Pore-forming peptides of Entamoeba dispar. Similarity and divergence to amoebapores in structure, expression and activity.
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Amoebapore, a 77-residue peptide with pore-forming activity from the human pathogen Entamoeba histolytica, is implicated in the killing of phagocytosed bacteria and in the cytolytic reaction of the amoeba against host cells. Previously, we structurally and functionally characterized three amoebapore isoforms in E. histolytica but recognized only one homolog in the closely related but non-pathogenic species Entamoeba dispar. Here, we identified two novel amoebapore homologs from E. dispar by molecular cloning. Despite strong resemblance of the primary structures of the homologs, molecular modeling predicts a species-specific variance between the peptide structures. Parallel isolation from trophozoite extracts of the two species revealed a lower amount of pore-forming peptides in E. dispar and substantially higher activity of the major isoform from E. histolytica towards natural membranes than that from E. dispar. Differences in abundance and activity of the lytic polypeptides may have an impact on the pathogenicity of amoebae. (+info)
Entamoeba histolytica and Entamoeba dispar: epidemiology and comparison of diagnostic methods in a setting of nonendemicity.
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Recent studies suggest that stool antigen assays are more sensitive and specific than microscopy for the diagnosis of Entamoeba histolytica infection. One hundred twelve patients presenting at 3 centers with symptoms or risk factors of E. histolytica infection were prospectively enrolled in this study to evaluate new diagnostic tests for infections with E. histolytica and Entamoeba dispar. Four ELISA-based stool antigen kits for detecting E. histolytica or E. dispar were blindly compared with stool microscopy. Amebic serology was assessed by indirect hemagglutination. When antigen assays were used as the reference standard, microscopy performed at referral centers was more specific (68.4% vs. 9.5%) but less sensitive (70.4% vs. 92.1%) than microscopy performed in community laboratories. Diagnosis with the E. histolytica test and Merlin Optimun S ELISA indicated that only 3 (4.2%) of 72 coproantigen-positive stools were positive for E. histolytica. Indirect hemagglutination was a good predictor of E. histolytica infection when titers of antibody to ameba were >/=1:512. (+info)
A DNA sequence corresponding to the gene encoding cysteine proteinase 5 in Entamoeba histolytica is present and positionally conserved but highly degenerated in Entamoeba dispar.
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Cysteine proteinases of Entamoeba histolytica are considered to be one of the most important classes of molecules responsible for the parasite's ability to destroy human tissues. Interestingly, one particular cysteine proteinase, located on the surface of E. histolytica trophozoites and designated cysteine proteinase 5 (CP5), is not expressed in the closely related but nonpathogenic species Entamoeba dispar. By comparing the E. histolytica and E. dispar genomic loci containing the gene for CP5 (cp5), it was found that the position of cp5 within the genomic context is conserved between the two organisms, but that the gene is highly degenerated in E. dispar, as it contains numerous nucleotide exchanges, insertions, and deletions, resulting in multiple stop codons within the cp5 reading frame. An alignment of all available orthologous E. histolytica and E. dispar DNA sequences suggested that cp5 started to degenerate in E. dispar coincidently when the two organisms began to diverge from a common ancestor. (+info)
Purification and biochemical characterization of a novel cysteine protease of Entamoeba histolytica.
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Cysteine proteases are important virulence factors of Entamoeba histolytica, the causative agent of amoebiasis. A novel cysteine protease from parasite extracts was purified 15-fold by a procedure including concanavalin A-Sepharose, hydroxylapatite and DEAE-Sepharose chromatography. The purification resulted in the obtainment of an homogeneous protein with a molecular mass of 66 kDa on native PAGE. In 10% SDS/PAGE, three bands of 60, 54 and 50 kDa were evident. Each of the three specific mouse antisera raised against these proteins showed cross-reactivity with the three bands obtained from the purified eluate. The N-terminal sequencing of the first 10 amino acids from the three proteins showed 100% identity. These results support the hypothesis of a common precursor for the 60, 54 and 50-kDa proteins. Protease activity of the purified enzyme was demonstrated by electrophoresis in a gelatine-acrylamide copolymerized gel. Its activity was quantified by cleaving a synthetic fluorogenic peptide substrate such as N-carbobenzyloxy-arginyl-arginyl-7-amido-4-methylcoumarin. The optimum pH for the protease activity was 6.5; however, enzymatic activity was observed between pH 5 and pH 7.5. Typical of cysteine proteases, the enzyme was inhibited by 4-[(2S, 3S)-carboxyoxiran-2-ylcarbonyl-L-leucylamido]butylg uanidine and iodoacetamide, and activated by free sulfhydryl groups. The cellular location of the enzyme was examined on trophozoites before and after contact with red blood cells using indirect immunofluorescence and cellular fractionation. The 60-kDa cysteine protease translocated to the amoebic surface upon the interaction of trophozoites with red blood cells. This result provided evidence for participation of the 60-kDa protease in erythrophagocytosis. (+info)
Characterization of the gene encoding serine acetyltransferase, a regulated enzyme of cysteine biosynthesis from the protist parasites Entamoeba histolytica and Entamoeba dispar. Regulation and possible function of the cysteine biosynthetic pathway in Entamoeba.
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The enteric protist parasites Entamoeba histolytica and Entamoeba dispar possess a cysteine biosynthetic pathway, unlike their mammalian host, and are capable of de novo production of L-cysteine. We cloned and characterized cDNAs that encode the regulated enzyme serine acetyltransferase (SAT) in this pathway from these amoebae by genetic complementation of a cysteine-auxotrophic Escherichia coli strain with the amoebic cDNA libraries. The deduced amino acid sequences of the amoebic SATs exhibited, within the most conserved region, 36-52% identities with the bacterial and plant SATs. The amoebic SATs contain a unique insertion of eight amino acids, also found in the corresponding region of a plasmid-encoded SAT from Synechococcus sp., which showed the highest overall identities to the amoebic SATs. Phylogenetic reconstruction also revealed a close kinship of the amoebic SATs with cyanobacterial SATs. Biochemical characterization of the recombinant E. histolytica SAT revealed several enzymatic features that distinguished the amoebic enzyme from the bacterial and plant enzymes: 1) inhibition by L-cysteine in a competitive manner with L-serine; 2) inhibition by L-cystine; and 3) no association with cysteine synthase. Genetically engineered amoeba strains that overproduced cysteine synthase and SAT were created. The cysteine synthase-overproducing amoebae had a higher level of cysteine synthase activity and total thiol content and revealed increased resistance to hydrogen peroxide. These results indicate that the cysteine biosynthetic pathway plays an important role in antioxidative defense of these enteric parasites. (+info)
Thermodynamics of target peptide recognition by calmodulin and a calmodulin analogue: implications for the role of the central linker.
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The thermodynamics of interaction of two model peptides melittin and mastoparan with bovine brain calmodulin (CAM) and a smaller CAM analogue, a calcium binding protein from Entamoeba histolytica (CaBP) in 10 mM MOPS buffer (pH 7.0) was examined using isothermal titration calorimetry (ITC). These data show that CAM binds to both the peptides and the enthalpy of binding is endothermic for melittin and exothermic for mastoparan at 25 degrees C. CaBP binds to the longer peptide melittin, but does not bind to mastoparan, the binding enthalpy being endothermic in nature. Concurrently, we also observe a larger increase in alpha-helicity upon the binding of melittin to CAM when compared to CaBP. The role of hydrophobic interactions in the binding process has also been examined using 8-anilino-1-naphthalene-sulphonic acid (ANS) binding monitored by ITC. These results have been employed to rationalize the energetic consequences of the binding reaction. (+info)
Development of a DeltaglnA balanced lethal plasmid system for expression of heterologous antigens by attenuated vaccine vector strains of Vibrio cholerae.
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We have previously shown that more prominent immune responses are induced to antigens expressed from multicopy plasmids in live attenuated vaccine vector strains of Vibrio cholerae than to antigens expressed from single-copy genes on the V. cholerae chromosome. Here, we report the construction of a DeltaglnA derivative of V. cholerae vaccine strain Peru2. This mutant strain, Peru2DeltaglnA, is unable to grow on medium that does not contain glutamine; this growth deficiency is complemented by pKEK71-NotI, a plasmid containing a complete copy of the Salmonella typhimurium glnA gene, or by pTIC5, a derivative of pKEK71-NotI containing a 1. 8-kbp fragment that directs expression of CtxB with a 12-amino-acid epitope of the serine-rich Entamoeba histolytica protein fused to the amino terminus. Strain Peru2DeltaglnA(pTIC5) produced 10-fold more SREHP-12-CtxB in supernatants than did ETR3, a Peru2-derivative strain containing the same fragment inserted on the chromosome. To assess immune responses to antigens expressed by this balanced lethal system in vivo, we inoculated germfree mice on days 0, 14, 28, and 42 with Peru2DeltaglnA, Peru2DeltaglnA(pKEK71-NotI), Peru2(pTIC5), Peru2DeltaglnA(pTIC5), or ETR3. All V. cholerae strains were recoverable from stool for 8 to 12 days after primary inoculation, including Peru2DeltaglnA; strains containing plasmids continued to harbor pKEK71-NotI or pTIC5 for 8 to 10 days after primary inoculation. Animals were sacrificed on day 56, and serum, stool and biliary samples were analyzed for immune responses. Vibriocidal antibody responses, reflective of in vivo colonization, were equivalent in all groups of animals. However, specific anti-CtxB immune responses in serum (P +info)
Innate immunity to amebic liver abscess is dependent on gamma interferon and nitric oxide in a murine model of disease.
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Evidence from in vitro studies suggests that gamma interferon (IFN-gamma) and nitric oxide (NO) are important in host defense against the protozoan parasite Entamoeba histolytica. We used SCID mice with targeted disruption of the IFN-gamma receptor gene and mice with targeted disruption of the gene encoding inducible NO synthase to show that IFN-gamma plays a role in the innate immunity to amebic liver abscess seen in SCID mice while NO is required for control of amebic liver abscess in immunocompetent mice. (+info)