Proteases from Entamoeba spp. and Pathogenic Free-Living Amoebae as Virulence Factors
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2.1.8. Proteins Containing Iron Are Degraded by Amoebic Proteases for Use as Iron Sources for Growth
Iron is a vital element for the survival of almost all organisms. However, under physiological conditions, Fe3+ is not soluble, and Fe2+ is soluble but toxic and readily oxidizes to Fe3+. To increase solubility, avoid toxicity, and keep iron away from intruders, this element is normally complexed to proteins; thus, the free iron concentration is far too low to sustain the growth of intruders. However, successful pathogens are able to scavenge iron from host proteins [179–183]. E. histolytica, as well as other amitochondriate protists (e.g., Tritrichomonas, Trichomonas, and Giardia), requires particularly high amounts of extracellular iron in vitro (~100 μM), surpassing that of the majority of both eukaryotic and prokaryotic cells (0.4–4 μM) [184]. This high iron requirement is attributable to the heavy reliance of their energy metabolism on Fe-S proteins [185–187].
We have reported that E. histolytica trophozoites are able to use four of the human iron-containing proteins as iron sources for the parasite’s growth in axenic culture medium in which ferric ammonium citrate was substituted by the ferrous- or ferric-protein under investigation. These proteins are hemoglobin, transferrin, lactoferrin, and ferritin [185]. In all cases, amoebae were able to endocytose and cleave the protein to obtain the needed iron (Figure 7). The use of these proteases by trophozoites could be considered a virulence factor because the pathogens seek out host iron to survive in the hostile host environment [179, 182, 183, 188, 189].
Hemoglobin. Hemoglobin (Hb) is a globular protein that is present at high concentrations in erythrocytes or red blood cells (RBCs). The function of Hb is to trap oxygen in the lungs and transport it through the blood to tissues and cells. In adult mammals, Hb is composed of two alpha and two beta chains, each containing one heme prosthetic group; therefore, there are four Fe2+ atoms in the Hb molecule, which has Mr of 64.5 kDa [190]. Hb uptake by E. histolytica trophozoites occurs by disrupting the RBC cytoplasmic membrane with surface hemolysins and phospholipases. The major amoebic hemolytic activity has been characterized in rat RBCs; this activity was detected in a vesicular fraction [191, 192]. An alkaline phospholipase has also been associated with virulence [193].
In E. histolytica, there is little information regarding how iron is obtained from Hb. This parasitic protist is extremely active as a phagocytic cell; once phagocytosed, human RBCs are broken down by amoebae. Chévez et al. [194] described complete RBC digestion in ~6–8 h by Perl’s staining experiments. Quantitative digestion assays using diaminobenzidine staining to visualize RBCs revealed that, after 9 h of RBC phagocytosis, Hb was thoroughly degraded [194–196]. Several researchers have studied the role of amoebic hemoglobinases in the cleavage of different types of Hb. The degradation of native bovine Hb at pH 7.6 by extracted proteinases from different monoxenic strains was observed [197]. Thirty-five years ago, two proteinases against native bovine Hb were purified [23]: one of 41 kDa, with optimal activity at pH 3.5, and another of 27 kDa, with optimal activity at pH 6.0. Subsequently, a cytotoxin of 22 kDa with strong proteolytic activity against denatured Hb at an optimal pH of 4.5 was described, and a cathepsin B of 16 kDa that was active against native and denatured Hb was purified [24–26]. Perez-Montfort et al. [24] identified two proteins of 32 and 40 kDa that were able to degrade denatured Hb.
Our group has described three proteases of 21, 82, and 116 kDa in extracts of E. histolytica HM-1:IMSS. These proteases were able to degrade human, bovine, and porcine Hbs, mainly at pH 7.0, and were inhibited by PHMB, E-64, NEM, and IA, all of which are specific CP inhibitors [27]. Becker et al. [198] reported a 30 kDa protease in vacuoles that previously contained phagocytosed RBCs; electrophoretic analysis revealed the incorporation of Hb monomers into trophozoites. In parallel to the decrease in human Hb during RBC digestion, X-ray analysis revealed a loss of iron content [198, 199]. In vitro assays have demonstrated that purified recombinant EhCP112 and EhCP5 are able to degrade Hb [8, 9].
Transferrin. In mammals, iron is mainly transported by transferrin (Tf), a protein found in serum and lymph that delivers iron to all sites, mainly to tissues with active cell division and bone marrow erythroid cells synthesizing Hb. Tf is part of a family that also contains lactoferrin. Tf has a for Fe3+ of M and is extremely stable against degradation when saturated. Tf is a glycoprotein of 80 kDa with two lobes, each containing one binding site with differing affinity for Fe3+. TfR1 is a dimeric glycoprotein of approximately 90 kDa per subunit that is expressed in nearly all cells [200, 201].
Interestingly, one of the amoebic receptors for holoTf is the acetaldehyde/alcohol dehydrogenase-2, an enzyme that requires iron. HoloTf is endocytosed through clathrin-coated vesicles and transported to lysosomes, likely losing the first iron in early endosomes and the second in lysosomes due to the acidic pH [28, 202]. To determine whether trophozoites possess cytoplasmic or secreted proteases that can degrade holoTf, total extracts and culture supernatants (SN) of medium with ferric citrate or in the absence of iron were analyzed for holoTf cleavage. Four bands of holoTf degradation corresponding to 130, 43, 20, and 6 kDa were observed in the extracts. In contrast, five bands of 130, 70, 50, 35, and 30 kDa were observed in the SN. All of the proteolytic activities were of the cysteine type. Secreted CPs could play a key role in cleaving Tf when amoebae travel by the portal vein to the liver and when, upon remaining in the liver, produce hepatic abscesses [28].
Lactoferrin. Lactoferrin (Lf) is a glycoprotein from the innate immune system that is secreted to mucosae; it is abundant in colostrum and milk. Lf is secreted without iron (apoLf) by the secondary granules of neutrophils at the infection site; thus, it is a marker of inflammatory bowel diseases (IBDs) [203]. One of the functions of Lf is to chelate iron to make it unavailable to intruders. Lf is a single polypeptide chain that is folded into two lobes; like Tf, each lobe can bind one Fe3+. The degree of Lf glycosylation determines its resistance to proteases and to very acidic conditions. Apo-Lf has a higher avidity for iron than apo-Tf. HoloLf releases iron only in very acidic environments (e.g., pH < 4), and its conformation changes according to the saturation state. When saturated, Lf is more stable and resistant to proteolysis [204–206].
HoloLf can be used as a sole iron source for in vitro growth by E. histolytica trophozoites in a similar fashion to that observed for ferric citrate. HoloLf was recognized by two proteins (45 and 90 kDa) located in the amoebic membrane, and its binding was specific [29]. HoloLf enters the amoeba by a clathrin-independent via (possibly caveolae-like structures). Following endocytosis, holoLf is found in vesicles similar to early endosomes and is then delivered to late endosomes and lysosomes. Delivery of holoLf to lysosomes may be required for its digestion by proteases and iron release, which only occurs in a very acidic milieu. CPs of 250, 100, 40, and 22 kDa from amoebic extracts cleaved holoLf at pH 7; however, the activity increased considerably at pH 4 [29]. In acidic lysosomes, the iron from holoLf is likely released, and the protein is degraded by CPs. Culture SN did not contain proteolytic activity against holoLf. Whether E. histolytica contains a reductase capable of changing the iron oxidation state remains unknown. This mechanism seems to be shared by other parasites, such as Tritrichomonas foetus [207]. As amoebae develop in the intestinal mucosa where Lf is found, this protein could be the iron source for the parasite at the beginning of infection, in addition to iron-containing bacterial proteins.
Ferritin. Due to the toxicity of iron, all life forms must have a mechanism to store/scavenge excess iron. Human ferritin is a major cytosolic protein with the capacity to capture up to 4,500 iron atoms. When the intracellular iron level increases, ferritin sequesters iron inside its cavity to detoxify the cell and prevent damage. Ferritin is abundant in the liver, which stores ~50% of the body’s total iron reserves. The mammalian ferritin family generally consists in spherical proteins. Each 474 kDa molecule consists of 24 subunits that are either heavy (H) or light (L) with a molecular mass of ~21 and ~19 kDa, respectively [208–211].
Ferritin uptake by amoeba may be mediated by a binding protein because it is concentration and time dependent, highly specific, and saturable at 46 nM ferritin. E. histolytica can cleave ferritin into several fragments. Three neutral CPs (100, 75, and 50 kDa) were observed to degrade ferritin in culture extracts. Ferritin entrance is constrained by inhibitors of clathrin-coated pits, and after 30 min of incubation, ferritin colocalized with an anti-rat LAMP-2 Ab in lysosomes [30]. The liver invasion by E. histolytica is poorly understood. Once liver cells are destroyed by amoebic enzymes, ferritin can be released and may be endocytosed by trophozoites and used as a source of iron and nutrients to form hepatic abscesses. In the liver, amoebae may also use Hb as an iron source; however, ferritin can provide up to 1,000-fold more iron than Hb. The capacity of E. histolytica to utilize ferritin as iron source may well explain its high pathogenic potential in the liver.
