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

Abstract

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 ...

Transferrin Binding Proteins as a Means to Obtain Iron in Parasitic Protozoa

“…This result is in agreement with the observation that TF internalization is unsaturable [Welter et al, 2006]. The presence of clathrin has been demonstrated in some protozoa and in E. histolytica [León-Sicairos et al, 2005;López-Soto et al, 2009a;Reyes-López et al, 2011]; clathrin may be important in parasites for the acquisition of nutrients. The gene encoding the clathrin protein has been identified in the E. histolytica genome [Loftus et al, 2005].…”

“…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]. This result is in agreement with the observation that TF internalization is unsaturable [Welter et al, 2006].…”

“…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].…”

“…This observation could be due to the presence of two binding proteins or only one protein with two different affinities [Reyes-López et al, 2011]. Comparing the affinity for TF in bacteria (0.7 a 4 x 10 -7 M) [Pintor et al, 1993] and Trypanosoma cruzi (2.8 X 10 -6 M) [Testa, 2002], the amoebic receptor presents the higher affinity.…”

TvMP50 is an Immunogenic Metalloproteinase during Male Trichomoniasis

“…Although EhADH3 is present on the plasma membrane surface of E. histolytica, the role of EhADH3 in the virulence of amoeba is still unknown (69). On the other hand, EhADH2 is a major adhesion factor only in pathogenic strains and is involved in the internalization of human transfer by E. histolytica (70). In T. vaginalis, the alcohol dehydrogenase activity is related to metronidazole-resistance.…”