Amoebic Liver Abscess Formation Research Articles

Genes differentially expressed between pathogenic and non-pathogenic Entamoeba histolytica clones influence pathogenicity-associated phenotypes by multiple mechanisms.

Recently, two genes involved in amoebic liver abscess formation in a mouse model were identified by their differential expression of non-pathogenic (A1np) and pathogenic (B2p) clones of the Entamoeba histolytica isolate HM:1-IMSS. While overexpression of a gene encoding the metallopeptidase EhMP8-2 reduces the virulence of the pathogenic clone B2p, overexpression of the gene ehi_127670 (ehhp127), encoding a hypothetical protein, increases the virulence of the non-pathogenic clone A1np, while silencing this gene in the pathogenic B2p reduces virulence. To understand the role of both molecules in determining the pathogenicity of E. histolytica, silencing, and overexpression transfectants were characterized in detail. Silencing of ehmp8-2, of the homologous gene ehmp8-1, or both in non-pathogenic A1np trophozoites significantly altered the transcript levels of 347, 216, and 58 genes, respectively. This strong change in the expression profiles caused by the silencing of ehmp8-1 and ehmp8-2 implies that these peptidases regulate the expression of numerous genes. Consequently, numerous phenotypic characteristics, including cytopathic, hemolytic, and cysteine peptidase activity, were altered in response to their silencing. Silencing of ehhp127 in pathogenic B2p trophozoites did not affect the expression of other genes, whereas its overexpression in non-pathogenic A1np trophozoites results in an altered expression of approximately 140 genes. EhHP127 is important for trophozoite motility, as its silencing reduces, while its overexpression enhances movement activity. Interestingly, the specific silencing of ehhp127 also significantly affects cytopathic, cysteine peptidase, and hemolytic activities. All three molecules characterized in this study, namely EhMP8-1, EhMP8-2, and EhHP127, are present in amoeba vesicles. The results show that ehmp8-2 and ehhp127 are not only differentially expressed between pathogenic and non-pathogenic amoebae, but that they also significantly affect amoeba pathogenicity-associated phenotypes by completely different mechanisms. This observation suggests that the regulation of amoeba pathogenicity is achieved by a complex network of molecular mechanisms rather than by single factors.

Multilocus sequence typing (MLST) of Entamoeba histolytica identifies kerp2 as a genetic marker associated with disease outcomes.

Amoebiasis caused by protozoan parasite Entamoeba histolytica has diverse infection outcomes. The relationship between parasite genotypes and outcome of amoebic infection is still a paradox and needs to be explored. Genome information of infecting strains from endemic areas throughout the world is essential to explore this relation. Comparative genetics between E. histolytica populations from different disease outcomes have been studied to identify potential genetic markers having single nucleotide polymorphisms (SNPs) significantly associated with specific clinical outcome. Coding and non-coding regions have significantly different rates of polymorphism. Non-synonymous base substitutions were significantly more frequent than synonymous within coding loci. Both synonymous and non-synonymous SNPs within lysine- and glutamic acid rich protein 2 (kerp2) locus were significantly associated with disease outcomes. An incomplete linkage disequilibrium (LD) value with potential recombination events and significant population differentiation (FST) value have also been identified at kerp2 locus within the study population. Presence of disease specific SNPs, potential recombination events, and significant FST value at kerp2 locus indicate that kerp2 gene and its gene product are under constant selection pressure exerted by host on parasite and could also be a potential determinant of disease outcome of E. histolytica infection. Furthermore, E. histolytica isolated from asymptomatic carriers are phylogenetically closer to those causing liver abscess in human and exhibit potential inter-population recombination among them. Individuals with persistent asymptomatic E. histolytica infection may be under high risk of developing amoebic liver abscess formation in future and detailed investigation of asymptomatic individuals from endemic areas should be always required.

EhRab21 associates with the Golgi apparatus in Entamoeba histolytica.

Rab proteins constitute the largest group of small GTPases and act as molecular switches in a wide variety of cellular processes, including proliferation, cytoskeleton assembly, and membrane trafficking in all eukaryotic cells. Rab21 has been reported in several eukaryotic cells, and our results suggest that in Entamoeba histolytica, Rab21 is involved in the vesicular traffic associated with the Golgi apparatus, where its function appears to be important to maintain the structure of this organelle. In addition, proteins such as Rab1A and Sec24, identified in this work associated with EhRab21, participate in the traffic of COPII vesicles from the endoplasmic reticulum to the Golgi apparatus and are necessary to maintain the latter's structure in human cells. In addition, EhRab21 probably affects the lysosome biogenesis, as indicated by an increase in the number of lysosomes as a result of the increase in EhRab21 activity. The participation of EhRab21 in the pathogenesis of amebiasis was verified on the amoebic liver abscess formation model using hamsters (Mesocricetus auratus), in which the overexpression of EhRab21Q64L (positive dominant mutant protein) decreased the number of liver abscesses formed.

Overexpression of Differentially Expressed Genes Identified in Non-pathogenic and Pathogenic Entamoeba histolytica Clones Allow Identification of New Pathogenicity Factors Involved in Amoebic Liver Abscess Formation.

We here compared pathogenic (p) and non-pathogenic (np) isolates of Entamoeba histolytica to identify molecules involved in the ability of this parasite to induce amoebic liver abscess (ALA)-like lesions in two rodent models for the disease. We performed a comprehensive analysis of 12 clones (A1–A12) derived from a non-pathogenic isolate HM-1:IMSS-A and 12 clones (B1–B12) derived from a pathogenic isolate HM-1:IMSS-B. “Non-pathogenicity” included the induction of small and quickly resolved lesions while “pathogenicity” comprised larger abscess development that overstayed day 7 post infection. All A-clones were designated as non-pathogenic, whereas 4 out of 12 B-clones lost their ability to induce ALAs in gerbils. No correlation between ALA formation and cysteine peptidase (CP) activity, haemolytic activity, erythrophagocytosis, motility or cytopathic activity was found. To identify the molecular framework underlying different pathogenic phenotypes, three clones were selected for in-depth transcriptome analyses. Comparison of a non-pathogenic clone A1np with pathogenic clone B2p revealed 76 differentially expressed genes, whereas comparison of a non-pathogenic clone B8np with B2p revealed only 19 differentially expressed genes. Only six genes were found to be similarly regulated in the two non-pathogenic clones A1np and B8np in comparison with the pathogenic clone B2p. Based on these analyses, we chose 20 candidate genes and evaluated their roles in ALA formation using the respective gene-overexpressing transfectants. We conclude that different mechanisms lead to loss of pathogenicity. In total, we identified eight proteins, comprising a metallopeptidase, C2 domain proteins, alcohol dehydrogenases and hypothetical proteins, that affect the pathogenicity of E. histolytica.

Morphological Findings in Trophozoites during Amoebic Abscess Development in Misoprostol-Treated BALB/c Mice.

During amoebic liver abscess (ALA) formation in susceptible animals, immune response is regulated by prostaglandin E2 (PGE2) dependent mechanisms. The aim of this study was to analyze the effect of misoprostol (MPL), a PGE1 analogue, on ALA formation in BALB/c mice. Male mice from BALB/c strain were intrahepatically infected with 7.5 × 105 trophozoites of E. histolytica strain HM1:IMSS and treated with 10−4 M of MPL daily until sacrifice at 2, 4, and 7 days postinfection (p.i.). ALA formation was evaluated at 2, 4, and 7 days postinfection; trophozoite morphology was analyzed using immunohistochemistry and image analysis. Results showed an increase in frequency of ALA formation in infected and MPL-treated mice only at 2 days p.i. (P = 0.03). A significant diminution in the size of trophozoites was detected in abscesses from mice independently of MPL treatment (from 5.8 ± 1.1 µm at 2 days p.i. to 2.7 ± 1.9 µm at 7 days p.i.) compared with trophozoites dimensions observed in susceptible hamsters (9.6 ± 2.7 µm) (P < 0.01). These results suggest that MPL treatment may modify the adequate control of inflammatory process to allow the persistence of trophozoites in the liver; however, natural resistance mechanisms cannot be discarded.

Overexpression of Specific Cysteine Peptidases Confers Pathogenicity to a Nonpathogenic Entamoeba histolytica Clone

ABSTRACTCysteine 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.

Obtaining of three recombinant antigens of Entamoeba histolytica and evaluation of their immunogenic ability without adjuvant in a hamster model of immunoprotection

A 30-kDa surface collagen binding protein peroxiredoxin of Entamoeba histolytica (EhCBP30) was evaluated either alone or fused to the chaperone (CHP) or ATPase (ATP) domains of heat shock protein 70 of Trypanosoma cruzi (TcHSP70) as a vaccine candidate in a hamster model of experimental amoebic liver abscess (ALA) development. Three constructs were produced containing the EhCBP30 DNA sequence, one expressing EhCBP30 and two expressing EhCBP30 fused to either CHP or ATP domains of TcHSP70. High purity recombinant proteins rEhCBP30, rEhCBP30-CHP and rEhCBP30-ATP with N-terminal His tag were obtained by single step affinity purification. Hamsters were immunized without adjuvant with the antigenic recombinant proteins and then challenged intrahepatically with E. histolytica trophozoites. A 70% decrease in ALA development was detected in hamsters immunized with rEhCBP30 and rEhCBP30-CHP, while animals immunized with rEhCBP30-ATP did not show a statistically significant decrease in ALA formation compared with non-immunized animals. Histological analysis of liver tissue showed that the inflammatory infiltrate was discrete or moderate in hamsters immunized with rEhCBP30 or rEhCBP30-CHP compared with that observed in control hamsters or hamsters immunized with rEhCBP30-ATP. These results suggest that rEhCBP30 and rEhCBP30-CHP are able to induce an effective immune response that may protect hamsters against ALA development.

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- γ response is required for protection against invasive amoebiasis.

Increased expression of the major cysteine proteinases by stable episomal transfection underlines the important role of EhCP5 for the pathogenicity of Entamoeba histolytica

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.

Entamoeba histolytica: Inflammatory process during amoebic liver abscess formation involves cyclooxygenase-2 expression in macrophages and trophozoites

It has been demonstrated that expression of cyclooxygenase-2 (COX-2) isoform is induced by Entamoeba histolytica in macrophages and polymorphonuclear cells during amoebic liver abscess (ALA) formation in hamsters. Trophozoites present in the lesion were also positive for COX-2 signal. However, no cross reactivity of the anti-COX-2 antibody with protein extract of cultivated trophozoites was found. To clarify if trophozoites are involved in PGE 2 production during ALA development, COX-2 expression was detected by in situ hybridization and RT-PCR in liver tissue from intrahepatically infected hamsters. COX-2 mRNA was in polymorphonuclear cells since 4 h postinfection, and subsequently, local macrophages expressed COX-2 mRNA in a similar way. Additionally, a positive signal for COX-2 mRNA expression was detected in E. histolytica trophozoites, suggesting that, in vivo, parasite COX expression may be an important mechanism to promote inflammation.

Antisense Inhibition of Entamoeba histolytica Cysteine Proteases Inhibits Colonic Mucus Degradation

The exact role Entamoeba histolytica cysteine proteases play in overcoming the colonic mucus barrier, as a prerequisite to epithelial cell disruption, is not known. Herein, we determined whether E histolytica trophozoites expressing the antisense transcript to cysteine protease 5 (EhCP5) could degrade colonic mucin and destroy epithelial cells. Cysteine protease-deficient amoebae were generated by antisense inhibition of EhCP5, and assayed for proteolytic activity against [(35)S]cysteine-labeled mucin from LS 174T, and HT-29F Cl.16E cells. Recombinant EhCP5 mucinase activity was also assessed. Disruption of an intact mucus barrier and epithelial cell invasion by amoebae were measured using high mucin producing LS 174T and HT-29 Cl.16E monolayers or Chinese hamster ovary (CHO) cells devoid of a mucus barrier. Trophozoites with reduced cysteine protease activity were ineffective at degrading [(35)S]cysteine-labeled colonic mucin compared to wild-type amoebae by >60%. However, bioactive recombinant EhCP5 degraded >45% of purified native mucin, which was specifically inhibited by the cysteine proteinase (CP) inhibitor, E-64. Cysteine protease-deficient trophozoites could not overcome a protective intact mucus barrier and disrupt LS 174T or HT-29F Cl.16 cell monolayers; however, they readily adhere to and disrupt CHO monolayers devoid of a mucus barrier. These findings unravel a central role for E histolytica CPs as key virulence factors in disrupting an intact mucus barrier in the pathogenesis of intestinal amoebiasis.

Expression of Inducible Nitric Oxide Synthase mRNA and Nitric Oxide Production During the Development of Liver Abscess in Hamster Inoculated with Entamoeba histolytica

The present study analyzed iNOS and eNOS mRNA expression and NO production during development of hepatic abscess caused by Entamoeba histolytica trophozoites. One 374-bp sequence, which displayed 88% identity to mammalian iNOS protein, was isolated from LPS-stimulated peritoneal hamster macrophages. A separate 365-bp cDNA sequence showed 99% identity with eNOS protein. iNOS mRNA was detected in hamsters during formation of amoebic liver abscesses, but not in control hamsters. eNOS mRNA expression was not modified. Serum nitrite concentration in hamsters infected with E. histolytica was 33 +/- 6 microM, in control hamsters was 20 +/- 3 microM. The study shows that iNOS mRNA expression and NO production are induced by E. histolytica trophozoites during amoebic liver abscess formation. However, in spite of iNOS mRNA expression and NO production, E. histolytica trophozoites induced liver abscess formation in hamster.

Entamoeba histolytica: induction of cyclooxygenase-2 expression during amoebic liver abscess formation in hamsters ( Mesocricetus auratus)

Experimental amoebic liver abscess in hamsters curses with an increase in both, systemic levels of prostaglandin E2 (PGE 2) and local cyclooxygenase activity in liver microsomes. The cellular source of PGE 2 and the isoform of cyclooxygenase responsible are not completely evidenced. Cyclooxygenase-2 (COX-2) protein and gene expression were demonstrated on macrophages and polymorphonuclear cells as a result of Entamoeba histolytica infection in hamsters at 2, 4, and 7 days postinfection by immunohistochemistry and RT-PCR. E. histolytica trophozoites located in the lesion showed a strong positive signal for COX-2, however the enzyme was not detected in cultured trophozoites by Western blot. Our results indicate that the increment in PGE 2 is the result of COX-2 activity from cells of the reticuloendothelial system and reinforce the possibility that PGE 2 production by enzyme induction in macrophages may be a mechanism by which E. histolytica modulates the host immune response in this parasitic infection. Index Descriptors and Abbreviations: ALA, amoebic liver abscess; AMAs, amoebic liver abscess-derived macrophages; COX-2, cyclooxygenase-2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IHC, immunohistochemical analysis; i.p., intraperitoneally; LPS, lipopolysaccharides; MOs, macrophages; PGE 2, prostaglandin E2; p.i., postinfection; PMNs, polymorphonuclear leukocytes; PMOs, peritoneal macrophages; PVDF, polyvinylidene difluoride; RT-PCR, reverse transcription and cDNA amplification by polymerase chain reaction

Liver function tests during amoebic liver abscess formation in indomethacin-treated hamsters.

Establishment of Entamoeba histolytica infection is facilitated through macrophage effector disruption by a prostaglandin E2 (PGE2)-mediated mechanism. Infection severity may be measured by weight of abscess formed. Indomethacin (Indo) treatment of infected hamsters reduced abscess weight by 30% at 7 days post-infection presumably by inhibition of PGE2. To explain reductions in abscess development by Indo treatment, we determined liver functionality in Indo-treated or untreated animals, either healthy or infected. Determinations of serum glutamic oxaloacetic (SGOT) and glutamic pyruvic (SGPT) transaminases, serum alkaline phosphatase (SAP), total serum protein (TSP), and bilirubinemia were done. SGOT, SGPT, and SAP activities showed a significant increase in their values by 600% at seven days post-infection in infected animals in both conditions; nonstatistical differences were found between animals treated or not. This increase did not correlate with the percentage of damage. Infected nontreated hamsters showed TSP levels 30% below normal group (P < 0.05). Infected Indo-treated hamsters had no significant differences compared to normal values. Infected nontreated animals showed an increase in bilirubin, particularly in indirect bilirubin, whereas infected Indo-treated hamsters showed total bilirubin values lower than normals (P < 0.05), with a decrease in direct bilirubin levels. Our results demonstrated that E. histolytica infection in hamsters produces similar abnormalities in liver function as it does in humans, and that the beneficial effect of Indo treatment on amoebic abscess development is not related with an improvement of liver functionality.

Overexpression of cysteine proteinase 2 in Entamoeba histolytica or Entamoeba dispar increases amoeba-induced monolayer destruction in vitro but does not augment amoebic liver abscess formation in gerbils.

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.

Role of Prostaglandin E 2 on Amoebic Liver Abscess Formation in Hamsters

Entamoeba histolytica can modulate macrophage functions and cytokine production by a prostaglandin E 2 (PGE 2) mechanism. To study the participation of PGE 2 on amoebic liver abscess formation, we tested the effect of the PG synthesis inhibitor indomethacin (INDO) on abscess development in hamsters infected intrahepatically with E. histolytica trophozoites. Male infected animals had higher levels of plasma PGE 2 (5.7 ± 0.7 pg/ml pre-infection; 26.0 ± 2.0 pg/ml 7 days postinfection; p < 0.001). INDO prevented this increase, so that infected-treated and control non-infected animals had similar levels of plasma PGE 2. INDO reduced liver and abscess weight by 18% and 30% respectively (p < 0.05). Cyclooxygenase (COX) activity determination by thin layer chromatography using (1- 14C) arachidonic acid (AA) showed that liver microsomes from infected animals produced more PGE 2 than controls. COX activity was considerably inhibited in infected INDO-treated animals. Our data suggest that E. histolytica can stimulate the hepatic production of PGE 2 which contributes to pathogenesis of amoebic abscesses through generation and support of the inflammation. The partial effect of INDO treatment suggests that additional factors are involved.