Entamoeba histolytica: Transferrin Binding Proteins

Reyes-López, Magda; Serrano-Luna, José de Jesús; Negrete-Abascal, Erasmo; León-Sicairos, Nidia; Guerrero-Barrera, Alma L.; Garza, Mireya de la

doi:10.1006/expr.2001.4647

Cited by 23 publications

(20 citation statements)

References 34 publications

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“…A low-iron medium was obtained by using Chelex-100 resin (BioRad, Richmond, VA). 18,19 Amoebas grown for 48 hours in glass tubes with screw caps containing BI-S-33 medium were chilled on ice baths. This allowed for removal of the amoebas from the tube walls.…”

Section: Amoeba Strain and Growth Conditionsmentioning

confidence: 99%

Amoebicidal Activity of Milk, Apo-lactoferrin, sIgA and Lysozyme

León-Sicairos¹,

López-Soto²,

Reyes-López³

et al. 2006

Clinical Medicine & Research

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Entamoeba histolytica is a pathogenic, parasitic protozoan that can infect the large intestine and cause amoebiasis, an illness responsible for at least 100,000 deaths annually worldwide. The capacity of human milk to kill amoebas 6,15 and the amoebicidal action of secretory immunoglobulin type A (sIgA) 16 have been reported. However, it is unknown which other components of milk are amoebicides. As milk contains several microbicidal components, we fractionated milk and tested each fraction against trophozoites in axenic cultures to describe any amoebicidal effects of other components of human and bovine milk such as lactoferrin, lysozyme and sIgA. We also sought to identify any combined effect among these molecules. Objectives: To identify amoebicidal components in human milk and the effect of iron on the amoebicidal activity. Original ResearchDesign: Investigation in axenic cultures of Entamoeba histolytica trophozoites.Methods: Amoebas were treated with 5%-20% of human, bovine and swine milk, with 10% of human milk fractions (i.e., casein, proteins except casein and fat) or with 1 mg/ml of human milk apo-lactoferrin, human secretory immunoglobulin type A (sIgA) and chicken egg-white lysozyme (i.e., purified proteins). Milk proteins were detected using immunoblot. Confocal microscopy was used to define the interaction of milk proteins (100 µM each) and amoebas. Experiments were done at least three times in triplicate, and mean and standard deviations were calculated.Results: Human and bovine milk were amoebicidal showing a concentration-dependent effect. The amoebicidal effect was increased in the absence of iron. Milk protein fractions, with the exception of casein, were the components responsible for the amoebicidal activity found. Apo-lactoferrin, sIgA and lysozyme were identified in the amoebicidal milk protein fraction. Apo-lactoferrin showed the major amoebicidal effect. These proteins, either alone or in combination, showed a killing effect on the trophozoites.They bound to the amoebic membrane causing cell rounding, lipid disruption and damage. Conclusions:Milk proteins such as apo-lactoferrin, sIgA and lysozyme are able to kill Entamoeba histolytica trophozoites.This study confirms the importance of feeding breast milk to newborns.

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Section: Amoeba Strain and Growth Conditionsmentioning

confidence: 99%

Amoebicidal Activity of Milk, Apo-lactoferrin, sIgA and Lysozyme

León-Sicairos¹,

López-Soto²,

Reyes-López³

et al. 2006

Clinical Medicine & Research

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“…Amoebiasis affects the human population worldwide, mainly in developing countries. This parasite requires about 100 mM iron for growth; thus it has developed mechanisms to scavenge iron, for example through the removal of iron from Hb, holoTf, holoLf and ferritin (Leó n-Sicairos et al, 2005; Ló pez-Soto et al, 2009a, b;Reyes-Ló pez et al, 2001;Serrano-Luna et al, 1998). Previously, we reported the detection of E. histolytica holoTf-binding proteins (EhTfbp) in extracts from E. histolytica trophozoites cultured in vitro; they were detected using the anti-human transferrin receptor B3/25 mAb.…”

mentioning

confidence: 99%

Acetaldehyde/alcohol dehydrogenase-2 (EhADH2) and clathrin are involved in internalization of human transferrin by Entamoeba histolytica

et al. 2011

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Transferrin (Tf) is a host glycoprotein capable of binding two ferric-iron ions to become holotransferrin (holoTf), which transports iron in to all cells. Entamoeba histolytica is a parasitic protozoan able to use holoTf as a sole iron source in vitro. The mechanism by which this parasite scavenges iron from holoTf is unknown. An E. histolytica holoTf-binding protein (EhTfbp) was purified by using an anti-human transferrin receptor (TfR) monoclonal antibody. EhTfbp was identified by MS/MS analysis and database searches as E. histolytica acetaldehyde/alcohol dehydrogenase-2 (EhADH2), an iron-dependent enzyme. Both EhTfbp and EhADH2 bound holoTf and were recognized by the anti-human TfR antibody, indicating that they correspond to the same protein. It was found that the amoebae internalized holoTf through clathrin-coated pits, suggesting that holoTf endocytosis could be important for the parasite during colonization and invasion of the intestinal mucosa and liver. INTRODUCTIONIron is a vital element for nearly all living organisms but it is bound to proteins to prevent tissue damage by oxygen free-radical formation. In mammals, iron is an enzyme cofactor and forms part of haemoproteins. In addition, iron is sequestered by proteins such as the iron-transporter transferrin (Tf) in serum and the iron-withholding defence lactoferrin (Lf) in mucosae. Thus, the free ionic-iron concentration (~10 218 M) is far too low to sustain the growth of pathogens (Bullen et al., 1978).Bacterial pathogens have developed several mechanisms for obtaining host iron; one of these is the use of receptors that can bind iron-containing proteins, such as holoTf, holoLf, and haemoglobin, with very high host specificity (Weinberg, 2009). In Gram-negative bacteria, receptors for holoTf generally consist of two iron-regulated outer-membrane proteins, termed TbpA and TbpB (Genco & Desai, 1996), and iron is removed from its receptor in an energydependent process. A transferrin-binding protein A (StbA, also known as IsdA), has been reported in Staphylococcus aureus strain RN6390 (Maresso & Schneewind, 2006;Mazmanian et al., 2003;Taylor & Heinrichs, 2002). Homologues of the isd gene are also found in Gram-positive bacilli (Bierne et al., 2004; Gat et al., 2008;Maresso & Schneewind, 2006). These pathogens have several elaborate mechanisms for iron sequestration.Furthermore, it has been reported that S. aureus strain BB and Staphylococcus epidermidis strain 138 utilize the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Gap or Tpn) to bind holoTf (Modun & Williams, 1999). Interestingly, the parasite Trypanosoma brucei also makes use of GAPDH for holoLf binding rather than for holoTf binding (Tanaka et al., 2004). Enzymes that bind holoTf have not previously been described in parasitic protozoa.Iron uptake in mammalian cells is initiated by the binding of holoTf to the TfR on the cell surface. Then, via clathrincoated pits, the Tf-TfR complex becomes trapped within endocytic vesicles, which are rapidly acidified, and the iron is removed from ...

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“…Several studies have shown responses of the parasite to the absence or excess of iron and to the presence of iron-containing proteins [Serrano-Luna et al, 1998;Reyes-López et al, 2001;León-Sicairos et al, 2005;López-Soto et al, 2009a]. The concerted use of strategies to bind and use iron from different sources provides the parasite with the ability to use various host proteins for its benefit.…”

Section: Iron and E Histolyticamentioning

confidence: 99%

“…This assertion is based on the parasite growth in vitro in media depleted of iron and to which different iron proteins have been added. Trophozoites have been tested in cultures with hemoglobin, LF, TF, ferritin, and as the sole iron sources, and all of them have been utilized by the parasite for growth [Serrano-Luna et al, 1998;Reyes-López et al, 2001;León-Sicairos et al, 2005;López-Soto et al, 2009b]. In this way, amoebae could ensure the presence of iron for the colonization of the different organs and tissues involved in amoebic infection.…”

Section: Use Of Host Iron-containing Proteinsmentioning

confidence: 99%

“…Using pharmacological and immunofluorescence microscopy studies, the participation of clathrin protein in the endocytic process was demonstrated. Once inside the vacuoles, TF is transported into the endolysosomal system [Reyes-López et al, 2001;Reyes-López et al, 2011]. However, when the endocytic process was followed using high TF concentrations, the TF was internalized independently of the binding protein [Reyes-López et al, 2011].…”

Section: E Histolytica Transferrin Binding Proteins Ehtfbpsmentioning

confidence: 99%

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