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Entamoeba histolytica: A novel cysteine protease and an adhesin form the 112 kDa surface protein
Abstract
Here, we present evidence that a cysteine protease (EhCP112) and a protein with an adherence domain (EhADH112) form the Entamoeba histolytica 112 kDa adhesin. Immunoelectron microscopy and immunofluorescence assays using monoclonal antibodies (mAbAdh) revealed that, during phagocytosis, the adhesin is translocated from the plasma membrane to phagocytic vacuoles. mAbAdh inhibited 54% adherence, 41% phagocytosis, and 35% and 62% destruction of MDCK cell monolayers by live trophozoites and their extracts respectively. We cloned a 3587 bp DNA fragment (Eh112 ) with two open reading frames (ORFs) separated by a 188 bp non-coding region. The ORF at the 5' end (Ehcp112 ) encodes a protein with a cysteine protease active site, a transmembranal segment and an RGD motif. The second ORF (Ehadh112 ) encodes a protein recognized by mAbAdh with three putative transmembranal segments and four glycosylation sites. Northern blot, primer extension and Southern blot experiments revealed that Ehcp112 and Ehadh112 are two adjacent genes in DNA. Ehcp112 and Ehadh112 genes were expressed in bacteria. The recombinant peptides presented protease activity and inhibited adherence and phagocytosis, respectively, and both were recognized by mAbAdh. The EhCP112 and EhADH112 peptides could be joined by covalent or strong electrostatic forces, which are not broken during phagocytosis.
Supplementary resource (1)
Nucleotide Sequence
February 1999
... The EhCPADH complex, integrated by the EhADH adhesin and the EhCP112 cysteine protease (Garcıá-Rivera et al., 1999), and the prostaglandin E2, secreted from the amoeba, disrupt tight junctions of epithelial cells (Lejeune et al., 2011;Betanzos et al., 2013). Phagocytosis and trogocytosis are also involved in the removal of epithelial cells and invasion into the tissue (Labruyère and Guilleń, 2006;Ralston et al., 2014). ...
... To perform phagocytosis, the trophozoites need first to be attracted to and make contact with the prey . Several ESCRT proteins associate to EhADH and Gal/GalNAc (Bañuelos et al., 2012;Avalos-Padilla et al., 2015;Avalos-Padilla et al., 2018;Galindo et al., 2021), both involved in the adherence of trophozoites to the target cells (Garcıá-Rivera et al., 1999;Lopez-Vancell et al., 2000). Cellular signals initiate the pseudopodia formation and the capture of the prey. ...
... Particularly, phagocytosis begins with the adherence of trophozoites to target cells (Christy and Petri, 2011). The most characterized molecules in the primary contact are the Gal/ GalNAc lectin (Singh et al., 2016) and the EhADH adhesin ( Figure 1) (Garcıá-Rivera et al., 1999). Other proteins involved in the contact to RBCs and human colorectal adenocarcinoma (Caco-2) cells, include the C2-domain-containing protein kinase (EhC2PK) (Babuta et al., 2020) and the lysine and glutamic acid rich protein (KERP1) (Seigneur et al., 2005), respectively. ...
Entamoeba histolytica, the causative agent of human amoebiasis, exhibits a continuous membrane remodelling to exert its virulence properties. During this dynamic process, the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery is a key player, particularly in phagocytosis, a virulence hallmark of this parasite. In addition to ESCRT, other molecules contribute to membrane remodelling, including the EhADH adhesin, EhRabs, actin, and the lysobisphosphatidic acid (LBPA). The endocytosis of a prey or molecules induces membrane invaginations, resulting in endosome and multivesicular bodies (MVBs) formation for cargo delivery into lysosomes. Alternatively, some proteins are recycled or secreted. Most of these pathways have been broadly characterized in other biological systems, but poorly described in protozoan parasites. Here, we encompass 10 years of ESCRT research in E. histolytica, highlighting the role of the ESCRT-I and ESCRT-III components and the EhADH and EhVps4-ATPase accessory proteins during phagocytosis. In particular, EhADH exhibits a multifunctional role along the endocytic pathway, from cargo recognition to endosome maturation and lysosomal degradation. Interestingly, the interaction of EhADH with EhVps32 seems to shape a concurrent route to the conventional one for MVBs biogenesis, that could optimize their formation. Furthermore, this adhesin is secreted, but its role in this event remains under study. Other components from the endosomal pathway, such as EhVps23 and LBPA, are also secreted. A proteomic approach performed here, using an anti-LBPA antibody, revealed that some proteins related to membrane trafficking, cellular transport, cytoskeleton dynamics, and transcriptional and translational functions are secreted and associated to LBPA. Altogether, the accumulated knowledge around the ESCRT machinery in E. histolytica, points it out as a dynamic platform facilitating the interaction of molecules participating in different cellular events. Seen as an integrated system, ESCRTs lead to a better understanding of E. histolytica phagocytosis.
... Proteins that conform these complexes differ in sequence along organisms through the evolutionary scale (Leung et al., 2008), but conserve their functional domains that allow them to participate in cell division, endocytosis, virus budding and other functions (Calistri et al., 2021). In E. histolytica, ESCRT machinery is involved in phagocytosis (Lopez-Reyes et al., 2010;Avalos-Padilla et al., 2015;Avalos-Padilla et al., 2018), a virulence landmark of the parasite (Garcıá-Rivera et al., 1999). Thus, this protozoan is an excellent model to study the ESCRT machinery and its role in events involving vesicular trafficking, such as phagocytosis and tissue invasion (Alfred and Vaccari, 2016;Liu et al., 2019;Vietri et al., 2020). ...
... It is formed by TSG101 (named Vps23 in yeast), Vps28, Vps37, and UBAP1 (MVB12 in yeast) proteins ( Figure 1A). Vps23 from Saccharomyces cerevisiae and Homo sapiens orthologues have been already crystallized and their role in vital cellular functions has been studied (Kostelansky et al., 2007;Im et al., 2010;Ren and Hurley, 2011;Flower et al., 2020). In E. histolytica, the ESCRT-III proteins have been studied, but little is known on the other complexes of the ESCRT machinery. ...
... Another fluctuation peak was located at the carboxy-terminus from N358 to E494 amino acids, where an a-helix is formed ( Figure 2C). Thus, these analysis showed that EhVps23 has the 3D structure described for its orthologues (Kostelansky et al., 2007;Im et al., 2010;Ren and Hurley, 2011;Flower et al., 2020) with fourteen alpha helices and six beta-sheets ( Figure 2D). ...
The endosomal sorting complex required for transport (ESCRT) is formed by ESCRT-0, ESCRT-I, ESCRT-II, ESCRT-III complexes, and accessory proteins. It conducts vesicular trafficking in eukaryotes through the formation of vesicles and membrane fission and fusion events. The trophozoites of Entamoeba histolytica, the protozoan responsible for human amoebiasis, presents an active membrane movement in basal state that increases during phagocytosis and tissue invasion. ESCRT-III complex has a pivotal role during these events, but ESCRT-0, ESCRT-I and ESCRT-II have been poorly studied. Here, we unveiled the E. histolytica ESCRT-I complex and its implication in vesicular trafficking and phagocytosis, as well as the molecular relationships with other phagocytosis-involved molecules. We found a gene encoding for a putative EhVps23 protein with the ubiquitin-binding and Vps23 core domains. In basal state, it was in the plasma membrane, cytoplasmic vesicles and multivesicular bodies, whereas during phagocytosis it was extensively ubiquitinated and detected in phagosomes and connected vesicles. Docking analysis, immunoprecipitation assays and microscopy studies evidenced its interaction with EhUbiquitin, EhADH, EhVps32 proteins, and the lysobisphosphatidic acid phospholipid. The knocking down of the Ehvps23 gene resulted in lower rates of phagocytosis. Our results disclosed the concert of finely regulated molecules and vesicular structures participating in vesicular trafficking-related events with a pivotal role of EhVps23.
... To explore this idea, we used two distinct polyclonal antibodies, directed against the full-length EhADH protein (α-EhADH) and another against the 18 amino acids epitope at the C-terminus of the protein. In agreement with earlier results [34,41], in the control trophozoites, α-EhADH antibodies recognized the 112-124 kDa bands corresponding to the EhCPADH complex [41], but free EhADH was not visible. However, the α-EhADH antibodies detected the 75 kDa band corresponding to the free EhADH and the EhCPADH complex ( Figure 10A). ...
... To explore this idea, we used two distinct polyclonal antibodies, directed against the full-length EhADH protein (α-EhADH) and another against the 18 amino acids epitope at the C-terminus of the protein. In agreement with earlier results [34,41], in the control trophozoites, α-EhADH antibodies recognized the 112-124 kDa bands corresponding to the EhCPADH complex [41], but free EhADH was not visible. However, the α-EhADH antibodies detected the 75 kDa band corresponding to the free EhADH and the EhCPADH complex ( Figure 10A). ...
... These differences could be interpreted as necessary events to carry out the distinct functions of the proteins. Our work with EhADH since many years ago [41], has evidenced its interaction with many other molecules, such as EhVps32 [14] and EhNPC1 and EhNPC2 [57] as it has been described for other ALIX family proteins [58], thus, it is possible that the protein conformation could be altered according to the target protein. Moreover, these four proteins presented putative SUMOylation sites in our in silico screening. ...
Posttranslational modifications provide Entamoeba histolytica proteins the timing and signaling to intervene during different processes, such as phagocytosis. However, SUMOylation has not been studied in E. histolytica yet. Here, we characterized the E. histolytica SUMO gene, its product (EhSUMO), and the relevance of SUMOylation in phagocytosis. Our results indicated that EhSUMO has an extended N-terminus that differentiates SUMO from ubiquitin. It also presents the GG residues at the C-terminus and the ΨKXE/D binding motif, both involved in target protein contact. Additionally, the E. histolytica genome possesses the enzymes belonging to the SUMOylation-deSUMOylation machinery. Confocal microscopy assays disclosed a remarkable EhSUMO membrane activity with convoluted and changing structures in trophozoites during erythrophagocytosis. SUMOylated proteins appeared in pseudopodia, phagocytic channels, and around the adhered and ingested erythrocytes. Docking analysis predicted interaction of EhSUMO with EhADH (an ALIX family protein), and immunoprecipitation and immunofluorescence assays revealed that the association increased during phagocytosis; whereas the EhVps32 (a protein of the ESCRT-III complex)-EhSUMO interaction appeared stronger since basal conditions. In EhSUMO knocked-down trophozoites, the bizarre membranous structures disappeared, and EhSUMO interaction with EhADH and EhVps32 diminished. Our results evidenced the presence of a SUMO gene in E. histolytica and the SUMOylation relevance during phagocytosis. This is supported by bioinformatics screening of many other proteins of E. histolytica involved in phagocytosis, which present putative SUMOylation sites and the ΨKXE/D binding motif.
... Then, trophozoites adhere to epithelial cells through specific molecules that are mobilized from the parasite cytoplasm to the plasma membrane (Christy and Petri 2011;Fig. 20.1 Confocal microscopy image showing a trophozoite with multiple vesicles and vacuoles of distinct size, labelled by the anti-EhRab7 antibody García-Rivera et al. 1999;Ravdin and Guerrant 1981). Besides, trophozoites also secret proteins that reach and damage target cells (Biller et al. 2014;Cuellar et al. 2017;Ujang et al. 2016). ...
... On the other hand, previous work has evidenced the contribution of EhADH, alone or forming part of the EhCPADH complex, at early and late stages of trophozoites interaction with host cells. EhADH participates in adherence to and phagocytosis of target cells, due to an adherence epitope present at its C-terminus (García-Rivera et al. 1999). Moreover, EhADH displays a N-terminal Bro1 domain, a hallmark of the ALIX family, which mediates protein sorting and trafficking along the MVBs pathway. ...
... Once trophozoites reach the epithelium, they adhere to the cells by the EhGal/GalNAc lectin, EhADH, EhCPADH and other molecules transported from the cytoplasm to the parasite membrane (Betanzos et al. 2013;García-Rivera et al. 1999;Goplen et al. 2013;Petri et al. 2002). The lectin is composed by the light (Lgl), intermediate (Igl) and heavy (Hgl) subunits (Petri et al. 2002). ...
Vesicular trafficking is based on membrane remodelling that produces vesicles with distinct composition. This allows the transport of nutrients and molecules, and the communication among intracellular organelles. Vesicular trafficking is one of the main mechanisms for pathogens virulence. Besides, multiple factors elicit parasite resistance to host defences, changing the environment during tissue invasion. Virulence factors are linked to the secretory (exocytosis) and importing (endocytosis) pathways. In this paper, we discuss the relevance of vesicular trafficking in the virulence of the protozoan Entamoeba histolytica. An efficient function of virulence factors during adherence to and phagocytosis of target cells and tissue invasion by trophozoites, requires the transport of specific molecules. The EhGal/GalNAc lectin, the EhCPADH complex, phosphoinositides, KERP1, EhRabs, EhCPs, LBPA and cholesterol are some of the molecules involved in the uptake, movement and digestion of the prey. Moreover, phagocytosis is an event that depends on active membranes fusion and fission, finely controlled by rearrangements of the actin cytoskeleton. During this process, a diversity of signalling transducers and effector molecules are implicated in endosomes and phagosomes formation and maturation, including the ESCRT machinery. Furthermore, some of these molecules participate in the exocytosis and others are secreted by the parasite and reach the target cell to initiate the invasion. For instance, proteins forming the EhCPADH complex, involved in adherence and phagocytosis, are secreted. EhCP112 and EhADH reach target cells and penetrate them by caveolae or clathrin-coated vesicles. These proteins destroy the intercellular junctions and provoke cell detachment and the impairment of epithelium homeostasis, eventually resulting in symptomatic manifestations of amoebiasis. In summary, vesicular trafficking results determinant for the transport of virulence factors during the whole E. histolytica pathogenesis process.
... EhCP112 couples with the EhADH adhesin to form the EhCPADH complex, which is involved in several virulence mechanisms [56,57]. The EhCPADH complex, as well as EhADH and EhCP112, are peripheral membrane proteins, also localized in cytoplasmic vesicles, susceptible of being secreted by trophozoites [54,58,59]. ...
... Trophozoites adhere to host cells mainly by adhesins and lectins [63]. The most well-known molecules are the EhCPADH complex and the EhGal/GalNAc lectin [51,56]. However, other molecules seem to be involved in this episode, such as the 220-lectin [64,65], a lysine and glutamic acid enriched protein (KERP1) [66], the pyruvate:ferredoxin oxidoreductase (PFO) [67] and the E. histolytica rhomboid protein 1 (EhROM1) [68]. ...
... Monoclonal antibodies against the EhCPADH complex diminish the ability of parasites to make contact with epithelial cells and erythrocytes [56]. EhADH and EhCP112 also contribute to the adherence to target cells. ...
The epithelium represents the first and most extensive line of defence against pathogens, toxins and pollutant agents in humans. In general, pathogens have developed strategies to overcome this barrier and use it as an entrance to the organism. Entamoeba histolytica, Naegleria fowleri and Acanthamoeba spp. are amoebae mainly responsible for intestinal dysentery, meningoencephalitis and keratitis, respectively. These amoebae cause significant morbidity and mortality rates. Thus, the identification, characterization and validation of molecules participating in host-parasite interactions can provide attractive targets to timely intervene disease progress. In this work, we present a compendium of the parasite adhesins, lectins, proteases, hydrolases, kinases, and others, that participate in key pathogenic events. Special focus is made for the analysis of assorted molecules and mechanisms involved in the interaction of the parasites with epithelial surface receptors, changes in epithelial junctional markers, implications on the barrier function, among others. This review allows the assessment of initial host-pathogen interaction, to correlate it to the potential of parasite invasion.
... All of the genes predict proteins with 34 to 39% identity to papain, 36 to 46% identity to cathepsin L, and complete conservation of all residues known to be critical for cysteine proteinase function. Orozco's group has recently described a novel surface cysteine proteinase which is fused with an adhesin (29). The cysteine proteinase has a transmembrane sequence and an RGD (integrin attachment) domain (29). ...
... Orozco's group has recently described a novel surface cysteine proteinase which is fused with an adhesin (29). The cysteine proteinase has a transmembrane sequence and an RGD (integrin attachment) domain (29). Whether the increased cysteine proteinase activity and invasive potential of E. histolytica can be attributed to the expression of specific cysteine proteinase genes or an overall increased level of cysteine proteinase activity remains to be determined. ...
... EhCP5 is thought to associate with membranes of E. histolytica by virtue of a hydrophobic patch sequence (43). The most recently identified surface cysteine proteinase, the 112-kDa cysteine proteinase-adhesin fusion, does have a transmembrane domain and an RGD sequence (29). During phagocytosis of erythrocytes, the entire protein is translocated to phagocytic vesicles (29). ...
Amebiasis is a major cause of morbidity and mortality throughout the tropical world. Entamoeba histolytica is now recognized as a separate species from the morphologically identical E. dispar, which cannot invade. Cysteine proteinases are a key virulence factor of E. histolytica and play a role in intestinal invasion by degrading the extracellular matrix and circumventing the host immune response through cleavage of secretory immunoglobulin A (sIgA), IgG, and activation of complement. Cysteine proteinases are encoded by at least seven genes, several of which are found in E. histolytica but not E. dispar. A number of new animal models, including the formation of liver abscesses in SCID mice and intestinal infection in human intestinal xenografts, have proven useful to confirm the critical role of cysteine proteinases in invasion. Detailed structural analysis of cysteine proteinases should provide further insights into their biochemical function and may facilitate the design of specific inhibitors which could be used as potential chemotherapeutic agents in the future.
... Subsequently, epithelial cells are detached from the substrate and phagocytosed by the parasite (Martínez-Palomo et al., 1985). Several molecules are involved in this process, such as Gal/GalNAc lectin, amoebapores, cysteine and serine proteases, prostaglandin E2 (PGE2), the EhCPADH complex, among others (Chadee et al., 1987;Leippe, 1997;García-Rivera et al., 1999;Meléndez-López et al., 2007;Lejeune et al., 2011;Cornick et al., 2016). ...
... The EhCPADH complex (Arroyo and Orozco, 1987), formed by an adhesin (EhADH) and a cysteine protease (EhCP112), participates in adhesion, cytolysis and phagocytosis of target cells (García-Rivera et al., 1999). EhCPADH, EhADH, and EhCP112 are secreted during trophozoite attack (Ocádiz et al., 2005;Bolaños et al., 2016). ...
... Moreover, EhADH associates to cholesterol-trafficking proteins EhNPC1 and EhNPC2, suggesting an extra role in the uptake and transport of this essential lipid toward cellular membranes (Bolaños et al., 2016). Monoclonal antibodies (mAbAdh) against the C-terminal adherence domain (residues 480-600) of this protein (Montaño et al., 2017), inhibit trophozoite adhesion to and phagocytosis of erythrocytes, as well as destruction of MDCK cell monolayers (García-Rivera et al., 1999). However, the specific role of EhADH on epithelium damage has not been fully studied. ...
In Entamoeba histolytica, the EhADH adhesin together with the EhCP112 cysteine protease, form a 124 kDa complex named EhCPADH. This complex participates in trophozoite adherence, phagocytosis and cytolysis of target cells. EhCPADH and EhCP112 are both involved on epithelium damage, by opening tight junctions (TJ) and reaching other intercellular junctions. EhADH is a scaffold protein belonging to the ALIX family that contains a Bro1 domain, expresses at plasma membrane, endosomes and cytoplasm of trophozoites, and is also secreted to the medium. Contribution of EhADH to TJ opening still remains unknown. In this paper, to elucidate the role of EhADH on epithelium injury, we followed two strategies: producing a recombinant protein (rEhADH) and transfecting the ehadh gene in MDCK cells. Results from the first strategy revealed that rEhADH reached the intercellular space of epithelial cells and co-localized with claudin-1 and occludin at TJ region; later, rEhADH was mainly internalized by clathrin-coated vesicles. In the second strategy, MDCK cells expressing EhADH (MDCK-EhADH) showed the adhesin at plasma membrane. In addition, MDCK-EHADH cells exhibited adhesive features, producing epithelial aggregation and adherence to erythrocytes, as described in trophozoites. Surprisingly, the adhesin expression produced an increase of claudin-1, occludin, ZO-1 and ZO-2 at TJ, and also the transepithelial electric resistance (TEER), which is a measure of TJ gate function. Moreover, MDCK-EhADH cells resulted more susceptible to trophozoites attack, as showed by TEER and cytopathic experiments. Overall, our results indicated that EhADH disturbed TJ from the extracellular space and also intracellularly, suggesting that EhADH affects by itself TJ proteins, and possibly synergizes the action of other parasite molecules during epithelial invasion.
... They are localized in the plasma membrane, in phagocytic cups (Arroyo and Orozco, 1987;Petri et al., 2002;Seigneur et al., 2005;Jain et al., 2008), and in the endosomes and internal membranes (Saito-Nakano et al., 2004;Loftus et al., 2005;Castellanos-Castro et al., 2016). Earlier, we identified the EhADH protein (an ALIX family member) that together with EhCP112 (a cysteine protease), forms the EhCPADH virulence complex (García-Rivera et al., 1999), which interacts with the Gal/Gal lectin at the trophozoites surface (Seigneur et al., 2005). EhADH possesses an adherence epitope at the C-terminus, which interacts with target cells (Arroyo and Orozco, 1987;García-Rivera et al., 1999) and a Bro1 domain at the N-terminus that faces the cytoplasm (Bañuelos et al., 2005). ...
... Earlier, we identified the EhADH protein (an ALIX family member) that together with EhCP112 (a cysteine protease), forms the EhCPADH virulence complex (García-Rivera et al., 1999), which interacts with the Gal/Gal lectin at the trophozoites surface (Seigneur et al., 2005). EhADH possesses an adherence epitope at the C-terminus, which interacts with target cells (Arroyo and Orozco, 1987;García-Rivera et al., 1999) and a Bro1 domain at the N-terminus that faces the cytoplasm (Bañuelos et al., 2005). The EhADH protein binds to the EhVps32 protein (Bañuelos et al., 2012;Avalos-Padilla et al., 2015), which is the most abundant member of the ESCRT machinery. ...
... In E. histolytica, the precise function for the majority of the ESCRT products is unknown, even when almost all ESCRT genes are present in the genome (López-Reyes et al., 2011). EhVps32, the ortholog of Snf7 in yeast and CHMP4 in human, as well as the accessory proteins EhADH (an ALIX family protein) and the EhVps4 ATPase are involved in the phagocytosis of trophozoites (García-Rivera et al., 1999;López-Reyes et al., 2010;Avalos-Padilla et al., 2015). Additionally, EhVps32 and EhADH are also involved in pinocytosis of dextran and phagocytosis of latex-coated beads (Avalos-Padilla et al., 2015;Castellanos-Castro et al., 2016) which implies that ESCRT-III proteins participate in different types of endocytosis. ...
The endosomal sorting complex required for transport (ESCRT) orchestrates cell membrane-remodeling mechanisms in eukaryotes, including endocytosis. However, ESCRT functions in phagocytosis (ingestion of ≥250 nm particles), has been poorly studied. In macrophages and amoebae, phagocytosis is required for cell nutrition and attack to other microorganisms and cells. In Entamoeba histolytica, the voracious protozoan responsible for human amoebiasis, phagocytosis is a land mark of virulence. Here, we have investigated the role of ESCRT-III in the phagocytosis of E. histolytica, using mutant trophozoites, recombinant proteins (rEhVps20, rEhVps32, rEhVps24, and rEhVps2) and giant unilamellar vesicles (GUVs). Confocal images displayed the four proteins located around the ingested erythrocytes, in erythrocytes-containing phagosomes and in multivesicular bodies. EhVps32 and EhVps2 proteins co-localized at the phagocytic cups. Protein association increased during phagocytosis. Immunoprecipitation and flow cytometry assays substantiated these associations. GUVs revealed that the protein assembly sequence is essential to form intraluminal vesicles (ILVs). First, the active rEhVps20 bound to membranes and recruited rEhVps32, promoting membrane invaginations. rEhVps24 allowed the detachment of nascent vesicles, forming ILVs; and rEhVps2 modulated their size. The knock down of Ehvps20 and Ehvps24genes diminished the rate of erythrophagocytosis demonstrating the importance of ESCRT-III in this event. In conclusion, we present here evidence of the ESCRT-III participation in phagocytosis and delimitate the putative function of proteins, according to the in vitro reconstruction of their assembling.
... On the other hand, the a-H4K20me2 and a-H4K20me3 detected a 16 kDa band only in the nuclear extracts of trophozoites and a 14 kDa band in nuclear extracts of HeLa cells (Fig. 6A). As a control for fractionation we incubated the nuclear and cytoplasmic extracts of E. histolytica with antibodies against unmethylated H4 and against the cytoplasmic EhCPADH protein (Garcia-Rivera et al., 1999). These antibodies detected the respective protein only in the corresponding fraction (Fig. 6A). ...
... Protein members of the Endosomal Sorting Complex Required for Transport (ESCRT), such as EhADH and EhVps32, participate in E. histolytica phagocytosis; and they are immunodetected also around ingested erythrocytes (Garcia-Rivera et al., 1999;Avalos-Padilla et al., 2015).Therefore, to support the hypothesis that EhHKMT2 and EhHKMT4 are involved in E. histolytica phagocytosis, we analyzed the co-localization of these enzymes with the EhADH protein after 20 min of phagocytosis, time where we observed a higher association of EhHKMTs with vesicles containing ingested cells. Results showed that EhHKMT2 and EhHKMT4 colocalized with EhADH surrounding some ingested erythrocytes, whereas EhHKMT1 did not co-localize with the complex (Fig. 9A). ...
... Lysine methylation may be important not only in epigenetic regulation of gene expression, but also in the regulation of cellular signal transduction pathways (Kurash et al., 2008;Van Duyne et al., 2008;Abu-Farha et al., 2011;Herz et al., 2013;Jakobsson et al., 2015;Zhang et al., 2015;Vershinin et al., 2016). During E. histolytica phagocytosis, an event related to its pathogenicity (Orozco et al., 1983), EhHKMT2 and EhHKMT4 were located around ingested erythrocytes and they colocalized and co-immunoprecipitated with EhADH, a protein involved in phagocytosis (Rodriguez et al., 1989;Garcia-Rivera et al., 1999), suggesting that these EhHKMTs could methylate some proteins that are implicated in this process. Cytoplasmic proteins that participate in or regulate the cytoskeleton could be targets of these enzymes, because some of they have been found to be methylated in lysine residues during neural crest migration (Vermillion et al., 2014), other event where cytoskeleton plays an essential role. ...
Lysine methylation of histones, a posttranslational modification catalyzed by lysine methyltransferases (HKMTs), plays an important role in the epigenetic regulation of transcription. Lysine methylation of non-histone proteins also impacts the biological function of proteins. Previously it has been shown that lysine methylation of histones of Entamoeba histolytica, the protozoan parasite that infects 50 million people worldwide each year and causing up to 100 000 deaths annually, is implicated in the epigenetic machinery of this microorganism. However, the identification and characterization of HKMTs in this parasite had not yet been determined. In this work we identified four HKMTs in E. histolytica (EhHKMT1 to EhHKMT4) that are expressed by trophozoites. Enzymatic assays indicated that all of them are able to transfer methyl groups to commercial histones. EhHKMT1, EhHKMT2 and EhHKMT4 were detected in nucleus and cytoplasm of trophozoites. In addition EhHKMT2 and EhHKMT4 were located in vesicles containing ingested cells during phagocytosis, and they co-immunoprecited with EhADH, a protein involved in the phagocytosis of this parasite. Results suggest that E. histolytica uses its HKMTs to regulate transcription by epigenetic mechanisms, and at least two of them could also be implicated in methylation of proteins that participate in phagocytosis. This article is protected by copyright. All rights reserved.
... The inactive enzyme pEhCP112 (426 residues) is generated when the native enzyme ppEhCP112 (446 residues) loses the signal peptide (20 residues) and the active form mEhCP112 (310 residues) is produced after losing the propeptide (96 residues) (Garcia-Rivera et al., 1999). It is unknown which one of the protease forms is present in the EhCPADH complex. ...
... It is unknown which one of the protease forms is present in the EhCPADH complex. The EhCP112 catalytic site is formed by C167, H329, and N348 residues, and it also possesses a RGD motif (R250, G251, and D252 residues), which is an integrin binding site (Garcia-Rivera et al., 1999). Ehcp112 gene overexpression confers pathogenic phenotype to non-pathogenic trophozoites (Matthiesen et al., 2013); while its silencing reduces the in vitro and in vivo trophozoites virulence (Ocádiz-Ruiz et al., 2013. ...
... According to earlier reports, the EhCPADH complex is located at plasma membrane, in cytoplasm, in vesicles and it is secreted (Garcia-Rivera et al., 1999;Ocadiz et al., 2005). Additionally, the EhCP112 protein has a hydrophobic region (K395 to L416 residues), suggesting that it could be a transmembrane protein (Garcia-Rivera et al., 1999). ...
EhCPADH is a protein complex involved in the virulence of Entamoeba histolytica, the protozoan responsible for human amoebiasis. It is formed by the EhCP112 cysteine protease and the EhADH adhesin. To explore the molecular basis of the complex formation, three-dimensional models were built for both proteins and molecular dynamics simulations (MDS) and docking calculations were performed. Results predicted that the pEhCP112 proenzyme and the mEhCP112 mature enzyme were globular and peripheral membrane proteins. Interestingly, in pEhCP112, the propeptide appeared hiding the catalytic site (C167, H329, N348); while in mEhCP112, this site was exposed and its residues were found structurally closer than in pEhCP112. EhADH emerged as an extended peripheral membrane protein with high fluctuation in Bro1 and V shape domains. 500 ns-long MDS and protein-protein docking predictions evidenced different heterodimeric complexes with the lowest free energy. pEhCP112 interacted with EhADH by the propeptide and C-terminal regions and mEhCP112 by the C-terminal through hydrogen bonds. In contrast, EhADH bound to mEhCP112 by 442 to 479 residues, adjacent to the target cell-adherence region (480 to 600 residues), and by the Bro1 domain (9 to 349 residues). Calculations of the effective binding free energy and per residue free energy decomposition showed that EhADH binds to mEhCP112 with a higher binding energy than to pEhCP112, mainly through van der Waals interactions and the nonpolar part of solvation energy. The EhADH and EhCP112 structural relationship was validated in trophozoites by immunofluorescence, TEM and immunoprecipitation assays. Experimental findings fair agreed with in silico results.
... Trophozoites are professional phagocytes and constitute the mobile and invasive phase of the parasite. Several proteins participating in phagocytosis have been identified, among them the Gal/GalNac lectin [2], EhC2PK, EhCaBP1, EhAK1 [3,4], several EhRab proteins [5][6][7][8][9] and the EhCPADH complex [10]. EhCPADH is formed by a protease (EhCP112) and an adhesin (EhADH) [10], a member of the ALIX family [11,12]. ...
... Several proteins participating in phagocytosis have been identified, among them the Gal/GalNac lectin [2], EhC2PK, EhCaBP1, EhAK1 [3,4], several EhRab proteins [5][6][7][8][9] and the EhCPADH complex [10]. EhCPADH is formed by a protease (EhCP112) and an adhesin (EhADH) [10], a member of the ALIX family [11,12]. Lipids also influence the endosome membrane properties by changing biophysical characteristics and by recruiting proteins involved in membrane remodeling [13]. ...
... Anti-EhADH and 6C4 antibodies recognized both immunoprecipitates (Fig. 11A,B). In western blot assays using these immunoprecipitates, anti-EhADH antibody recognized EhADH (75 kDa) and the EhCPADH complex (124 kDa), as described [10]. Immunoprecipitates produced using erythrocyte lysates gave negative results with anti-EhADH and 6C4 antibodies, as well as with pre-immune serum and with lecithin ( Fig. 11A, B, and D). ...
Phospholipids are essential for vesicle fusion and fission and both are fundamental events for Entamoeba histolytica phagocytosis. Our aim was to identify the lysobisphosphatidic acid (LBPA) in trophozoites and investigate its cellular fate during endocytosis. LBPA was detected by TLC in a 0.5 Rf spot of total lipids, which co-migrated with the LBPA standard. The 6C4 antibody, against LBPA recognized phospholipids extracted from this spot. Reverse phase LC-ESI-MS and MS/MS mass spectrometry revealed six LBPA species of m/z 772.58–802.68. LBPA was associated to pinosomes and phagosomes. Intriguingly, during pinocytosis, whole cell fluorescence quantification showed that LBPA dropped 84% after 15min incubation with FITC-Dextran, and after 60min, it increased at levels close to steady state conditions. Similarly, during erythrophagocytosis, after 15min, LBPA also dropped in 36% and increased after 60 and 90min. EhRab7A protein appeared in some vesicles with LBPA in steady state conditions, but after phagocytosis co-localization of both molecules increased and in late phases of erythrophagocytosis they were found in huge phagosomes or multivesicular bodies with many intraluminal vacuoles, and surrounding ingested erythrocytes and phagosomes. The 6C4 and anti-EhADH (EhADH is an ALIX family protein) antibodies and Lysotracker merged in about 50% of the vesicles in steady state conditions and throughout phagocytosis. LBPA and EhADH were also inside huge phagosomes. These results demonstrated that E. histolytica LBPA is associated to pinosomes and phagosomes during endocytosis and suggested differences of LBPA requirements during pinocytosis and phagocytosis.
... Although the corresponding proteins differ slightly in their molecular weight and enzymatic properties, it was postulated that all these are related [28]. Subsequent molecular cloning revealed a large number of cysteine peptidase genes in the E. histolytica genome [29][30][31][32][33][34][35]. Until 2003, in total 20 genes coding for cysteine peptidases of E. histolytica were described, all of which belonged to the C1 papain superfamily of cysteine peptidases [30]. ...
... EhCP-B9 forms a complex with an adherence domain protein (EhADH112). As a complex, both proteins bind to target cells and are translocated during phagocytosis from the plasma membrane to phagocytic vacuoles [32,52]. Furthermore, EhCP-B9 has a putative transmembrane domain and contains an RGD (Arg-Gly-Asp) motif at positions 249-251 between the active site cysteine-167 and histidine-328 [32]. ...
... As a complex, both proteins bind to target cells and are translocated during phagocytosis from the plasma membrane to phagocytic vacuoles [32,52]. Furthermore, EhCP-B9 has a putative transmembrane domain and contains an RGD (Arg-Gly-Asp) motif at positions 249-251 between the active site cysteine-167 and histidine-328 [32]. This RDG motif is also found in EhCP-B8 at a similar position (245-247), as well as in the pro-region of EhCP-A5 (positions 92-94). ...
Entamoeba histolytica is characterized by its extraordinary capacity to invade and destroy human tissues. The main lytic activity has been attributed to cysteine peptidases, and a number of studies have shown that cysteine peptidases constitute major pathogenicity factors in E. histolytica. Interestingly, although most of the classes of peptidases are present in E. histolytica, only cysteine peptidases, and on a lesser scale, metallo-peptidases and serine peptidases, have been adequately studied. In this chapter, the peptidase families of E. histolytica are introduced, and their involvement in colonic invasion and in liver abscess formation are discussed.
... The E. histolytica cysteine proteinase-adhesin (EhCPADH) complex, a surface membrane and vacuolar heterodimeric E. histolytica protein complex formed by a cysteine protease (EhCP112, 446 amino acids) and an adhesin (EhADH, 687 amino acids), has been shown to be involved in the cytopathic mechanism of this parasite (Arroyo and Orozco, 1987;Garcia-Rivera et al. 1999;Betanzos et al. 2013). In vitro, a recombinant EhCP112 polypeptide digests gelatin, type I collagen, fibronectin and hemoglobin, as well as binds to red blood cells and destroys Madin Darby Canine Kidney (MDCK) cell monolayers (Ocadiz et al. 2005). ...
... Interestingly, the subcutaneous immunization of hamsters with a recombinant polypeptide containing the last 243 amino acids of the EhADH carboxyterminus (rEhADH243) as well as DNA vaccination with a mixture of Ehcp112 and Ehadh genes decreased the formation of hepatic abscesses (Madriz et al. 2004;Martinez-Lopez et al. 2004;Martinez et al. 2009). Moreover, genes encoding EhCP112 and EhADH are located adjacent to each other in the E. histolytica genome, separated by only 188 bp (Garcia-Rivera et al. 1999). This close gene proximity suggests a possible co-regulation in their expression, although this assumption has not been experimentally tested. ...
... It has been shown previously that a cysteine protease and an adhesin form the EhCPADH complex, and that this complex is involved in the pathogenic mechanism of E. histolytica (Arroyo and Orozco, 1987;Madriz et al. 2004;Martinez-Lopez et al. 2004;Ocadiz et al. 2005;Betanzos et al. 2013). EhCP112 and EhADH have been shown to be expressed as separate proteins encoded by different genes, as well as being able to form the EhCPADH complex (Garcia-Rivera et al. 1999;Banuelos et al. Fig. 5. ...
Entamoeba histolytica is the protozoan parasite causative of human amoebiasis, disease responsible for 40 000-100 000 deaths annually. The cysteine proteinase-adhesin complex of this parasite (EhCPADH) is a heterodimeric protein formed by a cysteine protease (EhCP112) and an adhesin (EhADH) that plays an important role in the cytopathic mechanism of this parasite. The coding genes for EhCP112 and EhADH are adjacent in the E. histolytica genome, suggesting that their expression may be co-regulated, but this hypothesis has not yet been confirmed. Here, we performed the knockdown of EhCP112 and EhADH using gene-specific short-hairpin RNAs (shRNA), and the effect of these knockdowns on the expression of both complex components as well as on the in vitro and in vivo virulence was analysed. Results showed that the knockdown of one of the EhCPADH components produced a simultaneous downregulation of the other protein. Accordingly, a concomitant reduction in the overall expression of the complex was observed. The downregulation of each component also produced a significant decrease in the in vitro and in vivo virulence of trophozoites. These results demonstrated that the expression of EhCP112 and EhADH is co-regulated and confirmed that the EhCPADH complex plays an important role in E. histolytica virulence.
... Entamoeba histolytica is the protozoan responsible for human amoebiasis, considered the third cause of death in the world due to parasitic diseases [1]. Phagocytosis is a key factor in the parasite virulence and several proteins involved in this event have been already unveiled [2][3][4][5][6][7][8][9], among them the Gal/GalNac lectin [10], EhC2PK, EhCaBP1, EhAK1 [4,11,12] and the EhC-PADH complex, formed by a protease (EhCP112) and an adhesin (EhADH) [2], which is a member of the ALIX (apoptosis-linked gene 2-interacting protein X) family [13]. In addition to the Bro1 domain located at its N-terminus, EhADH possesses an adherence epitope at the C-terminus which functions as a receptor during adherence to and phagocytosis of erythrocytes [2,13,14]. ...
... Entamoeba histolytica is the protozoan responsible for human amoebiasis, considered the third cause of death in the world due to parasitic diseases [1]. Phagocytosis is a key factor in the parasite virulence and several proteins involved in this event have been already unveiled [2][3][4][5][6][7][8][9], among them the Gal/GalNac lectin [10], EhC2PK, EhCaBP1, EhAK1 [4,11,12] and the EhC-PADH complex, formed by a protease (EhCP112) and an adhesin (EhADH) [2], which is a member of the ALIX (apoptosis-linked gene 2-interacting protein X) family [13]. In addition to the Bro1 domain located at its N-terminus, EhADH possesses an adherence epitope at the C-terminus which functions as a receptor during adherence to and phagocytosis of erythrocytes [2,13,14]. ...
... Phagocytosis is a key factor in the parasite virulence and several proteins involved in this event have been already unveiled [2][3][4][5][6][7][8][9], among them the Gal/GalNac lectin [10], EhC2PK, EhCaBP1, EhAK1 [4,11,12] and the EhC-PADH complex, formed by a protease (EhCP112) and an adhesin (EhADH) [2], which is a member of the ALIX (apoptosis-linked gene 2-interacting protein X) family [13]. In addition to the Bro1 domain located at its N-terminus, EhADH possesses an adherence epitope at the C-terminus which functions as a receptor during adherence to and phagocytosis of erythrocytes [2,13,14]. BRO1 was described as endosome associated protein that functions in the multivesicular bodies (MVBs) pathway in Saccharomyces cerevisiae [15]. EhADH interacts with EhVps32 [16], a protein described in mammals as a member of the endosomal sorting complex required for transport (ESCRT). ...
Here, we investigated the role of EhVps32 protein (a member of the endosomal-sorting complex required for transport) in endocytosis of Entamoeba histolytica, a professional phagocyte. Confocal microscopy, TEM and cell fractionation revealed EhVps32 in cytoplasmic vesicles and also located adjacent to the plasma membrane. Between 5 to 30 min of phagocytosis, EhVps32 was detected on some erythrocytes-containing phagosomes of acidic nature, and at 60 min it returned to cytoplasmic vesicles and also appeared adjacent to the plasma membrane. TEM images revealed it in membranous structures in the vicinity of ingested erythrocytes. EhVps32, EhADH (an ALIX family member), Gal/GalNac lectin and actin co-localized in the phagocytic cup and in some erythrocytes-containing phagosomes, but EhVps32 was scarcely detected in late phagosomes. During dextran uptake, EhVps32, EhADH and Gal/GalNac lectin, but not actin, co-localized in pinosomes. EhVps32 recombinant protein formed oligomers composed by rings and filaments. Antibodies against EhVps32 monomers stained cytoplasmic vesicles but not erythrocytes-containing phagosomes, suggesting that in vivo oligomers are formed on phagosome membranes. The involvement of EhVps32 in phagocytosis was further study in pNeoEhvps32-HA-transfected trophozoites, which augmented almost twice their rate of erythrophagocytosis as well as the membranous concentric arrays built by filaments, spirals and tunnel-like structures. Some of these structures apparently connected phagosomes with the phagocytic cup. In concordance, the EhVps32-silenced G3 trophozoites ingested 80% less erythrocytes than the G3 strain. Our results suggest that EhVps32 participates in E. histolytica phagocytosis and pinocytosis. It forms oligomers on erythrocytes-containing phagosomes, probably as a part of the scission machinery involved in membrane invagination and intraluminal vesicles formation.
... With the cloning of the 112 kDa-adhesin in 1999, it was possible to identify and locate the cellular adhesion domain [13]. It was also determined that the 112 kDa adhesin is formed by two polypeptides of 49 and 75 kDa encoded by different genes separated by a non-coding sequence of 188 bp. ...
... On the other hand, the 75 kDa protein (EhADH112) has a domain involved in the adherence of trophozoites to target cells. The molecular weight of the whole molecule ranges between 109 and 124 kDa, due to the posttranslational addition of carbohydrate residues to EhCP112 [13]. ...
... With the use of the recombinant protein, it was confirmed that the 112 kDa adhesin is translocated during phagocytosis from the plasma membrane to phagocytic vacuoles and, after 30 min of interaction, it returns to the plasma membrane. Due to its direct involvement in phagocytosis, it was called phagosine [13]. ...
Entamoeba histolytica is the causative agent of amebiasis in humans and is responsible for 100,000 deaths annually, making it the third leading cause of death due to a protozoan parasite. Pathogenesis appears to result from the potent cytotoxic activity of the parasite, which kills host cells within minutes. Although the mechanism is unknown, it is well established to be contact-dependent. The life cycle of the parasite alternates with two forms: the resistant cyst and the invasive trophozoite. The adhesive interactions between the parasite and surface glycoconjugates of host cells, as well as those lining the epithelia, are determinants for invasion of human tissues, for its cytotoxic activity, and finally for the outcome of the disease. In this review we present an overview of the information available on the amebic lectins and adhesins that are responsible of those adhesive interactions and we also refer to their effect on the host immune response. Finally, we present some concluding remarks and perspectives in the field.
... La segunda molécula de adherencia más estudiada de este protozoo, se denomina adhesina EhADH112. Esta molécula de 75 kDa se encuentra en el complejo inmunogénico EhCPADH, complejo de proteínas heterodiméricas que tiene un peso molecular de 124,6 kDa, está codificado por dos genes diferentes que se encuentran separados por 188 pb (35) y se halla localizado en la membrana plasmática y en las vacuolas citoplasmáticas (36) . ...
... La adhesina EhADH112 es una glucoproteína que, además, está involucrada directamente en la ingestión de las células diana por los trofozoítos. Por esto se considera que es una fagosina (proteína involucrada en la fagocitosis) (35) . ...
... El complejo EhCPADH está involucrado en la adhesión, el efecto citopático y, por la EhADH112, en a fagocitosis, y a su vez, comprende, además de la adhesina, a otro polipéptido, la proteasa de cisteína EhCP112 (49 kDa) (36) . El complejo está unido potentemente por enlaces covalentes o fuerzas electroestáticas (35) y no se separa ni durante la fagocitosis, ni durante el efecto citopático (36) . ...
Entamoeba histolytica es un protozoo entérico causante de la amebiasis intestinal y extraintestinal. Se calcula que 10% de la población mundial está infectada por el complejo Entamoeba histolytica/Entamoeba dispar. Según la OMS, hay 500 millones de nuevas infecciones por año y, aproximadamente, 70.000 a 100.000 muertes a causas de ellas. Este parásito cumple un proceso de invasión muy elaborado, en el cual se secretan y expresan proteínas que le permiten adherirse al epitelio, degradar la matriz extracelular y producir citólisis de las células epiteliales para penetrar dentro de la mucosa. El entendimiento de estos factores de virulencia ha generado múltiples estudios en diferentes áreas de las ciencias biomédicas, desde métodos diagnósticos cada vez más sensibles y específicos hasta candidatos para vacunas, lo que abre nuevas expectativas terapéuticas a raíz de estos estudios.The enteric protozoan parasite Entamoeba histolytica is a human pathogen that causes widespread morbidity and mortality. It is estimated that 10% of the world’s population is infected with the complex Entamoeba histolytica/ Entamoeba dispar. According to the WHO there are 500 million new infections per year and it is the cause of approximately 70,000 - 100,000 deaths. This parasite has a very elaborate process of invasion, where there are expressed and secreted proteins that allow the parasite to adhere to the epithelium, to degrade extracellular matrix and to penetrate epithelial cells within the mucosa. Numerous studies have been carried out to understand how virulence factors work in diverse areas of biomedical sciences. The studies have proposed diagnostic tests to increase the sensitivity and specificity and to find vaccine candidates, which are an opening way to new therapeutic expectations.
... Thus, after TJ are disrupted by the parasite, tissues are invaded. E. histolytica secretes several molecules such as: (i) those involved in adhesion of amoebae to target cells 5 ; (ii) membraneactive factors participating in killing of host cells by exocytosis, for example the ion channel-forming peptides termed amoebapores 6,7 ; and (iii) proteinases that degrade extracellular matrix proteins and mediate tissue disintegration 5,8,9 . ...
... Thus, after TJ are disrupted by the parasite, tissues are invaded. E. histolytica secretes several molecules such as: (i) those involved in adhesion of amoebae to target cells 5 ; (ii) membraneactive factors participating in killing of host cells by exocytosis, for example the ion channel-forming peptides termed amoebapores 6,7 ; and (iii) proteinases that degrade extracellular matrix proteins and mediate tissue disintegration 5,8,9 . ...
... For these reasons, MDCK cells are often chosen by researchers when studying TJ functions. MDCK cells have also been widely used as a model for the study of mechanisms induced during amoebiasis 5,8,10,[25][26][27][28] . In a recent study, we reported that interaction of strain I MDCK cells and intestinal epithelial Caco-2 cells with live amoebas, lysates and amoeba products caused similar effects on TJ composition and TER 10 . ...
Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.
... Male hamsters were immunized three times (each 2 weeks) with 300 mg of this peptide diluted in TiterMax ® Gold Adjuvant liquid (Sigma), then, animals were bled and antibodies obtained. Other primary antibodies used were: rat a-EhVps23 (Galindo et al., 2021), mouse monoclonal a-HA (GeneTex), mouse a-EhVps32 (Avalos-Padilla et al., 2015), mouse monoclonal a-Ubiquitin (Santacruz), rabbit a-EhCP112 (Garcıá-Rivera et al., 1999) and mouse monoclonal a-human actin (kindly given by Dr. Manuel Hernańdez, Cell Biology Department, CINVESTAV) antibodies. Secondary antibodies were: HRP-labelled a-rabbit, a-mouse, a-hamster and a-rat IgGs (Zymed) for western blot. ...
... Many authors, including us (Garcıá-Rivera et al., 1999), have reported that phagocytosis is one of the landmark virulence factors of amoebae. Immunofluorescence experiments, revealed that EhVps23 is closed to the erythrocytes since the initial contact, thus, EhVps23 could be involved in phagocytosis (Galindo et al., 2021). ...
The EhVps23 protein, an orthologue of the yeast Vps23 and the mammalian TSG101 proteins, is the single member of the ESCRT-I complex of Entamoeba histolytica identified and characterized until now. EhVps23 actively participates in vesicular trafficking and phagocytosis, which influence several cellular events. In this paper, we investigated the role of EhVps23 in virulence-related functions, including the invasive capacity of trophozoites, using transfected trophozoites. Trophozoites overexpressing the EhVps23 protein (Neo-EhVps23) presented helical arrangements in the cytoplasm, similar to the ones formed by EhVps32 for scission of vesicles. By confocal and transmission electron microscopy, EhVps23 was detected in multivesicular bodies, vesicles, and the extracellular space. It was secreted in vesicles together with other proteins, including the EhADH adhesin. Probably, these vesicles carry molecules that participate in the prey capture or in cell-cell communication. Mass spectrometry of precipitates obtained using α-EhVps23 antibodies, evidenced the presence of proteins involved in motility, phagocytosis, vesicular trafficking and secretion. The study of cellular functions, revealed that Neo-EhVps23 trophozoites exhibit characteristics similar to those described for mammalian transformed cells: they grew 50% faster than the control; presented a significant higher rate of phagocytosis, and migrated five-fold faster than the control, in concordance with the low rate of migration exhibited by Ehvps23-knocked down trophozoites. In addition, Neo-EhVps23 trophozoites produced dramatic liver abscesses in experimental animals. In conclusion, our results showed that EhVps23 overexpression gave to the trophozoites characteristics that resemble cancer cells, such as increased cell proliferation, migration, and invasion. The mutant that overexpresses EhVps23 can be a good study model to explore different events related to the transformation of malignant cells.
... To explore this idea, we used a polyclonal antibody, directed against the full length EhADH protein (EhADH). In agreement with earlier results (34,41), in the control trophozoites, EhADH antibodies recognized the 112-124 kDa bands corresponding to the EhCPADH complex (41) , but free EhADH was not visible. Surprisingly, in KD trophozoites, the EhADH antibodies detected the 75 kDa band corresponding to free EhADH and the EhCPADH complex ( Fig. 10 A). ...
... These differences could be interpreted as necessary events to carry out the distinct functions of the proteins. Our work with EhADH since many years ago (41), has . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. ...
During phagocytosis, a key event in the virulence of the protozoan Entamoeba histolytica , several molecules in concert contact the target, generate pseudopodia, and internalize and digest the ingested prey. Posttranslational modifications provide proteins the timing and signaling to intervene in these processes. SUMOylation is a posttranslational modification that in several systems grants a fine tuning for protein functions, protein interactions and cellular location, but it has not been studied in E. histolytica. In this paper, we characterized the E. histolytica SUMO gene and its product (EhSUMO) and elucidated the EhSUMO 3D-structure. Furthermore, here we studied the relevance of SUMOylation in phagocytosis, particularly in its association with EhADH (an ALIX family protein) and EhVps32 (a protein of the ESCRT-III complex), both involved in phagocytosis. Our results indicated that EhSUMO has an extended N-terminus that differentiates other SUMO from ubiquitin. It also presents the GG residues at the C-terminus and the ΨKXE/D binding motif, both involved in target protein contact. Additionally, E. histolytica genome possesses the enzymes belonging to the SUMOylation-deSUMOylation machineries. Confocal microscopy assays, using a-EhSUMO antibodies disclosed a remarkable membrane activity with convoluted and changing structures in trophozoites during erythrophagocytosis. SUMOylated proteins appeared in pseudopodia, phagocytic channels, and around the adhered and ingested erythrocytes. Docking analysis predicted interaction of EhSUMO with EhADH, and immunoprecipitation and immunofluorescence assays revealed that the EhADH-EhSUMO association increased during phagocytosis, whereas the EhVps32-EhSUMO interaction appeared stronger since basal conditions. In EhSUMO knocked down trophozoites, the bizarre membranous structures disappeared, and EhSUMO interaction with EhADH and EhVps32 diminished. Our results evidenced the presence of a SUMO gene in E. histolytica and the SUMOylation relevance during phagocytosis.
Author’s Abstract
Phagocytosis is one of the main functions that Entamoeba histolytica trophozoites carry out during the invasion to the host. Many proteins are involved in this fascinating event, in which the plasmatic membrane undergoes to multiple and speedy changes. Posttraductional modifications activate proteins in the precise time that they must get involved. SUMOylation, that consists in the non-covalent binding of SUMO protein with target molecules, is one of the main changes suffered by proteins in order to enable them to participate in cellular functions. SUMOylation had not been studied in E. histolytica nor in phagocytosis, and our working hypothesis is that this event is deeply engaged in the ingestion of target molecules and cells. The results of this paper prove the presence of an intronless bona fide EhSUMO gene encoding for a predicted 12.6 kDa protein that is actively involved in phagocytosis. Silencing of the EhSUMO gene affected the rate of phagocytosis and interfered with the EhADH and EhVps32 function, two proteins involved in phagocytosis, strongly supporting the importance of SUMOylation in this event.
... Previously, our group evidenced the role of EhADH and EhVps32 in phagocytosis (García-Rivera et al., 1999;Bañuelos et al., 2005;Avalos-Padilla et al., 2015), whose genes appeared in our search as EhGATA targets. Here, we examined their promoters to look for putative EhGATA-binding sites along 1,000 bp of the upstream region from the Ehadh and Ehvps32 transcription starting points. ...
... Trophozoites were incubated for 5 and 10 min with human erythrocytes (already-existing collection) in TY1-S medium (1:25 ratio) at 4°C for adherence, or 37°C for phagocytosis experiments (García-Rivera et al., 1999). Noningested erythrocytes were lysed with a TYI-S:water (1:1) mixture. ...
In this paper, we explored the presence of GATA in Entamoeba histolytica and their function as regulators of phagocytosis‐related genes. Bioinformatics analyses evidenced a single 579 bp sequence encoding for a protein (EhGATA), smaller than GATA factors of other organisms. EhGATA appeared phylogenetically close to Dictyostelium discoideum and Schistosoma mansoni GATA proteins. Its sequence predicts the presence of a zinc‐finger DNA binding domain and an AT‐Hook motif; it also has two nuclear localization signals. By transmission electron and confocal microscopy, anti‐EhGATA antibodies revealed the protein in the cytoplasm and nucleus, and 65% of nuclear signal was in the heterochromatin. EhGATA recombinant protein and trophozoites nuclear extracts bound to GATA‐DNA consensus sequence. By in silico scrutiny, 1,610 gene promoters containing GATA‐binding sequences appeared, including Ehadh and Ehvps32 promoters, whose genes participate in phagocytosis. Chromatin immuniprecipitation assays showed that EhGATA interact with Ehadh and Ehvps32 promoters. In EhGATA‐overexpressing trophozoites (NeoGATA), the Ehadh and Ehvps32 mRNAs amount was modified, strongly supporting that EhGATA could regulate their transcription. NeoGATA trophozoites exhibited rounded shapes, high proliferation rates and diminished erythrophagocytosis. Our results provide new insights into the role of EhGATA as a non‐canonical transcription factor, regulating genes associated with phagocytosis.
... (b) Parasite poses at the intercellular space of epithelial cells (Martinez-Palomo et al., 1985). (c) Then, virulence molecules are exposed at the parasite plasma membrane, or secreted (Chavez-Munguia et al., 2012;Petri et al., 2002;Rosales-Encina et al., 1987), among them the EhCPADH complex including the EhCP112 protease (García-Rivera et al., 1999). (d) EhCPADH and the protease reach TJs (Figure 3 and 8), probably together with other molecules such as EhADH (Arroyo & Orozco, 1987) and PGE 2 (Lejeune et al., 2011). ...
... Trophozoites were washed with PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.4) and lysed by freezethawing in the presence of 20 mM p-hydroxymercuribenzoate and 40 μg/ml of E-64(García-Rivera et al., 1999). MDCK and Caco-2 cells were washed with PBS, scraped with a rubber policeman and lysed in cold RIPA buffer-40 mM Tris-HCl pH 7.6, 150 mM NaCl, 2 mM EDTA, 10% glycerol, 1% Triton X-100, 0.5% sodium deoxycholate, 0.2% SDS, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM Complete™ protease inhibitor cocktail (Roche)-under continuous and vigorous shaking. ...
Entamoeba histolytica trophozoites adhere to epithelium at the cell-cell contact and perturb tight junctions (TJs) disturbing the transepithelial electrical resistance. Behind TJs are the adherens junctions (AJs) that reinforce them; and the desmosomes (DSMs) that maintain the epithelium integrity. The damage produced to AJs and DMSs by this parasite is unknown. Here, we studied the effect of the trophozoites, the EhCPADH complex and the EhCP112 recombinant enzyme (rEhCP112) on AJ and DSM proteins. We found that trophozoites degraded β-cat, E-cad, Dsp l/ll and Dsg-2 with the participation of EhCPADH and EhCP112. After contact of epithelial cells with trophozoites, immunofluorescence and transmission electron microscopy assays revealed EhCPADH and rEhCP112 at the intercellular space where they co-localized with β-cat, E-cad, Dsp l/ll and Dsg-2. Moreover, our results suggested that rEhCP112 could be internalized by caveolae and clathrin-coated vesicles. Immunoprecipitation assays showed the interaction of EhCPADH with β-cat and Dsp l/ll. Besides, in vivo assays demonstrated that rEhCP112 concentrates at the cellular borders of the mouse intestine degrading E-cad and Dsp I/II. Our research gives the first clues on the trophozoite attack to AJs and DSMs and point out the role of the EhCPADH and EhCP112 in the multifactorial event of trophozoites virulence.
... Thin sections (4-6 μm) were obtained deparaffinized and treated with 10% H 2 O 2 at RT for 60 min to remove endogenous peroxidase. Sections were incubated first with normal goat serum at RT for 60 min and ON at 4°C with a rabbit antibody (1:100) produced by Invitrogen, against a 16 residues polypeptide (431-KYHSNSTYVQFYNHTI-446) obtained from the EhCP112 protease [34]. Then, preparations were incubated with goat antirabbit IgG biotinylated-labeled antibody (1:300; Molecular Probes), followed by incubation with streptavidin-biotin-peroxidase complex (Zymed) and diaminobenzidine (DAB) containing 0.04% H 2 O 2. Antibody biding was revealed by hematoxylin. ...
... To evaluate the parasitic burden in the liver of untreated and resveratrol-treated animals (using both protocols described here), we carried out immunostaining assays, using an antibody against an EhCP112 polypeptide. EhCP112 is a cysteine protease forming part of the EhCPADH complex, involved in E. histolytica virulence [34]. To evaluate the parasitic burden, intact antibody-positive trophozoites were counted in 15 liver sections. ...
Entamoeba histolytica causes amoebiasis, an infection that kills 100,000 individuals each year. Metronidazole and its derivatives are currently used against this protozoan, but these drugs present adverse effects on human health. Here, we investigated the effect of resveratrol (a natural compound) on E. histolytica trophozoites viability, as well as its influence on the parasite virulence. Trophozoites growth was arrested by 72 μM resveratrol and the IC50 was determined as 220 μM at 48 h. Cells appeared smaller, rounded and in clusters, with debris-containing vacuoles and with abnormally condensed chromatin. Resveratrol triggered reactive oxygen species production. It caused lipid peroxidation and produced phosphatidylserine externalization and DNA fragmentation this latter evidenced by TUNEL assays. It also provoked an increase of intracellular Ca2+ concentration, activated calpain and decreased superoxide dismutase activity, indicating that an apoptosis-like event occurred; however, autophagy was not detected. Cytopathic activity, phagocytosis, encystment and in vivo virulence were diminished dramatically by pre-incubation of trophozoites with resveratrol, evidencing that resveratrol attenuated the trophozoite virulence in vitro. Interestingly, after the inoculation of virulent trophozoites, animals treated with the drug did not develop or developed very small abscesses. Our findings propose that resveratrol could be an alternative to contend amoebiasis.
... Other terms have been used to describe enzymes closely related or identical to this enzyme such as Cathepsin B [43], Neutral proteinase [44], Histolysin [45] (later changed to Histolysain [46]) and Amoebapain [47]. Cysteine endopeptidase enzymes secreted by E. histolytica [48] were found localized in lysosome-like vesicles or on the cell surface [49,50]. Molecular cloning revealed a large number of Cysteine endopeptidase genes in the genome of E. histolytica [49,51]. ...
The study in this review aims to know and study the parasite Entamoebahistolytica that causes Amoebiasis worldwide. During its life cycle, E. histolytica passes through several phases, trophozoite stage, precyst stage, the cyst stage, the Metacyst stage, and Metacystictrophozoite stage. In this study, the spread of the parasite globally and in the Arab world and methods of treating the disease were also discussed. The Genomic structureof E. histolytica, like other organisms, is characterized by diversity and heterogeneity in its genetic content, which is one of the most important reasons for its survival and its ability to infect. Interestingly, the genome of the E. histolytica contains a large amount of genes presumed to be of bacterial origin. The study of the genetic diversity of E. histolytica gives paths to the developmental change that resulted in the emergence of evolutionary features or traits. Understanding amoebic virulence is important. Several studies have shown that genetic factors influence the virulence of parasitic infections. Studying the virulence of E. histolytica by Serine-rich protein (REHP) gene is among the important issues used in molecular epidemiological studies.
... In the early stages of intestinal invasion [148], once the protective mucus layer has been destroyed, virulent trophozoites penetrate the intestinal parenchyma, adhere and destroy the extracellular matrix (ECM) through the action of amoebic surface receptors recognizing ECM components and proteinase activities [149]. The trophozoite evolves in the colon epithelium by attaching to the mammalian cell by means of surface glycosylated components like the Gal-GalNAc lectin and the glycocalyx-bound lipopeptidophosphoglycan (LPPG) [18]; or by direct amoeba-cell contact involving proteins like KERP1, KERP2 [150]; serine, threonine-, and isoleucine-rich proteins STIRPs [151] and the adhesin ADH112 [152]. Cell-cell junctions of epithelial cells are disrupted by proteinases [153], mammalian cell killing, and phagocytosis occurs; alternatively, after attaching to host cells, amoebae "chew off" and ingest host cell fragments by trogocytosis [154,155]. ...
The amoeba parasite Entamoeba histolytica is the causative agent of human amebiasis, an enteropathic disease affecting millions of people worldwide. This ancient protozoan is an elementary example of how parasites evolve with humans, e.g. taking advantage of multiple mechanisms to evade immune responses, interacting with microbiota for nutritional and protective needs, utilizing host resources for growth, division, and encystation. These skills of E. histolytica perpetuate the species and incidence of infection. However, in 10% of infected cases, the parasite turns into a pathogen; the host-parasite equilibrium is then disorganized, and the simple lifecycle based on two cell forms, trophozoites and cysts, becomes unbalanced. Trophozoites acquire a virulent phenotype which, when non-controlled, leads to intestinal invasion with the onset of amoebiasis symptoms. Virulent E. histolytica must cross mucus, epithelium, connective tissue and possibly blood. This highly mobile parasite faces various stresses and a powerful host immune response, with oxidative stress being a challenge for its survival. New emerging research avenues and omics technologies target gene regulation to determine human or parasitic factors activated upon infection, their role in virulence activation, and in pathogenesis; this research bears in mind that E. histolytica is a resident of the complex intestinal ecosystem. The goal is to eradicate amoebiasis from the planet, but the parasitic life of E. histolytica is ancient and complex and will likely continue to evolve with humans. Advances in these topics are summarized here.
... Anti-SPCB-NF antibodies are probably inhibiting N. fowleri adhesion to nasal epithelium of mice. In a similar study, García-Rivera et al. (1999) pre-incubated E. histolytica trophozoites with antibodies directed against EhCP112 CP and found that these antibodies inhibit trophozoites adhesion to erythrocytes or epithelial cells. Campos-Rodríguez et al. (2004) observed that when Acanthamoeba polyphaga is incubated with antibodies purified from colostrum of healthy women, these antibodies can inhibit adhesion of these amoebae to canine kidney Content courtesy of Springer Nature, terms of use apply. ...
Naegleria fowleri causes primary amoebic meningoencephalitis in humans and experimental animals. It has been suggested that cysteine proteases of parasites play key roles in metabolism, nutrient uptake, host tissue invasion, and immune evasion. The aim of this work was to evaluate the presence, expression, and role of cathepsin B from N. fowleri in vitro and during PAM. Rabbit-specific polyclonal antibodies against cathepsin B were obtained from rabbit immunization with a synthetic peptide obtained by bioinformatic design. In addition, a probe was designed from mRNA for N. fowleri cathepsin B. Both protein and messenger were detected in fixed trophozoites, trophozoites interacted with polymorphonuclear and histological sections of infected mice. The main cathepsin B distribution was observed in cytoplasm or membrane mainly pseudopods and food-cups while messenger was in nucleus and cytoplasm. Surprisingly, both the messenger and enzyme were observed in extracellular medium. To determine cathepsin B release, we used trophozoites supernatant recovered from nasal passages or brain of infected mice. We observed the highest release in supernatant from recovered brain amoebae, and when we analyzed molecular weight of secreted proteins by immunoblot, we found 30 and 37 kDa bands which were highly immunogenic. Finally, role of cathepsin B during N. fowleri infection was determined; we preincubated trophozoites with E-64, pHMB or antibodies with which we obtained 60%, 100%, and 60% of survival, respectively, in infected mice. These results suggest that cathepsin B plays a role during pathogenesis caused by N. fowleri mainly in adhesion and contributes to nervous tissue damage.
... In addition, other amebic proteins have been identified as effectors of amebic adherence to mammalian cells; for example, the protein rich in lysine and glutamic acid KERP1 (Seigneur et al., 2005), the adhesin EhCAPDH (Garcia-Rivera et al., 1999); and many other surface molecules already identified (Biller et al., 2014;Ng et al., 2018) that may contribute to human cell cytotoxicity, which is important for pathogenesis. ...
Entamoeba histolytica is the etiological agent of amoebiasis in humans. This amoeba parasite resides as a commensal in the intestine where it shares intestinal resources with the bacterial microbiome. In the intestinal ecosystem, the amoeba encysts and eventually develops disease by invading the tissues. E. histolytica possesses cell surface receptors for the proper sensing of signals involved in encystation or sustaining parasite interaction with bacteria and human cells. Among those receptors are the Gal/GalNAc lectin, G protein‐coupled receptors, and transmembrane kinases. In addition there are recently discovered, promising proteins, including orthologs of Toll‐type receptors and β‐trefoil lectins. These proteins trigger a wide variety of signal transduction pathways; however, most of the players involved in the signaling pathways evoked in this parasite are unknown. This review provides an overview of amoebic receptors and their role in encystation, adherence to bacteria or human cells, as well as the reported intracellular signal transduction processes that they can trigger. This knowledge is essential for understanding the lifestyle of E. histolytica and its cytopathic effect on bacteria and human cells that are responsible for infection.
... EhCPADH112, a complex formed by the combination of EhCP7 and E. histolytica adhesin (EhADH112), is involved in cytoadherence, phagocytosis, and cytotoxicity of human intestinal epithelia cells [103] . Later, it was found that E. histolytica ADH112 possesses a protein domain-like (Pro1) which is involved in trafficking of proteins required for the biogenesis of multi-vesicular bodies (MVBs). ...
Parasites developed several strategies for their survival and host tissue invasion. Helminths express potent molecules mainly for immunomodulation, which is why they stay in their hosts for years. Helminths display several mechanisms not only to evade host immune response(s), but also to preserve the host for as long as they could live. In contrast, protozoa evolve several policies primarily for pathogenesis, and invasion. Therefore, variable clinical manifestations are reported in protozoal diseases. Both symptomatic and asymptomatic cases are commonly observed in amoebiasis, giardiasis, trichomoniasis, cryptosporidiosis and toxoplasmosis, while mild, moderate, and severe cases occur in malaria, leishmaniasis, African sleeping sickness and Chagas’ disease. This was primarily attributed to strains variability and to a lesser extent, to host immune response(s). With recent evolutionary technology in molecular parasitology and bioinformatics, several molecules are established as virulence factors. These factors encourage researchers and scientists to develop novel drug targets and/or vaccine candidates. The present review aims to highlight, and review virulence strategies adapted by parasites to invade host tissue, enhance its replication and spread, as well as other processes for immunomodulation or immunoevasion of host immune response(s).
... El complejo Proteico EhCPAH contiene dos proteínas de interés la EhDH112 es una adhesina de 75KDa glicoproteína involucrada en el proceso de fagocitosis y la cisteína EhCP112 involucrada en el proceso de fagocitosis y en el efecto citopático Garcia-Rivera, 1999 ; Azuara-Liceaga, 2005 . ...
Este libro tiene como propósito, recrear de manera sencilla, al lector de conceptos y descubrimientos sobre la amibiasis como enfermedad, clínica, patología, diagnóstico y tratamiento, así como los diversos aspectos de la biología de la entamoeba histolytica como agente causante de la enfermedad. Este libro está diseñado para los cursos de pre y posgrado sobre la amibiasis. Su base se fundamenta en la amplia trayectoria de los investigadores en el estudio y diagnóstico de la amibiasis tanto a nivel nacional como internacional. En el uso de una cepa axénica autóctona y con referencia mundial de Entamoeba histolytica agente causante de la amibiasis. El texto incluye una revisión sobre la estructura, taxonomía actual y ciclo de vida de esta especie.
Posteriormente se revisa la epidemiología y prevalencia mundial y regional, de esta infección seguidamente se muestra el desarrollo histórico y una reseña histórica estructurada sobre los hallazgos de la biología del parasito y la enfermedad, destacando algunos aspectos microbiológicos y conocimientos poco abordados, como el cultivo, y la patogenia amibiana, que actualmente son de gran interés. Luego una visión general sobre la respuesta inmunitaria contra el parasito tanto humoral como celular, destacando la capacidad de evasión de la respuesta inmunitaria del parasito y su acción sobre el complemento.
Seguidamente explicamos la capacidad de duplicación y ciclo celular del parasito, el secuenciamiento completo del genoma aspecto de interés para elucidación de nuevos descubrimientos sobre la biología del parásito. Por último mostramos las propiedades antigénicas del parásito y un esbozo de nuestras experiencias y avances sobre el diagnóstico, con información novedosa sobre los métodos, paso a paso para el diagnóstico inmunológico y molecular -como componente practico- en el texto. Por último el tratamiento y las nuevas investigaciones que apuntan a la resolución de esta enfermedad.
... It is characterized as an essential virulence factor in clinical amoebiasis as it contributes with EhADH112 (adhesin) in the formation of EhCPADH complex, which is an immunogenic, heterodimeric protein involved in cytopathic effect, cytoadherence and phagocytosis. It was reported that EhCPADH complex is located in the plasma membrane and cytoplasmic vacuoles with a MW 124 kDa, encoded by two adjacent genes, cp112 and adh112 [32] . It is documented as virulence factor in amoebiasis, as its encoding gene was silenced (RNA interference). ...
... Unexpectedly, as seen in Figure 2A, the EhDbr1 enzyme was localized mainly in the cytoplasm and was nearly absent in the nucleus. As controls of nuclear and cytoplasmic fractionation, histones and the EhCPADH complex (Garcia- Rivera et al., 1999), respectively, were evaluated by western blot, confirming appropriate fractionation. ...
The RNA lariat debranching enzyme (Dbr1) has different functions in RNA metabolism, such as hydrolyzing the 2′-5′ linkage in intron lariats, positively influencing Ty1 and HIV-1 retrotransposition, and modulating snRNP recycling during splicing reactions. It seems that Dbr1 is one of the major players in RNA turnover. It is remarkable that of all the studies carried out to date with Dbr1, to our knowledge, none of them have evaluated the expression profile of the endogenous Dbr1 gene. In this work, we describe, for the first time, that Entamoeba histolytica EhDbr1 mRNA has a very short half-life (less than 30 min) and encodes a very stable protein that is present until trophozoite cultures die. We also show that the EhDbr1 protein is present in the nuclear periphery on the cytoplasmic basal side, contrary to the localization of human Dbr1. Comparing these results with previous hypotheses and with results from different organisms suggests that Dbr1 gene expression is finely tuned and conserved across eukaryotes. Experiments describing the aspects of Dbr1 gene expression and Dbr1 mRNA turnover as well as other functions of the protein need to be performed. Particularly, a special emphasis is needed on the protozoan parasite E. histolytica, the causative agent of amoebiasis, since even though it is a unicellular organism, it is an intron-rich eukaryote whose intron lariats seem to be open to avoid intron lariat accumulation and to process them in non-coding RNAs that might be involved in its virulence.
... It can form pseudopodia to phagocyte epithelial cells. The three parasites have adherence molecules and cysteine proteases that function in colonization and damage production to tissues [8][9][10]. They do not have bona fide mitochondria and peroxisomes, organelles found in most eukaryotes, neither canonical mitochondrial processes. ...
Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis, and other opportunistic protozoa infect over one billion individuals worldwide. Poverty and lack of public health policies are grounds for their morbidity and mortality. E. histolytica and G. lamblia are intestinal protozoa. Their cysts come with feces of infected individuals and when ingested by others produce intestinal diseases; beside the gut, amoebae also invade liver and other tissues. T. vaginalis is a sexually transmitted parasite and trichomonosis is linked to HIV and cytomegalovirus transmission. All these protozoa lack bona fide mitochondria and present singular facts in their energy production metabolism that make them susceptible to 5-nitroimidazole-derived drugs. Metronidazole, the drug of election against them, is activated by the electrons that PFOR transfers to ferredoxins and hydrogenases. The three protozoa are capable to develop resistance to 5-nitroimidazoles and other drugs. In addition to these three major anaerobic protozoan parasites, the emergence of opportunistic protozoa such as Blastocystis hominis, Cryptosporidium parvum, Isospora spp., Cyclospora spp., and Microsporidia also appears as medical problems. Novel compounds, molecules isolated from plants, and discovering of specific parasite biochemical targets are the main strategies to overcome the development of drug resistance in these parasites. Here, we review the current status of drug resistance in these anaerobic protozoa parasites.
... Other Cell Surface Molecules A number of other cell surface molecules with roles in amoebic pathogenesis have been identified and partially characterized. Among these, the EhCP-ADH complex has been studied most extensively [62][63][64][65][66][67]. EhCP-ADH complex encodes two proteins, a cysteine protease, EhCP112, and an adhesion protein, EhADH112 [68]. ...
Amoebiasis is one of the major public health problems in developing countries. In spite of the availability of an effective drug and absence of overt drug resistance, the disease is still prevalent among large population and spread over a number of countries. It is caused by the protist parasite Entamoeba histolytica that essentially infects humans, though other species that infect a few animals have been reported. A number of molecular techniques have recently been developed. These have helped in understanding biological processes in E. histolytica and in the identification of key molecules that are involved in amoebic virulence and invasion. Moreover, developments in the area of disease and invasion models have allowed understanding of these processes at molecular level and circumvented lack of a good animal model of amoebiasis. All these knowledge will help us to design better therapeutics and allow us to control this important disease.
... These results suggest G. duodenalis trophozoites possess multiple types of cathepsin cysteine proteases, and each may have a different role in disease pathogenesis. Indeed, single-celled parasites can possess both surface-associated and secreted cathepsin cysteine proteases which may have unique roles in pathogenesis [89][90][91][92]. ...
Giardia duodenalis infections are among the most common causes of waterborne diarrhoeal disease worldwide. At the height of infection, G. duodenalis trophozoites induce multiple pathophysiological processes within intestinal epithelial cells that contribute to the development of diarrhoeal disease. To date, our understanding of pathophysiological processes in giardiasis remains incompletely understood. The present study reveals a previously unappreciated role for G. duodenalis cathepsin cysteine proteases in intestinal epithelial pathophysiological processes that occur during giardiasis. Experiments first established that Giardia trophozoites indeed produce cathepsin B and L in strain-dependent fashion. Co-incubation of G. duodenalis with human enterocytes enhanced cathepsin production by Assemblage A (NF and S2 isolates) trophozoites, but not when epithelial cells were exposed to Assemblage B (GSM isolate) trophozoites. Direct contact between G. duodenalis parasites and human intestinal epithelial monolayers resulted in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin; these effects were abolished when parasite cathepsin cysteine proteases were inhibited. Interestingly, inhibition of parasite proteases did not prevent degradation of the intestinal tight junction-associated protein zonula occludens 1 (ZO-1), suggesting that G. duodenalis induces multiple pathophysiological processes within intestinal epithelial cells. Finally, this study demonstrates that G. duodenalis-mediated disruption of villin is, at least, in part dependent on activation of myosin light chain kinase (MLCK). Taken together, this study indicates a novel role for parasite cathepsin cysteine proteases in the pathophysiology of G. duodenalis infections.
... Another gene family involved in attachment is the family of predicted transmembrane serine-, threonine, and isoleucine rich proteins, known as EhSTIRP [82]. Additionally, the 112 kDa cysteine protease and adhesin complex, EhCPADH, contributes to attachment [83]. Finally, the amoeba protein KERP1 may also be involved in host cell attachment [84,85]. ...
Entamoeba histolytica was named 'histolytica' (from histo-, 'tissue'; lytic-, 'dissolving') for its ability to destroy host tissues. Direct killing of host cells by the amoebae is likely to be the driving factor that underlies tissue destruction, but the mechanism was unclear. We recently showed that, after attaching to host cells, amoebae bite off and ingest distinct host cell fragments, and that this contributes to cell killing. We review this process, termed 'amoebic trogocytosis' (trogo-, 'nibble'), and how this process interplays with phagocytosis, or whole cell ingestion, in this organism. 'Nibbling' processes have been described in other microbes and in multicellular organisms. The discovery of amoebic trogocytosis in E. histolytica may also shed light on an evolutionarily conserved process for intercellular exchange.
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... Finally, reactions were developed by chemiluminescence (ECL Plus, Amersham Biosciences). To demonstrate the enrichment of nuclear and cytoplasmic fractions, these proteins were probed with antibodies against the unmethylated H4 (Santa Cruz Biotechnology) and against the EhCPADH complex [23]. ...
Background
In eukaryotes, histone arginine methylation associates with both active and repressed chromatin states depending on the residues involved and the status of methylation. Even when the amino-terminus of Entamoeba histolytica histones diverge from metazoan sequences, these regions contain arginine residues that are potential targets for methylation. However, histone arginine methylation as well as the activity of arginine methyltransferases (PRMTs) has not been studied in this parasite. The aim of this work was to examine the dimethylation of arginine 3 of H4 histone (H4R3me2) and to identify the parasite PRMT that could be responsible for this modification (EhPRMT1).
Methods
To examine the presence of H4R3me2 in E histolytica, we performed Western blot and immunofluorescence assays on trophozoites using an antibody against this epigenetic mark. To recognize the PRMT1 enzyme of this parasite that possibly perform that modification, we first performed a phylogenetic analysis of E. histolytica and human PRMTs. RT-PCR assays were carried out to analyze the expression of the putative PRMT1 genes. One of these genes was cloned and expressed in Escherichia coli. The recombinant protein was tested by its recognition by an antibody against human PRMT1 and in its ability to form homodimers and to methylate commercial histones.
Results
The arginine 3 of human H4, which is subjected to post translational methylation, was aligned with the arginine 8 of E. histolytica H4, suggesting that this residue could be methylated. The recognition of an 18 kDa nuclear protein of E. histolytica by an antibody against H4R3me2 confirmed this assumption. We found that this parasite expresses three phylogenetic and structural proteins related to PRMT1. Antibodies against the human PRMT1 detected E. histolytica proteins in cytoplasm and nuclei and recognized a recombinant PRMT1 of this parasite. The recombinant protein was able to form homodimers and homotetramers and displayed methyltransferase activity on arginine 3 of chicken H4.
Conclusion
All these results suggest that E. histolytica contains as a minimum one structural and functional protein ortholog to PRMT1, enzyme that potentially dimethylates H4R8. This modification may play an important role in the gene expression regulation of this microorganism.
... Among these cell surface Gal/GalNAc lectin has been studied most extensively [1][2][3]. Some of the other cell surface molecules involved in pathogenesis are an unusual 112 kDa adhesin, an as yet unidentified mannose-containing amoebic surface molecule that interacts with bacterial mannose binding proteins [4] and a serine rich protein, as a dominant surface antigen with an apparent role in amoebic adherence to apoptotic cells [5]. Recently, cell surface localized transmembrane kinases (TMK) have also been identified as important molecules in amoebic pathogenesis. ...
... La adhesión observada en nuestro estudio entre T. vaginalis y E. histolytica no mostró preferencia por alguna área específica en ambos parásitos. Es probable que este reconocimiento sea mediado por moléculas del glicocalix de los dos protozoarios[14,15,16] o por proteínas de E. histolytica involucradas en la adhesión, como ocurre en la interacción de estos parásitos con los eritrocitos de humano[17,18,19,20].La segunda fase se caracterizó, al parecer, por la formación de un conducto en el estoma y el citoplasma de E. histolytica por donde fue internalizada T. vaginalis. ...
... E. histolytica has a remarkable ability to destroy tissues, thus showing its pathogenic behavior. As this behavior is not common in all strains, molecular basis of the cytotoxicity process has been largely investigated [3][4][5][6][7][8]. Studies on the analysis of gene expression and differentiation in amoebas isolated from different clinical cases may contribute to a better understanding of the parasite's biology. ...
Entamoeba histolytica is a parasite which presents capacity to degrade tissues and therefore has a pathogenic behavior. As this behavior is not shown by all strains, there have been several studies investigating molecular basis of the cytotoxicity process. Using the suppression subtractive hybridization (SSH) technique, differential gene expressions of two E. histolytica strains, one virulent (EGG) and one nonvirulent (452), have been analyzed with the purpose of isolating genes which may be involved with amoebic virulence. Nine cDNA fragments presenting high homology with E. histolytica previously sequenced genes were subtracted. Of these, four genes were confirmed by RT-PCR. Two coding for hypothetical proteins, one for a cysteine-rich protein, expressed only in the virulent strain, EGG and another one, coding for grainin 2 protein, exclusive from 452 strain. This study provided new insight into the proteins differences in the virulent and nonvirulent E. histolytica strains. We believe that further studies with these proteins may prove association of them with tissue damage, providing new perceptions to improve treatment or diagnosis of the invasive disease.
... In Entamoeba histolytica cysteine proteases are potential virulence factors (Olivos-Garcia et al., 2004; and play a role in adhesion of trophozoites to the mucosal lining (Garcia-Rivera et al., 1999). Bruchhaus et al., (2003) reported a total of 20 cysteine protease genes in E. histolytica with only EhCP1, EhCP2 and EhCP5 possessing proteolytic activity. ...
Animal trypanosomosis is a major constraint in livestock production in Sub-Saharan Africa. With the emergence of resistance against trypanocidal drugs, the cost and environmental concerns raised by vector control, and the challenge of antigenic variation in vaccine development, alternative control measures are being sought. An anti-disease strategy, whereby the immune response or chemotherapy is aimed towards pathogenic factors rather than the parasite itself, constitutes such a novel approach. Congopain is the major cysteine protease in Trypanosoma congolense, and upon release in the bloodstream of infected cattle, acts as a pathogenic factor. It is therefore an attractive candidate for an anti-disease vaccine. It was hence deemed necessary to investigate the variability of congopain-like cysteine proteases before attempting to design drugs and vaccines based on the inhibition of congopain. Most congopain-like cysteine protease genes of T. congolense exist in a single locus of 12-14 copies organised as tandem repeats of 2 kb gene units. A gene unit library of 120 clones was constructed out of several cosmid clones selected in a previous study that contained various lengths of the congopain locus. Some 24 gene unit clones were sequenced, and it was found that congopain genes cluster in three sub-families, named CP1 (8 clones), CP2 (12 clones) and CP3 (4 clones). The latter most characteristically shows a substitution of the active site cysteine by a serine. Isoform specific primers were designed and used to verify the proportions of the three isoforms (one third CP1, half CP2 and a sixth CP3) in the remaining clones of the library. Since this first study was conducted in one isolate, IL 3000, the results were subsequently validated in a large array of isolates, of T. congolense, as well as T. vivax and T. brucei subspecies, by a PCR approach. Finally, to gain access to copies of congopain genes that are not present in the locus, but rather scattered in the genome, an attempt was made to construct a 2 kb size-restricted genomic library. Only 206 clones could be produced, of which a mere 8 coded for congopain-like proteases. The fact that 7 out of 8 of these clones belong to CP3 (thought to be inactive) suggested a cloning artefact, possibly related to the activity of the cloned proteases.
Overall, all congopain genes appear very conserved in a given species, with 87-99% identity at protein level. The pre- and pro-region were the most conserved, while the catalytic domain was the most variable, especially around the active site cysteine, with frequent replacement by a serine residue, and in one instance by phenylalanine. The histidine residue of the
v
catalytic triad was also substituted by either a serine or a tyrosine in some instances. The proenzyme cleavage site sequence was also variable, with APEA being the predominant N-terminal sequence. RT-PCR analyses indicated that CP1, CP2 and CP3 mRNA are all present in the bloodstream forms of T. congolense, showing that these variants are likely to be expressed. The conclusion of this study is that, given the high overall conservation of congopain genes in the genome, for the purpose of anti-disease vaccine, it is likely that a single immunogen will suffice to raise antibody able to inhibit all circulating congopain-like cysteine proteases. For chemotherapy however, a more in-depth enzymatic characterisation of the mutants, involving functional recombinant expression, will have to be undertaken.
... Although we did not detect Vps4, we identified proteins (EHI_167710 and EHI_165220) that share homologies with mammalian Alix (a direct interactor with Snf7), which has been found to be necessary for membrane abscission during cytokinesis and virus budding in association with ESCRT-III [57]. Another E. histolytica protein identified and characterized with homology to Alix [58] ( Fig. 7), is the adhesin protein ADH112 (EHI_181220) that has a role in phagocytosis [59], [60] and was found in our analysis. The Alix-homologue (EHI_167710) appeared in all three independent experiments and has differences at the amino acid sequence level (Fig. 7A-B) indicating that in addition to the BRO1 domain (IPR004328), characteristic of the Alix family, it bears specific domains (Fig. 7C). ...
Unlabelled:
Entamoeba histolytica is the protozoan parasite agent of amoebiasis, an infectious disease of the human intestine and liver. Specific active pathogenic factors are secreted toward the external milieu upon interaction of the parasite with human tissue. Trafficking dynamics and secretion of these factors is not known and characterization of the dynamics interplay of subcellular compartments such as the ER or Golgi apparatus is still pending. In this work, we took advantage of cell fractionation and a wide proteomic analysis to search for principal components of the endomembrane system in E. histolytica. Over 1500 proteins were identified and the two top categories contained components of trafficking machinery and GTPases. Trafficking related proteins account for over 100 markers from the ER, Golgi, MVB, and retromers. The lack of important components supporting Golgi polarization was also highlighted. The data further describe principal components of the endosomal traffic highlighting Alix in isolated vesicles and during parasite division.
Biological significance:
This work represents the first in-depth proteomics analysis of subcellular compartments in E. histolytica and allows a detailed map of vesicle traffic components in an ancient single-cell organism that lacks a stereotypical ER and Golgi apparatus to be established.
The intestinal microbiota, the collection of all microorganisms residing in our gastrointestinal tract, provides a plethora of microbial diversity. Bacteria, viruses, fungi, worms and protozoa are the primary microorganisms comprising our microbiota. These microbes include representative species capable of both causing severe harm or symbiotic benefits to their host. In relation, the host immune system has evolved complex detection systems to identify members of each microbial faction and interpret their harmful or peaceful capabilities. As a consequence, our immune system mounts appropriate responses to either eliminate or tolerate members of our microbiota. Protozoa are an underappreciated kingdom within our microbiota and the interactions of these microbes with our immune system remain understudied. Several recent reports have demonstrated that the presence of Tritrichomonas spp. in the intestinal tract of mice and men facilitates novel interactions with our host immune system. Within this chapter, we are summarizing the most recent knowledge on how Tritrichomonads, as a newly emerging group of intestinal protozoan commensals, shape and communicate with our intestinal immune system.
em>Eimeria tenella est l’un des parasites apicomplexes à l’origine de la coccidiose aviaire, l’une des plus importantes maladies parasitaires de l’industrie avicole. Dans le but de caractériser les facteurs de pathogénicité d’ E. tenella , nous nous sommes intéressés aux protéases et plus particulièrement aux aminopeptidases N. Nous avons caractérisé Et-ApN1 et identifié Et-ApN3, deux aminopeptidases d’ E. tenella . Et-ApN1 présente de fortes homologies avec PfA-M1, l’homologue de Plasmodium falciparum, au niveau des séquences, des structures, des propriétés biochimiques, du clivage et de la localisation. L’ensemble des résultats suggèrent qu’Et-ApN1 est impliquée dans le développement parasitaire. Pour évaluer son rôle de cible thérapeutique potentielle, nous avons criblé une bibliothèque de molécules et identifié une nouvelle molécule le C36, qui inhibe directement l’activité d’Et-ApN1 et entraîne un arrêt du développement d’ E. tenella in vitro. Cet effet inhibiteur est également observé chez Toxoplasma gondi i et P. falciparum . Dans le but d’améliorer la solubilité du C36 pour de futures études in vivo, le C36 a été pharmaco-modulé. Les perspectives de ces travaux viseront à prouver l’implication directe des Et-ApN dans le développement d’ E. tenella</em
Giardia duodenalis is one of the most prevalent human enteropathogens and a major cause of diarrheal disease worldwide. Cysteine proteases (CPs) have been identified as major virulence factors in protozoan parasites, playing important roles in disease pathogenesis and in parasitic life cycles. G. duodenalis exhibits high proteolytic activity, and CPs play significant roles in giardiasis. Giardia CPs are directly involved in intestinal epithelial junctional complex disruption, intestinal epithelial cell apoptosis, and degradation of host immune factors, including chemokines and immunoglobulins. Giardia CPs have also been implicated in mucus depletion and microbiota dysbiosis induced by the parasite. This review discusses the most recent advances in characterization of Giardia Assemblage A and B CPs, including cathepsin B (catB)-like proteases.
Amebiasis is one of the twenty major causes of disease in Mexico; however, the diagnosis is difficult due to limitations of conventional microscopy-based techniques. In this study, we analyzed stool samples using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) to differentiate between Entamoeba histolytica (pathogenic) and E. dispar (non-pathogenic). The target for the PCR amplification was a small region (228 bp) of the adh112 gene selected to increase the sensitivity of the test. The study involved 62 stool samples that were collected from individuals with complaints of gastrointestinal discomfort. Of the 62 samples, 10 (16.1%) were positive for E. histolytica while 52 (83.9%) were negative. No sample was positive for E. dispar. These results were validated by nested PCR-RFLP (restriction fragment length polymorphism) and suggest that PCR-DGGE is a promising tool to differentiate among Entamoeba infections, contributing to determine the specific treatment for patients infected with E. histolytica, and therefore, avoiding unnecessary treatment of patients infected with the non-pathogenic E. dispar.
Entamoeba histolytica is an obligate human intestinal parasite causing amoebic dysentery and liver abscess. Entamoeba dispar is its close largely nonpathogenic relative. Both species are well adapted to the human host. The motile amoebae called trophozoites dwell in the lumen of the large intestine; in its distal part, they differentiate to cysts, which are excreted and can survive in the environment and infect new hosts. Although metronidazole and other nitroimidazoles represent an efficient treatment, there are more than 55,000 deaths worldwide every year.
The E. histolytica genome project led to a better understanding of the molecular equipment; two surprises were the significant number of genes acquired from bacteria by lateral gene transfer and a very high number of protein kinases and phosphatases underscoring the complex signal transduction pathways in this parasite.
The parasite attacks human cells by first attaching to them via a galactose and N-acetylgalactose (Gal/GalNAc)-specific lectin. The host cell membrane is pierced by a pore-forming protein called amoebapore, and cysteine proteinases destroy cellular components, antibodies and complement.
The human host mounts a massive humoral and cellular immune response against E. histolytica with macrophages and NKT cells being the most efficient defenders. The antibody response is limited in its impact through the ability of the amoebae to cap and shed antibodies bound to their surface. In addition, E. histolytica has developed a number of mechanisms to cope with immune cells.
E. histolytica belongs to the microaerophilic microorganisms devoid of classical mitochondria and devoid of oxidative phosphorylation. So the main pathway to extract energy is by glycolysis, and when not needed, the energy is stored in the form of glycogen. The biosynthetic abilities are quite crippled; most useful molecules, sugars, amino acids, lipids and nucleotides are acquired from the host or from phagocytosed bacteria.
Cysteine proteases of the protozoan parasite Entamoeba histolytica are key virulence factors involved in overcoming host defences. These proteases are cathepsin-like enzymes with a cathepsin-L like structure, but cathepsin-B substrate specificity. In the host intestine, amoeba cysteine proteases cleave colonic mucins and degrade secretory immunoglobulin (Ig) A and IgG rendering them ineffective. They also act on epithelial tight junctions and degrade the extracellular matrix to promote cell death. They are involved in the destruction of red blood cells and the evasion of neutrophils and macrophages and they activate pro-inflammatory cytokines IL-1 beta and IL-18. In short, amoeba cysteine proteases manipulate and destroy host defences to facilitate nutrient acquisition, parasite colonization and/or invasion. Strategies to inhibit the activity of amoeba cysteine proteases could contribute significantly to host protection against E. histolytica.
Phagocytosis is one of the essential processes that is necessary for the survival and pathogenicity of Entamoeba histolytica. E. histolytica is known to phagocytose red blood cells (RBC), bacteria and other unicellular organisms, immune cells, and apoptotic cells during either proliferation in the gut or invasion in intestinal and extraintestinal tissues. Molecular pathways are not clear about molecular details in E. histolytica and are thought to be different from those known in other systems. In this chapter we describe our current understanding of molecular mechanisms of phagocytosis in E. histolytica and highlight new avenues for research for future drug development.
Virulence of the human parasite Entamoeba histolytica is related to adherence to, killing, and phagocytosis of host cells, and contact between trophozoites and host cells is necessary for amoebic effectors to act. Virulence also relies on parasite motility in extracellular matrices and human tissues and on the capacity of the parasite to escape the host defense response and to trigger infl ammation. Amoebic surface components implicated in all these processes have been identifi ed as important virulence factors. This chapter presents current knowledge on the major surface molecules of E. histolytica critically involved in parasite interactions with healthy or apoptotic cells (Gal/GalNAc lectin, LPG/LPPG proteophosphoglycans, KERP1, CPADH, STIRP, proform of CP-A5, ROM1), in recognition of apoptotic cells (calreticulin, M17, C2PK, SREHP, TMK96), host cell phagocytosis (CPADH, SREHP, TMK96, ROM1), attachment to extracellular matrices (β1FNR, β2 integrinlike), and immune evasion or triggering the infl ammatory response (Gal/GalNAc lectin, KERP1, proform of CP-A5, LPG/LPPG).
Tight junctions and microvilli constitute an anti-invasive barrier at the luminal side of enteric cell layers. Both subcellular structures are disrupted following adhesion of Entamoeba histolytica trophozoites to enteric cell layers in vitro. It was our aim to analyse the molecular mechanism underlying this disruption. Therefore, we cocultured enteric T84 cell layers established on filter inserts with E. histolytica trophozoites and tested various modulators of enteric molecules, involved in the functional regulation of tight junctions, as well as inhibitors of trophozoite virulence factors on their capacity to maintain the transepithelial electrical resistance. Pretreatment of trophozoites with the proteinase inhibitor N-Tosyl-Phenylalanine chloromethyl ketone or N-Tosyl-l-Lysine chloromethyl ketone prevented the decrease in transepithelial electrical resistance whereas none of the modulators used to pretreat enterocytes were successful. Moreover, zymography and Western blot analysis revealed that both N-Tosyl-Phenylalanine chloromethyl ketone and N-Tosyl-l-Lysine chloromethyl ketone inhibited E. histolytica cysteine proteinases and prevented proteolysis of tight junction molecules ZO-1 and ZO-2 and of villin, the major actin bundling molecule in microvilli. Immunocytochemistry with an antibody against ezrin, an actin-binding molecule in microvilli, and phase contrast microscopy demonstrated that pretreatment of trophozoites with N-Tosyl-Phenylalanine chloromethyl ketone or N-Tosyl-l-Lysine chloromethyl ketone also prevented disturbance of microvilli and destruction of Caco-2 enteric cell layers in cocultures. Taken together, our results indicate that trophozoites use their proteinases to overcome microvilli and tight junction barriers during the invasion of enteric cell layers, that these phenomena could be prevented by pretreatment of trophozoites with N-Tosyl-Phenylalanine chloromethyl ketone or N-Tosyl-l-Lysine chloromethyl ketone, and that such pretreatment disabled trophozoites to destroy enteric cell layers in vitro.
In this paper, we attempted to define the role of phagocytosis in the virulence of Entamoeba histolytica. We have isolated, from a highly phagocytic and virulent strain, a clone deficient in phagocytosis. Trophozoites of wild-type strain HM1:IMSS were fed with Escherichia coli strain CR34-Thy- grown on 5-bromo,2'-deoxyuridine. The trophozoites that had incorporated the base analog through phagocytosis of the bacteria were killed by irradiation with 310 nm light. The survivors, presumably trophozoites defective in phagocytosis, were grown until log phase and submitted two more times to the selection procedure. Clone L-6, isolated from a subpopulation resulting from this selection procedure, showed 75-85% less erythrophagocytic activity than the wild-type strain. The virulence of clone L-6 and strain HM1:IMSS was measured. The inoculum required to induce liver abscesses in 50% of the newborn hamsters inoculated (AD50) of HM1:IMSS was 1.5 X 10(4) trophozoites. Clone L-6 trophozoites failed to induce liver abscesses in newborn hamsters even with inocula of 5 X 10(5) trophozoites. Virulence revertants were obtained by successive passage of L-6 trophozoites through the liver of young hamsters. The trophozoites that recovered the ability to produce liver abscesses simultaneously recuperate high erythrophagocytic rates. These results show that phagocytosis is involved in the aggressive mechanisms of E. histolytica.
We report here the cloning and transcriptional characterization of the EhPgp5 multidrug resistance gene promoter isolated from the drug-resistant clone C2 of Entamoeba histolytica. The EhPgp5 promoter has the TATA-like motif at −31 base pairs; transcription initiates three nucleotides upstream from the ATG in trophozoites
grown in 225 μmemetine (clone C2(225)), whereas in those grown without the drug (clone C2) a product with no open reading frame was detected.
The promoter was active in transfected clone C2 trophozoites, its activity increased when trophozoites were cultured in 40
μm emetine, while it was turned off in the drug-sensitive clone A. The first −235 base pair kept full promoter activity, suggesting
that it has important drug responsive elements. Gel shift assays detected the complex Ib in clone C2, which was augmented
in clone C2(225). Competition experiments suggested that complex Ib may be constituted by HOX and AP-1 like factors in clone
C2, whereas in clone C2(225), complex Ib was only competed by the HOX sequence. Complexes Ie, detected in clones A and C2
but not in C2(225), and Ia, present in all clones, were competed by the TATA box oligonucleotide. Our results suggest that
proteins forming complexes Ib and Ie may be participating in the regulation of theEhPgp5 gene expression.
We present here the cloning and characterization of the EhPgp1 multidrug resistance gene promoter isolated from the Entamoeba histolytica drug-resistant mutant clone C2. The EhPgp1 promoter lacks the typical TATA box and the transcriptional initiation sequences described for other E. histolytica promoters. The major transcription initiation site of the EhPgp1 gene was located at the ATG start codon. TheEhPgp1 core promoter located within the first 244 base pairs showed a higher chloramphenicol acetyltransferase expression in the
transfected trophozoites of clone C2 than in those of the sensitive clone A. Gel shift assays revealed three specific DNA-protein
complexes (Ia, IIa, and IIIc) using nuclear extracts from clone C2, whereas three main complexes (If, IIf, and IIg) were limited
to clone A. Competition assays suggested the presence of C/EBP-like and OCT-like proteins in complexes Ia and IIa, respectively,
probably involved in the expression of the EhPgp1 gene, whereas complex IIIc was competed by GATA-1, C/EBP, OCT, and HOX oligonucleotides. Thus, differential DNA-protein complexes
may be formed by transcriptional factors involved in the regulation of the EhPgp1 gene expression.
A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.
Entamoeba histolytica causes amebic dysentery and amebic liver abscess, major causes of morbidity and mortality worldwide. We have used differential hybridization screening to isolate an E. histolytica-specific cDNA clone. The cDNA was found to encode a serine-rich E. histolytica protein (SREHP) containing multiple tandem repeats. The structural motif of SREHP resembles some of the repetitive antigens of malarial species, especially the circumsporozoite proteins. A recombinant trpE fusion protein containing the tandem repeats of SREHP was recognized by immune serum from a patient with amebiasis, demonstrating that SREHP is a naturally immunogenic protein. An antiserum raised against the recombinant fusion protein specifically bound to two distinct bands with apparent molecular masses of 46 and 52 kDa in a crude preparation of E. histolytica trophozoite membranes. This antiserum also inhibited E. histolytica trophozoite adhesion to Chinese hamster ovary cells in vitro. The ability to isolate E. histolytica-specific genes, and to express those genes in Escherichia coli, may be important in studying the molecular basis of E. histolytica pathogenesis and for the future development of vaccines.
Monoclonal antibodies have been developed and used as specific probe to locate and identify a 29-kDa molecule of axenic Entamoeba histolytica trophozoites. Monoclonal antibody produced by clone C8 (MoAb C8) strongly agglutinated the amoebic trophozoites. The immunofluorescence of live E. histolytica trophozoites and surface fluorescence of acetone-fixed trophozoites by MoAb C8 indicated existence of a 29-kDa molecule on surface-associated plasma membrane of E. histolytica. The monoclonal antibody belonged to IgG1 isotype. The prior treatment of E. histolytica trophozoites with MoAb C8 resulted in significant (P less than 0.01) reduction in adherence of amoebic trophozoites to cultured Chinese Hamster Ovary cells and significant (P less than 0.01) reduction in cytotoxicity to cultured Baby Hamster Kidney cells. Pretreatment of amoebic trophozoites with MoAb C8 prior to cultivation in TPS-1 medium resulted in significant (P less than 0.01) reduction in growth of the parasite. Thus, the data suggested that the surface-exposed 29-kDa molecule may be one of the receptors involved in E. histolytica host cell interactions and may possibly modulate amoebic disease processes.
The galactose and N-acetyl-D-galactosamine-inhibitable adherence lectin of Entamoeba histolytica is a cell surface protein which mediates parasite adherence to human colonic mucus, colonic epithelial cells, and other target cells. The amebic lectin was purified in 100-micrograms quantities from detergent-solubilized trophozoites by monoclonal antibody affinity chromatography. The adherence lectin was purified 500-fold as judged by radioimmunoassay. The nonreduced lectin had a molecular mass of 260 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of pH 6.2. The amebic lectin reduced with beta-mercaptoethanol consisted of 170- and 35-kDa subunits. Both subunits could be labeled on the cell surface with 125I, and both were metabolically labeled with [3H]glucosamine. The amino termini of the subunits had unique amino acid sequences, and polyclonal antisera to the heavy subunit did not cross-react with the light subunit. The yield of phenylthiohydantoin derivatives from the second and third positions in the sequence of the heavy and light subunits gave a molar ratio of one 170- to one 35-kDa subunit. Antibodies directed to the heavy subunit inhibited amebic adherence to Chinese hamster ovary cells by 100%, suggesting that the heavy subunit is predominantly responsible for mediating amebic adherence.
The role of cysteine proteinases in adherence of Trichomonas vaginalis NYH 286 to HeLa and human vaginal epithelial cells was evaluated. Only pretreatment of trichomonads, but not epithelial cells, with N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), an inhibitor of trichomonad cysteine proteinases, greatly diminished the ability of T. vaginalis to recognize and bind to epithelial cells. Leupeptin and L-1-tosylamide-2-phenylethyl chloromethyl ketone, other cysteine proteinase inhibitors, also decreased T. vaginalis cytadherence. Parasites incubated with TLCK and washed extensively still did not adhere to cells at levels equal to those seen for control trichomonads treated with phosphate-buffered saline or culture medium alone. Exposure of TLCK-treated organisms with other cysteine proteinases restored cytadherence levels, indicating that proteinase action on the parasite surface is prerequisite for host cell attachment. Concentrations of TLCK which inhibited cytadherence did not alter the metabolism of T. vaginalis, as determined by metabolic labeling of trichomonad proteins; the protein patterns of T. vaginalis in the presence and absence of TLCK were identical. Kinetics of TLCK-mediated inhibition of cytadherence of other T. vaginalis isolates with different levels of epithelial-cell parasitism were similar to the concentration-dependent inhibition seen for isolate NYH 286. Incubation of TLCK-treated, washed organisms in growth medium resulted in regeneration of adherence. Finally, treatment of T. vaginalis organisms with proteinase inhibitors for abrogation of cytadherence effectively rendered the trichomonads unable to kill host cells, which is consistent with the contact-dependent nature of host cytotoxicity. These data show for the first time the involvement of T. vaginalis cysteine proteinases in parasite attachment to human epithelial cells. These results have implications for future pharmacologic intervention at a key step in infection.
A 220-kilodalton (kDa) protein with lectin properties was isolated from Entamoeba histolytica strain HM1:IMSS and was purified by Sepharose 4B chromatography and electroelution from 5% SDS-polyacrylamide gels. The protein
contains 9% carbohydrate by weight; is rich in hydrophobic residues; and is very immunogenic in mice, hamsters, and rabbits.
The protein binds to fixed monolayers of MDCK cells and inhibits trophozoite attachment to the cultured cells. The 220-kDa
protein agglutinates human erythrocytes, and agglutination is inhibited by micro molar concentrations of hyaluronic acid,
chitin, chitinderived products (chitotriose), and antibodies to the purified protein. The 220-kDa protein is recognized by
an antibody to the membrane but not by antibodies to other subcellular fractions. We therefore suggest that this 220-kDa protein
with lectin properties is a component of the plasma membrane and could be one of the putative “receptor” molecules involved
in cell and/or matrix attachment.
Entamoeba histolytica adheres to human colonic mucus, colonic epithelial cells, and other target cells via a galactose (Gal) or N-acetyl-D-galactosamine (GalNAc) inhibitable surface lectin. Blockade of this adherence lectin with Gal or GalNAc in vitro prevents amebic killing of target cells. We have identified and purified the adherence lectin by two methods: affinity columns derivatized with galactose monomers or galactose terminal glycoproteins, and affinity columns and immunoblots prepared with monoclonal antibodies that inhibit amebic adherence. By both methods the adherence lectin was identified as a 170-kD secreted and membrane-bound amebic protein. The surface location of the lectin was confirmed by indirect immunofluorescence. Purified lectin competitively inhibited amebic adherence to target cells by binding to receptors on the target Chinese hamster ovary cells in a Gal-inhibitable manner.
We have developed three computer programs for comparisons of protein and DNA sequences. They can be used to search sequence data bases, evaluate similarity scores, and identify periodic structures based on local sequence similarity. The FASTA program is a more sensitive derivative of the FASTP program, which can be used to search protein or DNA sequence data bases and can compare a protein sequence to a DNA sequence data base by translating the DNA data base as it is searched. FASTA includes an additional step in the calculation of the initial pairwise similarity score that allows multiple regions of similarity to be joined to increase the score of related sequences. The RDF2 program can be used to evaluate the significance of similarity scores using a shuffling method that preserves local sequence composition. The LFASTA program can display all the regions of local similarity between two sequences with scores greater than a threshold, using the same scoring parameters and a similar alignment algorithm; these local similarities can be displayed as a "graphic matrix" plot or as individual alignments. In addition, these programs have been generalized to allow comparison of DNA or protein sequences based on a variety of alternative scoring matrices.
Ultracryotomy of fixed tissue has been investigated for a number of years but, so far, success has been limited for several reasons. The simple technique herein reported allows the ultracryotomy not only of a variety of tissues but also of single cells in suspension, with a preservation and visualization of ultrastructural detail at least equivalent to that obtained with conventional embedding procedures. In this technique, sucrose is infused into glutaraldehyde-fixed tissue pieces before freezing for the purpose of controlling the sectioning consistency. By choosing the proper combinations of sucrose concentration and sectioning temperature, a wide variety of tissues can be smoothly sectioned. Isolated cells, suspended in a sucrose solution, are sectioned by sectioning the frozen droplet of the suspension. A small liquid droplet of a saturated or near-saturated sucrose solution, suspended on the tip of an eyelash probe, is used to transfer frozen sections from the knife edge onto a grid substrate or a water surface. Upon melting of the sections on the surface of the sucrose droplet, they are spread flat and smooth due to surface tension. When the section of a suspension of single cells melts, individual sections of cells remain confined to the small area of the droplet surface. These devices make it possible to cut wide dry sections, and to avoid flotation on dimethyl sulfoxide solutions. With appropriate staining procedures, well-preserved ultrastructural detail can be observed. The technique is illustrated with a number of tissue preparations and with suspensions of erythrocytes and bacterial cells.
The association of axenically grown trophozoites of Entamoeba histolytica strains HK-9 or HM-1:IMSS with various types of gram-negative bacteria for relatively short periods markedly increased their virulence, as evidenced by their ability to destroy monolayers of tissue-cultured cells. Interaction of trophozoites with bacteria that were heat inactivated, glutaraldehyde fixed, or disrupted by sonication, or bacteria treated with inhibitors of protein synthesis, did not augment amebic virulence. Lethally irradiated bacteria, however, retained their stimulative properties and trophozoites that ingested bacteria were protected from the toxic effects of added hydrogen peroxide. An increase in virulent properties of amebae was also found in experiments carried out under microaerobic conditions (5% O2, 10% CO2). The augmentation of amebic virulence due to association with bacteria was specifically blocked by metronidazole, but not by tetracycline or aminoglycosides, and the rate of metronidazole uptake in stimulated trophozoites was two to three times higher. The results obtained suggest that virulence of axenically grown E. histolytica trophozoites may depend to a considerable extent on the cell's reducing power. Both microaerobic conditions and the association with bacteria apparently stimulate the electron transport system of the ameba. Bacteria may function as broad range scavengers for oxidized molecules and metabolites through the contribution of enzymatic systems, components, or products.
In this paper, we attempted to define the role of phagocytosis in the virulence of Entamoeba histolytica. We have isolated, from a highly phagocytic and virulent strain, a clone deficient in phagocytosis. Trophozoites of wild-type strain HM1:IMSS were fed with Escherichia coli strain CR34-Thy- grown on 5-bromo,2'-deoxyuridine. The trophozoites that had incorporated the base analog through phagocytosis of the bacteria were killed by irradiation with 310 nm light. The survivors, presumably trophozoites defective in phagocytosis, were grown until log phase and submitted two more times to the selection procedure. Clone L-6, isolated from a subpopulation resulting from this selection procedure, showed 75-85% less erythrophagocytic activity than the wild-type strain. The virulence of clone L-6 and strain HM1:IMSS was measured. The inoculum required to induce liver abscesses in 50% of the newborn hamsters inoculated (AD50) of HM1:IMSS was 1.5 X 10(4) trophozoites. Clone L-6 trophozoites failed to induce liver abscesses in newborn hamsters even with inocula of 5 X 10(5) trophozoites. Virulence revertants were obtained by successive passage of L-6 trophozoites through the liver of young hamsters. The trophozoites that recovered the ability to produce liver abscesses simultaneously recuperate high erythrophagocytic rates. These results show that phagocytosis is involved in the aggressive mechanisms of E. histolytica.
An alignment/phylogeny of the papain superfamily of cysteine proteases was created using an initial structure-based alignment followed by successive iterations of sequence alignment and phylogenetic inference. The iterative approach resulted in significant improvements in the alignment/phylogeny. There were three groups of cysteine proteases that were distantly related and which could be aligned against each other only in the active site regions: the papain group, which included such stereotypical cysteine proteases as cathepsins B, C, H, L and S; and the bleomycin hydrolase and calpain groups. There was one bacterial sequence in each of the bleomycin hydrolase and calpain groups. The former probably arose by lateral gene transfer, the latter possibly by direct evolution from an ancestral protease predating the eukaryote/prokaryote divergence. The phylogeny of the papain group indicated that many families diverged almost simultaneously early during eukaryotic evolution. In mammals there are at least 12 distinct families of cysteine proteases, possibly many more, including at least two as yet uncharacterized enzymes.
We have analyzed three independent genomic loci of the protozoan parasite Entamoeba histolytica that contain coding regions for the iron-containing superoxide dismutase, the pore-forming peptide, and the galactose-inhibitable lectin. All of the three structural genes were found to be closely linked unidirectionally to other coding sequences. The intergenic regions did not exceed 1,350 nucleotides. Nuclear run-on data demonstrated that at least the galactose-inhibitable lectin gene is transcribed in a monocistronic fashion. Comparison of the genomic sequences described here with several others reported previously for E. histolytica revealed a number of invariable peculiarities for the gene organization of this parasite: (i) Coding sequences are not interrupted by introns; (ii) 5' untranslated regions are rather short and transcription starts at the consensus sequences ATTCA or ATCA; (iii) an unusual TATA-motif is located about 30 nucleotides upstream of the start of transcription and comprises the sequence TATTTAAA, which reveals protein binding activity as determined by gel retardation assays; (iv) the conserved pentanucleotide motif TAA/TTT is found within the relatively short 3' untranslated regions and functions putatively as the transcription termination signal; and (v) a stretch of up to 12 pyrmidine residues is located at the end of transcribed sequences.
A cDNA clone for a physiologically regulated Tetrahymena cysteine protease gene was sequenced. The nucleotide sequence predicts that the clone encodes a 336-amino acid protein composed of a 19-residue N-terminal signal sequence followed by a 107-residue propeptide and a 210-residue mature protein. Comparison of the deduced amino acid sequence of the protein with those of other cysteine proteases revealed a highly conserved interspersed amino acid motif in the propeptide region of the protein, the ERFNIN motif. The motif was present in all of the cysteine proteases in the data base with the exception of the cathepsin B-like proteins, which have shorter propeptides. Differences in the propeptides and in conserved amino acids of the mature proteins suggest that the ERFNIN proteases and the cathepsin B-like proteases constitute two distinct subfamilies within the cysteine proteases.
Adhesins from oral bacteria perform an important function in colonizing target tissues within the dentogingival cavity. In Porphyromonas gingivalis certain of these adhesion proteins exist as a complex with either of two major proteinases referred to as gingipain R (arginine-specific gingipain) and gingipain K (lysine-specific gingipain) (R. N. Pike, W. T. McGraw, J. Potempa, and J. Travis, J. Biol. Chem. 269:406-411, 1994). With specific proteinase inhibitors, it was shown that hemagglutination by either proteinase-adhesin complex could occur independently of proteinase activity. Significantly, low concentrations of fibrinogen, fibronectin, and laminin inhibited hemagglutination, indicating that adherence to these proteins and not the hemagglutination activity was a primary property of the adhesin activity component of complexes. Binding studies with gingipain K and gingipain R suggest that interaction with fibrinogen is a major function of the adhesin domain, with dissociation constants for binding to fibrinogen being 4 and 8.5 nM, respectively. Specific association with fibronectin and laminin was also found. All bound proteins were degraded by the functional proteinase domain, with gingipain R being more active on laminin and fibronectin and gingipain K being more effective in the digestion of fibrinogen. Cumulatively, these data suggest that gingipain R and gingipain K, acting as proteinase-adhesin complexes, progressively attach to, degrade, and detach from target proteins. Since such complexes appear to be present on the surfaces of both vesicles and membranes of P. gingivalis, they may play an important role in the attachment of this bacterium to host cell surfaces.
We have developed three computer programs for comparisons of protein and DNA sequences. They can be used to search sequence data bases, evaluate similarity scores, and identify periodic structures based on local sequence similarity. The FASTA program is a more sensitive derivative of the FASTP program, which can be used to search protein or DNA sequence data bases and can compare a protein sequence to a DNA sequence data base by translating the DNA data base as it is searched. FASTA includes an additional step in the calculation of the initial pairwise similarity score that allows multiple regions of similarity to be joined to increase the score of related sequences. The RDF2 program can be used to evaluate the significance of similarity scores using a shuffling method that preserves local sequence composition. The LFASTA program can display all the regions of local similarity between two sequences with scores greater than a threshold, using the same scoring parameters and a similar alignment algorithm; these local similarities can be displayed as a "graphic matrix" plot or as individual alignments. In addition, these programs have been generalized to allow comparison of DNA or protein sequences based on a variety of alternative scoring matrices.
The enteric pathogen, Entamoeba histolytica, appears to cause disease by adhering to and then destroying mucosal barriers. Using an in vitro method of studying the interaction of E. histolytica with target cells (Chinese hamster ovary [CHO] and human erythrocytes [RBC]), we examined the mechanism of amebic adherence and its role in lysis of target cells. Killing and phagocytosis of target cells by amebas ceases at 4°C, allowing observation of adherence. Amebas adhere to CHO cells at 4°C, 78.9% formed rosettes (amebas with ≥3 adherent CHO cells each) at 2 h. At 37°C, cytochalasins B and D inhibit adherence of amebas to CHO cells (P < 0.0005). Amebas adhere to and kill CHO cells in media with <0.1 μM calcium and magnesium plus 10 mM EDTA, indicating that divalent cations are not required in the medium. Adherence of amebas to human RBC was not ABO blood group specific and showed greater adherence to human than bovine or sheep RBC (P < 0.005). Neither Fc nor complement receptors were found on amebas by standard rosette studies. The amebic adherence receptor is not trypsin (0.125%) sensitive nor inhibited by trypan blue (1 mM). N-acetyl-d-galactosamine (GALNAc) inhibited the adherence of amebas to CHO cells and human RBC (0.1 g/100 ml or 4.5 mM GALNAc, P < 0.005) by binding to a receptor on the amebic surface. GALNAc abolishes amebic cytolysis of target CHO cells (determined by 111Indium oxine release from CHO cells, P < 0.001) but not amebic phagocytosis of CHO cells. By suspending ameba-CHO cells rosettes in dextran, we found that GALNAc (1%) reversibly inhibits amebic adherence (P < 0.0005) and that cytochalasins decrease amebic killing of adherent CHO cells (P < 0.025).
These findings indicate that the adherence of E. histolytica to target cells requires microfilament function, is via a specific amebic receptor that has affinity for GALNAc, and is required to lyse cells. Inhibition of the adherence of E. histolytica may alter the pathogenicity of this organism.
Inhibition of most of the expression of the cysteine proteinases of Entamoeba histolytica strain HM-1: IMSS was successfully performed by transcription of ehcp5 antisense RNA using the promoter of ehg34, which encodes a L21 ribosomal protein of E. histolytica. We have generated a stable transfectant in which the overall level of cysteine proteinase activity is strongly reduced (≈ 90%). This transfectant has a normal growth rate in Diamond's TYI-S-33 medium, a cytopathic and haemolytic activity similar to the control HM-1:IMSS pEhAct-Neo transfectant but with a significantly lower phagocytic activity.
In order to identify molecules that might be responsible for the difference in pathogenicity between the two closely related protozoan parasites Entamoeba histolytica and Entamoeba dispar, we focussed on cysteine proteinases because this class of enzymes has been considered important for pathogenic tissue destruction. By screening a genomic library derived from an E. histolytica isolate, a total of six distinct genes (ehcp1–ehcp6) encoding typical prepro-forms of cysteine proteinases were identified which differed from each other by 40% to 85% of their nucleotide sequences. Three of these genes, ehcp1, ehcp2, and ehcp5, which exhibited high levels of expression, were found to be responsible for approximately 90% of cysteine proteinase transcripts, whereas the remaining three were either not or only marginally expressed. Expression of the different genes directly correlated with the level of activity of the respective enzymes in trophozoite lysates. Purification of the enzymes and N-terminal sequencing revealed that virtually all cysteine proteinase activity of E. histolytica can be attributed to three enzymes namely EhCP1, EhCP2 and EhCP5. Southern blot analysis indicated that just two of these abundantly expressed genes are missing in E. dispar. On the other hand, genes analogous to four of the six genes identified in E.histolytica were found to be present in E. dispar, but only two of these are expressed within the trophozoite stage.
A gene encoding a novel cell wall-associated protein of Staphylococcus saprophyticus that binds fibronectin and to sheep erythrocytes has been cloned and sequenced. The 4392 bp open reading frame codes for an amino acid sequence that is quite similar to the Atl, an autolysin, of Staphylococcus aureus and to the AtlE of S. epidermidis. The two regions of most pronounced homology code for an N-acetyl-muramyl-L-alanine amidase and for an endo-β-N-acetyl-D-glucosaminidase. The cloned protein lysed cells of S. saprophyticus and Micrococcus luteus exogenously. Subcloning localized the enzymatic activities to the regions of high homology and demonstrated that the interposed sequence is responsible for the adhesive activities. Two allelic replacement mutants were constructed that lacked autolytic activity and adhesive properties. The N-terminal portion of the protein contains seven highly conserved, contiguous repeats with no similarity to published sequences. It lacks the motifs typical of Gram-positive surface proteins and shows a different overall organization. This autolysin/adhesin of S. saprophyticus (Aas) appears to represent a new class of staphylococcal adhesins.
Messenger RNA extracted from Carica papaya fruit was converted to cDNA and cloned into the PstI restriction site of plasmid pBR322. Subclones of the approximately 1.4-kb fragment were sequenced. The nucleotide sequence matched that expected, based on the amino acid (aa) sequence for papain, with the following exceptions: (l)at aa positions 47, 118 and 135 the codon for glutamate was found instead of glutamine; (2) at aa position 169 the codon for asparagine was found instead of glycine; (3) at aa positions 86–88, a difference in the order of the aa codons was observed, namely tyr-pro-tyr instead of the published pro-tyr-tyr. The upstream sequence revealed that papain is probably synthesized with a 133-aa prosegment, suggesting that the enzyme is synthesized as an inactive zymogen. The downstream segment revealed an unusual (AT)9AGAA sequence beginning 26 bp from the double TGA stop codon.
Orozco E., Suárez M. E. and Sánchez T. Differences in adhesion, phagocytosis and virulence of clones from Entamoeba histolytica, strain HM1: IMSS. International Journal for Parasitology15: 655–660. Clones isolated from Entamoeba histolytica, strain HM1: IMSS were tested for adhesion, phagocytosis and virulence after subculturing in liquid medium. Other clones were isolated from a subpopulation of strain HM1: IMSS, and highly phagocytic trophozoites were eliminated by irradiation, after incorporating bromodeoxiuridine into their DNA by phagocytosis of labelled bacteria. We thus obtained several clones from strain HM1: IMSS showing a different degree of phagocytosis. Some phagocytosis-deficient clones showed impairment in red blood cell adherence, while others showed a reduced intake of particles into their cytoplasm. The degree of phagocytosis always was associated with the virulence of the clone.
A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.
Proteolytic activity was measured in lysates of four axenic and five xenic cultures of different strains of pathogenic E. histolytica, and in five non-pathogenic strains ("E. dispar") growing in xenic culture. There was no significant difference in total proteolytic activity, using either gelatin or albumin as substrate, between the two sets of xenic cultures; the axenic pathogens however had significantly higher levels of activity than the xenic pathogens, the levels appearing to correlate with virulence. An axenic strain (NIH 200) reassociated with mixed bacterial flora reverted to close to xenic levels of activity. There was no evidence of secretion of an elevated proportion of the total enzymic activity by pathogenic strains, and expression levels may owe more to culture conditions than to genotype.
The 112 kDa adhesin of E. histolytica is directly involved in the cytopathogenic activity of the parasite. We describe here the purification of the 112 kDa protein by electroelution and immunoaffinity chromatography using a monoclonal antibody against the adhesin. Two proteins of 70 and 50 kDa were eluted from the immunoaffinity column along with the 112 kDa adhesin. The three proteins were recognized by monospecific polyclonal antibodies against the adhesin. The same peptides (72 and 56 kDa) were also observed after incubation of the purified intact adhesin in diethylamine buffer. Proteins of 112 and 72 kDa were found to have protease activity, evidenced by their ability to degrade gelatin. Our results indicate that the 112 kDa adhesin was specifically broken down into two polypeptides of 50-56 and 70-72 kDa. The significance of this in vivo is as yet unclear. The adhesin has proteolytic activity, which is retained in the 70-72 kDa polypeptide but not in the 50-56 kDa one.
The adherence of E. histolytica trophozoites to target cells was studied using a monoclonal antibody to a major surface antigen of 66 kDa. Preincubation of trophozoites with monoclonal antibody decreased their ability to adhere to and engulf erythrocytes and destruction of CHO cells. The monoclonal antibody was specific for the 66 kDa antigen which is possibly a major participant in adhesion which precedes other events in phagocytosis and cytopathic effects.
Extracellular neutral cysteine proteinases are an important virulence factor of E. histolytica. Experimental evidence supporting its role in invasion includes the ability to degrade components of the extracellular matrix and activate complement by specifically cleaving C3. We had previously reported the isolation of fragments encoding cysteine proteinase genes from HM-1 (ACP1) and a nonpathogenic strain (REF291, ACP2) by PCR using consensus sequences based on conserved structural motifs of eukaryotic cysteine proteinases. Using similar techniques, we have now identified a third gene encoding a cysteine proteinase which is present in both pathogenic and nonpathogenic strains and have correlated cysteine proteinase specific-mRNA levels with enhanced proteolytic activity and cytopathic effect on a fibroblast cell monolayer, a quantitative assay of virulence.
To examine the relationship between phagocytosis, proteinase activity and pathogenicity of axenically grown trophozoites of E. histolytica of strain HM-1:IMSS four different cultures were used: 1) a culture preserved in our laboratory for over 4 years, that lost its pathogenicity 3 years ago; 2) a culture passaged several times through hamster liver, that lost its pathogenicity recently; 3) a highly virulent culture supplied by another laboratory; and 4) amebas recovered from hamster liver abscesses caused by the latter culture. Phagocytosis was measured as erythrophagocytosis. Proteinase activity was determined on azocasein. Pathogenicity was defined as the capacity to cause liver abscesses in hamsters. A negative correlation was found between phagocytic activity and pathogenicity, since amebas unable to cause liver abscesses had the highest phagocytic activity whereas those recovered from liver abscesses had the lowest phagocytic activity. The percent of phagocytic amebas increased progressively in all cultures through a 2-month observation period. No correlation was found between the level of proteinase activity and pathogenicity. It is concluded that neither phagocytosis nor proteinase activity are adequate markers of amebic pathogenicity.
Two P-glycoprotein genes (EhPgp1 and EhPgp2) from the protozoan parasite Entamoeba histolytica were sequenced from a genomic library made with the DNA of an emetine-resistant ameba mutant, which overexpresses mRNAs homologous to segments of the human mdr1 (P-glycoprotein) gene. The open reading frames for EhPgp1 and EhPgp2 were 1302 and 1310 amino acids long, respectively, and showed a 67% positional identity with each other and 41% and 40% positional identities, respectively, with human mdr1 gene. Within each ameba P-glycoprotein were the ATP-binding sites found twice in eukaryotic P-glycoproteins and once in prokaryotic transport proteins. Hydropathy plots of the ameba P-glycoproteins were nearly superimposable on that of the human mdr 1, showing 2 homologous halves, each containing an ATP-binding site and 6 hydrophobic transmembrane domains that form the putative channel. A phylogenetic tree showed that the Entamoeba P-glycoproteins are more related to the human and mouse P-glycoproteins than to the Plasmodium and Leishmania P-glycoproteins. Also identified in the E. histolytica genomic library were 2 P-glycoprotein pseudogenes, each with a frame shift and stop codons in identical places within the amino ATP-binding site. In conclusion, the 2 E. histolytica P-glycoproteins encoded by the EhPgp1 and EhPgp2 genes are similar in structure to the mammalian P-glycoproteins and so may be involved in energy-dependent drug efflux by this human parasite.
Work from several laboratories suggests a correlation between expression of cysteine proteinase activity and the cytopathic effect of virulent HM1 strain Entamoeba histolytica trophozoites on cultured cell monolayers. Consistent with this relationship, we find that L-6 trophozoites, mutants cloned from the HM1 parent strain, are deficient in both proteinase expression and cytopathic effect. Three other clones, with proteinase expression equal to or greater than that of the HM1 strain, express the cytopathic effect. Furthermore, a nontoxic specific proteinase inhibitor, Z-phenylalanyl-alanyl-CH2F, inhibits the cytopathic effect of live trophozoites in a dose-dependent manner. These results support the hypothesis that expression and release of the cysteine proteinase is an important factor in producing the cytopathic effect, presumably by its degradation of cell anchoring proteins.
To detect molecules of Entamoeba histolytica involved in the trophozoite-target cell interaction, three different antisera were generated: (a) two rabbit antisera, one against total amebic proteins and another directed specifically to the 112-kDa adhesin; and (b) a mouse antiserum against amebic molecules adhering to the red blood cell (RBC) surface after incubation of RBCs with total soluble protein from trophozoites (anti-adhesion serum). All three antisera recognized the 112-kDa adhesion. Adhesion of this molecule to the RBC surface was temperature-dependent. More of the 112-kDa adhesion was found on the surface of RBCs incubated with trophozoites at 37 degrees C than on RBCs incubated at room temperature or at 0 degree C. Experiments using both anti-adhesin and anti-total ambebic protein sera revealed the presence of 210, 160, 112, 90, 70, 50, and 24-kDa proteins on RBC incubated with trophozoites. Surface proteins obtained from iodinated MDCK cells recognized amebic proteins of 112, 90, and 48-50 kDa. Virulence-deficient mutants presented a similar amount of the 112-kDa adhesin to the wild-type strain. However, in mutants, the adhesion was not functional, since they did not adhere to RBCs. 90- and 24-kDa proteins were also found to be altered in mutants.
The adherence of Entamoeba histolytica trophozoites to target cells was studied by using monoclonal antibodies (MAbs) and adhesion-deficient mutants of the parasite. MAbs Adh-1 and Adh-2 reacted with a surface protein of approximately 112 kDa of the total proteins of trophozoites from the wild type strain, clone A, strain HM1:IMSS. Both MAbs reacted weakly with the adhesion-deficient mutant clones, C-98, C-919 and C-923, all derived from HM1:IMSS. MAbs Adh-1 and Adh-2 incubated with trophozoites from clone A inhibited adherence to red blood cells, erythrophagocytosis and cytopathic effect on cell culture monolayers. Antibodies against a approximately 112 kDa polypeptide were found in the sera from patients with hepatic abscess. These results demonstrate that the adherence of trophozoites to target cells is a necessary event in order for cytopathogenicity to occur.
A cysteine proteinase has been isolated from the virulent strain HM1:IMSS of Entamoeba histolytica by two gel chromatography steps, ion exchange chromatography on DEAE-cellulose and affinity chromatography on organomercurial-Sepharose. The purified proteinase was homogeneous, as judged by polyacrylamide gel electrophoresis in the presence and in the absence of sodium dodecyl sulphate, and was a monomeric protein with a relative molecular mass of Mr 27000 +/- 2000 possessing an N-terminal alanine. The enzyme was inhibited by natural and synthetic inhibitors against cysteine proteinases. Split experiments employing blocked and unblocked peptide analogues with -2-naphthylamide moieties indicate that the cleavability was enhanced by the presence of basic residues, such as arginine or lysine, near the acyl end of the substrate. With unblocked tetrapeptides as substrates, exopeptidase activity of the amebic enzyme required an arginine at the P2-position, lysine could not substitute for arginine. The protease was able to digest native collagen type I, with an initial attack at the alpha 2-chain of the molecule.
Agarose can be activated by adding cyanogen bromide, dissolved in acetonitrile, to beads suspended in a solution of sodium carbonate. The necessity for manual titration and the use of a pH meter are thus eliminated. Activation for 1 min results in coupling capacities comparable to those reported for the titration method. The coupling capacity is influenced by the initial carbonate concentration and by the duration and temperature of the activation reaction. The coupling capacities of the activated gels are very reproducible.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
The enteric pathogen, Entamoeba histolytica, appears to cause disease by adhering to and then destroying mucosal barriers. Using an in vitro method of studying the interaction of E. histolytica with target cells (Chinese hamster ovary [CHO] and human erythrocytes [RBC]), we examined the mechanism of amebic adherence and its role in lysis of target cells. Killing and phagocytosis of target cells by amebas ceases at 4 degrees C, allowing observation of adherence. Amebas adhere to CHO cells at 4 degrees C, 78.9% formed rosettes (amebas with >/=3 adherent CHO cells each) at 2 h. At 37 degrees C, cytochalasins B and D inhibit adherence of amebas to CHO cells (P < 0.0005). Amebas adhere to and kill CHO cells in media with <0.1 muM calcium and magnesium plus 10 mM EDTA, indicating that divalent cations are not required in the medium. Adherence of amebas to human RBC was not ABO blood group specific and showed greater adherence to human than bovine or sheep RBC (P < 0.005). Neither Fc nor complement receptors were found on amebas by standard rosette studies. The amebic adherence receptor is not trypsin (0.125%) sensitive nor inhibited by trypan blue (1 mM). N-acetyl-d-galactosamine (GALNAc) inhibited the adherence of amebas to CHO cells and human RBC (0.1 g/100 ml or 4.5 mM GALNAc, P < 0.005) by binding to a receptor on the amebic surface. GALNAc abolishes amebic cytolysis of target CHO cells (determined by (111)Indium oxine release from CHO cells, P < 0.001) but not amebic phagocytosis of CHO cells. By suspending ameba-CHO cells rosettes in dextran, we found that GALNAc (1%) reversibly inhibits amebic adherence (P < 0.0005) and that cytochalasins decrease amebic killing of adherent CHO cells (P < 0.025). These findings indicate that the adherence of E. histolytica to target cells requires microfilament function, is via a specific amebic receptor that has affinity for GALNAc, and is required to lyse cells. Inhibition of the adherence of E. histolytica may alter the pathogenicity of this organism.
The levels of collagenolytic activity of strains HM-1:1 MSS (HM-1), (HM-1), 200-NIH, and HK-9 of Entamoeba histolytica were compared. Collagen degradation was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Conditioned media as well as extracts of the highly virulent strain HM-1 effectively degraded native type I collagen. Significantly lower activity was found in the analogous fractions of strains 200-NIH and HK-9, which are not as virulent. The collagenolytic activity of strain HM-1 was associated with the isolated plasma membrane fraction and could be eluted from the membranes by buffers of high ionic strength, indicating that it is not an integral membrane protein. Unlike the vertebrate and the clostridial collagenases, the collagenolytic activity of E. histolytica HM-1 was enhanced in presence of dithiothreitol and was inhibited by N-ethylmaleimide. The correlation between virulence of the individual strains and their collagenolytic activity suggests that collagenase might play a role in pathogenesis of amoebiasis. The localization of the enzyme on the plasma membrane and its presence in the extracellular medium favor this view.
The University of Wisconsin Genetics Computer Group (UWGCG) has been organized to develop computational tools for the analysis
and publication of biological sequence data. A group of programs that will interact with each research-article has been developed
for the Digital Equipment Corporation VAX computer using the VMS operating system. The programs available and the conditions
for transfer are described.
We have isolated and characterized an Entamoeba histolytica alpha-tubulin (alpha Tub)-encoding gene (Eh alpha tub). A 700-bp DNA fragment was amplified by PCR using primers derived from consensus alpha- and beta-Tub amino acid (aa) sequences from different organisms and E. histolytica DNA as the template. These PCR fragments were used to screen both genomic DNA and cDNA libraries in order to isolate an Eh alpha tub structural gene. Two overlapping clones containing the complete alpha tub ORF (1392 bp) were isolated from the genome and cDNA libraries. The deduced aa sequence of Eh alpha Tub has 55.5, 50 and 52% identity to Plasmodium falciparum alpha Tub 2, Saccharomyces cerevisiae alpha Tub 2 and human alpha Tub, respectively. Interestingly, the predicted Eh alpha Tub protein lacks a poly-acidic motif at its C terminus which is involved in Tub polymerization and microtubule-associated protein binding in other organisms. This fact may indicate a difference in tubule assembly in this organism and could provide a potential key for the development of therapeutic agents. According to Southern blot experiments and the sequences of several clones, at least two non-adjacent copies of alpha tub are present in the E. histolytica genome. A 1.5-kb transcript corresponding to the alpha tub mRNA was detected in mRNA from asynchronous E. histolytica trophozoites.
To examine the relationship between phagocytosis, proteinase activity and pathogenicity of axenically grown trophozoites of E. histolytica strain HM-1:IMSS four different cultures were used: (1) a culture preserved in our laboratory for over 4 years, which lost its pathogenicity 3 years ago; (2) a culture passaged several times through hamster liver, which lost its pathogenicity recently; (3) a highly virulent culture supplied by another laboratory; and (4) amebas recovered from hamster liver abscesses caused by culture 3. Phagocytosis was measured as erythrophagocytosis. Proteinase activity was determined on azocasein. Pathogenicity was defined as the capacity to cause liver abscesses in hamsters. A negative correlation was found between phagocytic activity and pathogenicity, since amebas unable to cause liver abscesses had the highest phagocytic activity, whereas those recovered from liver abscesses had the lowest phagocytic activity. The percent of phagocytic amebas showed wide variations through a 2-month observation period, with no change in amebic pathogenicity. No correlation was found between the level of proteinase activity and pathogenicity. It is concluded that neither phagocytosis nor proteinase activity is an adequate marker of amebic pathogenicity.
Entamoeba histolytica genomic organization and putative promoter elements appear to be distinct from both metazoan and better characterized protozoan organisms. The recent development of DNA-mediated transfection for E. histolytica enabled characterization of cis-acting promoter elements required for gene expression. A deletion and replacement analysis was conducted on the promoter of an E. histolytica gene encoding the heavy subunit of the N-acetyl-beta-D-galactosamine-specific adhesin (hgl5). Deletion of the DNA from -1000 bases to -272 bases upstream from the start of transcription of hgl5 did not decrease reporter gene expression. Subsequent nested deletions and 10-bp replacement mutagenesis identified four positive upstream regulatory elements between bases -219 to -200, -189 to -160, -69 to -60, and -49 to -40. A negative upstream regulatory element between bases -89 to -80 was conserved upstream of three other E. histolytica genes. Mutation of the previously unidentified 'GAAC' element conserved within the putative core promoter decreased reporter gene expression by 75%. Site directed mutagenesis of the putative TATA element decreased reporter gene expression by greater than 50%, while mutation of the putative initiator element resulted in a more modest decrease. This analysis suggests that E. histolytica promoters are unlike other protozoan promoters, with AT-rich upstream regulatory elements, a non-consensus TATA element, the "GAAC' element, and an unusual initiator element.