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Role of the Entamoeba histolytica cysteine proteinase in amebic liver abscess formation in severe combined immunodeficient (SCID) mice
Infection and Immunity
Abstract
Evidence from in vitro studies suggest that the Entamoeba histolytica cysteine proteinase plays a role in the tissue lysis and cytopathic effects seen in invasive amebiasis. We used affinity-purified antibodies against a recombinant E. histolytica cysteine proteinase to demonstrate that the proteinase is present extracellularly in amebic liver abscesses in mice with severe combined immunodeficiency (SCID mice). Treatment of E. histolytica trophozoites with specific cysteine proteinase inhibitor E-64 blocked or greatly reduced liver abscess formation at 48 h in SCID mice. Our study suggests an important role for a functional cysteine proteinase in amebic liver abscess formation.
... E. histolytica also has several unique genes encoding cysteine proteinases (11,84). In vivo studies by Stanley's and Mirelman's groups demonstrated that inhibition of cysteine proteinase activity with inhibitors or an antisense construct significantly decreased liver abscess formation in SCID mice (113) and hamsters (5). Taken together, the data supporting a key role of cysteine proteinases in virulence are extremely strong. ...
... They were able to decrease the size of amebic liver abscesses significantly by incubating E. histolytica trophozoites with laminin, which binds and blocks the activity of cysteine proteinases (54). Affinity-purified antibodies against a recombinant cysteine proteinase of E. histolytica localized the proteinase in amebic trophozoites and extracellularly in amebic liver abscesses of infected SCID mice (113). Pretreatment of E. histolytica trophozoites with the specific cysteine proteinase inhibitor E-64 blocked or greatly decreased the size of liver abscesses at 48 h (104). ...
... Therefore, E. dispar provides a unique comparative model to identify differences in cysteine proteinase regulation and processing in order to define their relationship to invasion and virulence. Finally, recent studies with SCID mice have firmly established the role of cysteine proteinases in the formation of amebic liver abscesses (113). The intestinal xenograft model will allow studies of early invasion of the human intestine for the first time (104). ...
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.
... Entamoeba histolytica is characterized by its enormous capacity to destroy and invade human tissues. This ability is mainly attributed to their cysteine peptidases, which have been shown by various in vitro and in vivo studies [1][2][3][4][5][6][7][8][9]: ...
... 1. Fibroblast monolayers are disrupted by purified cysteine peptidases [3], likely because of their ability to degrade extracellular matrix components [5,8,10,11]; 2. A direct correlation between cysteine peptidase activity and pathogenicity was observed [2,3,6,12]; 3. The abscess formation can be inhibited by the use of specific cysteine peptidase inhibitors [4,9]; 4. It has been postulated that secreted cysteine peptidases interfere with the host immune system by cleaving immune molecules such as IgG and IgA [11,13,14], processing of complement C3 [15,16], inactivation of complement C3a and C5a [17], inactivation of pro-IL-18 [18], and generation of mature IL-1β from its pro-form [19]; ...
... A direct correlation between the amount of cysteine peptidase activity and liver abscess formation in animal models was observed in several studies [2,6,12,66]. Furthermore, the inhibition of cysteine peptidases using specific cysteine peptidase inhibitors leads to a reduction in abscess formation [9,67]. It was shown that 25 Cysteine Peptidases in Pathogenesis incubation of trophozoites with laminin, which bind cysteine peptidases, leads to a reduction in liver abscess formation, which confirmed the assumption that cysteine peptidases play an important role in liver abscess formation [4]. ...
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.
... Effect of laminin on amebic liver abscess formation in SCID mice. SCID mice were inoculated with 10 6 E. histolytica trophozoites in either 100 l of BI-S-33 medium, 100 l of BI-S-33 plus 20 g of laminin, 100 l of BI-S-33 plus 20 g of fibronectin, or 100 l of BI-S-33 plus 20 g of collagen as described previously (14). After 48 h, SCID mice were sacrificed, their livers were removed and inspected for the presence of amebic liver abscesses, abscesses and livers were weighed, and the percentage of liver abscessed was calculated (14). ...
... SCID mice were inoculated with 10 6 E. histolytica trophozoites in either 100 l of BI-S-33 medium, 100 l of BI-S-33 plus 20 g of laminin, 100 l of BI-S-33 plus 20 g of fibronectin, or 100 l of BI-S-33 plus 20 g of collagen as described previously (14). After 48 h, SCID mice were sacrificed, their livers were removed and inspected for the presence of amebic liver abscesses, abscesses and livers were weighed, and the percentage of liver abscessed was calculated (14). ...
... Inhibition of amebic protease activity markedly reduces the inflammatory lesions resulting from highly virulent E. histolytica (11). We have previously reported immunohistochemical studies of tissue sections of a SCID mouse amebic liver abscess which indicate that significant quantities of extracellular E. histolytica cysteine proteinase are present within the amebic liver abscess and have shown that treatment of E. histolytica trophozoites with the cysteine proteinase inhibitor E-64 blocked or greatly reduced liver abscess formation in SCID mice (14). We have shown that E. histolytica cysteine proteinases bind to laminin tightly and that laminin inhibits the cleavage of other substrates. ...
The protozoan parasite Entamoeba histolytica is the caus- ative agent of human amebiasis. Invasive disease is character- ized by ulcerations of the intestinal wall and in some cases by invasion through the wall and dissemination to the liver, re- sulting in the clinical syndromes of amebic dysentery and liver abscess, respectively (8). Ultrastructural studies of experimen- talE. histolyticainfection revealed the degeneration of epithe- lial cells adjacent to invading trophozoites and the penetration of trophozoites into the lamina propria through the basement membrane (16). The interaction of amebic factors with com- ponents of the extracellular matrix potentially plays an impor- tant role in the penetration of trophozoites through the intes- tinal mucosa. In order to identify E. histolytica proteins that interact with components of the extracellular matrix, amebic lysates were fractionated over laminin-Sepharose,fibronectin- Sepharose, and collagen-Sepharose. We report here that E. histolytica27-kDa cysteine proteinases exhibit striking binding specificities for immobilized laminin over immobilized fi- bronectin or collagen. Furthermore, the coinjection of laminin but notfibronectin or collagen withE. histolyticatrophozoites greatly reduces liver abscess formation in severe combined immunodeficient (SCID) mice.
... However, occasionally E. histolytica penetrates the intestinal mucosa, which leads to ulcerative colitis or it disseminates to other organs, most commonly to the liver, where it induces abscess formation. Cysteine peptidases are considered to play a major role for the pathogenicity of E. histolytica as suggested by a large number of in vitro and in vivo studies [1][2][3][4][5][6][7][8][9]. Most convincing are results from infections of laboratory animals indicating that E. histolytica trophozoites that have reduced cysteine peptidase activity are greatly impaired in their ability to induce amoebic liver abscesses [8,9]. ...
... Cysteine peptidases are considered to play a major role for the pathogenicity of E. histolytica as suggested by a large number of in vitro and in vivo studies [1][2][3][4][5][6][7][8][9]. Most convincing are results from infections of laboratory animals indicating that E. histolytica trophozoites that have reduced cysteine peptidase activity are greatly impaired in their ability to induce amoebic liver abscesses [8,9]. In addition, overexpression of cysteine peptidases led to an increase in cytopathic activity, measured by in vitro monolayer disruption, as well as to a significant increase in amoebic liver abscess formation in laboratory animals in comparison to matching controls [10]. ...
... Trophozoites can damage host cells through direct contact or close proximity, as well as through phagocytic activity towards dead and dying host cells in a receptor-mediated fashion [14]. Of the molecules secreted by amoebas, cysteine proteases are particularly important [15][16][17][18][19][20]. They are responsible for a cytolytic effect on host cells [7], the modulation of the cellmediated immune response, and the proteolysis of the host extracellular matrix [16,[21][22][23][24][25]. ...
... Extensive tissue damage has been attributed to the cysteine proteinases of E. histolytica because they are (i) secreted in large quantities and can cleave extracellular matrix proteins, thus facilitating amebic invasion; (ii) secreted in higher quantities by virulent than nonvirulent trophozoites; and (iii) found to participate in the inflammation of the gut and ALA [15][16][17][18][19][20]27]. However, cysteine proteases are dispensable for phagocytosis and cytopathogenicity [28,29]. ...
The molecular mechanisms by which Entamoeba histolytica causes amebic liver abscess (ALA) are still not fully understood. Amebic mechanisms of adherence and cytotoxic activity are pivotal for amebic survival but apparently do not directly cause liver abscess. Abundant evidence indicates that chronic inflammation (resulting from an inadequate immune response) is probably the main cause of ALA. Reports referring to inflammatory mechanisms of liver damage mention a repertoire of toxic molecules by the immune response (especially nitric oxide and reactive oxygen intermediates) and cytotoxic substances released by neutrophils and macrophages after being lysed by amoebas (e.g., defensins, complement, and proteases). Nevertheless, recent evidence downplays these mechanisms in abscess formation and emphasizes the importance of peroxynitrite (ONOO(-)). It seems that the defense mechanism of amoebas against ONOO(-), namely, the amebic thioredoxin system (including peroxiredoxin), is superior to that of mammals. The aim of the present text is to define the importance of ONOO(-) as the main agent of liver abscess formation during amebic invasion, and to explain the superior capacity of amoebas to defend themselves against this toxic agent through the peroxiredoxin and thioredoxin system.
... Most studies have focused on the two proteinases specific to E. histolytica, EhCP1 and EhCP5. Antisense expressed to EhCP5 decreased liver abscesses in SCID mice [81] and hamsters [82]. The epoxide cysteine proteinase inhibitor, E-64, blocked amebic liver infection in SCID mice when preincubated with trophozoites [81], but it is too toxic to be used therapeutically. ...
... Antisense expressed to EhCP5 decreased liver abscesses in SCID mice [81] and hamsters [82]. The epoxide cysteine proteinase inhibitor, E-64, blocked amebic liver infection in SCID mice when preincubated with trophozoites [81], but it is too toxic to be used therapeutically. Vinyl sulfone-based inhibitors that modify the thiolate of the catalytic cysteine of cysteine proteases and irreversibly inactivate the enzymes look to be very promising parasitic cysteine protease inhibitors [83,84]. ...
Proteases are successfully targeted in many human pathophysiologic and infectious diseases, exemplified by drugs against hypertension, osteoporosis, type II diabetes, and HIV/AIDS. Proteases that are indispensable or that function at critical bottlenecks in the parasite lifecycle are sought after, and several from parasitic protozoa impacting public health and animal welfare are currently being studied. Malaria alone infects 300-500 million people and causes approximately 3000 deaths daily, most of which represent children less than five years of age in sub-Saharan Africa. It is estimated that a total of 500 million humans are exposed to Trypanosoma brucei, Leishmania, and Trypansoma cruzi, the causative agents, respectively, of African sleeping sickness, leishmaniasis, and Chagas disease. Advances in genetic and selective chemical targeting of proteases in parasitic protozoa have shed light on the discrete biological roles that these enzymes play, and serve as a necessary tool in the target validation process. This chapter highlights the current status in the target validation process of all classes of proteases from parasitic protozoa of human clinical and veterinary significance.
... Study of extraintestinal amoebasis in experimental models has shown that the activity of parasitic CPs is important for tissue pathology, especially for the cytolytic but not cytotoxic effect on liver cells [105]. Thus, for example, treatment of virulent E. histolytica trophozoites with the membrane-permeant specific inhibitor of CPs, E-64d, prevented the formation of liver abscesses in mice with severe combined immunodeficiency (SCID) and in golden hamsters (Mesocricetus auratus) [106,107]. The passage of axenic or xenic E. histolytica trophozoites through the liver of golden hamsters resulted in a progressive increase in proteinase activity of amoeba supernatants and lysates, which correlated well with increased virulence [108]. ...
Intestinal diseases caused by protistan parasites of the genera Giardia (giardiasis), Entamoeba (amoebiasis), Cryptosporidium (cryptosporidiosis) and Blastocystis (blastocystosis) represent a major burden in human and animal populations worldwide due to the severity of diarrhea and/or inflammation in susceptible hosts. These pathogens interact with epithelial cells, promoting increased paracellular permeability and enterocyte cell death (mainly apoptosis), which precede physiological and immunological disorders. Some cell-surface-anchored and molecules secreted from these parasites function as virulence markers, of which peptide hydrolases, particularly cysteine proteases (CPs), are abundant and have versatile lytic activities. Upon secretion, CPs can affect host tissues and immune responses beyond the site of parasite colonization, thereby increasing the pathogens’ virulence. The four intestinal protists considered here are known to secrete predominantly clan A (C1- and C2-type) CPs, some of which have been characterized. CPs of Giardia duodenalis (e.g., Giardipain-1) and Entamoeba histolytica (EhCPs 1-6 and EhCP112) degrade mucin and villin, cause damage to intercellular junction proteins, induce apoptosis in epithelial cells and degrade immunoglobulins, cytokines and defensins. In Cryptosporidium, five Cryptopains are encoded in its genome, but only Cryptopains 4 and 5 are likely secreted. In Blastocystis sp., a legumain-activated CP, called Blastopain-1, and legumain itself have been detected in the extracellular medium, and the former has similar adverse effects on epithelial integrity and enterocyte survival. Due to their different functions, these enzymes could represent novel drug targets. Indeed, some promising results with CP inhibitors, such as vinyl sulfones (K11777 and WRR605), the garlic derivative, allicin, and purified amoebic CPs have been obtained in experimental models, suggesting that these enzymes might be useful drug targets.
... As amoebae first establish themselves in the liver parenchyma and tissue necrosis spreads, parasites interact with liver parenchymal cells, leading to the destruction of hepatocytes and activation of host immune cells [103]. The experimental animal models revealed the responsible roles of CPs and Gal/GalNAc lectin in ALA formation [104,105]. Recent research indicates that peroxynitrite (ONOO−) plays a substantial role in the formation of abscesses, reducing the importance of other mechanisms. ...
The complexity of clinical syndromes of amebiasis, caused by the parasite Entamoeba histolytica, stems from the intricate interplay between the host immune system, the virulence of the invading parasite, and the surrounding environment. Although there is still a relative paucity of information about the precise relationship between virulence factors and the pathogenesis of Entamoeba histolytica, by accumulating data from clinical and basic research, researchers have identified essential pathogenic factors that play a critical role in the pathogenesis of amebiasis, providing important insights into disease development through animal models. Moreover, the parasite’s genetic variability has been associated with differences in virulence and disease outcomes, making it important to fully understand the epidemiology and pathogenesis of amebiasis. Deciphering the true mechanism of disease progression in humans caused by this parasite is made more difficult through its ability to demonstrate both genomic and pathological plasticity. The objective of this article is to underscore the heterogeneous nature of disease states and the malleable virulence characteristics in experimental models, while also identifying persistent scientific issues that need to be addressed.
... It has been well established that E. histolytica trophozoites secrete CPs via biosynthesis and receptor (CPBF1) [29]-mediated secretion via the default secretory pathway. CPs and their secretory process play an indispensable role in amoebic invasion and tissue destruction due to their hydrolytic and degradative activities towards host cells and extracellular matrix proteins [30]. EhLTP1gs line had 127 ± 41.8% (2.27 fold) higher intra-cellular CP activity in cell lysates as compared to vector control ( Fig 5A). ...
Lipid transfer proteins (LTPs) are the key contributor of organelle-specific lipid distribution and cellular lipid homeostasis. Here, we report a novel implication of LTPs in phagocytosis, trogocytosis, pinocytosis, biosynthetic secretion, recycling of pinosomes, and motility of the parasitic protist E. histolytica, the etiological agent of human amoebiasis. We show that two StAR-related lipid transfer (START) domain-containing LTPs (named as EhLTP1 and 3) are involved in these biological pathways in an LTP-specific manner. Our findings provide novel implications of LTPs, which are relevant to the elucidation of pathophysiology of the diseases caused by parasitic protists.
... The importance of CPs, particularly EhCP-A5, in ALA formation is evident in infections of laboratory animals, where overexpression of CPs leads to an increase in ALA size (Tillack et al., 2006). Conversely, reduced CP activity leads to a decrease in ALA formation ability in E. histolytica Stanley et al., 1995). In addition, an increase in the expression of some ehcp genes during ALA formation has previously been described, while non-pathogenic amoebae can be converted to pathogenic amoebae, simply by overexpression of some of specific ehcp genes (Matthiesen et al., 2013). ...
The human protozoan parasite Entamoeba histolytica can live in the human intestine for months or years without generating any symptoms in the host. For unknown reasons, amoebae can suddenly destroy the intestinal mucosa and become invasive. This can lead to amoebic colitis or extraintestinal amoebiasis whereby the amoebae spread to other organs via the blood vessels, most commonly the liver where abscesses develop. Entamoeba nuttalli is the closest genetic relative of E. histolytica and is found in wild macaques. Another close relative is E. dispar, which asyptomatically infects the human intestine. Although all three species are closely related, only E. histolytica and E. nuttalli are able to penetrate their host’s intestinal epithelium. Lineage-specific genes and gene families may hold the key to understanding differences in virulence among species. Here we discuss those genes found in E. histolytica that have relatives in only one or neither of its sister species, with particular focus on the peptidase, AIG, Ariel, and BspA families.
... Likewise, CP of E. histolytica are considered important pathogenicity mediators (Bruchhaus et al., 2003). Certainly, these proteolytic enzymes are essential in ALA formation (Stanley et al., 1995;Matthiesen et al., 2013). In particular, EhCP-A5 is directly involved in tissue damage and invasion through the degradation of extracellular matrix components; additionally, EhCP-A5 participates in the evasion of the host immune response by antibodies degradation and inhibition of the complement cascade (Freitas et al., 2009;Hou et al., 2010). ...
Entamoeba histolytica is an invasive enteric protozoan, whose infections are associated to high morbidity and mortality rates. However, only less than 10% of infected patients develop invasive amebiasis. The ability of E. histolytica to adapt to the intestinal microenvironment could be determinant in triggering pathogenic behavior. Indeed, during chronic inflammation, the vagus nerve limits the immune response through the anti-inflammatory reflex, which includes acetylcholine (ACh) as one of the predominant neurotransmitters at the infection site. Consequently, the response of E. histolytica trophozoites to ACh could be implicated in the establishment of invasive disease. The aim of this study was to evaluate the effect of ACh on E. histolytica virulence. Methods include binding detection of ACh to plasma membrane, quantification of the relative expression of virulence factors by RT-PCR and western blot, evaluation of the effect of ACh in different cellular processes related to E. histolytica pathogenesis, and assessment of the capability of E. histolytica to migrate and form hepatic abscesses in hamsters. Results demonstrated that E. histolytica trophozoites bind ACh on their membrane and show a clear increase of the expression of virulence factors, that were upregulated upon stimulation with the neurotransmitter. ACh treatment increased the expression of L220, Gal/GalNAc lectin heavy subunit (170 kDa), amebapore C, cysteine proteinase 2 (ehcp-a2), and cysteine proteinase 5 (ehcp-a5). Moreover, erythrophagocytosis, cytotoxicity, and actin cytoskeleton remodeling were augmented after ACh treatment. Likewise, by assessing the formation of amebic liver abscess, we found that stimulated trophozoites to develop greater hamster hepatic lesions with multiple granulomas. In conclusion, ACh enhanced parasite pathogenicity by upregulating diverse virulence factors, thereby contributing to disease severity, and could be linked to the establishment of invasive amebiasis.
... The cysteine proteases are a group of at least 50 endopeptidases, 36 of which form 3 major clades -'A', 'B' and 'C' (Clark et al. 2007;Casados-Vázquez et al. 2011). Whilst, collectively, the cysteine proteases are regarded as virulence factors, evidence suggests that approximately 90% of E. histolytica's cysteine protease-derived proteolytic activity is provided by just three proteins -EhCP-A1, EhCP-A2 and EhCP-A5 (Bruchhaus et al. 1996;Ankri et al. 1999;Stanley et al. 1995;Meléndez-López et al. 2007). EhCP-A5 is of particular interest as no functional orthologue exists in the non-pathogenic E. dispar (Jacobs et al. 1998) and expression of the protein is thought to be necessary for E. histolytica to invade the human intestinal mucosa (Thibeaux et al. 2014). ...
Amoebiasis is the third-most common cause of mortality worldwide from a parasitic disease. Whilst the primary aetiological agent of amoebiasis is the obligate human parasite Entamoeba histolytica, other members of the genus Entamoeba can infect humans and may be pathogenic. Here, we present the first annotated reference genome for Entamoeba moshkovskii, a species that has been associated with human infections, and compare the genomes of E. moshkovskii, E. histolytica, the human commensal Entamoeba dispar and the non-human pathogen Entamoeba invadens. Gene clustering and phylogenetic analyses show differences in expansion and contraction of families of proteins associated with host or bacterial interactions. They intimate the importance to parasitic Entamoeba species of surface-bound proteins involved in adhesion to extracellular membranes, such as the Gal/GalNAc lectin and members of the BspA and Ariel1 families. Furthermore, E. dispar is the only one of the four species to lack a functional copy of the key virulence factor cysteine protease CP-A5, whilst the gene’s presence in E. moshkovskii is consistent with the species’ potentially pathogenic nature. Entamoeba moshkovskii was found to be more diverse than E. histolytica across all sequence classes. The former is approximately 200 times more diverse than latter, with the 4 E. moshkovskii strains tested having a most recent common ancestor nearly 500 times more ancient than the tested E. histolytica strains. A four-haplotype test indicates that these E. moshkovskii strains are not the same species and should be regarded as a species complex.
... In EHI_127670-silencing transfectants, a lower activity for EhCP-A1 and EhCP-A5 can be detected. This result supports the major role for these peptidases (especially EhCP-A5) in tissue destruction, as previously shown (25,26,34,35). However, it has not been possible to clarify which mechanism underlies the inhibition identified hererin. ...
Recently, Entamoeba histolytica clones derived from isolate HM‐1:IMSS that differ in their pathogenicity were identified. Whereas some clones induce amoebic liver abscesses (ALAs) in animal models of amoebiasis, others provoke only minimal liver lesions. Based on transcriptome studies of pathogenic and nonpathogenic clones, differentially expressed genes associated with reduced or increased liver pathology can be identified. Here, to analyze the influence of these genes on ALA formation in more detail, an RNA interference‐trigger mediated silencing approach was used. Using newly identified trigger sequences, the expression of 15 genes was silenced. The respective transfectants were analyzed for their ability to induce liver destruction in the murine model for the disease. Silencing of EHI_180390 (encoding an AIG1 protein) increased liver pathology induced by a nonpathogenic parent clone, whereas silencing of EHI_127670 (encoding a hypothetical protein) decreased the pathogenicity of an initially pathogenic parent clone. Additional phenotypical in vitro analyses of EHI_127670 silencing as well as overexpression transfectants indicated that this molecule has an influence on size, growth, and cysteine peptidase activity of E. histolytica. This work describes an example of how the sole operational method for effective gene silencing in E. histolytica can be used for comprehensive analyses of putative pathogenicity factors.—Matthiesen, J., Lender, C., Haferkorn, A., Fehling, H., Meyer, M., Matthies, T., Tannich, E., Roeder, T., Lotter, H., Bruchhaus, I. Trigger‐induced RNAi gene silencing to identify pathogenicity factors of Entamoeba histolytica. FASEB J. 33, 1658–1668 (2019). www.fasebj.org
... In several studies, a direct correlation between CP activity and ALA formation was observed [19,[28][29][30][31]. In addition, ALA formation can be inhibited by specific cysteine peptidase inhibitors, and overexpression and silencing of individual E. histolytica cp genes can alter the ALAs-inducing ability of amoebae [3,6,[32][33][34][35][36]. Furthermore, several studies indicate that especially EhCP-A5 is involved in the invasion process into the intestinal mucosa [37][38][39]. ...
We here compared pathogenic (p) and non-pathogenic (np) isolates of Entamoeba histolytica to identify molecules involved in the ability of this parasite to induce amoebic liver abscess (ALA)-like lesions in two rodent models for the disease. We performed a comprehensive analysis of 12 clones (A1-A12) derived from a non-pathogenic isolate HM-1:IMSS-A and 12 clones (B1-B12) derived from a pathogenic isolate HM-1:IMSS-B. "Non-pathogenicity" included the induction of small and quickly resolved lesions while "pathogenicity" comprised larger abscess development that overstayed day 7 post infection. All A-clones were designated as non-pathogenic, whereas 4 out of 12 B-clones lost their ability to induce ALAs in gerbils. No correlation between ALA formation and cysteine peptidase (CP) activity, haemolytic activity, erythrophagocytosis, motility or cytopathic activity was found. To identify the molecular framework underlying different pathogenic phenotypes, three clones were selected for in-depth transcriptome analyses. Comparison of a non-pathogenic clone A1np with pathogenic clone B2p revealed 76 differentially expressed genes, whereas comparison of a non-pathogenic clone B8np with B2p revealed only 19 differentially expressed genes. Only six genes were found to be similarly regulated in the two non-pathogenic clones A1np and B8np in comparison with the pathogenic clone B2p. Based on these analyses, we chose 20 candidate genes and evaluated their roles in ALA formation using the respective gene-overexpressing transfectants. We conclude that different mechanisms lead to loss of pathogenicity. In total, we identified eight proteins, comprising a metallopeptidase, C2 domain proteins, alcohol dehydrogenases and hypothetical proteins, that affect the pathogenicity of E. histolytica.
... Stanley's et al. (Stanley et al., 1995) ...
Blastocystis spp. is a highly prevalent anaerobic Stramenopile parasite found in the intestinal tract of humans and various animals. This parasite is associated with non specific intestinal disorders, and could be involved in functional disorders such as the irritable bowel syndrome (IBS). In this work, the Blastocystis sp. ST7 genome sequencing project was carried out in collaboration with the Génoscope of Evry, the National University of Singapore, the Pasteur Institute of Lille and the University of Provence. This genome consists in a nuclear genome of 18,8 Mpb encoding 6020 genes, and a mitochondria‐like genome of 29 kpb localised in the mitochondrion‐like organelles. The analysis of this genome brings information about the evolution of this micro‐organism, its adaptation to the intestinal environment and its potential virulence factors. Blastocystis sp. ST7 was predicted to harbor several genes coding proteins that could act at the parasite‐host interface, and that are known to be involved in the pathogeny of many protozoa. They are PKS, NRPS, and hydrolases among them proteases. In addition, proteolytic activities were highlighted in the parasite culture supernatants. Two cysteine proteases (a cathepsin B and a legumain) were identified and characterized from the supernatants and could play a role in the physiopathology of the parasite, that confirm our in silico analyses. This work opens new ways to evaluate the impact of this parasite in human health.
... Extrazelluläre Matrix (ECM) des Wirtes zerstören und Gewebsverbände auflösen (Bruchhaus et al., 2003;Keene et al., 1986;Olivos-Garcia et al., 2004;Que et al., 2002;Scholze und Tannich, 1994;Stanley et al., 1995). ...
... Así, algunos de los factores de patogenicidad son: una lectina heterodimérica de 260 kDa, la cual se encuentra en la superficie celular y es esencial para la adhesión de la ameba a las mucinas de la pared del intestino y poder iniciar la invasión del huésped 58 , una adhesina con un peso molecular de 112 kDa que se une al hialuronato de los eritrocitos, un receptor a fibronectina de peso molecular de 37 kDa que participa en la adhesión a la matriz extracelular, una proteína de 77 kDa formadora de poros, conocida como amebaporo, que incrementa la permeabilidad de las bicapas lipídicas, serotonina que altera el transporte de electrolitos del intestino y una fosfolipasa A 2 que hidroliza diacilfosfolípidos 51,52 . Además, la E. histolytica posee una gran cantidad de proteasas para la degradación de los tejidos, de las cuales las más importantes son dependientes de cisteína 51,59 . Se ha observado que las cisteín proteasas amebianas son capaces de degradar las proteínas del complemento 60 e inmunoglobulinas humanas 61 . ...
Nitric oxide participation during amoebic liver abscess development. Nitric oxide participates in both physiological and pathophysiological functions, and it plays an important role in the mammalian immune system in killing or inhibiting the growth of many pathogens, including parasites, viruses and bacteria. En- tamoeba histolytica is a protozoan parasite that causes amoebiasis, which is characterized by intestinal damage and amoebic liver abscess development. The development of amoebic liver abscess in hamsters is similar to that in humans, whereas mice are resistant to amoebic liver abscess development due to an increase in nitric oxide produc- tion. Unlike in mice, amoebic liver abscess development in hamsters is due to an excess in nitric oxide production or possibly to a greater susceptibility of the hamster to damage caused by nitric oxide. Therefore, it could be important to elucidate if, in humans, an excess in nitric oxide production favors amoebic liver abscess development.
... Cysteine proteases are ubiquitous in nature and their implications at various stages of the lifecycle of the parasites make them promising potential drug targets. Three types of cysteine proteases gene-cpA, cpB and cpC have been identified [156,157] in Leishmania and are known to play a vital role in the parasite activities like destruction of host proteins [158], nutrition and neutralization of the host immune response [158,160]. Cysteine proteases also help in the intracellular survival of promastigotes during their differentiation in amastigotes by the evasion of the host immune response via destruction of effector molecules. ...
Leishmaniasis is a parasitic disease caused by hemoflagellate, Leishmania spp. The parasite is transmitted through the bites of an infected female phlebotomine sandfly. Leishmaniasis is prevalent throughout the world and in at least 88 countries. For its treatment, nearly 25 compounds are reported to have anti-leishmanial effects but not all are in use. Pentavalent antimony compounds had remained mainstay for nearly 75 years. However, emergence of resistance to this drug, led to the use of other compounds such as -Amphotericin B, Pentamidine, Paromomycin, Allopurinol etc. Amphotericin B, an antifungal macrolide polyene is characterized by the hydrophilic polyhydroxyl and hydrophobic polyene faces on it long axis which acts on membrane sterols resulting in parasite cell lysis. Presently, it is the only drug with highest cure rate. Other anti-fungals like ketoconazole, fluconazole and terbinafine are found less effective. Recently, anticancer alkylphosphocholines have been found to be the most effective oral compounds. These act as membrane synthetic ether-lipid analogues, and consist of alkyl chains in the lipid portions. Most promising of these are miltefosine (hexadecylphosphocholine), Edelfosine (ET-18-OCH3) and Ilmofosine (BM 41.440). However, the recent focus has been on identifying newer therapeutic targets in the parasite such as DNA topoisomerases. The present review describes the current understanding of different drugs against leishmaniasis, their chemistry, mode of action and the mechanism of resistance in the parasite. Future perspectives in the area of new anti-leishmanial drug targets are also enumerated. However, due to the vastness of the topic main emphasis is given on visceral leishmaniasis.
... The parasite infects over 50 million people annually resulting in 100,000 deaths, causing a significant impact on global health [2]. Several factors responsible for the pathogenesis of the disease have been elucidated in recent years including the parasite surface adhesion molecule Gal/GalNAc lectin (Gal-lectin) and secreted and membrane bound cysteine proteases and lipophosphopeptidoglycans [3,4,5]. Many of the defined virulence factors disrupt colonic mucosal barrier integrity, specifically tight junction proteins that result in diarrhea or more serious amebic dysentery [6]. ...
The exact mechanism by which Entamoeba histolytica disrupts the human colonic epithelium and invades the mucosa has yet to be clearly elucidated. E. histolytica produces a diverse array of putative virulent factors such as glycosidase, cysteine proteinases and amebapore that can modulate and/or disrupt epithelial barrier functions. However, it is currently thought that E. histolytica produces numerous other molecules and strategies to disrupt colonic mucosal defenses. In this study, we document a putative mechanism whereby the parasite alters the integrity of human epithelium by expressing a cognate tight junction protein of the host. We detected this protein as "occludin-like" as revealed by immunoblotting and immunoprecipitation studies and visualization by confocal microscopy using antibodies highly specific for human occludin. We propose that E. histolytica occludin-like protein might displace mucosal epithelial occludin-occludin tight junction interactions resulting in epithelial disruption analogous to sub mucosal human dendritic cells sampling luminal contents. These results indicate that E. histolytica occludin is a putative virulent component that can play a role in the pathogenesis of intestinal amebiasis.
... This relationship has been tested using trophozoites with low levels of CPs in an amoebic liver abscess hamster model [71] or in human colonic xenografts [22]. The inhibitor of CP activity E-64 reduces abscess formation in SCID mice [148], and specific inhibitors of EhCP4 reduce cecal inflammation [14], or specific inhibitors of EhCP1 in human colonic xenografts [17]. In the opposite strategy, overexpression of ehcp5 but not ehcp1 or ehcp2 significantly increased abscess formation in gerbils [149]. ...
... This relationship has been tested using trophozoites with low levels of CPs in an amoebic liver abscess hamster model [71] or in human colonic xenografts [22]. The inhibitor of CP activity E-64 reduces abscess formation in SCID mice [148], and specific inhibitors of EhCP4 reduce cecal inflammation [14], or specific inhibitors of EhCP1 in human colonic xenografts [17]. In the opposite strategy, overexpression of ehcp5 but not ehcp1 or ehcp2 significantly increased abscess formation in gerbils [149]. ...
The standard reference for pathogenic and nonpathogenic amoebae is the human parasite ; a direct correlation between virulence and protease expression has been demonstrated for this amoeba. Traditionally, proteases are considered virulence factors, including those that produce cytopathic effects in the host or that have been implicated in manipulating the immune response. Here, we expand the scope to other amoebae, including less-pathogenic species and highly pathogenic free-living amoebae. In this paper, proteases that affect mucin, extracellular matrix, immune system components, and diverse tissues and cells are included, based on studies in amoebic cultures and animal models. We also include proteases used by amoebae to degrade iron-containing proteins because iron scavenger capacity is currently considered a virulence factor for pathogens. In addition, proteases that have a role in adhesion and encystation, which are essential for establishing and transmitting infection, are discussed. The study of proteases and their specific inhibitors is relevant to the search for new therapeutic targets and to increase the power of drugs used to treat the diseases caused by these complex microorganisms.
... Studies with trophozoites, in which amoebapore expression was decreased, or even completely inhibited (Bracha et al., , 2003Zhang et al., 2004), have shown a considerable effect on amebic virulence in vitro or/and in vivo models. The same effect has been observed when cysteine proteinases were inhibited Stanley et al., 1995). ...
Precision-cut liver slices (PCLS) are mainly used to evaluate hepatotoxicity and metabolism of chemicals, as well as to study mechanisms of liver damage and repair. However, recently they have been used as a system to study amoebic infections. The aim of this study was to validate this model as an alternative for experimental amoebic liver absess (ALA) in animals. To do this, the PCLS was analyzed for the expression of amoebapore and cysteine proteinases 1 and 5, three of the most studied virulence factors of Entamoeba histolytica, as well as the induction of apoptosis and cytokines production in response to the ex vivo infection. PCHLS were prepared with the Brendel-Vitron tissue slicer and then, infected with 200,000 trophozoites of E. histolytica. Samples were taken at 0, 6, 12, 18, and 24h and compared to control non-infected slices. Morphological studies were performed in order to verify the infection; while apoptosis was studied by TUNEL and PAS techniques. The expression of cysteine proteinases (1 and 5), and amoebapore, was analyzed by real-time PCR. By using ELISA assays, the production of cytokines was also studied. PCHLS were found to be a reproducible infection system, and E. histolytica caused the expression of cysteine proteinases and amoebapore in infected slices. At the same time, trophozoites induce release of cytokines and apoptotic death of the hepatocytes close to them. PCHLS represent a new and suitable alternative model to study the pathogenesis of hepatic amoebiasis.
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.
Amebic liver abscess (ALA) is a focal destruction of the liver tissue due to infection with the protozoan parasite Entamoeba histolytica (E. histolytica). So far, it is generally accepted that pathogenicity factors of the parasite contribute to tissue damage. However, epidemiological studies as well as data that evolve from respective murine models suggest a contribution of host factors as well. Independent from the infection rates, men have a higher risk to develop ALA compared to women. The mouse model for ALA exhibits the same sex difference and based on this model, we found that IFNγ provided by Natural Killer T cells confers the female resistance towards ALA. This specific immune response is clearly modulated by androgens. Hence, female mice which were substituted with testosterone develop larger abscesses and are less able to control E. histolytica viability in the liver compared to males. In male mice, on the other side, an enhanced CCL2-dependent recruitment of inflammatory Ly6Chi monocytes via the IL-23/IL-17 immune pathological axis is responsible for tissue destruction during ALA development. Furthermore, TNFα, that exhibits a pivotal role in cytotoxicity, is an effector molecule also responsible for increased liver damage in this case. Interestingly, in human asymptomatically infected with E. histolytica, men exhibit higher CCL2 serum levels compared to women, suggesting a similar mechanism to mice underlying the immune response to the parasite. Indeed, there are a variety of phenotypical similarities within the inflammatory monocyte subsets between humans and mice including sex-dependent differences in the expression of the surface receptor for CCL2 as well as a male bias in the production of specific cytokines involved in the recruitment of innate immune cells. In summary, we assume that the host immune response is considerably involved in liver tissue damage due to the strong reactivity of inflammatory monocytes, which is significantly more distinct in male individuals.
This study was conducted in Samarra, city on patients with amoebic dysentery and diarrhea for the period (1/12/ 2017-30/8/2018). The study included the examination of (600) stool samples of patients with diarrhea who were coming to the general Samarra hospital and medical clinics and took 30 samples with amoebic dysentery and 30 samples with diarrhea and anather 30 healthy samples as control. The results showed that 205 of tested samples were infected with amoebic dysentery (34.16%). Also, we have been studying and examining some of biochemical parameters of blood in patients with amoebic dysentery and diarrhea and for control group, these parameters are percentage of Ferritin, Hepsidin and Iron in serum and total iron band capacity (TIBC) and the results showed significant decrease (P≤ ≤ ≤ ≤ ≤0.05) in percentage of Ferritin, Iron and TIBC in patients with amoebic dysentery in compared with control group, while itwas increased (P≤ ≤ ≤ ≤ ≤0.05) in Hepcidin percentage.
The Entamoeba histolytica 27-kDa cysteine proteinases exhibit striking binding specificities for immobilized laminin over other components of the extracellular matrix, such as collagen and fibronectin. Inactivation of the proteinase with the active-site inhibitor L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane abolishes laminin binding by the enzyme, and conversely, laminin inhibits cleavage of a fluorogenic dipeptide substrate of the amebic cysteine proteinase, suggesting that the substrate binding pocket of the enzyme is involved in the binding of laminin. The addition of laminin but not fibronectin or collagen to E. histolytica trophozoites significantly reduces amebic liver abscess formation in severe combined immunodeficient mice, further supporting the hypothesis that E. histolytica cysteine proteinases play an important role in amebic pathogenesis. The specific interaction of amebic proteinases with laminin may be exploited in designing new inhibitors of these enzymes.
Infection with the enteric protozoan Entamoeba histolytica is still a serious public health problem, especially in developing countries. Amoebic liver abscess (ALA) is the most common extraintestinal manifestation of the amoebiasis, and it can lead to serious and potentially life-threatening complications in some people. ALA can be cured by metronidazole (MTZ); however, because it has poor activity against luminal trophozoites, 40–60% of treated patients get repeated episodes of invasive disease and require repeated treatments that can induce resistance to MTZ, this may emerge as an important public health problem. Anti-virulence strategies that impair the virulence of pathogens are one of the novel approaches to solving the problem. In this study, we found that low doses of curcumin (10 and 50 μM) attenuate the virulence of E. histolytica without affecting trophozoites growth or triggering liver injury. Curcumin (CUR) decreases the expression of genes associated with E. histolytica virulence (gal/galnac lectin, ehcp1, ehcp5, and amoebapore), and is correlated with significantly lower amoebic invasion. In addition, oxidative stress is critically involved in the etiopathology of amoebic liver abscess; our results show no changes in mRNA expression levels of superoxide dismutase (SOD) and catalase (CAT) after E. histolytica infection, with or without CUR. This study provides clear evidence that curcumin could be an anti-virulence agent against E. histolytica, and makes it an attractive potential starting point for effective treatments that reduce downstream amoebic liver abscess.
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.
Several members of the genus Entamoeba infect humans including E. histolytica, E. moshkovskii, and E. dispar. Among these, only E. histolytica is considered pathogenic and is the causal agent of amebiasis. Amebiasis is the second most common cause of death from a parasite worldwide after malaria with considerable morbidity and mortality [1, 2]. Based on the site of infection, the clinical features of amebiasis range from asymptomatic colonization to amebic colitis (dysentery or diarrhea) and invasive extraintestinal amebiasis, which appears most commonly in the form of liver abscesses [2]. According to the World Health Organization, amebiasis occurs worldwide, with approximately 50 million people infected annually, causing close to 100,000 deaths per year. Therefore, it is important to understand the epidemiology, infection, and control of the disease. The aim of this chapter is to provide a current understanding of human amebiasis, which illustrates the parasite biology, mechanism of pathogenesis, diagnosis, and prevention.
Protozoan parasitic infections of the gastrointestinal (GI) tract are being increasingly recognized as an important public health problem in the United States. The intestinal protozoan pathogens include both parasites that are extracellular, such as Giardia lamblia, Blastocystis hominis, and Entamoeba histolytica, and the intracellular spore-forming parasites Cryptosporidium parvum, Cyclospora cayetanensis, and Isospora belli. The AIDS epidemic has played an important role in the recognition of intracellular spore-forming parasites as important gastrointestinal protozoan pathogens. Stool microscopic examinations may be performed in clinical laboratories only if specifically requested. In some cases in which infection is suspected on clinical grounds, stool examination may not be revealing, and endoscopy may be necessary to make the diagnosis. The recent rapid development of new molecular assays, will likely facilitate more rapid laboratory diagnoses of not only protozoan, but also viral and bacterial pathogens in the near future.
In October 2000, The Institute of Genomic Research (TIGR) received funding from the National Institutes of Health to sequence approximately 99% of the Entamoeba histolytica genome. There are many questions regarding the pathogenicity and biology of E. histolytica that will be fully addressed by the genome project. Practical applications, such as new diagnostic agents, vaccine candidates, and potential new drug targets, may also be revealed. This chapter updates the latest knowledge regarding these aspects of this parasite and indicates those areas in which the genome project is likely to have a particular impact.
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.
In the human intestine, evolutionary pressures have selected host and parasite mechanisms that maintain spatial separation of Entamoeba histolytica on the luminal side of the mucus–epithelial barrier. The function of the barrier is conferred by many systems acting on multiple levels. Mechanisms that strengthen and maintain stability of the epithelial barrier are critical for preventing disease and keeping E. histolytica infections asymptomatic. It is unknown why invasion happens. Intestinal epithelial cells are in close and continuous proximity to the parasite, and abnormal responses by epithelial cells are suspected to instigate disease. This interaction, however, is poorly understood. When invasion occurs the gut has a second line of innate defenses that rapidly eliminate the parasite: Sensing of invasion by resident cells, innate humoral immunity, and recruitment of competent immune cells to sites of invasion. The pathology that arises during invasion, which culminates as amebic dysentery or colitis, is a combined effect of direct damage by trophozoites and collateral damage from host defenses.
Amebic liver abscess (ALA) is a severe focal destruction of liver tissue caused by infection with the parasite Entamoeba histolytica (E. histolytica). In the past, tissue damage has been mainly attributed to pathogenicity factors of the parasite. However, the massive presence of innate immune cells raises the question whether host cells contribute to the destruction of the liver tissue as well. In this chapter, we discuss the role of neutrophils, monocytes, and macrophages during ALA in animal models for the disease. In brief, neutrophils contribute only partially to the observed pathology, whereas inflammatory monocytes and resident liver macrophages are substantially involved in tissue damage seen during E. histolytica infection. Therefore, we conclude beyond parasite-specific effector molecules, immune pathological mechanisms of the host substantially contribute to the development of ALA.
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.
Garlic (Allium sativum L. family Liliaceae), like other plants, has an exquisite defence system composed of as many different components as the human immune system. In order to protect itself from insects and fungi, garlic enzymatically produces an organosulfur compound known as allicin, when it is injured. And it is chemically known as 2-propene-1-sulfinothioc acid S-2-propenyl ester; thio-2-propene-1-sulfinic acid S-allyl ester. It prevents infectious agents or organisms from spreading of infection. This is found in its pure form to exhibit antiparasitic activity against many major human intestinal protozoan parasites such as Entamoeba histolytica which is the causative agent of human amebiasis. Infection with this parasite often results in amoebic colitis and liver abscess formation, causing significant morbidity and mortality. More than 500 million people are infected with the parasite worldwide, resulting in an estimated 50 million cases of diarrhoea and 11 thousand deaths per year. Evidence from in vitro studies suggests that the E.histolytica cysteine proteinase a 315 residue long globular protein plays a role in the tissue lysis and cytopathic effects seen in invasive amebiasis. Invasive E. histolytica trophozoites secrete 10–1000 fold more cysteine proteinases than non-invasive Entamoeba dispar. Since the crystal structure of E.histolytica cysteine proteinase is not been solved yet, our study involved in developing a reliable model and estimate the drugable activity of allicin to inhibit the proteolytic activity of cysteine protienase (Uniprot Q01957) from Entamoeba histolytica using the natural compound allicin as antagonist.
Garlic (Allium sativum L. family Liliaceae), like other plants, has an exquisite defence system composed of as many different components as the human immune system. In order to protect itself from insects and fungi, garlic enzymatically produces an organosulfur compound known as allicin, when it is injured. And it is chemically known as 2-propene-1-sulfinothioc acid S-2-propenyl ester; thio-2-propene-1-sulfinic acid S-allyl ester. It prevents infectious agents or organisms from spreading of infection. This is found in its pure form to exhibit antiparasitic activity against many major human intestinal protozoan parasites such as Entamoeba histolytica which is the causative agent of human amebiasis. Infection with this parasite often results in amoebic colitis and liver abscess formation, causing significant morbidity and mortality. More than 500 million people are infected with the parasite worldwide, resulting in an estimated 50 million cases of diarrhoea and 11 thousand deaths per year. Evidence from in vitro studies suggests that the E.histolytica cysteine proteinase a 315 residue long globular protein plays a role in the tissue lysis and cytopathic effects seen in invasive amebiasis. Invasive E. histolytica trophozoites secrete 10–1000 fold more cysteine proteinases than non-invasive Entamoeba dispar. Since the crystal structure of E.histolytica cysteine proteinase is not been solved yet, our study involved in developing a reliable model and estimate the drugable activity of allicin to inhibit the proteolytic activity of cysteine protienase (Uniprot Q01957) from Entamoeba histolytica using the natural compound allicin as antagonist.
Entamoeba histolytica and E. dispar are genetically distinct but closely related protozoan species. Both colonize the human gut but only E. histolytica is able to invade tissues and cause disease. Comparison of the 2 species may help to elucidate the specific mechanisms involved
in the pathogenicity of E. histolytica. During the last few years, various amoeba molecules considered to be important for pathogenic tissue invasion have been
identified and characterized, such as a galactose-inhibitable surface lectin, pore-forming peptides and cysteine proteinases.
This review summarizes present knowledge about the structure and function of these molecules, with emphasis on the differences
between E. histolytica and E. dispar.
Cysteine peptidases (CPs) of Entamoeba histolytica are considered to be important pathogenicity factors. Previous studies have found that under standard axenic culture conditions, only four (ehcp-a1, ehcp-a2, ehcp-a5, and ehcp-a7) out of 35 papain-like ehcp genes present in the E. histolytica genome are expressed at high levels. Little is known about the expression of CPs in E. histolytica during amoebic liver abscess (ALA) formation. In the current study, a quantitative real-time PCR assay was developed to determine the expression of the various ehcp genes during ALA formation in animal models. Increased expression of four ehcp genes (ehcp-a3, -a4, -a10, and -c13) was detected in the gerbil and mouse models. Increased expression of another three ehcp genes (ehcp-a5, -a6, and -a7) was detected in the mouse model only, and two other ehcp genes (ehcp-b8 and -b9) showed increased expression in the gerbil model only. Trophozoites of the nonpathogenic E. histolytica HM-1:IMSS clone A1, which was unable to induce ALAs, were transfected with vectors enabling overexpression of those CPs that are expressed at high levels under culture conditions or during ALA formation. Interestingly, overexpression of ehcp-b8, -b9, and -c13 restored the pathogenic phenotype of the nonpathogenic clone A1 whereas overexpression of various other peptidase genes had no effect on the pathogenicity of this clone.
Protease function is essential to many biological systems and processes. In parasites, proteases are essential for host tissue degradation, immune evasion, and nutrition acquisition. Helminths (worms) depend on several classes of proteases for development, host tissue invasion and migration, and for degradation of host hemoglobin and serum proteins. The protozoa, which cause malaria, depend on both cysteine and aspartic proteases to initiate host hemoglobin digestion. Other types of proteases are involved in erythrocyte cell invasion and cell exit. Surface metalloproteases in kinetoplastids are implicated in the evasion of complement-mediated cell lysis and cell entry. Cysteine proteases in Entamoeba facilitate invasion of the host colon. Giardia utilizes a cysteine protease for both encystation and excystation. This review will summarize published data using protease inhibitors as tools to identify the function of parasite proteases in the development, virulence, and pathogenesis of parasites; as well as the role of endogenous parasite protease inhibitors in regulation.
The human cathelicidin hCAP18/LL-37 is an antimicrobial protein consisting of a conserved N-terminal prosequence called the cathelin-like domain and a C-terminal peptide called LL-37. This peptide contains 37 amino acid residues, and several truncated variants obtained from natural sources or by chemical synthesis differ in their capability to damage Gram positive and Gram negative bacteria as well as Candida albicans. KR-12 is the shortest peptide (12 amino acids) of LL-37 that has conserved antibacterial activity. In addition to LL-37, other active cathelicidin-derived peptides have been reported; for instance, the peptides KR-20, a 20-aa derivative of LL-37, and KS-30, a 30-aa derivative of LL-37, have been found in human sweat. Both peptides exhibit an overall increased antibacterial and antifungal activity when compared with LL-37. We investigated the effect of LL-37 and three peptides derived from this antimicrobial molecule, KR-12, KR-20 and KS-30, on the integrity of Entamoeba histolytica trophozoites. The four peptides showed effects on Entamoeba histolytica integrity and viability in the concentration range of 10 - 50 μM. The peptides KR-12, KR-20, KS-30 and LL-37 differed in their capability to damage the parasite integrity, with KR-20 being the most effective and with KR-12 and LL-37 being less active. These results demonstrate the ability of antimicrobial peptides derived from human cathelicidin to damage Entamoeba trophozoites. Moreover, it was shown that the integrity of the peptides is altered in the presence of an ameba soluble fraction with cysteine protease activity.
Classical cancer treatments have focused on the use of cytotoxic agents and/or radiation therapy that target both tumour and normal cells. Consequently, current cancer treatments with chemotherapeutic agents are subject to limitations associated with high toxicity and resistance. There is a need to develop new agents and therapies that will permit long-term administration without compromising the patient. The individual steps of angiogenesis and metastasis during cancer progression are now well understood and present new targets for chemotherapy. The cysteine protease cathepsin B has been linked to the invasive steps during the metastatic process and provides a new target for drug development. Cathepsin B has also been implicated in other disease states with aberrant protein turnover such as muscular dystrophy, inflammatory airway diseases, bone and joint disorders and pancreatitis. Examination of the x-ray crystal structure of cathepsin B reveals the presence of an insertion loop extending for 18 residues that obstructs part of the active-site cleft. This ‘occluding loop’ is a unique feature that can be targeted for the development of specific inhibitors of cathepsin B as potential therapeutic agents. Inhibitors can be classified as to whether they act in a reversible or irreversible manner. This review details members of both of these classes of inhibitors and their therapeutic applications.
AmebiasisBlastocystis hominisGiardiasisDientamoeba fragilisBalantidiasisCoccidia (Cryptosporidium, Isospora, and Cyclospora)
We used severe combined immunodeficient (SCID) mice to study resistance to invasive infection with Entamoeba histolytica. Seven of seven SCID mice developed liver abscesses when challenged intrahepatically with virulent HM1:IMSS strain E. histolytica trophozoites. Only one of seven similarly challenged immunocompetent congenic C.B-17 mice developed an abscess. Adoptive transfer of polyclonal rabbit anti-E. histolytica antiserum, but not preimmune rabbit serum, completely protected 7 of 12 SCID mice from intrahepatic challenge with ameba. These results demonstrate that lymphocyte-based immunity is important in protection against amebic liver abscess, and that anti-E. histolytica antibody can protect against amebic infection in this system. The SCID mouse may provide a powerful model for studying the components of protective immunity to invasive amebiasis.
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.
A cDNA clone derived from the gene encoding a cysteine proteinase of pathogenic Entamoeba histolytica was isolated using an antiserum to the purified enzyme. This clone was used to identify the homologous clone in a cDNA library from nonpathogenic E. histolytica. Sequence analysis and comparison of the predicted amino acid sequences revealed a sequence divergence of 16%. Southern blot analyses indicated that (i) pathogenic isolates may contain more genes coding for these or related enzymes than nonpathogenic isolates, (ii) the structure and organization of these genes are conserved within each group of amoebae, and (iii) none of the genes is found in both pathogenic and nonpathogenic E. histolytica, underlining the notion that the two groups are genetically distinct. Northern blot analyses suggested that the cysteine proteinase is expressed by pathogenic isolates in substantially higher amounts than by nonpathogenic isolates. Overexpression of this enzyme may be an important factor in the pathogenicity of E. histolytica.
The gastrointestinal tract is lined with a monolayer of cells that undergo perpetual and rapid renewal. Four principal, terminally differentiated cell types populate the monolayer, enterocytes, goblet cells, Paneth cells, and enteroendocrine cells. This epithelium exhibits complex patterns of regional differentiation, both from crypt-to-villus and from duodenum-to-colon. The "liver" fatty acid binding protein (L-FABP) gene represents a useful model for analyzing the molecular basis for intestinal epithelial differentiation since it exhibits cell-specific, region-specific, as well as developmental stage specific expression. We have previously linked portions of the 5' nontranscribed domain of the rat L-FABP gene to the human growth hormone (hGH) gene and analyzed expression of the fusion gene in adult transgenic mice. High levels of hGH expression were noted in enterocytes as well as cells that histologically resembled enteroendocrine cells. In the present study, we have used immunocytochemical techniques to map the distribution of enteroendocrine cells in the normal adult mouse gut and to characterize those that synthesize L-FABP. In addition, L-FABP/hGH fusion genes were used to identify subsets of enteroendocrine cells based on their ability to support hGH synthesis in several different pedigrees of transgenic mice. The results reveal remarkable differences in transgene expression between, and within, enteroendocrine cell populations previously classified only on the basis of their neuroendocrine products. In some cases, these differences are related to the position occupied by cells along the duodenal-to-colonic and crypt-to-villus axes of the gut. Thus, transgenes appear to be sensitive tools for examining the cellular and regional differentiation of this class of intestinal epithelial cells.
FPLC anion-exchange and chromatofocusing chromatography were used to purify the major neutral proteinase from secretions of axenically cultured Entamoeba histolytica trophozoites. HM-1 strain trophozoites, which were more proteolytically active than the less virulent HK-9 strain, were used for purification of the enzyme. It is a thiol proteinase with a subunit Mr of approximately 56,000, a neutral pH optimum, and a pI of 6. The importance of this enzyme in extraintestinal amoebiasis is suggested by its ability to degrade a model of connective tissue extracellular matrix as well as purified fibronectin, laminin, and type I collagen. The enzyme caused a loss of adhesion of mammalian cells in culture, probably because of its ability to degrade anchoring proteins. Experiments with a peptide substrate and inhibitors indicated that the proteinase preferentially binds peptides with arginine at P-1. It is also a plasminogen activator, and could thus potentiate host proteinase systems.
Previously, we described the isolation of a cDNA clone and the gene encoding a protective antigen of the protozoan parasite Entamoeba histolytica, the serine-rich Entamoeba histolytica protein (SREHP). The derived amino acid sequence of the SREHP cDNA clone was remarkable for a high serine content (52/233 amino acids), a putative signal sequence, multiple hydrophilic dodecapeptide and octapeptide tandem repeats, and a hydrophobic C-terminal putative membrane-spanning region. Here, we show that SREHP is modified by the addition of phosphate at serine residues, O-linked terminal N-acetylglucosamine residues, and by acylation. When the SREHP gene is expressed in baculovirus transformed Sf-9 cells, the product is also phosphorylated and glycosylated and is localized to the plasma membrane of the insect cells. The native SREHP molecule also serves as a potent chemoattractant for amebic trophozoites. The data presented here suggest that SREHP is a unique membrane protein with phosphorylation and glycosylation patterns usually associated with nuclear or cytoplasmic proteins.
To model the initial pathogenic effects of Entamoeba histolytica trophozoites on intestinal epithelial cells, the interactions of E. histolytica HM1-IMSS trophozoites with polarized human intestinal Caco-2 cell monolayers grown on permeabilized filters were examined. Trophozoites, when incubated with the apical surface of the monolayers at 37 degrees C, induced a rapid decrease in transepithelial resistance over 15 to 60 min. The transmonolayer resistance response was not associated with changes in short-circuit current but was associated with an increase in mannitol flux, suggesting that the drop in resistance reflected a nonselective increase in epithelial permeability rather than stimulation of electrogenic ion transport. This response preceded the earliest detection of morphologic disruption of monolayer integrity by light or electron microscopy. Apical injury to the monolayer was detected by ultrastructural studies which revealed a loss of brush border in regions of contact between epithelial cells and amebas and by chromium release assays where a small increase in the apical release of 51Cr from the monolayer (6% over background) was observed. The transmonolayer resistance response was inhibited when the temperature was reduced to 4 degrees C and by addition of cytochalasin D (1 microgram/ml) to the medium at concentrations that did not directly affect transmonolayer resistance. Application of amebic lysates or medium conditioned by coincubation of amebas with Caco-2 monolayers failed to lower transmonolayer resistance, suggesting that this effect was not mediated by soluble amebic cytotoxins. Polarized Caco-2 monolayers grown on permeable filters provide a useful model for studying the initial interactions of E. histolytica trophozoites with intestinal epithelial cells.
Cysteine proteinases are hypothesized to be important virulence factors of Entamoeba histolytica, the causative agent of amebic dysentery and liver abscesses. The release of a histolytic cysteine proteinase from E. histolytica correlates with the pathogenicity of both axenic strains and recent clinical isolates as determined by clinical history of invasive disease, zymodeme analysis, and cytopathic effect. We now show that pathogenic isolates have a unique cysteine proteinase gene (ACP1). Two other cysteine proteinase genes (ACP2, ACP3) are 85% identical to each other and are present in both pathogenic and nonpathogenic isolates. ACP1 is only 35 and 45% identical in sequence to the two genes found in all isolates and is present on a distinct chromosome-size DNA fragment. Presence of the ACP1 gene correlates with increased proteinase expression and activity in pathogenic isolates as well as cytopathic effect on a fibroblast monolayer, an in vitro assay of virulence. Analysis of the predicted amino acid sequence of the ACP1 proteinase gene reveals homology with cysteine proteinases released by activated macrophages and invasive cancer cells, suggesting an evolutionarily conserved mechanism of tissue invasion. The observation that a histolytic cysteine proteinase gene is present only in pathogenic isolates of E. histolytica suggests that this aspect of virulence in amebiasis is genetically predetermined.
We used severe combined immunodeficient (SCID) mice to study resistance to invasive infection with Entamoeba histolytica. Seven of seven SCID mice developed liver abscesses when challenged intrahepatically with virulent HM1:IMSS strain E. histolytica trophozoites. Only one of seven similarly challenged immunocompetent congenic C.B-17 mice developed an abscess. Adoptive transfer of polyclonal rabbit anti-E. histolytica antiserum, but not preimmune rabbit serum, completely protected 7 of 12 SCID mice from intrahepatic challenge with ameba. These results demonstrate that lymphocyte-based immunity is important in protection against amebic liver abscess, and that anti-E. histolytica antibody can protect against amebic infection in this system. The SCID mouse may provide a powerful model for studying the components of protective immunity to invasive amebiasis.
The gastrointestinal tract is lined with a monolayer of cells that undergo perpetual and rapid renewal. Four principal, terminally differentiated cell types populate the monolayer, enterocytes, goblet cells, Paneth cells, and enteroendocrine cells. This epithelium exhibits complex patterns of regional differentiation, both from crypt-to-villus and from duodenum-to-colon. The "liver" fatty acid binding protein (L-FABP) gene represents a useful model for analyzing the molecular basis for intestinal epithelial differentiation since it exhibits cell-specific, region-specific, as well as developmental stage specific expression. We have previously linked portions of the 5' nontranscribed domain of the rat L-FABP gene to the human growth hormone (hGH) gene and analyzed expression of the fusion gene in adult transgenic mice. High levels of hGH expression were noted in enterocytes as well as cells that histologically resembled enteroendocrine cells. In the present study, we have used immunocytochemical techniques to map the distribution of enteroendocrine cells in the normal adult mouse gut and to characterize those that synthesize L-FABP. In addition, L-FABP/hGH fusion genes were used to identify subsets of enteroendocrine cells based on their ability to support hGH synthesis in several different pedigrees of transgenic mice. The results reveal remarkable differences in transgene expression between, and within, enteroendocrine cell populations previously classified only on the basis of their neuroendocrine products. In some cases, these differences are related to the position occupied by cells along the duodenal-to-colonic and crypt-to-villus axes of the gut. Thus, transgenes appear to be sensitive tools for examining the cellular and regional differentiation of this class of intestinal epithelial cells.
FPLC anion-exchange and chromatofocusing chromatography were used to purify the major neutral proteinase from secretions of axenically cultured Entamoeba histolytica trophozoites. HM-1 strain trophozoites, which were more proteolytically active than the less virulent HK-9 strain, were used for purification of the enzyme. It is a thiol proteinase with a subunit Mr of approximately 56,000, a neutral pH optimum, and a pI of 6. The importance of this enzyme in extraintestinal amoebiasis is suggested by its ability to degrade a model of connective tissue extracellular matrix as well as purified fibronectin, laminin, and type I collagen. The enzyme caused a loss of adhesion of mammalian cells in culture, probably because of its ability to degrade anchoring proteins. Experiments with a peptide substrate and inhibitors indicated that the proteinase preferentially binds peptides with arginine at P-1. It is also a plasminogen activator, and could thus potentiate host proteinase systems.
The partial purification of two intracellular proteinases from the protozoan parasite Entamoeba histolytica is reported. One of these enzymes is an acid proteinase exhibiting maximum activity at pH 3.5 (hemoglobin substrate), is little affected by a range of inhibitors or activators, and is presumed to be similar to cathepsin D. Also present is a neutral proteinase exhibiting optimum activity at pH 6.0 (azocasein) but only poorly hydrolyzing either hemoglobin or serum albumen. This latter enzyme displayed no metal ion requirement, but was markedly inhibited by thiol-blocking agents and activated by free sulhydryl-containing compounds.
A heat-labile material with cytotoxic and enterotoxic activities was isolated from axenically cultivated Entamoeba histolytica. The cytotoxin-enterotoxin was partially purified from the particulate-free supernatant of sonicated trophozoites by ammonium
sulfate precipitation and gel filtration. Cytotoxic activity, assayed on monolayers of HeLa or BHK-21 cells. was restricted
to proteins that eluted in the molecular weight range of 25,000–35,000 daltons. Cytotoxicity was demonstrated at protein concentrations
as low as 2–4 µg/ml, was heat-labile (75 C, 30 min), and was inhibited by specific immune IgG and by an undetermined factor
in nonimmune serum. Enterotoxic activity of the partially purified toxin was demonstrated by induction of fluid secretion
in ligated rabbit ileal loops. The cytotoxin-enterotoxin of E. histoIytica may play an important role in the production of diarrhea and mucosal injury in amoebic colitis.
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.
The major secreted proteinase of Entamoeba histolytica, a 56-kDa neutral cysteine proteinase, activates C by cleaving C3. The action of the proteinase is similar to C-derived C3 convertases because it produces a single cleavage of the alpha-chain in a dose- and time-dependent manner and cleaves C3 between residues 78 and 79, only one amino acid residue distal to the natural site acted on by the C3 convertases. C3a generation was detected by RIA. The 105-kDa fragment produced by the cleavage of the alpha-chain was structurally and functionally equivalent to the alpha'-chain of C3b, as demonstrated by susceptibility to the action of factors I and H and participation in the activation of the alternative pathway of C. Activation of C by the 56-kDa neutral cysteine proteinase may play a role in the early inflammatory response in amebic lesions and thus contribute to the pathogenesis of invasive amebiasis.
Expression of the 56-kilodalton (kDa) neutral thiol proteinase has been shown to correlate with the potential of clinical isolates of Entamoeba histolytica to produce invasive disease. A 56-kDa band was identified by gelatin substrate gel electrophoresis in 10 of 10 isolates from patients with colitis or amebic liver abscesses, but in only 1 of 10 isolates from asymptomatic patients. Pathogenic isolates appear capable of releasing significantly larger quantities of the proteinase, as measured by cleavage of a synthetic peptide substrate, ZRR-AMC (benzyloxy-carbonyl-arginine-arginine-4-amino-7-methylcoumarin). We have also shown that the proteinase is released during the course of clinical invasive amebic disease, as demonstrated by the presence of circulating antibodies detectable by enzyme-linked immunosorbent assay. These studies support the importance of the 56-kDa thiol proteinase in the pathogenesis of invasive amebiasis.
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.
The injection of 1 x 10(6) trophozoites of axenically grown Entamoeba histolytica strain HM-1 in the subcutaneous tissue of the rat results in an acute and self-limited inflammatory process, characterized by the early onset of conspicuous tissue necrosis and focal hemorrhage in the vicinity of the parasites, followed by infiltration with polymorphonuclear leukocytes. The process develops for 5-10 hr but during that period amebic trophozoites progressively disappear, leukocytes undergo degenerative changes, and the lesion tends to heal in 72-96 hr. In leukopenic animals (less than 1000 white blood cells/ml) tissue necrosis and hemorrhage are equally conspicuous in the neighborhood of amebas. Inhibition of amebic proteinase activity prior to injection by heat denaturation, p-hydroxy-mercuri-benzoate (PHMB), soybean trypsin inhibitor (STI), and human alpha-2-macroglobulin (alpha 2M), alone or in various combinations, results in absence or notorious decrease in tissue necrosis as well as in clearly diminished inflammatory reaction. This effect is particularly evident when cysteine proteinases are either specifically or generally inhibited. On the other hand, amebic proteinase inhibition with alpha 2M and STI does not interfere with the cell-killing capacity of trophozoites co-incubated in vitro for 2 hr with rat peritoneal cells enriched for macrophages. We conclude that in acute experimental amebiasis produced in the subcutaneous tissue of the rat, amebic cysteine (and perhaps other) proteinases are primarily responsible for necrosis and are also important, but not essential, for inflammation. We also suggest that in this model polymorphonuclear leukocytes are not required for tissue necrosis. Finally, in an in vitro model, the cell-killing capacity of amebas is not influenced by the proteinase activity of the parasite.
We previously reported partial purification of a proteinaceous substance with cytotoxic and enterotoxic activity isolated from the soluble fraction of sonicated axenically cultivated Entamoeba histolytica trophozoites. Demonstration of cytotoxic activity of the preparation (amebal toxin) was dependent on removal of serum from the tissue culture assay system. The objective of the present study was to identify the factor(s) in non-immune sera responsible for producing in vitro inhibition of amebal toxin cytotoxicity on HeLa cells. Gel filtration of non-immune sera from adult humans or bovines demonstrated that two portions of the eluate had significant inhibitory against the toxin. A high molecular weight inhibitory fraction was identified as predominantly alpha-2 macroglobulin and a low molecular weight inhibitory fraction was identified as predominantly alpha-1 antiprotease. Preparative isoelectric focusing of human serum isolated inhibitory fractions containing these same alpha globulins. Alpha-2 macroglobulin was purified and alpha-1 antiprotease was partially purified from human serum by other methods and shown to have high inhibitory activity against the amebal cytotoxin. Substances that were inhibitory to the cytotoxic activity of the amebal toxin also mediated reattachment of toxin treated HeLa cells. We conclude that the characteristics of the serum inhibitors, especially their ability to reverse the cytotoxic effects of amebal toxin on HeLa cells, suggests that the amebal toxin has protease activity.
A heat-labile cytotoxin was isolated from virulent strains of axenically cultivated Entamoeba thistolytica. Strains of E. histolytica representing a spectrum of virulence as determined in animal and in vitro models of disease were examined for cytotoxic activity.
Extracts of virulent strain HMI possessed marked cytotoxic activity, those of moderately virulent strain 200 showed intermediate
activity, and those of avirulent strains 303 and Rahman showed no activity. The cytotoxin was partially purified from the
cell-free supernatant of sonicated E. histolytica HMI trophozoites by ammonium sulfate precipitation and gel filtration. Cytotoxic activity was stable in a narrow pH range
(6–7.2) and in 1 m NaCI, urea, and guanidine. Specific immune rabbit and human antiserum as wellas the protease inhibitors aprotinin, pepstatin,
and leupeptin inhibited cytotoxicity. The partially purified cytotoxin did not have any detectable degradative enzymatic activities.
Thus, virulent strains of E. histolytica possess an immunogenic cytotoxic protein which may be important in the pathophysiology of amoebiasis.
Intact trophozoites of the virulent Entamoeba histolytica strain HM-1:IMSS (HM-1) destroyed a monolayer of baby hamster kidney (BHK) cells at a higher rate and efficiency than trophozoites of the nonvirulent strain HK-9. The destructive effect could be partially attributed to the proteolytic activity of the amoeba, since quantitative differences were found in the enzymatic activity of the two strains tested. Crude extracts or secreted enzymes of HM-1 trophozoites digested Azocoll, as well as the bovine cold-insoluble globulin fraction, at a much higher rate than the corresponding preparations from HK-9. This proteolytic activity was found to be activated by free sulfhydryl groups. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the BHK cell proteins of pre- and postamoebic activities showed patterns similar to the trypsin effect on the same target cells. These enzymes were found to digest the proteins participating in the attachment of the target cells to the substrate and, consequently, cause detachment of these cells.
The proteinase activity of the low molecular weight cytotoxin of Entamoeba histolytica was correlated with its cytotoxicity. Gel-filtered amebal toxin (mol wt 10-30,000) proteinase activities could be assayed on azocasein at pH 6 or on hemoglobin at pH 4.5. Proteinase activity was inhibited by serum fractions, thiol reagents, heavy metals, leupeptin, and antipain. The cytotoxic activity of gel-filtered amebal toxin was inhibited by serum fractions, leupeptin, and antipain. Increased proteinase and cytotoxic activity was produced by treatment with cysteine. These data support the action of a thiol proteinase in the production of cytopathic effects by gel-filtered amebal toxin in vitro. The cytotoxic and proteinase activities were further purified using a combination of column chromatography and preparative isoelectric focusing. Two low molecular weight cytotoxins with proteinase activity on both substrates were isolated. The major cytotoxin had an isoelectric point of 4.5 and a molecular weight of 22,000; the other cytotoxin had a basic isoelectric point. These substances may be cathepsin B-like proteinase and elastase or cathepsin G-like proteinases of E. histolytica. The major proteinase activity in the high molecular weight fraction was not cytotoxic. The isoelectric points of the high molecular weight proteinase activities corresponded to that of mammalian cathepsin D. The major cell rounding cytotoxic activity of E. histolytica extracts in vitro is probably due to the activity of a thiol-containing cathepsin B-like proteinase.