[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to establish a noninvasive tracking of the pathogenic parasite Entamoeba histolytica (Eh) after superparamagnetic iron oxide (SPIO) labeling by magnetic resonance imaging (MRI) on a single-cell level in vitro and in vivo in a mouse model for amebic liver abscess (ALA).
Local institutional review committee on animal care approved all animal experiments. Entamoeba histolytica trophozoites were labeled with SPIO nanoparticles (SPIO-Eh). The uptake of SPIO by Eh was optimized using flow cytometry and visualized by bright field, fluorescence, and transmission electron microscopy. The viability of SPIO-Eh was assessed in vitro by determination of growth and ingestion rate of red blood cells. Migration of SPIO-Eh was proven by in vitro MRI in a preclinical 7 T MRI system using continually repeated MRI scans. In vivo distribution of SPIO-Eh within the mouse liver was assessed qualitatively and quantitatively by serial respiration-triggered T2*-weighted MRI, T2-weighted MRI, and R2* MR relaxometry up to 5 days after injection and correlated with immunohistology of the liver sections after removal.
Entamoeba histolytica can be efficiently labeled with SPIO without influence on parasite growth rate or phagocytic capacity. In vitro dynamic MRI allowed real-time migration monitoring and determination of velocity of single SPIO-Eh. In vivo SPIO-Eh showed signal decrease in T2*-weighted and increase of R2* in ALA formations. Motility of SPIO-Eh was necessary to induce ALA formations.
The present study demonstrates the feasibility of an efficient magnetic labeling and a noninvasive in vitro and in vivo MR tracking of the pathogenic protozoan Eh in a mouse model for ALA, thus representing in future a noninvasive imaging tool to study parasite, as well as on host-specific pathomechanisms.
[Show abstract][Hide abstract] ABSTRACT: Amebic liver abscess (ALA) is a severe focal destruction of liver tissue caused by infection with the parasite Entamoeba histolytica (E. histolytica). In the past, tissue damage has been mainly attributed to pathogenicity factors of the parasite. However, the massive presence of innate immune cells raises the question whether host cells contribute to the destruction of the liver tissue as well. In this chapter, we discuss the role of neutrophils, monocytes, and macrophages during ALA in animal models for the disease. In brief, neutrophils contribute only partially to the observed pathology, whereas inflammatory monocytes and resident liver macrophages are substantially involved in tissue damage seen during E. histolytica infection. Therefore, we conclude beyond parasite-specific effector molecules, immune pathological mechanisms of the host substantially contribute to the development of ALA.
[Show abstract][Hide abstract] ABSTRACT: Background
The protozoan parasite Entamoeba histolytica (E. histolytica) usually asymptomatically colonizes the human intestine. In the minority of the cases, the parasite evades from the gut and can induce severe symptoms like colitis or amebic liver abscess (ALA). Interestingly, ALA predominates in adult men despite a higher prevalence of the parasite in women. The present study aimed to identify characteristic serum markers in a unique cohort of clearly defined asymptomatically infected E. histolytica individuals in comparison to patients with an E. histolytica liver manifestation of both sex.Methods
The following study groups were investigated: ALA patients (n¿=¿38), healthy asymptomatic E. histolytica carriers (AC) (n¿=¿44), and healthy E. dispar-infected controls (n¿=¿24) out of an amebiasis endemic area. E. histolytica-specific immunoglobulin G (IgG) and the IgG subclasses against proteinaceous and non-proteinaceous amebic antigens were measured by ELISA. Serum cytokine and chemokine levels were investigated using a flow cytometry bead-based multiplex immunoassay.ResultsThe IgG results revealed that not only ALA patients, but also AC, developed high E. histolytica-specific titers of IgG and all IgG subclasses as well as IgA. IgG and IgG2 titers against the glycolipid E. histolytica lipophosphoglycan were highest in ALA patients. As in ALA patients, high cytokine levels of interleukin (IL-) 4 were detected in AC compared to E. dispar infected individuals, while IL-6 was exclusively elevated in ALA patients. IL-10 was lower in AC compared to ALA patients. Equal serum levels of CCL2 were found in all study groups but ALA patients showed decreased levels of CCL3. Sex dependent analysis of the data indicated significantly higher IgG and IgG1 titers in female AC compared to male AC. CCL2, the chemokine involved in immunopathology in the mouse model for the disease, was higher in male AC compared to female AC.Conclusion
In this study we characterize for the first time an asymptomatic carrier stage in amebiasis that is associated with a significant immune reaction and provide immunological markers that might give first hints towards an understanding of immune mechanisms underlying the control or development of invasive amebiasis.
[Show abstract][Hide abstract] ABSTRACT: Surface molecules are of major importance for host-parasite interactions. During Entamoeba histolytica infections, these interactions are predicted to be of prime importance for tissue invasion, induction of colitis and liver abscess formation. To date, however, little is known about the molecules involved in these processes, with only about 20 proteins or protein families found exposed on the E. histolytica surface. We have therefore analyzed the complete surface proteome of E. histolytica. Using cell surface biotinylation and mass spectrometry, 694 putative surface-associated proteins were identified. In silico analysis predicted that approximately 26% of these proteins are membrane-associated, as they contain transmembrane domains and/or signal sequences, as well as sites of palmitoylation, myristoylation or prenylation. An additional 25% of the identified proteins likely represent non-classical secreted proteins. Surprisingly, no membrane-association sites could be predicted for the remaining 49% of the identified proteins. To verify surface localization, 23 proteins were randomly selected and analyzed by immunofluorescence microscopy. Of these 23 proteins, 20 (87%) showed definite surface localization. These findings indicate that a far greater number of E. histolytica proteins than previously supposed are surface-associated, a phenomenon that may be based on the high membrane turnover of E. histolytica.
[Show abstract][Hide abstract] ABSTRACT: Cysteine peptidases (CPs) of Entamoeba histolytica are considered to be important pathogenicity factors. Previous studies have found that under standard axenic culture conditions, only four (ehcp-a1, ehcp-a2, ehcp-a5, and ehcp-a7) out of 35 papain-like ehcp genes present in the E. histolytica genome are expressed at high levels. Little is known about the expression of CPs in E. histolytica during amoebic liver abscess (ALA) formation. In the current study, a quantitative real-time PCR assay was developed to determine the expression of the various ehcp genes during ALA formation in animal models. Increased expression of four ehcp genes (ehcp-a3, -a4, -a10, and -c13) was detected in the gerbil and mouse models. Increased expression of another three ehcp genes (ehcp-a5, -a6, and -a7) was detected in the mouse model only, and two other ehcp genes (ehcp-b8 and -b9) showed increased expression in the gerbil model only. Trophozoites of the nonpathogenic E. histolytica HM-1:IMSS clone A1, which was unable to induce ALAs, were transfected with vectors enabling overexpression of those CPs that are expressed at high levels under culture conditions or during ALA formation. Interestingly, overexpression of ehcp-b8, -b9, and -c13 restored the pathogenic phenotype of the nonpathogenic clone A1 whereas overexpression of various other peptidase genes had no effect on the pathogenicity of this clone.
[Show abstract][Hide abstract] ABSTRACT: Amebic liver abscess (ALA), a parasitic disease due to infection with the protozoan Entamoeba histolytica, occurs age and gender dependent with strong preferences for adult males. Using a mouse model for ALA with a similar male bias for the disease, we have investigated the role of female and male sexual hormones and provide evidence for a strong contribution of testosterone. Removal of testosterone by orchiectomy significantly reduced sizes of abscesses in male mice, while substitution of testosterone increased development of ALA in female mice. Activation of natural killer T (NKT) cells, which are known to be important for the control of ALA, is influenced by testosterone. Specifically activated NKT cells isolated from female mice produce more IFNγ compared to NKT cells derived from male mice. This high level production of IFNγ in female derived NKT cells was inhibited by testosterone substitution, while the IFNγ production in male derived NKT cells was increased by orchiectomy. Gender dependent differences were not a result of differences in the total number of NKT cells, but a result of a higher activation potential for the CD4(-) NKT cell subpopulation in female mice. Taken together, we conclude that the hormone status of the host, in particular the testosterone level, determines susceptibility to ALA at least in a mouse model of the disease.
PLoS ONE 02/2013; 8(2):e55694. DOI:10.1371/journal.pone.0055694 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Author Summary
Amebic liver abscess (ALA), an infectious disease caused by the protozoan parasite Entamoeba histolytica is characterized by severe focal liver damages. According to its name giving activity, destruction of liver tissue by E. histolytica has been attributed to parasite-specific effector molecules. However, abscess lesions contain considerable numbers of innate immune cells, such as neutrophils and macrophages, raising the question whether these host cells might contribute to the disease as well. We have investigated the role of the host immune response during ALA development using a mouse model for the disease. The results indicated that despite the presence of considerable numbers of neutrophils within the abscess lesions, these cells were dispensable for both the control of the disease and the tissue damage. On the other hand, two subsets of immune cells, the liver resident Kupffer cells and the inflammatory Ly6C-expressing monocytes were identified as the main effector cells responsible for liver tissue destruction. Furthermore, TNFα produced by the Ly6C-expressing monocytes, was found to be a cytokine that is critically involved in abscess development. Thus, our finding that host immune mechanisms are indeed responsible for liver tissue destruction during ALA development may change the view on the pathological mechanism of amebic disease.
[Show abstract][Hide abstract] ABSTRACT: The availability of two genetically very similar cell lines (A and B) derived from the laboratory isolate Entamoeba histolytica HM-1:IMSS, which differ in their virulence properties, provides a powerful tool for identifying pathogenicity factors of the causative agent of human amoebiasis. Cell line A is incapable inducing liver abscesses in gerbils, whereas interaction with cell line B leads to considerable abscess formation. Phenotypic characterization of both cell lines revealed that trophozoites from the pathogenic cell line B have a larger cell size, an increased growth rate in vitro, an increased cysteine peptidase activity and higher resistance to nitric oxide stress. To find proteins that may serve as virulence factors, the proteomes of both cell lines were previously studied, resulting in the identification of a limited number of differentially synthesized proteins. This study aims to identify additional genes, serving as virulence factors, or virulence markers.
To obtain a comprehensive picture of the differences between the cell lines, we compared their transcriptomes using an oligonucleotide-based microarray and confirmed findings with quantitative real-time PCR. Out of 6242 genes represented on the array, 87 are differentially transcribed (> or = two-fold) in the two cell lines. Approximately 50% code for hypothetical proteins. Interestingly, only 19 genes show a five-fold or higher differential expression. These include three rab7 GTPases, which were found with a higher abundance in the non-pathogenic cell line A. The aig1-like GTPasesare of special interest because the majority of them show higher levels of transcription in the pathogenic cell line B. Only two molecules were found to be differentially expressed between the two cell lines in both this study and our previous proteomic approach.
In this study we have identified a defined set of genes that are differentially transcribed between the non-pathogenic cell line A and the pathogenic cell line B of E. histolytica. The identification of transcription profiles unique for amoebic cell lines with pathogenic phenotypes may help to elucidate the transcriptional framework of E. histolytica pathogenicity and serve as a basis for identifying transcriptional markers and virulence factors.
[Show abstract][Hide abstract] ABSTRACT: Entamoeba histolytica is known for its extraordinary capacity to destroy human tissues, leading to invasive diseases such as ulcerative colitis or extra-intestinal abscesses. In order to identify the virulence factors of this parasite phenotypes and proteomes of two recently identified genetically related cell lines (A and B), derived from the laboratory E. histolytica isolate HM-1:IMSS, were compared. Both cell lines are indistinguishable on the basis of highly polymorphic tandem repeat DNA sequences. However, cell line A is incapable to induce liver abscesses in experimentally infected rodents, whereas cell line B provokes considerable abscesses. Phenotypic analyses revealed increased hemolytic activity, lower growth rate, smaller cell size, reduced cysteine peptidase activity and lower resistance to nitric oxide stress for cell line A. In contrast, no differences between the two cell lines were found for cytopathic activity, erythrophagocytosis, digestion of erythrocytes or resistance to complement, hydrogen peroxide and superoxide radical anions. Proteomic comparison by 2-D DIGE followed by MS, identified a total of 21 proteins with higher abundance in cell line A and ten proteins with higher abundance in cell line B. Remarkably, three differentially up-regulated antioxidants were exclusively found in the pathogenic cell line B. Notably, only for two differentially regulated proteins, namely a Fe-hydrogenase and a C2 domain protein, a similar type was found at the level of transcription. Summarized, a defined set of different proteins could be identified between cell lines A and B. These molecules may have an important role in amoeba pathogenicity.
[Show abstract][Hide abstract] ABSTRACT: Author Summary
Amoebiasis is a widespread human parasitic disease caused by the intestinal protozoan Entamoeba histolytica. There are two major clinical manifestations of the disease, amebic colitis and amebic liver abscess. Interestingly, only a small proportion of E. histolytica-infected individuals develop invasive disease, whereas the majority harbors the parasite within the gut without clinical symptoms. So far, cells of the innate immune system have been described to constitute the main host defense mechanism for the control of amoebiasis, relying largely on the early production of interferon-γ (IFN-γ), a protein, which activates macrophages to kill microorganisms including parasites. However, the IFN-γ-producing cells as well as the amebic antigen involved in the activation have not been identified. Here we demonstrate that mice challenged with live E. histolytica, and which are deficient of a specific lymphocyte population known as natural killer T cells (NKT cells), have a reduced capacity to control ameba infection and develop much larger amebic liver abscesses compared to normal mice. In addition, we isolated a molecule from the surface membrane of E. histolytica, which constitutes a lipopeptidophosphoglycan, and which activates NKT cells for the production of protective IFN-γ. Thus, our study provides a mechanism for the innate control of ameba invasion that might explain why the majority of E. histolytica-infected individuals do not develop amebic disease.
[Show abstract][Hide abstract] ABSTRACT: 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.
International journal of medical microbiology: IJMM 02/2008; 298(1-2):79-86. DOI:10.1016/j.ijmm.2007.07.001 · 3.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The protozoan Entamoeba histolytica causes intestinal inflammation and liver abscess. Cysteine proteinases (CPs) have been proposed as important virulence factors for amoebiasis. To test the role of the various CPs for amoeba induced pathology, the three major enzymes of the parasite, namely EhCP1, EhCP2 and EhCP5 accounting for about 90% of total proteinase activity, were overexpressed by stable episomal transfection. Total CP activity of recombinant amoebae increased by three- to six-fold depending on the gene transfected. Interestingly, overexpression of the genes for EhCP1 or EhCP2 increased the activity of the corresponding enzyme only, whereas overexpression of the gene for EhCP5 increased the activity of all three enzymes, which is consistent with enzyme-converting activity of EhCP5. Cytopathic activity, measured by in vitro monolayer disruption, was dramatically increased in ehcp5-transfectants (five-fold) but showed only a modest increase in ehcp1- or ehcp2-transfectants (1.5-2-fold). In addition, overexpression of ehcp5 but not of ehcp1 or ehcp2 significantly increased amoebic liver abscess formation in laboratory animals. Moreover, transfection and overexpression of ehcp5 was able to compensate the reduction of in vivo pathogenicity in parasites, which have been silenced for the gene encoding the pore-forming protein amoebapore A. In summary, these results further support the important role of EhCP5 in E. histolytica pathogenicity.
[Show abstract][Hide abstract] ABSTRACT: Efficient control of infectious diseases requires the development and application of suitable vaccines. Development of vaccines against amebiasis is still in its infancy. However, in recent years progress has been made in the identification of possible vaccine candidates, the route of application and the understanding of the immune response that is required for protection against amebiasis.
Archives of Medical Research 03/2006; 37(2):292-6. DOI:10.1016/j.arcmed.2005.09.004 · 2.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Amebic liver abscess (ALA) is the most common extraintestinal manifestation of human infection by the enteric protozoan parasite Entamoeba histolytica. In contrast to intestinal infection, ALA greatly predominates in males but is rare in females. Since humans are the only relevant host for E. histolytica, experimental studies concerning this sexual dimorphism have been hampered by the lack of a suitable animal model. By serial liver passage of cultured E. histolytica trophozoites in gerbils and mice, we generated amebae which reproducibly induce ALA in C57BL/6 mice. Interestingly, all animals developed ALA, but the time courses of abscess formation differed significantly between the genders. Female mice were able to clear the infection within 3 days, whereas in male mice the parasite could be recovered for at least 14 days. Accordingly, male mice showed a prolonged time of recovery from ALA. Immunohistology of abscesses revealed that polymorphonuclear leukocytes and macrophages were the dominant infiltrates, but in addition, gamma,delta-T cells, NK cells, and natural killer T (NKT) cells were also present at early times during abscess development, whereas conventional alpha,beta-T cells appeared later, when female mice had already cleared the parasite. Interestingly, male and female mice differed in early cytokine production in response to ameba infection. Enzyme-linked immunospot assays performed with spleen cells of infected animals revealed significantly higher numbers of interleukin-4-producing cells in male mice but significantly higher numbers of gamma interferon (IFN-gamma)-producing cells in female mice. Early IFN-gamma production and the presence of functional NKT cells were found to be important for the control of hepatic amebiasis as application of an IFN-gamma-neutralizing monoclonal antibody or the use of NKT knockout mice (Valpha14iNKT, Jalpha 18(-/-)) dramatically increased the size of ALA in female mice. In addition, E. histolytica trophozoites could be reisolated from liver abscesses of Jalpha18(-/-) mice on day 7 postinfection, when wild-type mice had already cleared the parasite. These data suggest that the sexual dimorphism in the control of ALA is due to gender-specific differences in early cytokine production mediated at least in part by NKT cells in response to E. histolytica infection of the liver.
Infection and Immunity 02/2006; 74(1):118-24. DOI:10.1128/IAI.74.1.118-124.2006 · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Protection against invasive amebiasis was achieved in the gerbil model for amebic liver abscess by oral immunization with
live attenuated Yersinia enterocolitica expressing the Entamoeba histolytica galactose-inhibitable lectin that has been fused to the Yersinia outer protein E (YopE). Protection was dependent on the presence of the YopE translocation domain but was independent from
the antibody response to the ameba lectin.
Infection and Immunity 01/2005; 72(12):7318-21. DOI:10.1128/IAI.72.12.7318-7321.2004 · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cysteine proteinases (CPs) have been considered suitable targets for the development of antiparasitic drugs. To assess the importance of CPs for the growth and pathogenicity of the protozoan parasite Entamoeba histolytica we have cultured amoebae in the presence of various cysteine proteinase inhibitors (CPIs). It was found that broad range CPIs, which are membrane permeable and rapidly enter the cell, are highly toxic at micromolar concentrations, and all attempts to generate E. histolytica mutants resistant to these CPIs were unsuccessful. In contrast, the broad range CPI E64, which does not permeate membranes as well, was deleterious at much higher concentrations, and amoebae rapidly developed resistance to this inhibitor. Compared with sensitive wild-type cells, E64-resistant E. histolytica were substantially reduced in the expression of various CP genes and were able to secrete unprocessed enzyme into the culture medium. Moreover, E64 resistance was associated with a significant reduction in virulence, because these cells were greatly impaired in the ability to generate liver abscesses in experimentally infected gerbils.
[Show abstract][Hide abstract] ABSTRACT: The majority of human infections with the intestinal protozoan parasite Entamoeba histolytica remain asymptomatic. In a small proportion of infections, however, E. histolytica trophozoites penetrate the intestinal mucosa and disseminate to other organs, most commonly to the liver, where they induce abscess formation. It is believed that the ability of E. histolytica trophozoites to destroy host tissues and to survive within the liver is accomplished by a strong adaptive response, which requires the specific regulation of a number of amoeba proteins. Using differential display polymerase chain reaction (DD-PCR), we compared RNA expression between E. histolytica trophozoites isolated from liver abscesses of infected gerbils and those grown under normal culture conditions. A total of 3000 cDNA-derived amplicons were compared between the two groups of amoebae, which were calculated to represent about one-third of all E. histolytica mRNA species (transcriptome). Among these, 55 were found to be specifically present or absent in abscess-derived amoebae, of which 42 were successfully cloned and sequenced. Database searches and Northern blot analyses revealed that the 42 amplicons correspond to 29 independent E. histolytica genes, of which at least seven are specifically upregulated and five are downregulated in abscess-derived amoebae. Specific expression of most of these genes was not simply the result of a heat shock response, which might be expected during abscess formation, as only five of the genes revealed an expression profile similar to that found in amoebae cultured under elevated temperatures. The two genes specifically downregulated in abscess-derived amoebae encode members of a family of so far unknown proteins, which contain repetitive stretches of sequences that are rich in lysine and glutamic acid residues. In contrast, a diverse set of genes is specifically upregulated, encoding ribosomal proteins (S30, L37A), cyclophilin, ferredoxin 2 and GTP-binding protein RAB7D, supporting the notion that liver abscess formation requires the regulation and concerted action of a variety of amoeba proteins. These proteins are associated with stress response, signal transduction, regulation of transcription and vesicular trafficking. However, transcriptome analysis will not be sufficient to identify all proteins specifically upregulated during abscess formation, as at least an increase in the expression of actin was found to be regulated at the post-transcriptional level.
[Show abstract][Hide abstract] ABSTRACT: To study the role of cysteine proteinases in the pathogenicity of Entamoeba histolytica, we have attempted to overexpress the three main cysteine proteinases (EhCP1, EhCP2, EhCP5) of this parasite in trophozoites of E. histolytica as well as in non-pathogenic Entamoeba dispar by episomal transfection. Although each of the corresponding coding sequences were cloned in identical expression plasmids, we were unable to overexpress EhCP1 and EhCP5, respectively, but could substantially induce expression of EhCP2 in both amoeba species by sevenfold, leading to a threefold increase in total cysteine proteinase activity. Overexpression of EhCP2 did not influence expression of other cysteine proteinases and could be attributed to an increase of a single 35 kDa activity band in substrate gel electrophoresis. In contrast to previous findings, which indicated that amoeba cysteine proteinases are involved in erythrophagocytosis and liver abscess formation, cells overexpressing EhCP2 showed no difference in erythrophagocytosis or liver abscess formation compared with respective controls. However, overexpression of EhCP2 in both amoeba species resulted in a marked increase of in vitro monolayer destruction.