The 29-kilodalton thiol-dependent peroxidase of Entamoeba histolytica is a factor involved in pathogenesis and survival of the parasite during oxidative stress

The 29-kDa surface antigen (thiol-dependent peroxidase; Eh29) of Entamoeba histolytica exhibits peroxidative and protective antioxidant activities. During tissue invasion, the trophozoites are exposed to oxidative stress and need to deal with highly toxic reactive oxygen species (ROS). In this investigation, attempts have been made to understand the role of the 29-kDa peroxidase gene in parasite survival and pathogenesis. Inhibition of eh29 gene expression by antisense RNA technology has shown approximately 55% inhibition in eh29 expression, maximum ROS accumulation, and significantly lower viability in 29-kDa downregulated trophozoites during oxidative stress. The cytopathic and cytotoxic activities were also found to decrease effectively in the 29-kDa downregulated trophozoites. Size of liver abscesses was substantially lower in hamsters inoculated with 29-kDa downregulated trophozoites compared to the normal HM1:IMSS. These findings clearly suggest that the 29-kDa protein of E. histolytica has a role in both survival of trophozoites in the presence of ROS and pathogenesis of amoebiasis.     

Antisense inhibition of amoebapore expression in Entamoeba histolytica causes a decrease in amoebic virulence

Amoebapores have been proposed to be a major pathogenicity factor of the protozoan parasite Entamoeba histolytica, which is responsible for the killing of target cells. These 77-residue peptides are structural and functional analogues of NK-lysin and granulysin of porcine and human cytotoxic lymphocytes. Inhibition of amoebapore gene expression in amoebae was obtained following transfection with a hybrid plasmid construct (pAP-R2) containing the Neo resistance gene and the gene coding for amoebapore A, including its 5' and 3' untranslated region (UTR) sequences, in reverse orientation under a promoter (g34) taken from one of the E. histolytica ribosomal protein (RP-L21) gene copies. Transfectants of virulent E. histolytica strain HM-1:IMSS, in which the expression of amoebapore was inhibited by approximately 60%, were significantly less pathogenic. Cytopathic and cytolytic activities of viable trophozoites against mammalian nucleated cells, as well as lysis of red blood cells, were markedly inhibited. Moreover, trophozoite extracts of pAP-R2 transfectant displayed lower pore-forming activity and were less potent in inhibiting bacterial growth compared with controls. Notably, liver abscess formation in hamsters by the pAP-R2 transfectant was substantially impaired. These results demonstrate for the first time that amoebapore is one of the pathogenicity factors by which trophozoites of E. histolytica exert their remarkable cytolytic and tissue destructive activity.     

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.     

Systemically delivered antisense oligomers upregulate gene expression in mouse tissues

Systemically injected 2'-O-methoxyethyl (2'-O-MOE)-phosphorothioate and PNA-4K oligomers (peptide nucleic acid with four lysines linked at the C terminus) exhibited sequence-specific antisense activity in a number of mouse organs. Morpholino oligomers were less effective, whereas PNA oligomers with only one lysine (PNA-1K) were completely inactive. The latter result indicates that the four-lysine tail is essential for the antisense activity of PNA oligomers in vivo. These results were obtained in a transgenic mouse model designed as a positive readout test for activity, delivery, and distribution of antisense oligomers. In this model, the expressed gene (EGFP-654) encoding enhanced green fluorescence protein (EGFP) is interrupted by an aberrantly spliced mutated intron of the human beta-globin gene. Aberrant splicing of this intron prevented expression of EGFP-654 in all tissues, whereas in tissues and organs that took up a splice site-targeted antisense oligomer, correct splicing was restored and EGFP-654 expression upregulated. The sequence-specific ability of PNA-4K and the 2'-O-MOE oligomers to upregulate EGFP-654 provides strong evidence that systemically delivered, chemically modified oligonucleotides affect gene expression by sequence-specific true antisense activity, validating their application as potential therapeutics.     

Entamoeba histolytica: identification of a distinct beta2 integrin-like molecule with a potential role in cellular adherence

Entamoeba histolytica infection causes dysentery, intestinal colitis, and hepatic abscess in an estimated 50 million people worldwide. Attachment of E. histolytica trophozoites to intestinal epithelium and vascular endothelium during liver metastasis results in an inflammatory process. We report the identification of a distinct amebic beta2 integrin (CD18)-like molecule which affords adherence to TNF-alpha-activated endothelial cells. Data from flow cytometry and indirect immunofluorescence assays suggest the amebic beta2 integrin was localized to focal adhesion plates and was present in both E. histolytica and Entamoeba dispar. The amebic beta2 integrin appeared to be distinct from the amebic Gal/GalNAc lectin based on recombinant expression, amebic colocalization, and ELISA studies. Trophozoite adherence to endothelial cells expressing ICAM-1 (CD54) following activation with TNF-alpha or ICAM-1-transfected CHO cells was specifically inhibited with anti-CD18 or anti-CD54 MAbs. In summary, evidence in support of a distinct beta2 integrin-like molecule participating in amebic adherence to TNF-alpha-activated endothelial cells expressing ICAM-1 is presented. The presence of integrin-dependent binding may allow trophozoites to opportunistically adhere to activated intestinal epithelium or vascular endothelium expressing ICAM-1 during amebic colitis or hepatic abscess.     

The lysine- and glutamic acid-rich protein KERP1 plays a role in Entamoeba histolytica liver abscess pathogenesis

The parasite Entamoeba histolytica colonizes the large bowel where it may persist as an asymptomatic luminal gut infection, which changes to virulence. Parasite invasion of the intestine leads to dysentery and spreads to the liver, where amoebae form abscesses. We took advantage of changes in virulence that occurs after long-term in vitro culture of E. histolytica strains. Using microarrays, we concluded that virulence correlates with upregulation of key genes involved in stress response, including molecular chaperones, ssp1 and peroxiredoxin; as well as the induction of unknown genes encoding lysine-rich proteins. Seven of these were retained with respect to their lysine content higher than 25%. Among them, we found KERP1, formerly identified as associated to parasite surface and involved in the parasite adherence to host cells. Experimentally induced liver abscesses, using molecular beacons and protein analysis, allowed us to draw a parallel between the intricate upregulation of kerp1 gene expression during abscess development and the increased abundance of KERP1 in virulent trophozoites. Following its characterization as a marker for the progression of infection, KERP1 was also seen to be a virulence marker as trophozoites affected in kerp1 expression by an antisense strategy were unable to form liver abscesses.     

Entamoeba histolytica: intracellular distribution of the sec61alpha subunit of the secretory pathway and down-regulation by antisense peptide nucleic acids

The Sec61alpha protein is defined as a highly conserved essential integral component of the endoplasmic reticulum in eukaryotic cells. We report a detailed immunolocalization of the Entamoeba histolytica homologue of the Sec61alpha subunit (EhSec61alpha), which shows an irregular pattern throughout the cell and is also found on the cell surface, its effective down-regulation by means of antisense peptide nucleic acids and its effects on cell proliferation, subcellular distribution of two virulence factors, and the ability of the trophozoites to cause liver abscess in hamsters. Although Sec61alpha levels are specifically decreased in antisense PNA-treated trophozoites, which proliferate more slowly than the controls, mobilization of the cysteine protease 5 and amoebapore to the cell surface is not significantly impeded and the capacity to induce liver abscess in hamsters is largely unaffected. The implications of these findings are discussed in the context of the peculiar cell biology of E. histolytica.     

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 4h 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.     

Entamoeba dispar: molecular characterization of the galactose/N-acetyl-d-galactosamine lectin

Amebiasis contributes to approximately 50 million cases of life-threatening dysentery worldwide. Comparison of the lectins from Entamoeba histolytica (pathogenic) and Entamoeba dispar (nonpathogenic) was undertaken to elucidate the differential roles of this molecule in invasion versus colonization. Surface lectin was less abundant on axenic E. dispar than on axenic E. histolytica, commensurate with differences in lectin (heavy and light subunits) RNA when assessed by semiquantitative RT-PCR. The 1G7 epitope, which falls within the immunodominant and immunoprotective cysteine-rich region (480-900), was absent on axenic E. dispar. Indirect immunofluorescence, transient transection of COS7, and immunoprecipitation demonstrated that the 1G7 epitope was conserved in the nonpathogenic lectin homologue but not exposed on live E. dispar trophozoites. Hgl2 (E. histolytica) and Dhgl2 (E. dispar) lectin homologues demonstrated comparable high-affinity binding to multivalent GalNAc(19) BSA. These data provide evidence for relative gene and conformational regulation of the E.dispar lectin.     

Recognition of 29 kDa surface-associated adhesive molecule of Entamoeba histolytica by monoclonal antibodies

Abstract Monoclonal antibodies have been developed and used as specific probe to locate and identify a 29-kDa molecule of axenic Entamoeba histolytica trophozoites. Monoclonal antibody produced by clone C8 (MoAb C8) strongly agglutinated the amoebic trophozoites. THe immunofluorescence of live E. histolytica trophozoites and surface fluorescence of acetone-fixed trophozoites by MoAb C8 indicated existence of a 29-kDa molecule on surface-associated plasma membrane of E. histolytica . The monoclonal antibody belonged to IgG₁ isotype. The prior treatment of E. histolytica trophozoites with MoAb C8 resulted in significant ( P < 0.01) reduction in adherence of amoebic trophozoites to cultured Chinese Hamster Ovary cells and significant ( P < 0.01) reduction in cytotoxicity to cultured Baby Hamster Kidney cells. Pretreatment of amoebic trophozoites with MoAb C8 prior to cultivation in TPS-1 medium resulted in significant ( P < 0.01) reduction in growth of the parasite. Thus, the data suggested that the surface-exposed 29-kDa molecule may be one of the receptors involved in E. histolytica host cell interactions and may possibly modulate amoebic disease processes.     

Recognition of 29 kDa surface-associated adhesive molecule of Entamoeba histolytica by monoclonal antibodies

Monoclonal antibodies have been developed and used as specific probe to locate and identify a 29-kDa molecule of axenic Entamoeba histolytica trophozoites. Monoclonal antibody produced by clone C8 (MoAb C8) strongly agglutinated the amoebic trophozoites. The immunofluorescence of live E. histolytica trophozoites and surface fluorescence of acetone-fixed trophozoites by MoAb C8 indicated existence of a 29-kDa molecule on surface-associated plasma membrane of E. histolytica. The monoclonal antibody belonged to IgG1 isotype. The prior treatment of E. histolytica trophozoites with MoAb C8 resulted in significant (P less than 0.01) reduction in adherence of amoebic trophozoites to cultured Chinese Hamster Ovary cells and significant (P less than 0.01) reduction in cytotoxicity to cultured Baby Hamster Kidney cells. Pretreatment of amoebic trophozoites with MoAb C8 prior to cultivation in TPS-1 medium resulted in significant (P less than 0.01) reduction in growth of the parasite. Thus, the data suggested that the surface-exposed 29-kDa molecule may be one of the receptors involved in E. histolytica host cell interactions and may possibly modulate amoebic disease processes.     

Inhibition of gene expression with double strand RNA interference in Entamoeba histolytica

In order to inhibit gene expression in Entamoeba histolytica, we have developed a method based on expressing double strand RNA interference constructs in stable transformants. The 5' end of Eh Dia was cloned head to head with an intervening non-specific stuffer fragment in the E. histolytica expression vector pJST4. This construct was transformed in E. histolytica HM1:IMSS trophozoites and stable transformants were selected with 20microg/ml G418. Our results show that expression of Eh Dia was completely inhibited in these transformants. These stable transformants could be maintained indefinitely without expression of Eh Dia. This method therefore provides an effective tool to study the phenotypic changes, which occur due to inhibition of gene expression in the absence of mutants and other microbiological manipulations in this protozoan parasite.     

Mg2+, Mn2+, and Co2+ stimulate Entamoeba histolytica to produce chitin-like material

The mechanism of Entamoeba histolytica cyst cell wall synthesis is not well understood. Previous research has shown that cyst-like structures formed in the presence of chitin synthase cofactors (Mg2+, Mn2+, and Co2+) resist 1% sodium dodecyl sulfate lysis (RCLS), whereas those formed in the absence of cofactors (CLS) do not, and trophozoites are immediately destroyed. This suggests that E. histolytica is able to synthesize chitin, initiating a differentiation process under axenic conditions. To test this hypothesis, polysaccharide hydrolysates from E. histolytica trophozoites, CLS, or RCLS were analyzed with high-performance liquid chromatography. The major components found in all 3 preparations were N-acetylglucosamine (NAG) and glucose (GLC), with RCLS possessing 129 and 180 times more NAG and 2.4 and 2.0 more GLC than trophozoites and CLS, respectively. After 36 hr of incubation with chitinase (16 U/ml) in a hypotonic medium (50 mOsm/kg), 68% of RCLS was lysed, and 100% lost affinity for calcofluor white M2R. The RCLS polysaccharides bound wheat germ agglutinin and appeared as long and thin or short and thick fibers. Accordingly, Mg2+, Mn2+, and Co2+ stimulated E. histolytica to synthesize a chitin-like material.     

Comparative proteomic analysis of two Entamoeba histolytica strains with different virulence phenotypes identifies peroxiredoxin as an important component of amoebic virulence

Entamoeba histolytica is a protozoan intestinal parasite that causes amoebic colitis and amoebic liver abscess. To identify virulence factors of E. histolytica, we first defined the phenotypes of two E. histolytica strains, HM-1:IMSS, the prototype virulent strain, and E. histolytica Rahman, a strain that was reportedly less virulent than HM-1:IMSS. We found that compared with HM-1:IMSS, Rahman has a defect in erythrophagocytosis and the ability to cause amoebic colitis in human colonic xenografts. We used differential in-gel 2D electrophoresis to compare the proteome of Rahman and HM-1:IMSS, and identified six proteins that were differentially expressed above a fivefold level between the two organisms. These included two proteins with antioxidative properties (peroxiredoxin and superoxide dismutase), and three proteins of unknown function, grainin 1, grainin 2 and a protein containing a LIM-domain. Overexpression of peroxiredoxin in Rahman rendered the transgenic trophozoites more resistant to killing by H2O2 in vitro, and infection with Rahman trophozoites expressing higher levels of peroxiredoxin was associated with higher levels of intestinal inflammation in human colonic xenografts, and more severe disease based on histology. In contrast, higher levels of grainin appear to be associated with a reduced virulence phenotype, and E. histolytica HM-1:IMSS trophozoites infecting human intestinal xenografts show marked decreases in grainin expression. Our data indicate that there are definable molecular differences between Rahman and HM-1:IMSS that may explain the phenotypic differences, and identify peroxiredoxin as an important component of virulence in amoebic colitis.