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Degradation of Human IgA by Entamoeba histolytica
Abstract
To determine whether the virulent enteric pathogen Entamoeba histolytica degrades human IgA molecules, serum and secretory IgA was exposed to viable axenic trophozoites (strain HM1:IMSS), a parasite
sonicate, and medium conditioned by incubation with live trophozoites. IgA was completely degraded under all conditions, proteinase
activity was maximal at a neutral pH, and there was a four- to eightfold enrichment of amebic IgA proteolytic activity in
a soluble fraction of amebic sonicate. Degradation of serum IgA by amebic sonicate was completely inhibited by the cysteine
proteinase inhibitors trans-epoxysuccinyl-l-Ieucylamino(4-guanidino)butane (E-64, 100 µ,M) and benzyloxycarbonyl-phenyl-alanyl-alanyl-fluoromethyl ketone (Z-Phe-AlaCH2F, 12.5 µM). Secretion of degradative activity, the optimal pH, and the inhibition by E-64 and Z-Phe-Ala-CH2F indicates that cysteine proteinase activity is predominately responsible for the degradation of human IgA by E. histolytica.
... The colonized parasites are capable of extensive tissue destruction. Beside the pore-forming proteins, amoebapores (14,15), hydrolytic enzymes, particularly cysteine proteases (CP), are considered to be essential weapons of the parasite to penetrate the epithelium and destroy components of the host's extracellular matrix (ECM) (16)(17)(18)(19)(20). During and after penetration into the submucosal region, amebic trophozoites interact directly and indirectly with host immune and nonimmune cells. ...
... It was demonstrated that anti-Gal/GalNAc lectin IgA reduces trophozoite colonization in the colon (5,23,25,(113)(114)(115)(116)(117). Intriguingly, E. histolytica surface-associated CP [most likely EhCP-A5, (118)] cleaves human IgA (16,119). Amebic CPs are capable of cleaving both isotypes, i.e., IgA1 and IgA2 (119,120). ...
Entamoeba histolytica is a protozoan parasite and the causative agent of amebiasis. It is estimated approximately 1% of humans are infected with E. histolytica, resulting in an estimate of 100,000 deaths annually. Clinical manifestations of amebic infection range widely from asymptomatic to severe symptoms, including dysentery and extra-intestinal abscesses. Like other infectious diseases, it is assumed that only ~20% of infected individuals develop symptoms, and genetic factors of both the parasite and humans as well as the environmental factors, e.g., microbiota, determine outcome of infection. There are multiple essential steps in amebic infection: degradation of and invasion into the mucosal layer, adherence to the intestinal epithelium, invasion into the tissues, and dissemination to other organs. While the mechanisms of invasion and destruction of the host tissues by the amebae during infection have been elucidated at the molecular levels, it remains largely uncharacterized how the parasite survive in the host by evading and attacking host immune system. Recently, the strategies for immune evasion by the parasite have been unraveled, including immunomodulation to suppress IFN-γ production, elimination of immune cells and soluble immune mediators, and metabolic alterations against reactive oxygen and nitrogen species to fend off the attack from immune system. In this review, we summarized the latest knowledge on immune reaction and immune evasion during amebiasis.
... Interestingly, several lines of in vitro evidence support that E. histolytica strikes back by using its surface and secreted CPs to degrade host sIgA. When serum and sIgA are exposed to viable axenic trophozoites (strain HM-1:IMSS), a parasite sonicate, or conditioned medium by incubation with live amoebae, sIgA is completely degraded, and proteinase activity is maximal at a neutral pH and is completely inhibited by E-64 [16]. Serum and sIgA are susceptible to degradation by amoeba surface-associated CPs; both sIgA1 and sIgA2 are degraded in a similar fashion by surface amoeba proteases. ...
... However, sIgA2 is functionally more resistant to proteolysis than sIgA1 [156]. The CP identity was not determined, but it could be a 70 kDa protease [16]. EhCP4 was recently shown to degrade IgA in vitro [14]; however, it is unclear if it is the same protease or if E. histolytica has more than one IgA-degrading protease. ...
... Interestingly, several lines of in vitro evidence support that E. histolytica strikes back by using its surface and secreted CPs to degrade host sIgA. When serum and sIgA are exposed to viable axenic trophozoites (strain HM-1:IMSS), a parasite sonicate, or conditioned medium by incubation with live amoebae, sIgA is completely degraded, and proteinase activity is maximal at a neutral pH and is completely inhibited by E-64 [16]. Serum and sIgA are susceptible to degradation by amoeba surface-associated CPs; both sIgA1 and sIgA2 are degraded in a similar fashion by surface amoeba proteases. ...
... However, sIgA2 is functionally more resistant to proteolysis than sIgA1 [156]. The CP identity was not determined, but it could be a 70 kDa protease [16]. EhCP4 was recently shown to degrade IgA in vitro [14]; however, it is unclear if it is the same protease or if E. histolytica has more than one IgA-degrading protease. ...
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.
... Directed apoptosis and subsequent phagocytosis may serve to limit host inflammatory mechanisms by suppressing necrosis and subsequent Th1-type immunity [6]. Cysteine proteases that are known to degrade host extracellular matrix also protect Entamoeba histolytica from complement, secretory IgA, and serum IgG [7][8][9]. ...
... Strangely, this strain of Entamoeba histolytica had a decrease in phagocytosis, while having no apparent defect in hemolytic activity or monolayer destruction. This is in stark contrast to the known roles of cysteine proteases that include degradation of extracellular matrix, mucin, complement proteins, immunoglobulins, and cytokines [7][8][9]92]. EhCP5-attenuated parasites were also unable to penetrate the colonic lamina propria in an ex vivo human colonic model of invasion [93]. Targeted inhibitors to EhCP1 and EhCP4 have also been shown to be protective in the SCID mouse-human intestinal xenograph model and in the SCID mouse hepatic abscess model, respectively [94,95]. ...
The protozoan parasite Entamoeba histolytica is responsible for invasive intestinal and extraintestinal amebiasis. The virulence of Entamoeba histolytica is strongly correlated with the parasite's capacity to effectively kill and phagocytose host cells. The process by which host cells are killed and phagocytosed follows a sequential model of adherence, cell killing, initiation of phagocytosis, and engulfment. This paper presents recent advances in the cytolytic and phagocytic processes of Entamoeba histolytica in context of the sequential model.
... Furthermore, cysteine proteases interfere with both the complement pathway and the humoral response in the human immune system. Enzymes are able to cleave C3 in such a way as to activate the complement pathway, degrading E. dispar heterotrophs but not E. histolytica [54]. Gal/GalNAclectin inhibits the complement mediated complement mediated by E. histolytica mediated mechanisms, particularly via cross-reacting with CD59, and the C5b-9 membranous inhibitor is found in human blood cells [33]. ...
The study in this review aims to know and study the parasite Entamoebahistolytica that causes Amoebiasis worldwide. During its life cycle, E. histolytica passes through several phases, trophozoite stage, precyst stage, the cyst stage, the Metacyst stage, and Metacystictrophozoite stage. In this study, the spread of the parasite globally and in the Arab world and methods of treating the disease were also discussed. The Genomic structureof E. histolytica, like other organisms, is characterized by diversity and heterogeneity in its genetic content, which is one of the most important reasons for its survival and its ability to infect. Interestingly, the genome of the E. histolytica contains a large amount of genes presumed to be of bacterial origin. The study of the genetic diversity of E. histolytica gives paths to the developmental change that resulted in the emergence of evolutionary features or traits. Understanding amoebic virulence is important. Several studies have shown that genetic factors influence the virulence of parasitic infections. Studying the virulence of E. histolytica by Serine-rich protein (REHP) gene is among the important issues used in molecular epidemiological studies.
... Although it was suggested that the activation of the alternative complement pathway by this C3 disruption may play a role in the early inflammatory response in amebic lesions contributing to the pathogenesis of invasive amoebiasis, the same group subsequently demonstrated that CPs may also degrade the proinflammatory C3a and C5a anaphylatoxins into biologically inactive fragments, thus avoiding the host's immune response [118]. Likewise, the neutral CP of E. histolytica also rapidly degraded human IgA immunoglobulin, both in serum and in secretions, suggesting that it could be important during the process of intestinal invasion by amoeba [119]. Furthermore, both secretory IgA1 and IgA2, as well as their secretory component, were also degraded by a CP activity located on the surface of glutaraldehydefixed amoebae, which could be EhCP5 [63,120]. ...
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.
... CP-5 deficient trophozoites have been shown to induce less gut inflammation and damage to the intestinal barrier, while also being incapacitated of creating amoebic liver abscesses (Ankri, Stolarsky, and Mirelman 1998). Cysteine proteinases are capable of degrading human IgA (Kelsall and Ravdin 1993) and IgG (Tran et al. 1998), complement components C3 (Reed and Gigli 1990) and may also provide a mechanism to circumvent normal host immunity by inactivating the proinflammatory factors C3a and C5a (Reed et al. 1995). IFN-γ and macrophage activation are two of the most important host defensive mechanisms against invading trophozoites, and IL-18 is a key cytokine in this process. ...
Entamoeba histolytica is an intestinal protozoan parasite causing amoebiasis, a severe public health issue in impoverished countries. It affects up to 50 million individuals worldwide and is the second leading cause of parasite associated mortality after malaria. Despite significant progress in deciphering the pathobiological causes of the disease over the last several decades, knowledge regarding the molecular pathways involved in tissue invasion process and damage during both intestinal and extra intestinal amoebiasis remains scarce. The multiple complex pathways to achieve the pathogenesis seem to provide an evolutionary benefit to this organism. Recent findings revealed that adhesion to host cells, virulence factors, secreted proteinases, cell cycle regulatory mechanisms play a critical role in pathogenesis of this human pathogen. In this chapter, we have summarized the recent research findings related to the pathogenesis of E. histolytica. A comprehensive understanding of its pathogenesis might assist in the identification of possible anti-amoebic targets and strategies to combat the pathogen in the near future.
... 37 Furthermore, in vitro and in silico studies have demonstrated the presence of cysteine proteases in Giardia that can cleave several components of the human mucosal defence immune system, including IgA and IgG. 38,39 Other intestinal protozoa, such as Blastocystis hominis 40,41 and Entamoeba histolytica, 42,43 also showed IgA protease activity. ...
Background
Interactions between parasites during coinfections are often complex and can impact immunization and treatment programs, as well as disease outcomes and morbidity. However, little is known about these interactions and the mechanisms involved.
Methods
In this study, a coproparasitological survey was carried out in school-age children living in endemic areas of parasitic infection in the state of Sergipe, in Northeastern Brazil. Anti-helminth-specific and total secretory immunoglobulin-A (SIgA) levels were measured in stool and saliva samples and were compared in children presenting monoparasitism, polyparasitism (helminths and/or intestinal protozoa), and no infections.
Results
The survey showed that protozoa were more prevalent than helminths, and that there was a high frequency of polyparasitism in the studied population, mainly from combinations of protozoan species. Although less frequent, combinations between species of protozoa and helminths were also observed. The levels of salivary SIgA in these co-infected individuals were lower than the average observed in infections with helminths alone.
Conclusions
Although the children participating in this survey were asymptomatic, and it was, therefore, not possible to evaluate the impact of salivary SIgA reduction on the diseases, the study highlights the need for further investigations of coinfections by intestinal parasites and the effects on immune response induced by the interactions between different parasites.
... Other proteases with demonstrated IgA proteolytic activity have been reported but are not as well characterized. Proteases from Pseudomonas, Entamoeba, and Blastocytis are thought to degrade IgA, but little evidence exists regarding their selectivity and specificity [116][117][118]. In some cases, these proteases digest Fab and Fc fragments, suggesting multiple and possibly non-specific cleavage sites. ...
Epithelial surfaces throughout the body are coated by mucins, a class of proteins carrying domains characterized by a high density of O-glycosylated serine and threonine residues. The resulting mucosal layers form crucial host-microbe interfaces that prevent the translocation of microbes while also selecting for distinct bacteria via the presented glycan repertoire. The intricate interplay between mucus production and breakdown thus determines the composition of the microbiota maintained within these mucosal environments, which can have a large influence on the host during both homeostasis and disease. Most research to date on mucus breakdown has focused on glycosidases that trim glycan structures to release monosaccharides as a source of nutrients. More recent work has uncovered the existence of mucin-type O-glycosylation-dependent proteases that are secreted by pathogens, commensals, and mutualists to facilitate mucosal colonization and penetration. Additionally, immunoglobulin A (IgA) proteases promote bacterial colonization in the presence of neutralizing secretory IgA through selective cleavage of the heavily O-glycosylated hinge region. In this review, we summarize families of O-glycoproteases and IgA proteases, discuss known structural features, and review applications of these enzymes to glycobiology.
... In the invasive infection, motile trophozoites interact with the mucin layer of the human colon through the galactose and N-acetyl-D-galactosamine specific lectin (Gal/GalNAc lectin) and attach to host mucin and colonic epithelial cells [2] until arriving at the submucosa, constituted by loose connective tissue, blood vessels, and extracellular matrix components (ECM), including collagen and fibronectin [3]. Although the destruction of host tissue appears to be the basis of disease [4], others virulence factor are essential such as the secretion of cysteine proteases that degrade immunoglobulins and proteins of the complement system [5], pore-forming proteins, amoebapores [6], and the dephosphorylation in host cell's [7]. ...
The Entamoeba histolytica parasite is the causative agent of amebiasis, infecting approximately 1% of the world population and causing 100,000 deaths per year. It binds to Fibronectin (FN), activating signaling pathways regulated by kinases and phosphatases. EhLMW-PTPs genes from E. histolytica encode for Low Molecular Weight Tyrosine Phosphatases expressed in trophozoites and amoebic cysts. The role of these phosphatases in the virulence of the parasite has not yet been well characterized. Our results showed a differential expression of the EhLMW-PTPs, at the mRNA and protein levels, in an asynchronous trophozoites culture. Furthermore, we observed that trophozoites transfected that overexpressed EhLMW-PTP2 phagocytized fewer erythrocytes, possibly due to decreased phagocytic cups, and showed deficiencies in adherence to FN and less cytopathic effect. These analyzes suggest that the parasite's EhLMW-PTPs have an essential role in the mechanisms of proliferation, adhesion, and phagocytosis, regulating its pathogenicity.
... The pathogenicity of E. histolytica results from a complex set of events, including adhesion to the gut epithelium through the Gal-lectin, lysis of the epithelial cells via amoebapore action, degradation of matrix by the action of neutral cysteine proteinases and other lytic enzymes, and penetration into the mucosa. Another mechanism of immune evasion used by the amoeba, is the proteolytic digestion of IgA by proteases (24). During this process, the activation of the immune response is induced due to the release of inflammatory cytokines by host epithelial cells, initiating an acute inflammatory process that has been studied in several animal models (25) and in human intestinal xenografts (26). ...
Intestinal infection with the protozoan parasite Entamoebahistolytica elicits a local immune response with rising of spe-cific secretory IgA (sIgA) antibodies detectable in severalcompartments associated to mucosa. Anti-amoebic sIgAantibodies have been reported in faeces, saliva, bile and breastmilk from dysenteric patients and research trying to elucidatetheir role in protection has recently intensified. IgA antibodiesinhibit the in vitro adherence of E. histolytica trophozoitesto epithelial cell monolayers by recognizing several membraneantigens, including the galactose-binding lectin (Gal-lectin),main surface molecule involved in adherence, and the serine andcystein-rich proteins, all of them potential vaccine candidates.In fact, the presence of sIgA anti-Gal lectin in faeces of patientsrecovered from amoebic liver abscess (ALA) was associatedwith immunity to E. dispar . Moreover, the combined nasal andintraperitoneal vaccination of C3H/HeJ mice with native andrecombinant Gal-lectin protected mice against an intracecalchallenge with virulent E. histolytica trophozoites, protec-tion that seemed to be associated with the induction of specificintestinal sIgA antibodies. Therefore, the stimulation of intestinalsecretory response by mucosal delivery of amoebic antigenshas been positioned as a promising strategy for inducing pro-tection against human amoebiasis.
... a chemoattractant for neutrophils (9). Various virulence factors act on different stages of the invasion process, such as Gal/GalNAc lectin for adhesion to host cells (10), amebapores that cause cytolysis of immune cells (11), and cysteine proteases that degrade mucins (12) and immunoglobulins like IgA (13) and cause tissue destruction (14). ...
Entamoeba histolytica ( Eh ) is an anaerobic parasitic protozoan and the causative agent of amoebiasis. Eh expresses proteins that are structurally homologous to humans and use them as virulence factors. We have previously shown that Eh binds exogenous interferon gamma (IFN-γ) on its surface and in this study, we explored whether exogenous IFN-γ could modulate parasite virulence. We identified an IFN-γ-like receptor on the surface of Eh using IFN-γ R1 antibody by immunofluorescence, Western blot, protein sequencing and in silico analysis. Coupling of human IFN-γ to the IFN-γ-like receptor on live Eh significantly up regulated the expression of Eh Cysteine proteinase A1 ( Eh CP-A1), Eh CP-A2, Eh CP-A4, Eh CP-A5, Amebapore A ( AP-A ), Cyclooxygenase 1 ( Cox-1 ), Gal-lectin ( Hgl ) and peroxiredoxin ( Prx ) expression in a time-dependent fashion. IFN-γ signaling via the IFN-γ-like receptor enhanced Eh erythrophagocytosis of human red blood cells that was abrogated with the STAT1 inhibitor, fludarabine. Exogenous IFN-γ enhanced Eh chemotaxis and killing of Caco-2 colonic and Hep G2 liver cells and amebic liver abscess in hamsters. These results demonstrate that Eh expresses a surface IFN-γ-like receptor that is functional and may play a role in disease pathogenesis and/or immune evasion.
... The enzyme released into the host bloodstream has also been proposed to contribute to pathogenesis more directly. These extracellular cysteine proteinases may also interfere with the immune response by degrading IgA (47) and IgG (122). They also activate the alternative complement pathway (83) while circumventing the inflammatory reaction by inactivating anaphylatoxins C3a and C5a (122). ...
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.
... The other mechanism involved in the pathogenesis is apoptosis by non-Fas and non-tumor necrosis factor (TNF)-α1 receptor pathway [3]. The trophozoites had the ability to invade and lyse the tissue by cysteine proteinases enzyme that mediate inflammation by amplification IL-1 and cleavage IgA, IgG, C3a and C5a [4]. The host also had a defense mechanism against this protozoal represented by cell mediated immunity which is important in limiting and preventing recurrence of the disease. ...
Background
Entamoeba histolytica is the parasitic amoeba which caused amebiasis in human and responsible of 100,000 deaths every year. There is currently no vaccine against this parasite. The innate and adaptive immunity are important in protection against infection.
Aim of the study
To investigate the association between HLA-DRB1 and Entamoeba histolytica infection.
Patients and methods
The study is a case-controlled consisted from thirty Iraqi Arab Muslims patients with Entamoeba histolytica infection. The patients were consulted medical city and AL-Karama hospital for the period between May 2016 till February 2017. The control groups were sex and age matched with patient study group, they were consisted of thirty Iraqi Arab Muslims healthy individuals. HLA-DRB1 was done by SSOP method.
Results
A total of 30 patients with Entamoeba histolytica infection were participated in this study. Their ages were range from 21 to 55 years. Males were 83.3% and the rest were females. The other is 30 control group was sex and age matched with patient study group. There was an increased frequency of HLADRB1*03:0101 and *11:0101 in patients group compared to control group (P = 0.002, Odds ratio = 7.42, 95% CI:2.07 to 26.55) and (P = 0.01, Odd ratio = 4.29, 95% CI: 1.41 to 13.06) respectively.
Conclusions
HLA-DRB1*03:0101 and HLA-DRB1*110,101 may have a role in susceptibility to amebiasis.
... The pathogenicity of E. histolytica results from a complex set of events, including adhesion to the gut epithelium through the Gal-lectin, lysis of the epithelial cells via amoebapore action, degradation of matrix by the action of neutral cysteine proteinases and other lytic enzymes, and penetration into the mucosa. Another mechanism of immune evasion used by the amoeba, is the proteolytic digestion of IgA by proteases (Kelsall & Ravdin 1993). During this process, the activation of the immune response is induced due to the release of inflammatory cytokines by host epithelial cells, initiating an acute inflammatory process that has been studied in several animal models ( Tsutsumi V.1994) and in human intestinal xenografts (Seydel et al., 1997). ...
Parasites that inhabit the mammalian gastrointestinal tract remain one of the most prevalent groups of infectious microorganisms of humans (WHO 2001 ).
Intestinal parasitic infections are distributed throughout the world, with high prevalence in poor and socio-economically deprived communities in the tropics and subtropics. Amebiasis, Ascariasis, Hookworm infection and Trichuriasis are among the top ten most common infections in the world(Warren and Mahmoud 1984 ).
The gastrointestinal tract has the largest mucosal surface in our body. It houses diverse microorganisms that collectively form the commensal microbial community. The security of this community is kept by host-microbial interactions and is violated by foreign pathogens that induce local as well as systemic pathology(Sotolongo , et al 2012). The intestinal mucosa provides both a physiologic and immunologic barrier to a wide range of microorganisms and foreign substances.
In general, the mucosal immune system is homeostatic despite the considerable antigenic load in the intestine. When an imbalance does occur in the regulation of this response, gut barrier dysfunction and inflammatory bowel disease are observed. (Lloyd and Dominique 2001) .
Mucosal surfaces are lined by epithelial cells. These cells establish a barrier between sometimes hostile external environments and the internal milieu. However, mucosae are also responsible for nutrient absorption and waste secretion, which require a selectively permeable barrier. These functions place the mucosal epithelium at the center of interactions between the mucosal immune system and luminal contents, including dietary antigens and microbial products. Recent advances have uncovered mechanisms by which the intestinal mucosal barrier is regulated in response to physiological and immunological stimuli. ( Jerrold 2009).
The interface between the organism and the outside world, which is the site of exchange of nutrients, export of products and waste components, must be selectively permeable and at the same time, it must constitute a barrier equipped with local defense mechanisms against environmental threats (e.g. invading pathogens). The boundaries with the environment (mucosal and skin surfaces) are therefore covered (Helena etal., 2002)
Aside from acting as a barrier, the gut epithelial compartment can deploy a number of intrinsic mechanisms in the defense against invading pathogens. These include secretory diarrhea and the production of mucus and trefoil peptides by goblet cells and antimicrobial peptides, such as defensins and cathelicidins, by Paneth and epithelial cells ( Hecht, 1999.). Following Trichinella spiralis infection of the small intestine, goblet cells in the epithelial layer have been shown to secrete intelectin-2 (Pemberton et al., 2004) and resistin-like molecule (Artis et al., 2004), two molecules suspected of playing roles in the expulsion of the parasite. Studies of intestinal epithelial cells (IEC) have also shown altered expression of cytokines and chemokines in the small intestine in response to T. spiralis (Stadnyk, and Kearsey, 1996). These factors play important rolesin both the maturation and the migration of dendritic cells and the infiltration of leukocytes into the gut, and consequently the development of an adaptive immune response.
There is a need for human studies on the effects of parasitic infections on mucosal immunity and the potential for antiparasitic treatment to modify these effects. Such studies are likely to provide important insights into the regulation of mucosal immunity and inflammation, and the development of more effective mucosal vaccines.
... The pathogenicity of E. histolytica results from a complex set of events, including adhesion to the gut epithelium through the Gal-lectin, lysis of the epithelial cells via amoebapore action, degradation of matrix by the action of neutral cysteine proteinases and other lytic enzymes, and penetration into the mucosa. Another mechanism of immune evasion used by the amoeba, is the proteolytic digestion of IgA by proteases (Kelsall & Ravdin 1993). During this process, the activation of the immune response is induced due to the release of inflammatory cytokines by host epithelial cells, initiating an acute inflammatory process that has been studied in several animal models ( Tsutsumi V.1994) and in human intestinal xenografts (Seydel et al., 1997). ...
... 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]; ...
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.
... Purple lines indicate in vitro evidence. Other abbreviations: CP, cysteine protease; PGE2, prostaglandin 2; sIgA, secretory immunoglobulin A. 510 Marie · Petri on trophozoites (19), and E. histolytica CPs degraded the complement factors C3a and C5a (107) and host IgA and IgG (65,128) (Figure 4). ...
Entamoeba histolytica is the third-leading cause of parasitic mortality globally. E. histolytica infection generally does not cause symptoms, but the parasite has potent pathogenic potential. The origins, benefits, and triggers of amoebic virulence are complex. Amoebic pathogenesis entails depletion of the host mucosal barrier, adherence to the colonic lumen, cytotoxicity, and invasion of the colonic epithelium. Parasite damage results in colitis and, in some cases, disseminated disease. Both host and parasite genotypes influence the development of disease, as do the regulatory responses they govern at the host-pathogen interface. Host environmental factors determine parasite transmission and shape the colonic microenvironment E. histolytica infects. Here we highlight research that illuminates novel links between host, parasite, and environmental factors in the regulation of E. histolytica virulence. Expected final online publication date for the Annual Review of Microbiology Volume 68 is September 08, 2014. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
... Alguns estudos demonstraram o desprendimento das células epiteliais colônicas após contato com os trofozoítos, sugerindo que a degradação da membrana basal seja um importante passo na invasão tecidual (56,58,79) . Além disso, quando o parasita alcança a corrente circulatória, as moléculas de cisteína-proteinase são capazes de degradar tanto imunoglobulinas da classe A (IgA) humanas presentes nas mucosas como moléculas de IgG por meio de clivagem de sua cadeia pesada (47,79) . Assim, as proteinases não agem somente na degradação das barreiras naturais do hospedeiro como a fibronectina, o colágeno e a laminina, mas também exercem forte influência na imunidade específica pela clivagem de determinados isotipos de imunoglobulinas. ...
Amebiasis is the second cause of death among parasitary diseases in the world. Its etiologic agent is the protozoan Entamoeba histolytica, which destroys the host tissue by means of the secretion of proteinases, kills the target-cells by contact and phagocytizes erythrocytes. Accordingly, the trophozoites invade the intestinal mucosa, what causes amoebaean colitis. In some cases, they pass through the mucosa and reach the liver through the portal system, where they cause necrosis, which is composed of a few trophozoites surrounded by dead hepatocytes and liquefied cellular debris. This invasion is directly related to the synthesis capacity and secretion of molecules responsible for the virulence of trophozoites such as amoebapores, lectins and cysteine proteinases. The diagnosis of infection caused by this pathogen is routinely performed through optical microscopy of fresh samples or fixed specimens. However this methodology presents limitations insofar as it is unable to distinguish the specimens belonging to the complex E. histolytica /E. dispar. The research on coproantigens and the polymerase chain reaction (PCR) method have been used to differentiate these protozoa in fecal samples. However further studies are required for a better understanding of the host-parasite relationship, the proteomics and genomics of the protozoa, the development of vaccines and the real prevalence of this infection in Brazil and worldwide.
... In the late invasive phase, the inflammatory response leads to the recruitment of additional host defences molecules including the complement system and serum antibodies . E. histolytica trophozoites resist to secretory IgA and serum IgG by cleaving them with their cysteine proteinases (Kelsall and Ravdin 1993; Tran et al 1998). E. histolytica has the ability to activate the serum complement and to resist complement mediated lysis. ...
Molecules expressed by the pathogenic ameobaEntamoeba histolytica but weakly expressed or absent from the non-pathogenic ameobaEntamoeba dispar could be used by intestinal epithelial cells to discriminate between the two species and to initiate an appropriate inflammatory
response. Among the possible molecules involved in this identification are the Gal/GalNac lectin and the lipophosphoglycan.
Once the inflammatory response is initiated,E. histolytica trophozoites have to protect themselves against reactive nitrogen intermediates produced by intestinal epithelial cells,
oxygen intermediates, and cytotoxic molecules released by activated neutrophils. By screening theE. histolytica genome, we have identified proteins that may play a role in the defence strategy of the parasite. One of these proteins,
a serine proteinase inhibitor, inhibits human neutrophil cathepsin G, a key component of the host defence.
... A secreted cathepsin L-like protease from the liver fluke Fasciola hepatica cleaves host immunoglobulin into Fab and Fc fragments, which impairs adhesion of eosinophils and macrophages to the parasite [18]. Likewise, amoebic cysteine proteases from Entamoeba histolytica cleave human IgA and IgG in a dose-dependent manner [19,20] and a corresponding tapeworm cysteine protease from Taenia crassiceps degrades human IgG [21]. Due to the proteases' immunogenicity they have been used as immunodominant markers for infectious diseases and are well known as allergens in mammals including humans. ...
... Other correlates with invasion include the ability of CPs to degrade extracellular matrix components such as laminin, collagen types I and IV, fibronectin [22,52], and colonic mucin [53,54]. Furthermore, CPs enable E. histolytica to evade the host's immune defenses by activating and locally depleting complement [55], and by degrading anaphylotoxins C3a and C5a [56], human immunoglobulin [57,58], and interleukin-18 [59]. The completion of the E. histolytica genome project has revealed 50 cysteine, 4 aspartic, 10 serine, and 20 metallo peptidases [60]. ...
Entamoeba histolytica is able to invade human tissues by means of several molecules and biological properties related to the virulence. Pathogenic amebas use three major virulence factors, Gal/GalNAc lectin, amebapore and proteases, for lyse, phagocytose, kill and destroy a variety of cells and tissues in the host. Responses of the parasite to host components such as mucins and bacterial flora influence the behavior of pathogenic amebas altering their expression of virulence factors. The relative virulence of different strains of E. histolytica has been shown to vary as a consequence of changes in conditions of in vitro cultivation which implies substantial changes in basic metabolic aspects and factors directly and indirectly related to amebic virulence. Comparison of E. histolytica strains with different virulence phenotypes and under different conditions of growth will help to identify new virulence factor candidates and define the interplay between virulence factors and invasive phenotype. Virulence attenuate mutants of E. histolytica are useful also to uncover novel virulence determinants. The comparison of biological properties and virulence factors between E. histolytica and E. dispar, a non-pathogenic species, has been a useful approach to investigate the key factors involved in the experimental presentation of amebiasis and its complex regulation. The molecular mechanisms that regulate these variations in virulence are not yet known. Their elucidation will help us to better understand the gene expression plasticity that enables the effective adaptation of the ameba to changes in growth culture conditions and host factors.
... Inhibition of cysteine proteinases by specific inhibitors such as E-64 or laminin, prevents the disruption of the intestinal mucus and the subsequent damage to the mucosal cells (Moncada et al., 2003). Cleavage of the C3 fragment of complement as well as digestion of IgA and IgG were also attributed to the CPs (Reed et al., 1989Reed et al., , 1995Kelsall & Ravdin, 1993;Tran et al., 1998). Treatment of E. histolytica trophozoites with sublethal doses of the CP inhibitor E64 greatly reduced their ability to induce liver lesions in laboratory animals (Li et al., 1995;Stanley, 1995). ...
The human intestinal parasite Entamoeba histolytica causes amoebic colitis and amoebic liver abscesses. Three classes of amoebic molecules have been identified as the major virulence factors, the Gal/GalNAc inhibitable lectin that mediates adherence to mammalian cells, the amoebapores which cause the formation of membrane ion channels in the target cells and the cysteine proteinases which degrade the matrix proteins, the intestinal mucus and secretory IgA. Transcriptional silencing of the amoebapore (Ehapa) gene occurred after transfection of trophozoites with a plasmid containing a segment of the 5' upstream region of the gene. Transcriptional silencing of the Ehap-a gene continued even after the removal of the plasmid and the cloned amoebae were termed G3. Transfection of G3 trophozoites with a plasmid construct containing the cysteine proteinase (EhCP-5) gene and the light subunit of the Gal- lectin (Ehlgl1) gene, each under the 5' upstream sequences of the amoebapore gene, caused the simultaneous epigenetic silencing of expression of these two genes. The resulting trophozoites, termed RB-9, were cured from the plasmid and they do not express the three types of virulent genes. The RB9 amoeba are virulence attenuated and are incapable of killing mammalian cells, they can not induce the formation of liver abscesses and they do not cause ulcerations in the cecum of experimental animals. The gene-silenced amoebae express the same surface antigens which are present in virulent strains and following intra peritoneal inoculation of live trophozoites into hamsters they evoked a protective immune response. Further studies are needed to find out if RB-9 trophozoites could be used for vaccination against amoebaisis.
Colloidal stability and the regularity of the release kinetics benefit from the high circularity and the narrow size dispersion of polymeric particles as drug delivery carriers. A method for obtaining such particles composed of poly(lactide-co-glycolide) (PLGA), averaging at 1.0 ± 0.3 µm in size, is reported here, along with the analysis of the effects of different synthesis parameters on their morphological characteristics. As in agreement with the classical nucleation theory, the particle size and the degree of cohesion were inversely proportional to supersaturation. Consequently, the optimal conditions for the precipitation of small and narrowly dispersed particles involved an abrupt elevation of supersaturation. Owing to the high colloidal stability of the particles, centrifugation exhibited a counterintuitive effect on them, refining their morphological features and promoting their individuation. Polyvinyl alcohol (PVA) was used as a steric repulsion additive and its effect on the stability of PLGA spheres was concentration-dependent, with the particles aggregating, partially coalescing and losing their distinct features both with no PVA in the system and at PVA concentrations higher than the optimal. At its narrowest, the particle size distribution was bimodal, exhibiting the average circularity of 0.997 ± 0.003 and the average roundness of 0.913 ± 0.054. PLGA spheres were loaded with an inhibitor of EhCP4, a cysteine protease from E. histolytica, a parasite causing amoebic dysentery in the tropical and developing world. The burst release of the drug at early time points was followed by a zero-order release period, yielding a biphasic profile that can be a benefit for the delivery of anti-infective agents. The release profile fitted poorly with the Hixson-Crowell kinetic model and excellently with the Higuchi and the Korsmeyer-Peppas ones, indicating that the release is conditioned by diffusion rather than by the degradation of the polymer. The release and the erosion proceeded independently from one another, suggesting that the pore formation, water penetration and swelling are the primary driving forces for the release of the drug.
The amoeba and ciliates are protozoan parasites that have been causing illness in humans since antiquity. The most relevant foodborne parasites of these groups are Entamoeba histolytica and Balantidium coli. Entamoeba dispar has been identified mostly in asymptomatic patients however, reports demonstrating invasive amebiasis caused by E. dispar may indicate various levels of pathogenicity. In recent years, studies on the pathogenesis and immune responses to amoebiasis are providing insights on how to control this parasitosis that affects more than 50 million people annually.This chapter will address the various aspects of these parasites, from the biology, life cycle, clinical presentation, and treatment.
The introduction of molecular techniques allowed the differentiation of Entamoeba histomica from E. dispar, a comensal ameba morphologically indistinguisable from E. histolytica. Pathogenic mechanisms of E. histolytica inciudes the presence of a galactosa binding lectin that attachs trophozoites to intestinal epithelial cells. Also pore-forming peptides termed amebopores have been demonstrated, this peptides oligomerize after insertion in the cell membrane and form waterfilled channels through which ions and other molecules can pass, causing cell lysis. Cysteine proteasas have also been characterized as a viruience factor responsable of the degradation of the extracellular matrix and the evasion of the immune response by degradation of IgA, disruption of IgG, and degradation of anaphylotoxins C3a and C5a. The new epidemiological findings around the worid by the use of molecular technics are also discussed. In Costa Rica seroprevalence of 7,3% in E.histoyica / E.dispar cysts positive patients is taken an indication that the majority of the infections are caused by E. dispar. Antibody are present only in E. histoyica infection. As far as diagnosis, the finding of trofozoites with enguifed eritrocytes in fresh fecal samples or biopses correlates with E. histoyica. On the other hand, cysts of both protozoa are indistinguisable. Several immunological technics are available for the determination of antigen in stool samples or antibodies in sera.
Entamoeba histolytica is an obligate human intestinal parasite causing amoebic dysentery and liver abscess. Entamoeba dispar is its close largely nonpathogenic relative. Both species are well adapted to the human host. The motile amoebae called trophozoites dwell in the lumen of the large intestine; in its distal part, they differentiate to cysts, which are excreted and can survive in the environment and infect new hosts. Although metronidazole and other nitroimidazoles represent an efficient treatment, there are more than 55,000 deaths worldwide every year.
The E. histolytica genome project led to a better understanding of the molecular equipment; two surprises were the significant number of genes acquired from bacteria by lateral gene transfer and a very high number of protein kinases and phosphatases underscoring the complex signal transduction pathways in this parasite.
The parasite attacks human cells by first attaching to them via a galactose and N-acetylgalactose (Gal/GalNAc)-specific lectin. The host cell membrane is pierced by a pore-forming protein called amoebapore, and cysteine proteinases destroy cellular components, antibodies and complement.
The human host mounts a massive humoral and cellular immune response against E. histolytica with macrophages and NKT cells being the most efficient defenders. The antibody response is limited in its impact through the ability of the amoebae to cap and shed antibodies bound to their surface. In addition, E. histolytica has developed a number of mechanisms to cope with immune cells.
E. histolytica belongs to the microaerophilic microorganisms devoid of classical mitochondria and devoid of oxidative phosphorylation. So the main pathway to extract energy is by glycolysis, and when not needed, the energy is stored in the form of glycogen. The biosynthetic abilities are quite crippled; most useful molecules, sugars, amino acids, lipids and nucleotides are acquired from the host or from phagocytosed bacteria.
Cysteine proteases of the protozoan parasite Entamoeba histolytica are key virulence factors involved in overcoming host defences. These proteases are cathepsin-like enzymes with a cathepsin-L like structure, but cathepsin-B substrate specificity. In the host intestine, amoeba cysteine proteases cleave colonic mucins and degrade secretory immunoglobulin (Ig) A and IgG rendering them ineffective. They also act on epithelial tight junctions and degrade the extracellular matrix to promote cell death. They are involved in the destruction of red blood cells and the evasion of neutrophils and macrophages and they activate pro-inflammatory cytokines IL-1 beta and IL-18. In short, amoeba cysteine proteases manipulate and destroy host defences to facilitate nutrient acquisition, parasite colonization and/or invasion. Strategies to inhibit the activity of amoeba cysteine proteases could contribute significantly to host protection against E. histolytica.
The intestinal parasite Entamoeba histolytica (Eh) is a significant health risk in the developing world where infection occurs via ingestion of contaminated food or water. The resulting disease, amebiasis, claims 100,000 lives each year. In the colon, the parasite colonizes the mucous layer and interacts with the underlying mucosa to induce a variety of symptoms. The intestinal barrier has three main components: Bacteria and host proteins found within the lumen, a protective layer of mucus, and a single layer of intestinal epithelial cells (IEC) connected by tight junctions (TJ). Eh possesses a variety of virulence components that target the intestinal barrier to cause increased gut permeability, resulting in diarrhea. In the colon Eh can phagocytose pathogenic bacteria, which may increase the virulence of the parasite. Moreover, Eh binds colonic mucins via the parasite surface Gal/GalNAc lectin to facilitate colonization of the mucous layer. Subsequently, the virulence factor, Eh CP-A5, degrades the MUC2 polymer, allowing the parasite access to the epithelia where it binds to and destroys IEC by apoptosis or alters the expression of TJ proteins, causing secretory diarrhea. The sequence of events in intestinal amebiasis is not well understood and is based largely on in vitro and in vivo models of the disease.
Infection with Entamoeba histolytica provokes human diseases that range from amebic colitis to the life-threatening amebic liver abscess; nonetheless, about 90 % of infected people do not develop any symptom. What circumstances or which events in the dynamics of the host–parasite relationship define the outcome of infection? What are the causes that allow the apparently spontaneous clearance of the infection? How protective is the immune response against E. histolytica ? Why do some infected people remain asymptomatic and why do others develop clinical symptoms? What is the role of immunogenetic polymorphism in the final outcome of the infection? These questions are the major aims of many research groups around the world. In the present review, we analyze the potential participation of the immune system and the genetic variants of genes associated with immune response in the human host with its susceptibility or resistance to develop amebic invasive disease.
Entamoeba histolytica is an enteric tissue-invading protozoan parasite that causes amoebic colitis and occasionally liver abscess in humans. During tissue invasion, amoebic adhesion to host components is an important event for host cell death leading to successful invasion and infection. Among amoebic virulence factors, Gal/GalNAc lectin is known to be major adhesion factor to host cells. In this study, we investigated the role of amoebic secreted CP (Cysteine Proteases) in amoebic adhesion to extracellular matrix (ECM) protein using CP inhibitor and E. histolytica strains in which the endogenous inhibitor of cysteine protease (ICP) 1 gene was overexpressed (ICP1(+)) or repressed by antisense small RNA-mediated gene silencing (ICP1(-)). We found that pretreatment of wild-type amoebae with CP inhibitor E64, or thiol-group modifiers such as diamide and N-Ethylmaleimide resulted in a significant decrease in adhesion to laminin and collagen ECM proteins. Furthermore, ICP1(+) strain, with a reduction of secreted CP activity, exhibited reduced ability by 40 percent to adhere to laminin. In contrast, ICP1(-) strain, with an 1.9-fold increase of secreted CP activity, showed a two-fold increase in amoebic adherence to laminin compared to the control strain. In addition, total amount of secreted CP5 was decreased in ICP1(+) amoeba. Conversely, total amount of secreted CP1 and mature-form CP5 were increased in ICP1(-) amoeba. We also found that ICP1 was secreted into extracellular milieu. These results suggest that secreted CP activity by E. histolytica may be an important factor affecting adhesion to host proteins, and regulation of CP secretion by ICP plays a major role in pathogenesis. This study provides insight into the CP-mediated tissue pathogenesis in amoeba-invaded lesions during human amoebiasis.
Copyright © 2014. Published by Elsevier Inc.
Entamoeba histolytica and it´s cell/parasite relationship Resumen En esta revisión, se describen algunos datos actuales de la serie de eventos que se producen en la relación huésped-parásito que se establece en la amibiasis, infección endémica en nuestro país. La amibiasis ha sido descrita en la literatura médica desde los tiempos de Hipócrates, y constituye una de las tres causas más comunes de muerte por enfermedades parasitarias. La enfermedad presenta diferentes cuadros clínicos, aunque todavía se desconoce mucho sobre la relación huésped-parásito que conducen a la producción de las lesiones. En la interacción huésped-parásito, la liberación de mucina de la mucosa intestinal, la presencia de la flora bac-teriana residente en el intestino, el sistema de complemento, y la respuesta inmune representan algunas de las barreras de defensa contra E. Histolytica. Sin embargo, varios reportes han documentado la modulación de esta respuesta por la amiba principalmente a través de sus cisteín-proteasas y lectinas. Por lo tanto, probablemente la patogénesis de la enfermedad se deba a la combinación de efectos de las condiciones que presenta el hospedero para controlar la infección, así como de las características del propio parásito. Abstract In this review, we describe some current data from the series of events that occur in the host parasite relationship that is set to amebiasis, infection endemic in our country, described in medical literature since the days of Hippo-crates, which is one of the three most common causes of death from parasitic diseases. This disease has different clinical pictures, but even today much is unknown about host parasite relationship leading to the production of lesions. In host-parasite interaction of mucin release from intestinal mucosa, the presence of the resident bacterial flora in the intestine, the complement system and immune response represent some of the barriers of defense against E. histolytica, although several reports have documented the modulation of this response by the amoeba primarily through its cysteine proteases and lectins. Therefore, probably the pathogenesis of the disease is due to the combined effects of the conditions presented by the host to control infection, as well as to the characteristics of the parasite itself.
Naegleria fowleri, a free-living ameba, is the causative agent of Primary Amebic Meningoencephalitis. Highly-pathogenic mouse-passaged amebae (Mp) and weakly-pathogenic axenically-grown (Ax) N. fowleri were examined for peptidase activity. Zymography and azocasein peptidase activity assays demonstrated that Mp and Ax N. fowleri exhibited a similar peptidase pattern. Prominent for whole cell lysates, membranes and conditioned medium from Mp and Ax amebae was the presence of an activity band of approximately 58kDa that was sensitive to E64, a cysteine peptidase inhibitor. However, axenically-grown N. fowleri demonstrated a high level of this peptidase activity in membrane preparations. The inhibitor E64 also reduced peptidase activity in ameba-conditioned medium consistent with the presence of secreted cysteine peptidases. Exposure of Mp amebae to E64 reduced their migration through matrigel that was used as an extracellular matrix, suggesting a role for cysteine peptidases in invasion of the central nervous system (CNS). The collective results suggest that the profile of peptidases is not a discriminative marker for distinguishing Mp from Ax N. fowleri. However, the presence of a prominent level of activity for cysteine peptidases in N. fowleri membranes and conditioned medium, suggests that these enzymes may serve to facilitate passage of the amebae into the CNS.This article is protected by copyright. All rights reserved.
Amoeba and ciliates are two groups of protozoan parasites that have long been known to infect humans. The amoeba are unicellular
organisms which are characterized by the pseudopodia, which are cytoplasmic protrusions that provide motility to the organism.
Amoeba are commonly found in the environment and a feware pathogenic to mammals.
Characterization of a purified proteinase from T4chomoncs uoginalis was carried out using bacitracin-sepharose affinity chromatography. Trichomonos uqginolis KT-9 isolate was used as a source of eye study Proteinase activity was determined using Bz-Pro- Phe-Arg-Nan as the substrate. Optimum pH for the purified proteinase activity was 7.0 and 6.0, 9.0 with DTT. Optimum temperature was 37℃ and isoelectric point was 7.2 Activity of this proteinase was inhibited by E-64, antipain, leupeptin, Hg2+ and Zn2+ and activated by DTT and cysteine. Activity of the purified proteinase was visualized by gelatin SDS- PAGE. The gelatinolytic activity of the purified proteinase was inhibited by E-64, antipain, leupeptin, and IAA, but not by PMSF and EDTA. On SDS-PAGE, the molecular weight of the purified proteinase was 60,000 daltons. Sera of rabbits infected with T. vaginalis reacted specifically in immunoblots with this proteinase. These results indicate that 60 kDa of purified proteinase was cysteine proteinase with antigenicity. Key words: Trichomonqs voginclis, bacitracin-sepharose affinity chromatography, cysteine proteinase
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.
On clinical and prophylactic indications, 7905 patients were parasitologically examined, and Blastocystis hominis was detected in 273 (3.4%) of them. Detailed investigations were carried out in 82 of the infected patients (19 males and 63 females), divided into two groups: individuals with clinical symptoms and asymptomatic carriers. A correlation was found between the number of parasites detected, the clinical presentation and the changes in the levels of total serum immunoglobulins (IgG, IgA, IgM) in the persons infected with B.hominis. Half of the patients investigated presented with gastrointestinal and allergic symptoms. Significant decrease of levels of serum immunoglobulin IgA, correlating with the number of parasites detected was found in 43 patients, irrespective of the presence or absence of clinical symptoms.
IgA1 protease activity, which allows bacteria to cleave human IgA1 in the hinge region, represents a striking example of convergent evolution of a specific property in bacteria. Although it has been known since 1979 that IgA1 protease is produced by the three leading causes of bacterial meningitis in addition to important urogenital pathogens and some members of the oropharyngeal flora, the exact role of this enzyme in bacterial pathogenesis is still incompletely understood owing to lack of a satisfactory animal model. Cleavage of IgA1 by these post-proline endopeptidases efficiently separates the monomeric antigen-binding fragments from the secondary effector functions of the IgA1 antibody molecule. Several in vivo and in vitro observations indicate that the enzymes are important for the ability of bacteria to colonize mucosal membranes in the presence of S-IgA antibodies. Furthermore, the extensive cleavage of IgA sometimes observed in vivo, suggests that IgA1 protease activity results in a local functional IgA deficiency that may facilitate colonization of other microorganisms and the penetration of potential allergens. It has been hypothesized that IgA1 protease activity of Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumoniae, under special immunological circumstances, allows these bacteria to take advantage of specific IgA1 antibodies in a strategy to evade other immune factors of the human body. The decisive factor is the balance between IgA antibodies against surface antigens of the respective bacteria and their IgA1 protease. Recent studies have shown that serine-type IgA1 proteases of H. influenzae, meningococci, and gonococci belong to a family of proteins used by a diverse group of Gramnegative bacteria for colonization and invasion.
Diseases caused by the parasite Entamoeba histolytica disproportionately affect residents of underdeveloped areas, afflicting places lacking sufficient sanitation, hygiene, and
water processing especially. While the recent differentiation between Entamoeba histolytica and the morphologically identical but completely asymptomatic Entamoeba dispar based on genetic and biochemical analyses has enhanced the study of the disease-causing parasite, a mystery still exists
as to the factors which still cause about 90% of Entamoeba histolytica infections to remain asymptomatic after colonization. Researchers delving into these areas have identified several features
of the parasite—including the galactose and N-acetylgalactosamine inhibitable adherence lectin, proteinases, and amoebapores,
all virulence factors in Entamoeba—and of the host, including the association of anti-lectin IgA with resistance to disease, which may determine whether infection
is invasive. As specific and easy diagnostics are developed to aid in identifying more precisely the health burden caused
by E. histolytica, vaccine candidates are under evaluation.
This chapter focuses on microbial strategies specifically related to evasion of immunoglobulin A (IgA) functions, in particular in humans. IgA1 proteases are unique in their specific ability to cleave human IgA1. By attacking a single peptide bond in the IgA1 hinge region, IgA1 proteases eliminate all secondary effector functions of IgA1 antibodies while enabling the bacteria to take advantage of the released monomeric antigen-binding fragments for colonization and evasion of other parts of the immune system. Several lines of direct and indirect evidence indicate that the activity of IgA1 proteases secreted by bacteria colonizing the upper respiratory tract may result in a local functional IgA1 deficiency that facilitates penetration or alternative processing of potential allergens. However, because of the lack of a satisfactory animal model, full appreciation of the biological significance of IgA1 proteases must await successful strategies to prevent their activity, for example, by active or passive immunization. Further studies are necessary to understand how bacterial IgA binding proteins and glycosidases that attack the carbohydrate moiety of IgA may interfere with its protective functions.
Trophozoites of E. histolytica are equipped with two chagasin-like cysteine protease inhibitors, EhICP1 and EhICP2, also known as amoebiasin 1 and 2. Expression studies using E. invadens as model organism showed that corresponding mRNAs were detectable in both life stages of the parasite, cyst and trophozoite state. Unlike EhICP1 known to act in the cytosol, EhICP2 co-localized with cysteine protease EhCP-A1 in lysosome-like vesicles, as demonstrated by immunofluorescence microscopy. Silencing or overexpressing of the two inhibitors did not show any effect on morphology and viability of the trophozoites. Overexpression of the EhICPs, however, although dramatically dampening the proteolytic activity of cell extracts from the corresponding cell lines, did not influence expression rate or localization of the major amoebic cysteine proteases as well as phagocytosis and digestion of erythrocytes. Activity gels of cell extracts from strains overexpressing ehicp1 showed a drastically reduced activity of EhCP-A1 suggesting a high affinity of EhICP1 towards this protease. From these data, we propose that EhCP-A1 accidentally released into the cytosol is the main target of EhICP1, whereas EhICP2, beside its role in house-keeping processes, may control the proteolytic processing of other hydrolases or fulfils other tasks different from protease inhibition.
Entamoeba histolytica cysteine proteinases (EhCPs) play a key role in disrupting the colonic epithelial barrier and the innate host immune response
during invasion of E. histolytica, the protozoan cause of human amebiasis. EhCPs are encoded by 50 genes, of which ehcp4 (ehcp-a4) is the most up-regulated during invasion and colonization in a mouse cecal model of amebiasis. Up-regulation of ehcp4 in vivo correlated with our finding that co-culture of E. histolytica trophozoites with mucin-producing T84 cells increased ehcp4 expression up to 6-fold. We have expressed recombinant EhCP4, which was autocatalytically activated at acidic pH but had
highest proteolytic activity at neutral pH. In contrast to the other amebic cysteine proteinases characterized so far, which
have a preference for arginine in the P2 position, EhCP4 displayed a unique preference for valine and isoleucine at P2. This
preference was confirmed by homology modeling, which revealed a shallow, hydrophobic S2 pocket. Endogenous EhCP4 localized
to cytoplasmic vesicles, the nuclear region, and perinuclear endoplasmic reticulum (ER). Following co-culture with colonic
cells, EhCP4 appeared in acidic vesicles and was released extracellularly. A specific vinyl sulfone inhibitor, WRR605, synthesized
based on the substrate specificity of EhCP4, inhibited the recombinant enzyme in vitro and significantly reduced parasite burden and inflammation in the mouse cecal model. The unique expression pattern, localization,
and biochemical properties of EhCP4 could be exploited as a potential target for drug design.
Amebiasis is the disease caused by the enteric dwelling protozoan parasite Entamoeba histolytica. The WHO considers amebiasis as one of the major health problems in developing countries; it is surpassed by only malaria and schistosomiasis for death caused by parasitic infection. E. histolytica primarily lives in the colon as a harmless commensal, but is capable of causing devastating dysentery, colitis and liver abscess. What triggers the switch to a pathogenic phenotype and the onset of disease is unknown. We are becoming increasingly aware of the complexity of the host-parasite interaction. During chronic stages of amebiasis, the host develops an immune response that is incapable of eliminating tissue resident parasites, while the parasite actively immunosuppresses the host. However, most individuals with symptomatic infections succumb only to an episode of dysentery. Why most halt invasion and a minority progress to chronic disease remains poorly understood. This review presents a current understanding of the immune processes that shape the outcome of E. histolytica infections during its different stages.
Trichomonas vaginalis is a protozoan parasite that causes a widely distributed sexually transmitted disease (STD). Since immunoglobulin G (IgG) antibodies to specific trichomonad immunogens are found in serum and vaginal washes (VWs) from patients with trichomoniasis, a potential mechanism of immune evasion by this parasite might be the ability of T. vaginalis proteinases to degrade human immunoglobulins (Igs). Incubation of human IgG with lysates of T. vaginalis organisms resulted in time- and concentration-dependent degradation of the heavy chain. Secretory IgA was degraded similarly. Inhibitors of cysteine proteinases, when added to trichomonal lysates, abolished IgG and IgA degradation, while EDTA, a metalloproteinase inhibitor, did not. Substrate-gel electrophoresis with human IgG, IgM, or IgA copolymerized with acrylamide revealed several distinct cysteine proteinases in both lysates and culture supernatants from logarithmically growing parasites that degraded all classes of human antibodies. Trichomonal lysates and supernatants of numerous isolates tested all had Ig-degrading activity. Finally, proteolytic activity against IgG was detected in most (26 of 33; 78%) VWs from patients with trichomoniasis. In contrast, 18 of 28 (65%) VWs from women without trichomoniasis or from patients infected with other STDs had no detectable proteinases when tested in an identical manner. The other 10 of these 28 VWs (35%) had smaller amounts of detectable Ig-degrading proteinases. These differences in Ig-degrading proteinase activity between patients with and without trichomoniasis, regardless of coinfecting STDs, were statistically significant (P = 0.001). These results illustrate that T. vaginalis is capable of degrading human Igs.
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.
Human isolates of Streptococcus pneumoniae tested by traditional immunochemical methods produce a protease that cleaves human immunoglobulin A1 (IgA1) into Fab and Fc fragments. The protease may be an important virulence factor, but studies of its pathogenetic significance have been hampered by lack of a suitable animal model. Since S. pneumoniae is a respiratory pathogen for several species of animals, we sought to determine whether isolates of this organism from animals with pneumococcal infection, including fatal diplococcal pneumonia, produced an IgA protease. Isolates from six animal species including the mouse, rat, dog, guinea pig, rhesus monkey, and chimpanzee were tested for protease activity against IgA preparations from the mouse, rat, dog, guinea pig, rabbit, rhesus and cynomolgus monkeys, gorilla, and human. Cleavage of IgA was demonstrated by the appearance of Fc fragments in Western blots (immunoblots) treated with specific antisera. All these isolates except that from the guinea pig produced a protease that cleaved IgA of human, rhesus monkey, and gorilla origin. Cleavage was inhibited by 5 mM EDTA. IgA cleavage from the other species could not be demonstrated. Although S. pneumoniae can colonize the respiratory tracts of several animal species, it is a significant pathogen principally of humans and some other primates. Our data suggest that some species of nonhuman primates including the rhesus monkey could be suitable for experimental studies on the significance of IgA protease in the pathogenesis of pneumococcal disease.
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.
Establishment of adherence by Entamoeba histolytica is mediated by a 170-kD Gal/GalNAc inhibitable lectin and is required for cytolysis and phagocytosis of mammalian target cells. We studied the biochemical mechanisms of the in vitro interaction between rat and human colonic mucins and axenic E. histolytica trophozoites. Crude mucus prevented amebic adherence to Chinese hamster ovary (CHO) cells by up to 70%. Purification of the colonic mucins by Sepharose 4B chromatography, nuclease digestion, and cesium chloride gradient centrifugation resulted in a 1,000-fold enrichment of the inhibitory mucins. Purified rat mucin inhibited amebic adherence to and cytolysis of homologous rat colonic epithelial cells. Oxidation and enzymatic cleavage of rat mucin Gal and GalNAc residues completely abrogated mucin inhibition of amebic adherence. The binding of rat 125I-mucin to amebae was galactose specific, saturable, reversible, and pH dependent. A monoclonal antibody specific for the 170-kD amebic Gal/GalNAc lectin completely inhibited the binding of rat 125I-mucin. Rat mucin bound to Affigel affinity purified the amebic lectin from conditioned medium. Colonic mucin glycoproteins act as an important host defense by binding to the parasite's adherence lectin, thus preventing amebic attachment to and cytolysis of host epithelial cells.
It has previously been shown that secretory immunoglobulin A (S-IgA) influences the sorption of oral streptococci to hydroxyapatite as well as to cell surfaces. The present experiments demonstrate that bacterial IgA proteases, which cleave S-IgA in the hinge region, are capable of interfering with this mechanism. This result was obtained with an IgA1 specific protease from Haemophilus influenzae and with a protease from Clostridium ramosum that cleaves IgA1 as well as IgA2 of A2m(1) allotype. The modulation of S-IgA-mediated effects by IgA proteases were studied by means of an in vitro method which permits quantitative determination of the sorption of radiolabeled oral bacteria to hydroxyapatite beads. Other authors have suggested that IgA protease-mediated effects may be explained by a strongly reduced antigen-binding capacity of released Fab alpha fragments. Here we present evidence that streptococci, after exposure to specific S-IgA and IgA protease, are coated with Fab alpha fragments.
The majority of immunoglobulin A (IgA) produced in the human body is secreted onto the vast area of mucosal surfaces, becoming the principal mediator of humoral immunity at these sites. The serum and mucosal IgA systems differ with regard to ontogeny, regulation, cellular origin, and molecular configuration of the respective immunoglobulin molecules. The functional properties of IgA from both systems depend on the Fc portion of the IgA molecule. Unlike IgG and IgM, which activate the complement and phagocytic systems, both serum and secretory IgA are relatively ineffective in interacting with these inflammatory effector systems. However, the polymeric configuration, ability to bind to a secretory component, hydrophilicity, and charge, conferred by the Fc part of secretory IgA, give this immunoglobulin isotype special mucosal defense properties. Serum IgA may have a unique function in regulating immune effector mechanisms mediated by the delivery of null or negative signals by its Fc region. Several microbial pathogens that colonize or invade through mucosal surfaces can interfere with such Fc-mediated functions of IgA by releasing proteases that specifically cleave the hinge region of human IgA subclass 1 (IgA1). These IgA1 proteases are virulence factors that enable mucosal pathogens to counteract secretory IgA-mediated defense mechanisms; they probably also affect the complex microbial ecology of mucosal surfaces, and they may cause temporary or local deficiencies in mucosal immunity. Furthermore, IgA1 proteases are hypothesized to provide a unique mechanism by which the three principal agents of bacterial meningitis can evade immune defense mechanisms and cause invasive disease.
We report a one-step method for the purification to homogeneity of a cysteine proteinase of Entamoeba histolytica (histolysin) by affinity chromatography of the soluble extract of the parasite on immobilized phenylalanyl(2-phenyl)aminoacetaldehyde semicarbazone. The enzyme has an apparent Mr of 26,000 by SDS/polyacrylamide-gel electrophoresis and 29,000 by gel chromatography. Its pH optimum varies widely, from 5.5 with azocasein to approx. 7 with other protein substrates and benzyloxycarbonylphenylalanyl-L-citrullylaminomethylcourmarin++ + (Z-Phe-Cit-NHMec), and to 9.5 with benzyloxycarbonylphenylalanylarginylaminomethylcoumarin (Z-Phe-Arg-NHMec) and benzyloxycarbonylarginylarginylaminomethylcourmarin (Z-Arg-Arg-NHMec). Values of Km, kcat. and kcat/Km are 1.5 microM, 130 s-1 and 87 X 10(6) M-1.s-1 for Z-Arg-Arg-NHMec, and 32 microM, 0.4 s-1 and 0.012 x 10(6) M-1.s-1 for Z-Phe-Arg-NHMec, respectively, at pH 7.5 and 37 degrees C. The enzyme is inhibited by leupeptin and such inhibitors of cysteine proteinases as L-transepoxysuccinyl-L-leucylamido-4-(guanidino)butane, peptidyldiazomethanes, iodoacetic acid and chicken cystatin. The tentative N-terminal amino acid sequence of the enzyme closely resembles that of papain. Histolysin does not degrade type I collagen or elastin, but it is active against cartilage proteoglycan and kidney glomerular basement-membrane collagen. It also detaches cells from their substratum in vitro, and could well play a role in tissue invasion.