Glycan moieties in Entamoeba histolytica ubiquitin are immunodominant.

The ubiquitin-proteasome system plays a central role performing several functions to maintain parasite homeostasis. We have reported the partial characterization of N-linked glycosylation profile in E. histolytica ubiquitin (EhUb). Here we examined the immunogenicity and antigenicity of carbohydrates in EhUbiquitin. Rabbits were immunized with purified EhUbiquitin or purified recombinant rUb expressed by E. coli. Using Western Blot, we explored the immunogenicity and antigenicity of protein portion and carbohydrates moiety. Interestingly, immunized rabbits produced antibodies to both Ub glycoprotein and rUb; but antibodies against carbohydrates were immunodominant, rather than antibodies to the protein moiety of EhUbiquitin. In addition, we observed that antibodies to protein moiety are not conserved in serum unless antigen is continually administrated. Conversely, anti-Ub glycoprotein antibodies are well maintained in circulation. In humans, infection with Entamoeba histolytica induces strong IgG anti-Ub response. The human antibodies recognize both, the protein moieties and the glycosylated structure. Entamoeba histolytica ubiquitin is immunogenic and antigenic. The glycan moieties are immunodominant and induces IgG. These data open the door to use carbohydrates as potential targets for diagnose tests, drugs and vaccine to prevent this parasitic disease.

Rabeprazole inhibits several functions of Entamoeba histolytica related with its virulence.

Amoebiasis is a human parasitic disease caused by Entamoeba histolytica. The parasite can invade the large intestine and other organs such as liver; resistance to the host tissue oxygen is a condition for parasite invasion and survival. Thioredoxin reductase of E. histolytica (EhTrxR) is a critical enzyme mainly involved in maintaining reduced the redox system and detoxifying the intracellular oxygen; therefore, it is necessary for E. histolytica survival under both aerobic in vitro and in vivo conditions. In the present work, it is reported that rabeprazole (Rb), a drug widely used to treat heartburn, was able to inhibit the EhTrxR recombinant enzyme. Moreover, Rb affected amoebic proliferation and several functions required for parasite virulence such as cytotoxicity, oxygen reduction to hydrogen peroxide, erythrophagocytosis, proteolysis, and oxygen and complement resistances. In addition, amoebic pre-incubation with sublethal Rb concentration (600 µM) promoted amoebic death during early liver infection in hamsters. Despite the high Rb concentration used to inhibit amoebic virulence, the wide E. histolytica pathogenic-related functions affected by Rb strongly suggest that its molecular structure can be used as scaffold to design new antiamoebic compounds with lower IC50 values.

Molecular biology research to benefit patients with Entamoeba histolytica infection.

The development of molecular microbiology has made it possible for us to deepen our understanding of the pathogenesis of amebiasis. Research using the trophozoite form of Entamoeba histolytica has clearly shown us the importance of the interface between the parasite and host cells in vitro. Immuno-pathogenesis after excystation was similarly well advanced by the use of a novel murine model of amebic colitis. However, it is still challenging to apply these findings to clinical and epidemiological settings. This is mainly because of the lack of a complete infection animal model of amebiasis by oral-fecal infection. Moreover, in vitro experiments have predominantly been performed using the same axenic cultured strain HM-1: IMSS isolated about 50 years ago, whereas highly diverse strains are prevalent all over the world. Translational research informed by clinical observations has the greatest potential for the development of effective interventions. Here, we highlight discoveries of the experiments designed from cohort observation and discuss remaining problems to be solved.

Immunization with the Entamoeba histolytica surface metalloprotease EhMSP-1 protects hamsters from amebic liver abscess.

Diarrhea and amebic liver abscesses due to invasive Entamoeba histolytica infections are an important cause of morbidity and mortality in the developing world. Entamoeba histolytica adherence and cell migration, two phenotypes linked to virulence, are both aberrant in trophozoites deficient in the metallosurface protease EhMSP-1, which is a homologue of the Leishmania vaccine candidate leishmanolysin (GP63). We examined the potential of EhMSP-1 for use as a vaccine antigen to protect against amebic liver abscesses. First, existing serum samples from South Africans naturally infected with E. histolytica were examined by enzyme-linked immunosorbent assay (ELISA) for the presence of EhMSP-1-specific IgG. Nine of 12 (75%) people with anti-E. histolytica IgG also had EhMSP-1-specific IgG antibodies. We next used a hamster model of amebic liver abscess to determine the effect of immunization with a mixture of four recombinant EhMSP-1 protein fragments. EhMSP-1 immunization stimulated a robust IgG antibody response. Furthermore, EhMSP-1 immunization of hamsters reduced development of severe amebic liver abscesses following intrahepatic injection of E. histolytica by a combined rate of 68% in two independent animal experiments. Purified IgG from immunized compared to control animals bound to the surface of E. histolytica trophozoites and accelerated amebic lysis via activation of the classical complement cascade. We concluded that EhMSP-1 is a promising antigen that warrants further study to determine its full potential as a target for therapy and/or prevention of invasive amebiasis.

Immunogenicity and safety of an Entamoeba histolytica adjuvanted protein vaccine candidate (LecA+GLA-3M-052 liposomes) in rhesus macaques.

Entamoeba histolytica, the causative agent of amebiasis, is one of the top three parasitic causes of mortality worldwide. However, no vaccine exists against amebiasis. Using a lead candidate vaccine containing the LecA fragment of Gal-lectin and GLA-3M-052 liposome adjuvant, we immunized rhesus macaques via intranasal or intramuscular routes. The vaccine elicited high-avidity functional humoral responses as seen by the inhibition of amebic attachment to mammalian target cells by plasma and stool antibodies. Importantly, antigen-specific IFN-γ-secreting peripheral blood mononuclear cells (PBMCs) and IgG/IgA memory B cells (BMEM) were detected in immunized animals. Furthermore, antigen-specific antibody and cellular responses were maintained for at least 8 months after the final immunization as observed by robust LecA-specific BMEM as well as IFN-γ+ PBMC responses. Overall, both intranasal and intramuscular immunizations elicited a durable and functional response in systemic and mucosal compartments, which supports advancing the LecA+GLA-3M-052 liposome vaccine candidate to clinical testing.

Breast milk parasite-specific antibodies and protection from amebiasis and cryptosporidiosis in Bangladeshi infants: a prospective cohort study.

In this prospective cohort study, the presence of parasite-specific immunoglobulin A in breast milk was associated with protection of Bangladeshi infants from cryptosporidiosis and amebiasis. Our findings suggest that passive immunity could be harnessed for the prevention of Entamoeba histolytica and Cryptosporidium species infection in children living in endemic regions.

Evaluation of the efficacy of a recombinant Entamoeba histolytica cysteine proteinase (EhCP112) antigen in minipig.

Cysteine proteinases 112 (EhCP112) of Entamoeba histolytica are considered important for ameba pathogenicity. The recombinant gene was obtained by cloning and expression of the EhCP112 gene in heterologous host Escherichia coli BL-21 (DE3), were used to evaluate their ability to induce immune protective responses in minipig against challenge infection in a minipig-E. histolytica model. There was a 46.29% reduction (P<0.001) in the group of recovery of challenged E. histolytica compared with that in the control group. Specific anti-EhCP112 antibodies from immune protected minipig had significantly higher levels of immunoglobulin G (IgG) (P<0.001). This is a first report demonstrating that a recombinant form of EhCP112 generated in E. coli, to immunize a minipig model of E. histolytica, and there is significant protection. This study may help to understand the EhCP112 for human in the future.

Evaluation of the efficacy of a recombinant Entamoeba histolytica cysteine proteinase gene (EhCP5) antigen in Minipig.

Entamoeba histolytica cysteine proteinase gene 5(EhCP5) is one of the major proteinase genes of all EhCP-transcripts. The amebiasis cysteine proteinase gene encoding an antigen from E. histolytica, as well as the recombinant EhCP5, obtained by cloning and expression of the EhCP5 gene in heterologous host Escherichia coli BL-21 (DE3), were used to evaluate their ability to induce immune protective responses in Minipig against challenge infection in a minipig-E. histolytica model. There was a 52.27% reduction (P<0.001) in the group of recovery of challenged E. histolytica compared with that in the control group. Specific anti-EhCP5 antibodies from immune protected minipig had significantly higher levels of immunoglobulin G (IgG) (P<0.0001). Our data will help to know the mechanism of vaccinal protection of E. histolytica.

Cloning, expression and evaluation of the efficacy of a recombinant Entamoeba histolytica cysteine proteinase (EhCP4) antigen in minipig.

Cysteine proteinases 4 (EhCP4) of Entamoeba histolytica are considered important for ameba pathogenicity. The recombinant gene was obtained by cloning and expression of the EhCP4 gene in heterologous host Escherichia coli BL-21 (DE3), were used to evaluate their ability to induce immune protective responses in minipig against challenge infection in a minipig-E. histolytica model. There was a 53.16% reduction (P<0.001) in the group of recovery of challenged E. histolytica compared with that in the control group. Specific anti-EhCP4 antibodies from immune protected minipig had significantly higher levels of immunoglobulin G (IgG) (P<0.001). This is a first report demonstrating that a recombinant form of EhCP4 generated in E. coli, to immunize a minipig model of E. histolytica, and there is significant protection. This study may help to understand the EhCP4 for human in the future.

Entamoeba histolytica: cloning, expression and evaluation of the efficacy of a recombinant amebiasis cysteine proteinase gene (ACP1) antigen in minipig.

The amebiasis cysteine proteinase gene (ACP1) encoding an antigen from Entamoeba histolytica, as well as the recombinant ACP1, obtained by cloning and expression of the ACP1 gene in heterologous host Escherichia coli BL-21 (DE3), were used to evaluate their ability to induce immune protective responses in minipig against challenge infection in a minipig -E. histolytica model. There was a 64.52% reduction (P<0.001) in the group of recovery of challenged E. histolytica compared with that in the control group. Specific anti-ACP1 antibodies from immune protected minipig had significantly higher levels of immunoglobulin G (IgG) (P<0.001). Our data indicate recombinant ACP1 may be a potential target as a vaccine antigen.

Host Protective Mechanisms to Intestinal Amebiasis.

The protozoan parasite Entamoeba histolytica is the causative agent of amebiasis, an infection that manifests as colitis and, in some cases, liver abscess. A better understanding of host protective factors is key to developing an effective remedy. Recently, significant advances have been made in understanding the mechanisms of MUC2 production by goblet cells upon amebic infection, regulation of antimicrobial peptide production by Paneth cells, the interaction of commensal microbiota with immune stimulation, and host genetics in conferring protection from amebiasis. In addition to host pathways that may serve as potential therapeutic targets, significant progress has also been made with respect to development of a vaccine against amebiasis. Here, we aim to highlight the current understanding and knowledge gaps critically.

Optimizing a Multi-Component Intranasal Entamoeba Histolytica Vaccine Formulation Using a Design of Experiments Strategy.

Amebiasis is a neglected tropical disease caused by Entamoeba histolytica. Although the disease burden varies geographically, amebiasis is estimated to account for some 55,000 deaths and millions of infections globally per year. Children and travelers are among the groups with the greatest risk of infection. There are currently no licensed vaccines for prevention of amebiasis, although key immune correlates for protection have been proposed from observational studies in humans. We previously described the development of a liposomal adjuvant formulation containing two synthetic TLR ligands (GLA and 3M-052) that enhanced antigen-specific fecal IgA, serum IgG2a, a mixed IFNγ and IL-17A cytokine profile from splenocytes, and protective efficacy following intranasal administration with the LecA antigen. By applying a statistical design of experiments (DOE) and desirability function approach, we now describe the optimization of the dose of each vaccine formulation component (LecA, GLA, 3M-052, and liposome) as well as the excipient composition (acyl chain length and saturation; PEGylated lipid:phospholipid ratio; and presence of antioxidant, tonicity, or viscosity agents) to maximize desired immunogenicity characteristics while maintaining physicochemical stability. This DOE/desirability index approach led to the identification of a lead candidate composition that demonstrated immune response durability and protective efficacy in the mouse model, as well as an assessment of the impact of each active vaccine formulation component on protection. Thus, we demonstrate that both GLA and 3M-052 are required for statistically significant protective efficacy. We also show that immunogenicity and efficacy results differ in female vs male mice, and the differences appear to be at least partly associated with adjuvant formulation composition.

Identification of an epitope on the Entamoeba histolytica 170-kD lectin conferring antibody-mediated protection against invasive amebiasis.

The emergence of multidrug-resistant organisms and the failure to eradicate infection by a number of important pathogens has led to increased efforts to develop vaccines to prevent infectious diseases. However, the nature of the immune response to vaccination with a given antigen can be complex and unpredictable. An example is the galactose- and N-acetylgalactosamine-inhibitable lectin, a surface antigen of Entamoeba histolytica that has been identified as a major candidate in a vaccine to prevent amebiasis. Vaccination with the lectin can induce protective immunity to amebic liver abscess in some animals, but others of the same species exhibit exacerbations of disease after vaccination. To better understand this phenomenon, we used recombinant proteins corresponding to four distinct domains of the molecule, and synthetic peptides to localize both protective and exacerbative epitopes of the heavy chain subunit of the lectin. We show that protective immunity after vaccination can be correlated with the development of an antibody response to a region of 25 amino acid residues of the lectin, and have confirmed the importance of the antibody response to this region by passive immunization studies. In addition, we show that exacerbation of disease can be linked to the development of antibodies that bind to an NH2-terminal domain of the lectin. These findings are clinically relevant, as individuals who are colonized with E. histolytica but are resistant to invasive disease have a high prevalence of antibodies to the protective epitope(s), compared to individuals with a history of invasive amebiasis. These studies should enable us to develop an improved vaccine for amebiasis, and provide a model for the identification of protective and exacerbative epitopes of complex antigens.

Protection against invasive amebiasis by a single monoclonal antibody directed against a lipophosphoglycan antigen localized on the surface of Entamoeba histolytica.

A panel of monoclonal antibodies was raised from mice immunized with a membrane preparation from Entamoeba histolytica, the pathogenic species causing invasive amebiasis in humans. Antibody EH5 gave a polydisperse band in immunoblots from membrane preparations from different E. histolytica strains, and a much weaker signal from two strains of the nonpathogenic species Entamoeba dispar. Although the exact chemical structure of the EH5 antigen is not yet known, the ability of the antigen to be metabolically radiolabeled with [32P]phosphate or [3H]glucose, its sensitivity to digestion by mild acid and phosphatidylinositol-specific phospholipase C, and its specific extraction from E. histolytica trophozoites by a method used to prepare lipophosphoglycans from Leishmania showed that it could be classified as an amebal lipophosphoglycan. Confocal immunofluorescence and immunogold labeling of trophozoites localized the antigen on the outer face of the plasma membrane and on the inner face of internal vesicle membranes. Antibody EH5 strongly agglutinated amebas in a similar way to concanavalin A (Con A), and Con A bound to immunoaffinity-purified EH5 antigen. Therefore, surface lipophosphoglycans may play an important role in the preferential agglutination of pathogenic amebas by Con A. The protective ability of antibody EH5 was tested in a passive immunization experiment in a severe combined immunodeficient (SCID) mouse model. Intrahepatic challenge of animals after administration of an isotype-matched control antibody or without treatment led to the development of a liver abscess in all cases, whereas 11 out of 12 animals immunized with the EH5 antibody developed no liver abscess. Our results demonstrate the importance and, for the first time, the protective capacity of glycan antigens on the surface of the amebas.

Mass infection with Entamoeba histolytica in a Japanese institution for individuals with mental retardation: epidemiology and control measures.

After two cases of amoebic colitis were detected at an institution for the mentally retarded in the Yamagata prefecture of Japan, the prevalence and epidemiology of Entamoeba histolytica infection at the institution were investigated. When the 76 residents with mental retardation were checked by serology and stool examinations, 40 (53%) showed evidence of infection with E. histolytica (i.e. E. histolytica-specific antibodies in their serum, Entamoeba cysts in their stools, and/or E. histolytica-specific antigens in their stools). The cysts were all assumed to be those of E. histolytica since all nine of the 18 cyst-positive stool samples investigated using a PCR (that distinguishes E. histolytica from E. dispar) were found positive for this species. The E. histolytica found in the institution in Yamagata appears to have been brought into the institution, from a similar institution in Kanagawa prefecture, by a mentally retarded individual who relocated from Kanagawa to Yamagata. Isolates of E. histolytica recovered during an outbreak in the institution in Kanagawa appear genotypically identical to the genotyped isolates collected in the outbreak investigated in the present study. The 40 infected individuals in Yamagata were each treated for 10 days with metronidazole or diloxanide furoate. The residents and staff of the institution were encouraged to wash their hands more frequently and more thoroughly, and the staff were asked to clip residents' fingernails and to improve the cleanliness/sterilization of the surfaces in the institution that were most likely to be contaminated with E. histolytica (lavatories, handrails, doors, doorknobs, washrooms, clothing etc). In the last 5 years of follow-up since the instigation of these and other infection-control measures, and the last treatments, no cases of E. histolytica infection have been found in the institution. This encouraging result offers hope and guidance to those attempting to control outbreaks of E. histolytica infection in other institutions.

Role of kinases in virulence and pathogenesis of protozoan parasite E. Histolytica.

Protein kinases are known to regulate several cellular processes like metabolism, motility and endocytosis through phosphorylation of specific target proteins which forms a communication system relaying extracellular signals to intracellular milieu for an adaptive response. One of the protozoan parasite Entamoeba histolytica, which causes amoebiasis and is one of the prominent reason for causing diarrhoea in infants of developing countries, where it remains the third leading cause of deaths in infants(1). The genome of this parasite codes for 331 putative protein kinases which accounts for 3.7% of the proteome. The kinome of the parasite is composed of several conserved and as well as kinase with unusual domain architecture. About one-third of kinome codes for transmembrane kinases (TMK) which is proposed to help the parasite to sense and adapt to the gut environment which is constantly changing. Many kinases are known to be involved in virulence but, the kinome of this important parasite is unexplored. In this review, we present an overview of E. histolytica kinases and their role in amoebic biology understood till now.

Human milk kills parasitic intestinal protozoa.

Giardia lamblia, a common pathogenic intestinal parasite of humans, was rapidly killed by exposure to normal human milk in vitro. The killing did not depend on secretory immunoglobulin A. Entamoeba histolytica, the dysentery amoeba, was also killed by normal human milk. Giardia-cidal activity cochromatographed with an unusual lipase that is present in the milk of humans but not of lower mammals. Human milk may play a protective role in infants exposed to this parasite.

Attenuated recombinant Yersinia as live oral vaccine carrier to protect against amoebiasis.

Various attenuated Yersinia enterocolitica strains expressing different sections of the Entamoeba histolytica surface lectin via the type III protein secretion system (T3SS) were assessed for their use to orally vaccinate rodents against invasive amoebiasis. The T3SS was found to efficiently express and secrete or translocate subfragments as well as the entire heavy subunit of the lectin. Oral vaccination with recombinant Yersinia conferred significant protection against amoebic liver abscess formation when the antigen was expressed as a fusion molecule with the translocation domain of Yersinia outer protein E. However, effectiveness of vaccination was dependent on gender and the rodent species used. Protection was mediated primarily by cellular immune mechanisms as it was independent from the antibody titre against the amoeba lectin but correlated with an antigen-specific Th1-cytokine response. The results suggest that gram-negative bacteria expressing E. histolytica antigens via T3SS may constitute a suitable oral vaccine carrier against amoebiasis and that an effective IFN-gamma response is required for protection against invasive amoebiasis.

Inhibitors of Escherichia coli serine acetyltransferase block proliferation of Entamoeba histolytica trophozoites.

The protozoan parasite Entamoeba histolytica is the etiologic agent of amebiasis, a major global public health problem, particularly in developing countries. There is an effective anti-amoebic drug available, however its long term use produces undesirable side effects. As E. histolytica is a micro-aerophilic organism, it is sensitive to high levels of oxygen and the enzymes that are involved in protecting against oxygen-stress are crucial for its survival. Therefore serine acetyltransferase, an enzyme involved in cysteine biosynthesis, was used as a target for identifying potential inhibitors. Virtual screening with Escherichia coli serine acetyltransferase was carried out against the National Cancer Institute chemical database utilizing molecular docking tools such as GOLD and FlexX. The initial analysis yielded 11 molecules of which three compounds were procured and tested for biological activity. The results showed that these compounds partially block activity of the E. coli enzyme and the growth of E. histolytica trophozoites but not mammalian cells.

Introducing reptiles into a captive collection: the role of the veterinarian.

The successful introduction of reptiles into a captive collection depends on providing optimal husbandry and veterinary attention. An important role of the veterinarian in this process is the prevention of disease introduction, which may affect both the introduced and the resident animals. This review focuses on preventive veterinary medicine in reptiles, emphasising quarantine measures, disinfection and entry control for infectious agents. Agents discussed include those that are likely give rise to severe clinical problems on introduction into a collection of reptiles, or, in the case of Salmonella, those that pose a significant public health risk. Aetiology, clinical signs and diagnosis are discussed for the most relevant endo- and ectoparasites, bacteria and viruses including Cryptosporidium and Entamoeba, Salmonella, Dermabacter, Chlamydiales, Mycoplasma, Herpesvirus, Adenovirus, Paramyxovirus and inclusion body disease.