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The intestinal protozoan parasite Entamoeba histolytica remains a significant cause of morbidity and mortality worldwide. However, almost nothing is known about the molecules secreted by the parasite that modulate host immune responses or epithelial barrier function in the colon. Herein, we describe the isolation and characterization of a cyclooxyg...
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Citations
... The existence of isoforms of leishmanolysin or the gp63 protein would explain the different functions attributed to this molecule, which would imply an adhesin function, as well as that of metalloprotease and cyclooxygenase-like (COX-like) proteins. Particularly, adhesin and metalloprotease activities have been localized in the plasma membrane [61], and the COX-like activity has been localized in the nuclear membrane [62]. According to our results, we suggest that a large part of the leishmanolysin-like with COX-like activity is secreted as cargo in the EV of the trophozoites, which could explain the decrease in this activity in trophozoites. ...
Several species of Acanthamoeba genus are potential pathogens and etiological agents of several diseases. The pathogenic mechanisms carried out by these amoebae in different target tissues have been documented, evidencing the relevant role of contact-dependent mechanisms. With the purpose of describing the pathogenic processes carried out by these protozoans more precisely, we considered it important to determine the emission of extracellular vesicles (EVs) as part of the contact-independent pathogenicity mechanisms of A. culbertsoni, a highly pathogenic strain. Through transmission electronic microscopy (TEM) and nanoparticle tracking analysis (NTA), EVs were characterized. EVs showed lipid membrane and a size between 60 and 855 nm. The secretion of large vesicles was corroborated by confocal and TEM microscopy. The SDS-PAGE of EVs showed proteins of 45 to 200 kDa. Antigenic recognition was determined by Western Blot, and the internalization of EVs by trophozoites was observed through Dil-labeled EVs. In addition, some EVs biological characteristics were determined, such as proteolytic, hemolytic and COX activity. Furthermore, we highlighted the presence of leishmanolysin in trophozites and EVs. These results suggest that EVs are part of a contact-independent mechanism, which, together with contact-dependent ones, allow for a better understanding of the pathogenicity carried out by Acanthamoeba culbertsoni.
... In some environmental fungi, COX inhibitors stop the growth or prevent the sexual maturation of these microorganisms (Noverr et al. 2001). In the case of protozoan parasites, molecules responsible for COX-like activity have been reported only for E. histolytica and L. mexicana (Dey et al. 2003;Estrada-Figueroa et al. 2018). ...
... In the case of parasites, COX activity has been poorly explored. However, this enzyme's presence has recently been demonstrated (Dey et al. 2003;Estrada-Figueroa et al. 2018). Figure 3A shows the subcellular distribution of the positive signal of the D12 mAb, both in L mexicana promastigotes (a) and amastigotes (b). ...
... In trophozoites of E. histolytica, the presence of COX activity has been reported and identified as α-actinin (Dey et al. 2003). Interestingly, analysis by confocal microscopy using the D12 mAb evidenced cross-reactivity. ...
In Leishmania mexicana, the protease gp63 has been documented as the protein responsible for cyclooxygenase (COX) activity. The present work aimed to obtain a monoclonal antibody capable of recognizing this protein without blocking the COX-like enzymatic activity. The antibody produced by the selected hybridoma was named D12 mAb. The antigen recognized by the D12 mAb was characterized by the determination of COX activity associated with immune complexes in the presence of exogenous arachidonic acid (AA) using the commercial Activity Assay Abcam kit. LSM-SMS analysis validated the identity of the antigen associated with the D12 mAb as the L. mexicana protease gp63. Confocal microscopy assays with the D12 mAb detected, by cross-recognition, similar proteins in other protozoan parasites. COX-like molecules are located in vesicular structures, homogeneously distributed throughout the cytoplasm in amastigotes (intracellular infectious phase) and promastigotes of L. mexicana, and trophozoites of Entamoeba histolytica, Acanthamoeba castellanii, and Naegleria fowleri. However, in Giardia duodenalis trophozoites, the distribution of the COX-like molecule was also in perinuclear areas. In comparison, in Trypanosoma cruzi trypomastigotes, the distribution was mainly observed in the plasma membrane. Structural analyses of COX-2-like antigens revealed continuous and discontinuous epitopes for B cells, which could be relevant in the cross-reaction of D12 mAb with the analyzed parasites. These results indicate that the D12 mAb against the L. mexicana gp63 also recognizes a COX-like molecule in several protozoan parasites, suggesting that this D12 mAb could potentially be used in combined therapies against infectious diseases.
... Recent studies have also found higher ACE2 receptor expression in cholangiocytes than in hepatocytes; in fact, ACE2 levels in cholangiocytes are similar to those in lung type-2 alveolar cells, suggesting that the liver may be a potential target of SARS-CoV-2 [29]. Moreover, elevated prostaglandin E2 (PGE2) levels have been associated with ALA formation due to an immunosuppressive effect of PGE2 [30]. We can hypothesise that, in our patient, elevated PGE2 levels are secondary to both direct stimulation of cyclooxygenase-2 (COX-2) by SARS-CoV2-2 [31] and presence of a COX-2-like protein present in E. histolytica trophozoites [30], which could influence the clinical course; however, the available knowledge is still insufficient to correlate them with each other. ...
... Moreover, elevated prostaglandin E2 (PGE2) levels have been associated with ALA formation due to an immunosuppressive effect of PGE2 [30]. We can hypothesise that, in our patient, elevated PGE2 levels are secondary to both direct stimulation of cyclooxygenase-2 (COX-2) by SARS-CoV2-2 [31] and presence of a COX-2-like protein present in E. histolytica trophozoites [30], which could influence the clinical course; however, the available knowledge is still insufficient to correlate them with each other. In this case, the patient required an exploratory laparotomy which showed secondary peritonitis due to ALA rupture. ...
Background
Amoebiasis is a parasitic disease caused by Entamoeba histolytica , which affects people living in low- and middle-income countries and has intestinal and extraintestinal manifestations. To date, knowledge on coronavirus disease 2019 (COVID-19) coinfection with enteric parasites is limited, and E. histolytica coinfection has not been previously described. Here we present the case of a patient with COVID-19 who, during hospitalisation, presented a clinical picture consistent with an amoebic liver abscess (ALA).
Case presentation
A 54-year-old man, admitted as a suspected case of COVID-19, presented to our hospital with dyspnoea, malaise, fever and hypoxaemia. A nasopharyngeal swab was positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by reverse-transcription polymerase chain reaction. After 7 days, he developed diarrhoea, choluria and dysentery. An abdominal ultrasound showed a lesion compatible with a liver abscess; stool examination revealed E. histolytica trophozoites, and additional serology for E. histolytica was positive. After 12 days of treatment with metronidazole, ceftazidime and nitazoxanide, the patient reported acute abdominal pain, and an ultrasound examination revealed free liquid in the abdominal cavity. An emergency exploratory laparotomy was performed, finding 3000 mL of a thick fluid described as “anchovy paste”. Computed tomography scan revealed a second abscess. He ended up receiving 21 days of antibiotic treatment and was discharged with satisfactory improvement.
Conclusion
Here we present, to the best of our knowledge, the first report of ALA and COVID-19 co-presenting. Based on their pathophysiological similarities, coinfection with SARS-CoV-2 and E. histolytica could change the patient’s clinical course; however, larger studies are needed to fully understand the interaction between these pathogens.
... Infection and Immunity intestinal amebiasis in children (37). In tissues, E. histolytica evades host immune cells by phagocytosis or apoptosis (38), by trogocytosis (39), or by inhibiting IFN-␥ production with prostaglandin E 2 (PGE 2 ) from the cyclooxygenase (Cox)-like enzyme (5,40,41). By Western blot analysis, using an anti-human IFN-␥R1 antibody, we detected the presence of an E. histolytica protein of ϳ200 kDa in membrane fractions. ...
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.
... These data suggest that the cyclooxygenase pathway was present in early invertebrate species and in the animal kingdom. Recently a cyclooxygenase enzyme from the protozoan Entamoeba histolytica has been identified that lacks structural similarity with other COXs enzymes, but produce PGE 2 from arachidonic acid (Dey et al., 2003). ...
... Likewise, higher levels of IFN-γ are related to a lower incidence of E. histolytica infection [143,146]. Secreted EhPGE 2 leads to the il-8 mRNA expression in human colonic cells via EP 4 receptor [147]. EhCPs also increase the expression of IL-8 via a protease activated receptor (PAR) 2-independent mechanism [148]. ...
... Trophozoites also induce neutrophil apoptosis through the activation of extracellular signal-regulated kinase (ERK) 1/2, by the generation of NADPH oxidase-derived ROS [154]. When E. histolytica is exposed to macrophages, the parasite cyclooxygenase-like enzyme synthetizes EhPGE 2 [147], which inhibits the respiratory burst (ROS: H 2 O 2 , O 2− , OH − ) and NO production by macrophages [155,156]. Moreover, E. histolytica resists complement activation by the similarity and antigenic cross reactivity of the EhGal/GalNAc lectin with the host cluster of differentiation (CD)59 protein, inhibiting the complement-mediated lysis [157]. ...
The epithelium represents the first and most extensive line of defence against pathogens, toxins and pollutant agents in humans. In general, pathogens have developed strategies to overcome this barrier and use it as an entrance to the organism. Entamoeba histolytica, Naegleria fowleri and Acanthamoeba spp. are amoebae mainly responsible for intestinal dysentery, meningoencephalitis and keratitis, respectively. These amoebae cause significant morbidity and mortality rates. Thus, the identification, characterization and validation of molecules participating in host-parasite interactions can provide attractive targets to timely intervene disease progress. In this work, we present a compendium of the parasite adhesins, lectins, proteases, hydrolases, kinases, and others, that participate in key pathogenic events. Special focus is made for the analysis of assorted molecules and mechanisms involved in the interaction of the parasites with epithelial surface receptors, changes in epithelial junctional markers, implications on the barrier function, among others. This review allows the assessment of initial host-pathogen interaction, to correlate it to the potential of parasite invasion.
... The intestinal amebiasis-causing agent Entameoba histolytica produces PGE2 via a nuclear COX-like protein and this production is sensitive to the non-selective COX inhibitor aspirin (Dey et al., 2003). COX activity of the protease Gp63 in the Leishmania causing Leishmania mexicana was reported, using a chemiluminescence-based kit for COX activity (Estrada-Figueroa et al., 2018). ...
Oxylipins, or oxygenated lipids, are universal signaling molecules across all kingdoms of life. These molecules, either produced by microbial pathogens or their mammalian host, regulate inflammation during microbial infection. In this review, we summarize current literature on the biosynthesis pathways of microbial oxylipins and their biological activity towards mammalian cells. Collectively, these studies have illustrated how microbial pathogens can modulate immune response and disease outcome via oxylipin‐mediated mechanisms.
... Eh-derived PGE 2 not only induces pro-inflammatory IL-8 production but also disrupts colonic epithelial cell tight junction by coupling through EP4 receptors (Dey et al., 2003;Dey and Chadee, 2008;Lejeune et al., 2011). To analyze the biological functions of endogenous EhCox and EhPGE 2 mediated proinflammatory responses, we silenced the expression of the gene by small RNA-mediated transcriptional gene silencing in the G3 strain (Bracha et al., 2006). ...
... Complete silencing of EhCox was achieved in comparison to the Eh control ( Figures 1A,B, Supplementary Figure 1). Immunoblot analysis detected the 72 and 66 Kda protein band in the lysate of control Eh as previously described (Dey et al., 2003). As predicted, EhCox enzymatic assay using nuclear fractions isolated from log-phase Eh showed almost no aspirin (ASA) inhibited PGE 2 release (Dey et al., 2003) by EhCoxgs in comparison to control Eh incubated with arachidonic acid (AA) substrate ( Figure 1C). ...
... Immunoblot analysis detected the 72 and 66 Kda protein band in the lysate of control Eh as previously described (Dey et al., 2003). As predicted, EhCox enzymatic assay using nuclear fractions isolated from log-phase Eh showed almost no aspirin (ASA) inhibited PGE 2 release (Dey et al., 2003) by EhCoxgs in comparison to control Eh incubated with arachidonic acid (AA) substrate ( Figure 1C). We used ASA inhibited PGE 2 release to accurately quantify PGE 2 levels as EhCoxgs showed modest non-specific binding by enzyme immunoassay (EIA) that was not inhibited with ASA. ...
The intestinal protozoan parasite Entamoeba histolytica (Eh) causes amebiasis associated with severe diarrhea and/or liver abscess. Eh pathogenesis is multifactorial requiring both parasite virulent molecules and host-induced innate immune responses. Eh-induced host pro-inflammatory responses plays a critical role in disease pathogenesis by causing damage to tissues allowing parasites access to systemic sites. Eh cyclooxygenase (EhCox) derived prostaglandin E2 stimulates the chemokine IL-8 from mucosal epithelial cells that recruits neutrophils to the site of infection to exacerbate disease. At present, it is not known how EhCox is regulated or whether it affects the expression of other proteins in Eh. In this study, we found that gene silencing of EhCox (EhCoxgs) markedly increased endogenous cysteine protease (CP) protein expression and virulence without altering CP gene transcripts. Live virulent Eh pretreated with arachidonic acid substrate to enhance PGE2 production or aspirin to inhibit EhCox enzyme activity or addition of exogenous PGE2 to Eh had no effect on EhCP activity. Increased CP enzyme activity in EhCoxgs was stable and significantly enhanced erythrophagocytosis, cytopathic effects on colonic epithelial cells and elicited pro-inflammatory cytokines in mice colonic loops. Acute infection with EhCoxgs in colonic loops increased inflammation associated with high levels of myeloperoxidase activity. This study has identified EhCox protein as one of the important endogenous regulators of cysteine protease activity. Alterations of CP activity in response to Cox gene silencing may be a negative feedback mechanism in Eh to limit proteolytic activity during colonization that can inadvertently trigger inflammation in the gut.
... Even though some components of the route of production of prostaglandins in parasites are known, little or nothing is known about the enzymes that metabolize arachidonic acid (AA) in parasites. So far, only in E. histolytica, a COX-like enzyme has been described and isolated [15]; however it possess neither the arachidonate-binding domain, nor the heme-coordinating and catalytic sites, which are conserved in other species from higher organisms. COX like enzymes represent a missing link of prostaglandins production in parasites, and their relevance relays on the fact that their products modulate the immune system and most probably help parasites to produce these components to survive into their hosts. ...
... We demonstrate the presence of a protein with an approximate molecular weight of 66 kDa, which presents cross-reaction with an anti-mouse COX-2 polyclonal antibody. This type of cross-reaction was reported also by Dey et al. [15], using an anti-sheep COX-1 antibody that recognized a protein in E. histolytica. ...
Cyclooxygenase-2 (COX-2) is an enzyme responsible of prostaglandins production, such as prostaglandin E2 (PGE2), an immune response modulator that regulates the immune system to inhibit Th1 and to promote Th2 cytokines production. Many parasites modulate their host immune response through PGE2 effects; however, in parasites, only one protein with COX activity has been described, the α-actinin of Entamoeba histolytica. Prostanoids production has been reported in some species of Leishmania but not the enzymes responsible of their production. To identify the protein responsible for COX activity in Leishmania mexicana, we examined total extracts of promastigotes and samples with COX activity were subjected to ion exchange column purification and precipitation with ammonium sulphate; fractions with activity were analyzed by SDS-PAGE and Western blot using an anti-mouse COX-2 polyclonal antibody. Results showed that in those samples with enzymatic activity, the anti-mouse COX-2 polyclonal antibody recognized a protein with an approximate molecular weight of 66 KDa. Bands recognized by the antibody were subjected to mass spectrometry analysis and the results showed that several peptides from the bands purified by two different methods, and that were recognized by the anti-mouse COX-2 polyclonal antibody corresponded to the Leishmania mexicana gp63 surface protease. L. mexicana gp63 was purified by a Concanavalin A (Con-A) affinity column and subjected to immunoprecipitation with a commercial anti-Leishmania gp63 polyclonal antibody; the immunoprecipitated sample was analyzed for COX activity showing that the anti-gp63 antibody did immunoprecipitate the COX activity. The presence of COX activity was further confirmed in amastigotes extracts of L. mexicana. Moreover, a recombinant gp63 protein was produced and its COX activity tested, confirming that gp63 is the molecule responsible for COX activity.
... 66 There is also evidence that the parasites can produce serotonin and prostaglandin E 2 , which are direct chloride secretagogues. 67,68 Finally, C parvum causes a loss of absorptive villous enterocytes in the small intestine with an associated reduction in SGLT1 abundance and function, which has been assumed to underlie diarrheal pathogenesis along with host cell prostaglandin production and diminished barrier function. [69][70][71] Inflammatory Diarrhea ...
Every year, enteric infections and associated diarrhea kill millions of people. The situation is compounded by increases in the number of enteric pathogens that are acquiring resistance to antibiotics, as well as (hitherto) a relative paucity of information on host molecular targets that may contribute to diarrhea. Many forms of diarrheal disease depend on the dysregulation of intestinal ion transporters, and an associated imbalance between secretory and absorptive functions of the intestinal epithelium. A number of major transporters have been implicated in the pathogenesis of diarrheal diseases and thus an understanding of their expression, localization, and regulation after infection with various bacteria, viruses, and protozoa likely will prove critical in designing new therapies. This article surveys our understanding of transporters that are modulated by specific pathogens and the mechanism(s) involved, thereby illuminating targets that might be exploited for new therapeutic approaches.
