctx␤).

Lipid Microdomains in Tritrichomonas foetus 297

with MBCD, this alteration was not statistically significant (Fig. 9B).


Figure 6. (A) One-dimensional high performance thin layer chromatography (HPTLC) of neutral lipids in fractions obtained during isolation of DRMs from T. foetus cells. CHOE: cholesterol ester; TAG: triacylglycerol; FA: free fatty acids; CHO: Cholesterol; PL: phospholipids. (B) Quantitative analyses of the cholesterol (CHO) present in each fraction using GelQuant software.

significantly higher in treated cells than in untreated cells. Although the percentage of the cells with ruptured membrane was higher in parasites treated

Microdomains have been identified in several protozoa parasites, including E. histolytica (Welter et al. 2011), G. lamblia (Humen et al. 2011), Leishmania spp. (Tyler et al. 2009), Plasmodium spp. (Proellocks et al. 2007), T. gondii (Johnson et al. 2007) and Trypanosoma cruzi (Corrêa et al. 2008). In addition, previous studies have demonstrated that different pathogens, such as viruses (Sharma et al. 2012), bacteria (Fine-Coulson et al. 2012), fungi (Long et al. 2012) and protists (Koshino and Takakuwa 2009), interact with host lipid rafts to promote invasion into host cells, maintenance within host cells and evasion of the immune system. In this study, we demonstrated that the fluidity of the plasma membrane of the T. foetus was altered, as indicated by a pattern change of labeling with fluorescent lipid analogs and cholera toxin after treatment with the raft-disrupting agents MBCD and filipin. The lipid analog FAST-DiI is comprised of two 18-carbon chains, which each have two cis double bonds. Lipids with unsaturated tails preferentially enter fluid domains in model membranes containing coexisting gel and fluid phases (Mouritsen and Jørgensen 1995). In contrast, DilC16 is a lipophilic carbocyanine, whose tails have 16 carbons and no double bonds to facilitate incorporation into membranes. This feature makes DilC16 a powerful marker for raft-like membranes (Hao et al.

Figure 7. One-dimensional high performance thin-layer chromatography (HPTLC) analysis of neutral lipids in DRM fractions from T. foetus. Total lipids in fractions 6 and 7 were quantified gravimetrically using GelQuant software. Data are expressed as the observed percentage for each lipid component. The results are represented as the mean of two experiments. PHL: phospholipids; CHO: cholesterol; FA: free fatty acids; TG: triacylglycerol; CHOE: cholesterol ester.

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Figure 8. Involvement of raft-like microdomains in the plasma membrane of T. foetus in adhesion to MDCK cells. Both untreated T. foetus (A) and MBCD-treated T. foetus (B-D) adhered to MDCK cells. Note that MDCK cells incubated with untreated T. foetus exhibited a lower confluence than MDCK cells incubated with MBCDtreated parasites. Some T. foetus was observed with disruptions of the plasma membrane (arrows, C-D) and arrested cell division (D). Note the presence of two recurrent flagella (arrowheads, D).

2001; Mukherjee et al. 1999). The alteration or abolishment of DilC16 labeling on the membranes of parasites treated with MBCD or filipin demonstrated that the plasma membrane structure was altered, suggesting the presence of lipid microdomains in T. foetus. Similar results were observed in previous studies with E. histolytica (Laughlin et al. 2004). Moreover, we demonstrated the presence of GM1like, a lipid raft marker that is specifically recognized by the ␤-chain of the cholera toxin pentamer in the outer leaflet of the plasma membrane (Lafont and van der Goot 2005), in T. foetus. Although GM1 distribution has been reported primarily in lipid rafts, some studies have demonstrated that this ganglioside is concentrated in caveolae, which are specialized plasma membrane invaginations rich in the protein caveolin, cholesterol and glycolipids. Previous studies demonstrated that the partition of

molecules as GM1 in plasma membrane lipid rafts favors their internalization via a process called raftdependent endocytosis, generally characterized as clathrin-independent and sensitive to cholesterol depletion (Kirkham and Parton 2005; Lajoie and Nabi 2007; Lajoie et al. 2009; Nabi and Le 2003; Rodríguez et al. 2006). In addition, GM1 has been demonstrated to play a role in pathogen-host cell interactions (Barrias et al. 2007; Fernandes et al. 2007). In addition to lipid, rafts are highly enriched in glycosphingolipids, sphingomyelins, and cholesterol membrane microdomains, existing in a liquidordered phase of the plasma membrane. Lipid rafts are also functionally defined by their low density and their insolubility in cold 1% Triton X-100 (Brown and Rose 1992; Pike 2003) and this feature has given rise to the acronyms DRM (detergent-resistant

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Figure 9. (A) Quantitative analysis of the adhesion of untreated and raft-disrupted T. foetus to MDCK cells. The number of adhered parasites was calculated after counting 100 randomly chosen fields using SEM. Data are expressed as the number of cells/field. Results are represented as the mean ± SD of two experiments. (B) Quantitative analysis of morphological alterations observed in T. foetus after treatment with MBCD. Note that the percentage of cells displaying membrane rupture and blocked mitosis was significantly higher in treated cells than in untreated cells. Normal: normal parasite morphology; Endoflagellar: endoflagellar parasite forms; Ruptured: parasites with ruptured membranes; Mitosis: parasites with blocked mitosis.

membrane), TIM (Triton-insoluble membranes) and TIFF (Triton-insoluble floating fraction) (Pike 2003). These properties enabled their separation from the rest of the phospholipid bilayer, providing new insights into the structure of the plasma membrane, which until then was believed to represent a twodimensional liquid structure with proteins uniformly solubilized in the lipid solvent (Skwarek 2004). However, there are still controversies whether DRMs are identical to lipid rafts. Therefore, the data on proteins detected within DRMs from various types of cells should be treated with caution and not always considered to be functionally raft localized (Lichtenberg et al. 2005; Chen et al. 2009). In this study, DRMs from T. foetus were isolated according to protocols previously described for other types of cells (Corrêa et al. 2008; Harris et al. 2001). After the centrifugation step of the DRM isolation protocol, a band was observed in the obtained fraction. In this study, fractions 6 and 7 displayed characteristics of lipid rafts. These fractions were cholesterol-enriched and positive for GM1. Similar results have also been reported for other protists, such as T. cruzi (Corrêa et al. 2008) and Dictyostelium discoideum (Harris et al. 2001). The participation of lipid rafts in parasite-host cell adhesion has been investigated in the extracellular

parasites E. histolytica and G. lamblia. Exposure of these parasites to MBCD reduced or abolished their ability to adhere to mammalian cells (Humen et al. 2011; Mittal et al. 2008; Welter et al. 2011). In this study, no changes in adhesion to host cells were observed after treatment of T. foetus with MBCD. In addition, parasite adhesion to host cells was not affected when MDCK cells were exposed to MBCD. Although lipid microdomains do not participate in parasite adhesion, morphological alterations, such as cells with eruptions on the plasma membrane and cells with duplicated structures, were observed in T. foetus cultures treated with MBCD. The depletion of cholesterol directly affected plasma membrane structure, but did not affect cell viability, as these parasites maintained their ability to adhere to host cells. However, T. foetus cytotoxicity was affected, because untreated T. foetus caused more damage to MDCK cells than T. foetus treated with MBCD (Fig. 10B). In addition, the structural disorganization of the lipid microdomains may influence on parasite mitosis, which would explain the higher number of dividing cells. Studies with endothelial cells demonstrated that the phosphorylation of the proteins present in DRMs is able to modulate the mitosis (Sowa et al. 2008), however, more experiments are necessary

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Figure 10. (A) Competition assays using the DRMs fractions in adhesion experiments. Note that the MDCK cells sites that were blocked by the components present on DRM fractions of the parasite do not interfere on adhesion. The number of adhered parasites was calculated after counting 100 randomly fields using SEM. Data are expressed as the number of cells/field. Results are represented as the mean ± SD of two experiments. (B) Cytotoxicity assay of the T. foetus treated with MBCD or filipin. The experiments demonstrate that parasites without treatment provoke more damage to mammalian cells than T. foetus treated with some disrupting agents. Interactions performed with MDCK cells treated with MBCD and parasites without treatment showed similar results.

to verify the participation of the DRMs in T. foetus division. Endoflagellar forms of T. foetus appear when flagella are internalized under unfavorable environmental conditions. Because a cell wall is not formed, this form of T. foetus is called pseudocyst (Pereira-Neves et al. 2003). However, a small percentage of pseudocysts are naturally found in T. foetus cell cultures. In this study, a reduction in the percentage of natural endoflagellar forms in MBCD-treated cultures suggests that the ability of the trophozoites to maintain their rounded shape and internalized flagella was also affected by cholesterol depletion. This result suggests that the two different forms of T. foetus possess distinctly organized plasma membranes. However, we cannot eliminate the possibility that a reduction in membrane cholesterol content and a subsequent disorganization of lipid rafts could also affect intracellular signaling pathways. Further studies are needed to evaluate these possibilities.

Methods Cell cultures: The K strain of T. foetus was isolated from the urogenital tract of a bull by Dr. H. Guida (EMBRAPA, Rio de

Janeiro, Brazil) and has been maintained in culture for several years. Trophozoites of T. foetus were cultivated in TYM Diamond’s medium (Diamond 1957) supplemented with 10% fetal calf serum (FCS). The cells (1 x 106 cells/ml) were grown for 24 h at 36.5 ◦ C, washed in PBS to remove medium and used in the experiments described below. Madin-Darby canine kidney (MDCK) cells were cultured in flasks in Dulbecco s modified Eagle s medium (DMEM) (Sigma, St. Louis, MO, USA) supplemented with 10% fetal calf serum and incubated until a confluent monolayer of cells was achieved. Cholesterol detection: T. foetus were washed and fixed with 4% formaldehyde in phosphate buffer (pH 7.2). The cells were incubated for 30 min with 50 ␮g/ml filipin complex from Streptomyces filipinensis (Sigma, St. Louis, MO, USA). Images were acquired using a Zeiss Axiophot II microscope (Zeiss, Hamburg, Germany) equipped with a C5985 CCD camera (Hamamatsu, Hamamatsu city, Japan). Lipid microdomain disruption: Raft-like microdomains were chemically disrupted via cholesterol depletion using methyl-␤-cyclodextrin (MBCD) (Sigma) or cholesterol sequestration using filipin (Fluka, Seelze, Germany). MBCD was dissolved in serum-free TYI-33 medium at the appropriate concentration. Filipin was stored as a 5 mg/ml stock solution in ethanol and diluted into medium as required. Cells were treated for 30 min at 36.5 ◦ C with 7.5 mM MBCD or 3.8 ␮M filipin. For all experiments, untreated cells were used as controls. Fluorescent lipid analog staining: To stain lipid rafts and non-raft regions of the membrane, cells were disrupted by treatment with MBCD (7.5 mM) or filipin (3.8 ␮M) during the last 30 min of incubation, as described above. The medium was removed, and the cells were incubated with 1.1 ␮M dialkyindocarbocyanine (DiIC16) (Molecular Probes, Eugene, OR, USA)

Lipid Microdomains in Tritrichomonas foetus 301 or 0.9 ␮M 1,1 -dilinoleyl-3,3,3 ,3 -tetramethylindocarboxyanine (FAST-DiI) (Molecular Probes) for 2 min at room temperature. Cells were fixed with 1% formaldehyde in phosphate buffer, pH 7.2, overnight at room temperature. The fixed parasites were washed in PBS and allowed to adhere to poly-L-lysinecoated (mol wt 300,000) glass coverslips. The slides were rinsed twice with phosphate-buffered saline (PBS) and mounted in the antifade reagent SlowFade (Molecular Probes) in PBS. The samples were observed using a Zeiss Axiophot II microscope (Zeiss), and images were acquired using a chilled C5985 CCD camera. The Adobe Photoshop CS2 software programs were used to colorize images. GM1 detection: To stain the glycosphingolipid GM1 in the plasma membrane, T. foetus was cultured for 2 h in serum-free medium at 37 ◦ C. For some experiments, lipid rafts were disrupted by MBCD treatment during the last 30 min of incubation. The medium was removed, and the cells were fixed with 1% formaldehyde in phosphate buffer. The cells were allowed to adhere to poly-L-lysine coated coverslips and incubated in the dark with 20 ␮g/ml recombinant cholera toxin subunit B Alexa Fluor® 488 conjugate (Molecular Probes) in PBS for 1 h at room temperature. DRM purification: DRMs were purified as previously described (Chung et al. 2005). Briefly, the parasites were washed in PBS, pH 7.2 and TNE buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.2 mM PMSF, 1 ␮g/ml leupeptin, 1 ␮g/ml pepstatin, 1 ␮g/ ml aprotinin, 1 mM Na3 VO4 , and 1 mM NaF). Aliquots of 5 x 109 cells in TNE buffer were carefully disrupted on ice with a Branson Sonifier B15 cell disruptor (Sonifier Cell Disruptors, Danbury, CT, USA) using a standard probe (13 mm radiating diameter) operating at 15% of total amplitude, with 5 cycles of 30 s and 1 s rest between cycles, yielding a total homogenate. The resulting homogenate was centrifuged at 20,000 x g, and the supernatant was incubated for 20 min at 4 ◦ C in TNE buffer containing 1% Triton X-100. After incubation, the sample was mixed 1:1 with 80% sucrose (w/v) in TNE buffer and transferred to a Beckman SW41 centrifuge tube (Beckman Coulter Inc., Fullerton, CA, USA). This mixture was overlaid on 35% sucrose, followed by 5% sucrose, and centrifuged at 100,000 x g for 16 h at 4 ◦ C. Twelve 1 ml fractions were sequentially collected from the top of the gradient. Cholesterol content in the isolated DRM fractions was measured using a cholesterol oxidase-based fluorimetric assay (Amplex Red Cholesterol Kit) purchased from Molecular Probes. The cholesterol content of the purified lipid rafts was normalized to the total protein of each fraction, which was measured by the Lowry method. Slot blotting: To determine which gradient fractions contained cholesterol, 0.1 ml from each fraction was diluted to 1 ml with PBS and adsorbed onto a nitrocellulose filter using a slot-blot apparatus (Schleicher & Schuell, Keene, NH, USA). The membranes were blocked with 5% non-fat dry milk and probed with a 1/1,000 dilution of horseradish peroxidaseconjugated Cholera-␤-Toxin (HRP-Ctx␤) (Molecular Probes) for 1 h at room temperature. The blots were developed using a chemiluminescence assay (Pierce, Rockford, IL, USA). Lipid analysis: The twelve fractions collected from the gradient were submitted to lipid extraction (Bligh and Dyer 1959) for 2 h in a stoppered tube containing 5 ml of a chloroform-methanol-water solution (2:1:0.8, v/v), with intermittent agitation. After centrifugation, the supernatant was collected, and the pellet was subjected to a second lipid extraction for 1 h. The amount of total lipids was determined gravimetrically. Extracted lipids were analyzed by one-dimensional high performance thin-layer chromatography (HPTLC) for neutral lipids (Vogel et al. 1962). HPTLC plates were stained by spraying with a charring solution consisting

of 10% CuSO4 , 8% H3 PO4 and heated to 180 ◦ C for 5–10 min (Ruiz and Ochoa 1997). The charred HPTLC plate was then digitized, and densitometric analysis of lipid bands was conducted using “ImageMaster TotalLab v1.11” software. Adhesion of T. foetus to MDCK cell monolayers: To determine the influence of raft-like microdomains on T. foetus adhesion to host cells, MDCK cells were plated and grown to confluence in a 24-well culture plate. Control T. foetus (incubated with a 0.0475% ethanol diluent control) or raft-disrupted T. foetus (incubated with 7.5 mM MBCD or 3.8 ␮M filipin) were suspended in serum-free TYM:DMEM (1:1), added to MDCK cell monolayers at a ratio of 5:1 (5 T. foetus per MDCK cell) and incubated for 30 min at 37 ◦ C. Experiments using MDCK cells that were treated with raft-disrupting agents were performed using the same protocol. After incubation, the wells were gently washed with pre-warmed medium twice to remove non-adherent parasites. The cells were then fixed in 2.5% glutaraldeyde in 0.1 M sodium cacodylate buffer (pH 7.2), postfixed for 15 min in 1% OsO4 , dehydrated in ethanol, critical point-dried with CO2 and sputter-coated with gold-palladium. The number of adherent T. foetus observed in 100 randomly chosen fields was determined at a magnification of 1,000x using a JEOL 5800 scanning electron microscope. To quantify the morphological changes, the cellular alterations were counted in 100 cells of the two independent experiments in triplicate. The results were expressed on percentage. The statistical comparisons were performed using two-way ANOVA, and p-values were established according with the table below:

P value



> 0.05 0.01 to 0.05 0.001 to 0.01 < 0.001

not significant Significant Very significant Extremely significant

* ** ***

Competition assays: To confirm the participation of the raftlike microdomains on T. foetus adhesion to host cells, MDCK cells were plated, grown to confluence in a 24-well culture plate and incubated with DRM fraction of the T. foetus for 1 h in DMEM. Afterwards, the medium was replaced by serum-free TYM: DMEM (1:1), T. foetus were added to MDCK cells monolayer at a ratio of 5:1 (parasites: host cell)T. foetus per MDCK cell) and incubated for 30 min at 37 ◦ C. The control was performed without addition of the DRM fraction. The samples were processed for SEM and analyzed as described above. Cytotoxicity assays: To verify whether the disorganization of the lipid microdomains is able to affect the T. foetus cytotoxicity, MDCK cells were plated, and grown to confluence in a 24-well culture plate. Afterwards, cells were incubated with parasites for 1 h in different conditions: (1) T. foetus without treatment, (2) T. foetus MBCD treated, (3) T. foetus filipin-treated. In addition, experiments with MDCK cells MBCD treated incubated with parasites control were also performed. After the interaction, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) (0.5 mg/mL in DMEM) was added to each well, and the cells were incubated at 37 ◦ C for 1 h. The medium was discarded, and 1 mL of acid isopropanol solution (4 M HCl:isopropanol PA, 1:99, v/v) was added to each well to solubilize the formazan product, which exhibits a purple crystal color, produced by the activity of active hydrogenases. Absorbance was read at 590 nm, and the background at 630 nm was subtracted using a scanning ELISA microplate reader (BioTek - ELX800). The results were expressed as absorbance (A590-630). In all cases two

302 I. de Andrade Rosa et al. independent experiments were performed in triplicate. The statistical comparisons were performed using two-way ANOVA, and p-values were established as p < 0.001.

Acknowledgements This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), the Fundac¸ão Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), the Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES), the Programa de Núcleos de Excelência (PRONEX) and the Associac¸ão Universitária Santa Úrsula (AUSU).

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Tritrichomonas foetus displays classical detergent-resistant membrane microdomains on its cell surface.

Tritrichomonas foetus is a serious veterinary parasite that causes bovine trichomoniasis, a sexually transmitted disease that results in reproductive ...
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