1 Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del IPN, Apdo. 14-740, México, D F 07000, Mexico
2 Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del IPN, Apdo. 14-740, México, D F 07000, Mexico
3 Departamento de Patología Experimental, Centro de Investigación y de Estudios Avanzados del IPN, Apdo. 14-740, México, D F 07000, Mexico
Correspondence
Mireya de la Garza
mireya{at}cell.cinvestav.mx
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ABSTRACT |
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INTRODUCTION |
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In bacteria, iron-acquisition systems have been widely studied (Alderete et al., 1988; Gray-Owen & Schryvers, 1996
; Jarosik & Land, 2000
). However, the mechanisms of iron acquisition in protozoa are less well understood (Loo & Lalonde, 1984
; Murray et al., 1991
; Rodríguez & Jungery, 1986
; Tachezy et al., 1998
). Some of these primitive eukaryotes acquire iron from Tf through receptor-mediated endocytosis (Coppens et al., 1987
; Reyes-López et al., 2001
). A receptor on Trypanosoma brucei binds and endocytoses Tf; inside vesicles, iron is released, apotransferrin (apoTf) is degraded, and the receptor is recycled (Steverding, 2000
). Lf is also taken up by Try. brucei (Coppens et al., 1987
). Other parasites possess lactoferrin-binding proteins (Lfbp), and use Lf-iron for growth (Britigan et al., 1998
; Tachezy et al., 1996
; Weinberg, 1999
).
Entamoeba histolytica is an extracellular parasitic protozoan that causes amoebiasis, a human intestinal and hepatic disease that is a significant source of morbidity and mortality in developing countries. E. histolytica cysts transform into trophozoites in the terminal ileum, and reproduce and invade the colonic mucosa, resulting in ulcerative lesions and dysentery. An inflammatory process develops in the large intestine, with abundant infiltration of neutrophils around amoebas. In further stages of the disease, amoebas can migrate to the liver and other organs (Espinosa-Cantellano & Martínez-Palomo, 2000).
E. histolytica trophozoites depend on exogenous iron sources for growth in axenic culture, and they can use both ferric and ferrous ions (Latour & Reeves, 1965; Serrano-Luna et al., 1998a
; Smith & Meerovitch, 1982
). The capability of amoebas to use haemoglobin (Hb) as an iron source, and its cleavage by cysteine proteases, has been documented (Serrano-Luna et al., 1998b
). Also, the utilization of holotransferrin (holoTf) as an iron source, and iron acquisition through receptor-mediated endocytosis, have been described (Reyes-López et al., 2001
). Lf has been observed in E. histolytica tubular invaginations that did not show a typical clathrin coat (Batista et al., 2000
); however, it is not known whether human Lf supports growth of the amoeba.
The aim of this work was to determine how E. histolytica trophozoites take up and use human holoLf. We established that iron acquisition from this ferric protein can support growth of the amoeba. E. histolytica hololactoferrin-binding protein (EhLfbp) specifically recognized holoLf; this ferric protein was endocytosed by filipin-sensitive vesicles, which were recognized by an anti-caveolin mAb.
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METHODS |
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Growth of E. histolytica in holoLf as an iron source
Media and iron concentrations used are shown in Table 1. The low-iron medium listed in Table 1
is BI-S-33 medium without ammonium ferric citrate that was treated with 5 g ml1 of the chelating resin Chelex-100 in order to remove iron from the trace reagents in the medium. The resin was subsequently removed by filtration, and the medium was sterilized. This medium is henceforth referred to as low-iron. In order to determine whether holoLf sustains trophozoite growth, the following methods were used.
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Successive transfers.
Amoebas (104) were inoculated into BI-S-33, low-iron and low-iron containing human holoLf (50 or 100 µM total iron). Amoebas were subcultured in each medium at least three times, and incubated for 48 h each time. Viability was measured by trypan blue exclusion.
Specific holoLf binding to the E. histolytica cell surface
The interaction of the amoebic cell surface and holoLf was investigated using the following methods.
Dot blot.
Amoebas suspended in PBS (106 ml1) were fixed (4 % paraformaldehyde in PBS, pH 7·4, for 1 h at 37 °C). Cell suspensions (10 µl) were vacuum blotted onto a nitrocellulose membrane (Sigma) in a blotter apparatus (Bio-Rad). The membrane was blocked with PBS-T (5 % non-fat milk in PBS/0·05 % Tween 20, pH 7·4, for 1 h) at room temperature, washed, and incubated for 1 h with 3·0 µg HRP-holoLf ml1 [holoLf was coupled to horseradish peroxidase (HRP) by using the method of Avrameas & Ternynck, 1971]. The reaction was revealed with 3',3-diaminobenzidine. BS and Tri. vaginalis were used as negative and positive controls, respectively.
Confocal microscopy.
Trophozoites (2x105) were suspended for 30 min in BI-S-33, or low-iron lacking BS and AFC. The trophozoites were then washed, fixed, and incubated for 1 h with 100 µg ml1 FITC-holoLf or FITC-apoLf [FITC-holoLf was prepared as described for holoTf (Reyes-López et al. 2001), and FITC-apoLf was prepared by depleting iron from FITC-holoLf, as described by Mazurier & Spik (1980)
]. Samples were mounted in Vectashield on glass slides, and examined in a Leica TCS-SP2 confocal laser-scanning microscope, observing 1020 optical sections from each cell.
Competition assays.
To determine the specificity of the E. histolytica holoLf-binding sites, nitrocellulose membranes from the dot blot, and fixed amoebas from the confocal experiments, were preincubated for 1 h with 1 mg ml1 of other iron-containing proteins (Tf and Hb), and with apoLf. Evidence of an EhLfbp was obtained by preincubating the membrane or the fixed cells with 1 mg holoLf ml1 for 1 h, or by treatment of fixed amoebas with 24 µg trypsin ml1 (1 h at 37 °C), before incubation with HRP-holoLf or FITC-holoLf, as indicated for dot blot or confocal microscopy, respectively.
Detection of EhLfbp in low-iron-containing medium, with and without holoLf.
An amoebic total extract was obtained as reported by Serrano-Luna et al. (1998b) from 5x106 cells grown for 6 h in BI-S-33, low-iron, and low-iron plus holoLf (50 µM iron), or grown in BI-S-33, and then fixed and incubated with trypsin (see above). Proteins (30 µg per well) were separated (12 % SDS-PAGE) and stained with Coomassie blue. Protein concentration was determined by the method of Bradford (1976)
. For overlay assays, proteins were electrotransferred to a nitrocellulose membrane (1·5 h, 400 mA) (Towbin et al., 1979
), which was blocked (5 % non-fat milk in PBS-T, 3 h), incubated for 12 h with 3·0 µg holoLf ml1, washed, and incubated with rabbit anti-human Lf antibody (Ab) (Sigma, catalogue no. L 3262; 1 : 100). A secondary HRP-anti-rabbit IgG (1 : 1000) was added. The blotted dried membrane was then scanned and quantified by densitometry using SigmaGel software. Competition assays were performed in a similar way to the dot blots, but the membrane was incubated with HRP-holoLf for 12 h. Expression of holoLf-binding sites from E. histolytica grown in different iron sources was measured as follows: amoebas (1x106) were incubated for 30 min in BI-S-33 or low-iron, fixed (see above), washed, and incubated for 30 min with 100 µg FITC-holoLf ml1, and processed for flow cytometry. In other experiments, amoebas grown in low-iron or BI-S-33 were simultaneously exposed to FITC-holoLf and BI-S-33, fixed, washed, and processed. Fluorescence intensity (FI) was measured in 104 cells by flow cytometry, and the statistical significance of the difference between the two conditions was evaluated with the KolmogorovSmirnov test (Young, 1977
).
The holoLf endocytic pathway in E. histolytica.
To investigate whether E. histolytica is able to endocytose Lf, amoebas (2x105) were incubated in low-iron (without BS) containing FITC-apoLf or FITC-holoLf, for 5 or 30 min, fixed (see above), and processed for confocal microscopy. To determine the endocytosis pathway, amoebas (106) were incubated for 30 min in BI-S-33 without BS, but containing one of the following endocytosis-inhibitors: 100 µg filipin ml1, 8 µM chlorpromazine, 2 % (w/v) sucrose, 200 nM wortmannin or 100 mM chloroquine. Next, cells were incubated for 30 min with the inhibitor plus 100 µg ml1 FITC-holoLf, and prepared for scanning by flow cytometry and confocal microscopy. To investigate whether caveolin or clathrin was participating in the holoLf endocytosis process, amoebas (2x105) were incubated for 30 min in BI-S-33 without BS, and then for 15 min in the presence of 200 µg FITC-holoLf ml1, fixed, permeabilized (0·2 % Triton X-100, 15 min), and finally incubated for 1 h with anti-chick embryo fibroblast caveolin-1 mAb (Zymed, catalogue no. 03-6000, clone Z034; 1 : 20) or goat anti-bovine brain clathrin Ab (Sigma, catalogue no. C 8034; 1 : 40). Some amoebas were also treated with 100 µg filipin ml1 for 30 min before adding the Abs. Amoebas were then incubated for 1 h with secondary Abs: rabbit TRITC-anti-mouse IgG (for caveolin; Zymed; 1 : 50), and rabbit RITC-anti-goat IgG (for clathrin; Zymed; 1 : 50). The presence of caveolae-like vesicles in E. histolytica was determined by staining the following caveolae components: lipids (10 µg ml1 Nile red for 30 min, as described by Kimura et al., 2004; and Klinkner et al., 1997
) and caveolin [using anti-caveolin mAb as above, and, as a secondary Ab, goat anti-mouse IgG coupled to CY5 (Zymed; catalogue no. 62-6516; 1 : 100)]. Filipin was used to disrupt caveolae. In other assays, the traffic of endocytosed holoLf was visualized using Lucifer yellow (LY), which stains acidic vesicles. Cells were incubated for 35 or 45 min in BI-S-33 containing 1 mg LY ml1 and 100 µg holoLf ml1. Amoebas were then fixed, permeabilized, and incubated for 1 h with rabbit anti-human Lf (see above), and then for 1 h with a secondary Ab coupled to CY5 (Zymed; mouse anti-rabbit IgG; 1 : 100). Samples were processed for confocal microscopy, and for fluorescence quantification by flow cytometry.
Immunodetection of clathrin and caveolin-like protein in E. histolytica.
Lysates of HEp-2 cells or E. histolytica trophozoites (30 µg protein each) were separated by 13 % SDS-PAGE, and electrotransferred to a nitrocellulose membrane for 45 min at 4 °C. The blot was blocked with PBS-T at room temperature (see dot blot), and incubated for 2 h with anti-caveolin-1 or anti-clathrin Ab (1 : 300). HRP-conjugated goat anti-mouse IgG, or rabbit anti-goat IgG (Zymed) (each at 1 : 2000), was used as a secondary Ab. Blots were developed by chemiluminiscence (Amersham Pharmacia Biotech).
Substrate gel electrophoresis.
To determine whether amoebic proteases cleave holoLf, the method of Heussen & Dowdle (1980) was followed. Cells were placed for 6 h in BI-S-33 or in low-iron (both without BS), then centrifuged, and total extracts were obtained from the pellet. Supernatants were precipitated with neat 2-propanol. In order to characterize the proteolytic activity, total-extract proteins from BI-S-33 cultures were incubated with an equal volume of protease inhibitor [p-hydroxymercuribenzoate (pHMB), N-ethylmaleimide (NEM), PMSF or EGTA], as reported by Serrano-Luna et al. (1998b)
. Samples (10 µg protein per well) were run in 12 % polyacrylamide gels copolymerized with 0·1 % holoLf (3 h, 100 V, 4 °C). Gels were washed, and incubated for 1 h with 2·5 % Triton X-100, rinsed, and incubated for 12 h with 10 mM CaCl2 in either 1 M Tris-OH, pH 7·0, or 0·1 M sodium acetate-Tris/HCl, pH 4·0. Gels were rinsed one more time, and stained with Coomassie R-250.
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RESULTS |
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Taken together, these results show that the mechanism responsible for internalizing holoLf in E. histolytica trophozoites is not clathrin dependent; however, caveolin-like containing vesicles might be involved in this process.
Traffic of holoLf in acidic vesicles, and proteases that cleave holoLf
Since the E. histolytica trophozoite is a multivesicular cell in which non-acidified and acidified vesicles have been found (Aley et al., 1984; Swanson, 1989
; Batista et al., 2000
), we investigated whether holoLf reaches acidic vesicles, an environment in which Lf-iron is probably released. LY (green and red) (Fig. 6
a, 1
and 2
) and holoLf (blue) (Fig. 6a, 3
) co-localized in low amounts in acidic vesicles (purple and light blue) after 35 min incubation (Fig. 6a, 4
); at 45 min, co-localization was clearly observed (Fig. 6a
, 8, light blue). This co-localization was quantified by flow cytometry (not shown). These results suggest that holoLf may follow the vesicular traffic from endosomes, and reach the acidic vesicles in amoebas. In parasites, the Lf-iron release inside acidic vesicles is only partial, therefore alternative mechanisms, such as proteases, could be involved in the holoLf cleavage; thus, to determine whether proteases cleave holoLf, amoebic extracts and culture supernatants were run in substrate gels with holoLf or gelatin (positive control). Proteases of 250, 100, 40 and 22 kDa from amoebic extracts cleaved holoLf at pH 7 (Fig. 6b
, lane 2); however, the activity increased considerably at pH 4 (Fig. 6b
, lane 9). Similar results were found in extracts of iron-starved amoebas (Fig. 6b
, lane 3). No activity was found in supernatants (not shown). All activities were inhibited by the cysteine protease inhibitors pHMB and NEM (Fig. 6b
, lanes 4 and 10, and 5 and 11, respectively). There was no inhibition by the serine (PMSF) and metallo- (EGTA) protease inhibitors (Fig. 6b
, lanes 6 and 7). The data suggest that cysteine proteases could cleave holoLf in acidic vesicles, where the pH allows the release of Lf-iron, which is then utilized by E. histolytica for growth.
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DISCUSSION |
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The iron content of BI-S-33 medium supplemented with BS ranged between 90 and 105 µM depending on the reagent batch; however, the minimum iron requirement for amoebas is >50 µM. This requirement surpasses that of the majority of eukaryotic and prokaryotic cells, and is due to the presence of amoebic iron-containing proteins (Weinbach et al., 1980). Human Lf-iron (>25 µM) supported E. histolytica growth, and 50100 µM Lf-iron sustained growth through several passages. Lf-iron was the growth support, since a low-iron environment and absence of this ferric protein promote amoebic death. Thus, Lf-iron seems to be more efficient than ferric citrate for amoebic growth. In addition, pathogens grown in vitro require higher iron concentrations from ferric citrate, ferric nitrilotriacetate (Fe-NTA), FeSO4 and other sources than from host proteins, such as Hb, Lf and Tf (Wilson et al., 1994
; Tachezy et al., 1996
; Jarosik et al., 1998
; Serrano-Luna et al., 1998b
; Reyes-López et al., 2001
). The results also show that amoebas have a constitutive level of expression of binding sites for holoLf. However, in conditions of iron deficiency, the number of binding sites for holoLf is increased. Interestingly, in the presence of holoLf plus ferric citrate, amoebas expressed the basal level of binding sites, which could be due to Lf-iron acquisition requiring receptor expression and synthesis, endocytosis and iron release at acidic pH, whereas iron ferric citrate acquisition may consist of a more straightforward iron release and entry process.
Suchan et al. (2003) reported that human Lf-iron (up to 100 µM) was unable to support E. histolytica HM-1 : IMSS growth in TYI-S-33 containing the iron chelator 2,2-dipyridyl (100 µM). A possible explanation could be the characteristics of Lf in its N-terminal arginine-rich part, which is important for binding to some receptors (van Veen et al., 2002
). We used the Chelex-100 resin to eliminate ferrous and ferric iron from traces of reagents in the media, since the resin can be subsequently removed from media, and then the culture is not affected. Chelex-100 also sequesters calcium, magnesium and zinc (Giles & Czuprynski, 2004
), but they are restored by the serum added after chelation. Our data also show that bovine holoLf can sustain amoebic growth, but to a lower extent than human holoLf. It has been reported that Lf glycans are specific for different animal species (Spik et al., 1988
), however, since we tested human and bovine Lf only, it remains to be determined whether or not the use of Lf-iron by E. histolytica is species specific. It is important to state that the holoLf concentrations used by amoebas in this work can be found in physiological conditions. HoloLf present in the large intestine could be providing iron to amoebic cells, which is important for amoebic colonization and invasion.
We found specific holoLf-binding sites distributed in a patch-like pattern on the amoebic surface, which is in agreement with the observations of Batista et al. (2000) by electron microscopy. Other iron-containing proteins, such as Tf and Hb, and apoLf, were unable to prevent holoLf recognition by the E. histolytica sites located on the cell surface. HoloLf recognition was affected by unlabelled holoLf only, or by degradation of amoebic membrane proteins with trypsin, a serine protease. Therefore, different receptors for all these proteins may be present in E. histolytica. Protozoa such as Tritrichomonas foetus, Leishmania donovani and Tri. vaginalis bind ferric Lf in a specific receptor-mediated fashion to use it as iron source (Peterson & Alderete, 1984
; Lehker & Alderete, 1992
; Tachezy et al., 1996
). However, Leishmania chagasi binds Tf and Lf through the same protein, indicating that a specific receptor is not present (Wilson et al., 2002
). All these results corroborate that E. histolytica possesses specific holoLf-binding proteins on the surface. E. histolytica Lfbp might be recognizing amino acid residues or structural motifs unique to the holoLf molecule. In mammalian cells, Lf receptors bind equally to apoLf and holoLf (Testa, 2002
). As a result of its conformation, ApoLf (iron-free Lf) displays different properties compared with the ferric-saturated form of Lf (Mazurier & Spik, 1980
; Testa, 2002
). Since human apoLf did not compete with holoLf for the binding sites in amoebas, this suggests that amoebas could have different receptors for the two Lfs. In addition, apoLf was able to bind to amoebas, but the cells became round and died; we presume that apoLf had an amoebicidal effect that damaged the cellular membrane, and we are currently studying this possibility.
Amoebic proteins of 70 and 140 kDa that bind Tf have been reported (Reyes-López et al., 2001). Here, we report an E. histolytica surface protein of 90 kDa that is specifically recognized by holoLf. This recognition was not affected by Tf, Hb or apoLf; it was only inhibited with an excess of holoLf, or by treatment of amoebas with trypsin. On the other hand, since the 90 kDa band was detected in amoebas grown with ferric citrate, in low-iron, and with holoLf, this protein could be constitutively synthesized; however, its expression could be stimulated by holoLf or iron stress. Whether the EhLfbp is involved in the virulence of the parasite remains to be determined. With these results, it is tempting to speculate that amoebas could bind and use several iron-containing proteins at the time of invasion, e.g. Lf from mucosal surfaces, Tf from serum, and Hb from destroyed erythrocytes. Use of multiple iron sources by E. histolytica explains its successful survival in different organs, and it should be highly advantageous at sites with different iron environments.
The ability of amoebas to bind and endocytose holoLf, and the presence of a specific E. histolytica 90 kDa protein on the cell surface, provide some indication of the mechanism of Lf-iron acquisition. Protein endocytosis may occur through several different pathways, such as clathrin-coated pits, caveolar structures, and macropinocytosis (Mellman, 1996; Pelkmans & Helenius, 2002
). By using specific inhibitors of vesicular traffic and endocytosis, we found that only filipin inhibited the holoLf entry in amoebas. Filipin disrupts cholesterol, a major component of membrane glycolipid microdomains, and thus affects the structure and organization of caveolar components (Schnitzer et al., 1994
; Orlandi & Fishman, 1998
). Filipin has been recently used in E. histolytica to detect the presence of raft-like vesicles, and investigate their participation in virulence events (Laughlin et al., 2004
). We localized caveolae-like structures in E. histolytica by using an anti-caveolin mAb and lipid staining (Fig. 5b
); this mAb recognizes both
and
isoforms of caveolin-1 in fibroblasts from humans, mice and rats (Okamoto et al., 1998
), and, in amoebas, it recognized two proteins of 22 and 24 kDa that could correspond to caveolin-1-like isoforms. Further evidence of caveolae-like structures in E. histolytica included the inhibition of co-localization between lipids and caveolin when amoebas were treated with filipin. This inhibition could be due to loss of integrity of caveolae-like structures, as a result of cholesterol disruption and caveolin dissolution. The participation of E. histolytica caveolae-like vesicles in holoLf internalization was further supported by endocytosis experiments, which showed co-localization of holoLf and the anti-caveolin signal in vesicles after 15 min of holoLf endocytosis. Clathrin-coated pits did not participate in the holoLf endocytosis.
After endocytosis of holoLf via amoebic caveolae-like structures, we found that this protein reached acidic vesicles. In the environment of these vesicles, holoLf probably releases iron. The vesicular traffic of caveolae has been the subject of some controversy; however, recent studies have shown that caveolar structures can interact with both the caveosome and the lysosome pathway, and that caveolin-enriched regions can incorporate cargo in a regulated manner (Escriche et al., 2003; Peters et al., 2003
; Parton, 2004
; Schnitzer et al., 1995
). Furthermore, in amoebic extracts, we found proteases that cleave holoLf. It has been reported that E. histolytica endocytic vesicles are enriched in cysteine proteases which contain acid phosphatase (Temesvari et al., 1999
); the presence of holoLf in acid-phosphatase-containing vacuoles has been described in Trt. foetus and E. histolytica (Affonso et al., 1994
; Batista et al., 2000
). E. histolytica acidic vesicles show a pH below 4, (Aley et al., 1984
), a pH at which Lf-iron is partially released. These results suggest that holoLf could be releasing iron in the amoeba cysteine-protease-enriched acidic vesicles.
Together, the results indicate that human holoLf can be used by the parasite as an iron source for growth. The amoeba might acquire Lf-iron in a specific receptor-mediated endocytosis mechanism via caveolae-like filipin-sensitive vesicles. The acidic environment of amoebic vesicles, and the presence of cysteine proteases, may be factors that contribute to Lf-iron release.
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ACKNOWLEDGEMENTS |
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Received 14 April 2005;
revised 27 July 2005;
accepted 16 August 2005.
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