Recombinant expression and purification of an enzymatically active cysteine proteinase of the protozoan parasite Entamoeba histolytica.

Cysteine proteinases and in particular cysteine proteinase 5 (EhCP5) of Entamoeba histolytica are considered important for ameba pathogenicity. To study EhCP5 in more detail a protocol was elaborated to produce considerable amounts of the enzyme in its active form. The protein was expressed in Escherichia coli as a histidine-tagged pro-enzyme and purified to homogeneity under denaturing conditions in the presence of guanidine-HCl using nickel affinity chromatography. Renaturation was performed by 100-fold dilution in a buffer containing reduced and oxidized thiols, which led to soluble but enzymatically inactive pro-enzyme. Further processing and activation was achieved in the presence of 10 mM DTT and 0.04% SDS at 37 degrees C. Recombinant enzyme (rEhCP5) was indistinguishable from native EhCP5 purified from E. histolytica lysates. Both runs in SDS-PAGE under reducing and nonreducing conditions at positions corresponding to 27 and 29 kDa, respectively, had the same pH optima and displayed similar specific activity against azocasein. Moreover, both enzymes were active against a broad spectrum of biological and synthetic substrates such as mucin, fibrinogen, collagen, human hemoglobin, bovine serum albumin, gelatin, human IgG, Z-Arg-Arg-pNA, and Z-Ala-Arg-Arg-pNA, but not against Z-Phe-Arg-pNA. The identity of rEhCP5 as a cysteine proteinase was confirmed by inhibition with specific cysteine proteinase inhibitors. In contrast, various compounds known to specifically inhibit aspartic, metallo, or serine proteinases had no effect on rEhCP5 activity.     

A novel protease from Entamoeba histolytica homologous to members of the family S28 of serine proteases

Serine proteases are one of the biologically most important and widely distributed enzyme families. A protease capable of degrading the substrate Suc-AAF-AMC was isolated from axenically grown trophozoites of Entamoeba histolytica. The enzyme was purified by ion-exchange chromatography and electroelution, and appeared on 2D-PAGE as a spot of 60 kDa and pI of 4.65. Data obtained from zymogram suggest the active protease is present either as homodimer (130 kDa) or homotetramer (250 kDa). The optimal temperature of the enzyme was 37 degrees C, and it exhibited activity over a broad pH range. The protease was strongly inhibited by TPCK and chelating agents. The enzymatic activity was restored upon addition of calcium. BLAST analysis with the sequence of internal peptides of the protein revealed two open reading frames within the genome of E. histolytica, homologous to members of the family S28, clan SC of serine proteases.     

Characterization of Chloramphenicol Acetyltransferase from Chloramphenicol-resistant Staphylococcus aureus

Chloramphenicol-resistant strains of Staphylococcus aureus contain an inducible enzyme which inactivates chloramphenicol by acetylation in the presence of acetyl coenzyme A. The products of acetylation are chromatographically indistinguishable from those obtained with chloramphenicol-resistant Escherichia coli harboring an R factor. The kinetics of induction of chloramphenicol acetyltransferase are complicated by the inducer's effect on protein biosynthesis and its fate as chloramphenicol 3-acetate, which is not an inducer of the enzyme. The E. coli and S. aureus enzymes have been compared, with the conclusion that they are identical with respect to molecular weight (approximately 78,000) and pH optimum (7.8), but differ with respect to heat stability, substrate affinity, electrophoretic mobility, and immunological reactivity. Antiserum prepared against enzyme from E. coli contains precipitating antibody, which inactivates the E. coli enzyme, but neither precipitates nor neutralizes the activity of S. aureus enzyme.     

Entamoeba histolytica: molecular characterization of an aldose 1-epimerase (mutarotase)

In cells, the alpha-anomers of aldoses are the preferred metabolizable substrates, while beta-anomers of aldoses play their role in glycan structure. In the cytoplasm, alpha- and beta-anomers of aldoses interconvert through the enzyme termed aldose 1-epimerase or mutarotase (EC 5.1.3.3). We have identified a mutarotase gene in Entamoeba histolytica, the causative agent of non-bacterial dysentery in humans. Cloning and characterization of this gene in two strains of the parasite (HM-1:IMSS and Rahman) that differ in their pathogenicity, revealed that the sequence is identical in both strains. A recombinant E. histolytica mutarotase was produced as well as specific antibodies that recognized a 38 kDa protein in trophozoite lysates of both strains. Mutarotase activity was observed with the recombinant protein as well as in lysates of both HM-1:IMSS and Rahman, the former exhibiting a slightly higher mutarotase activity. Finally, we have shown by complementation that overexpression of the E. histolytica mutarotase in a mutarotase defective Escherichia coli strain restores the ability of these bacteria to grow in minimal medium with phenyl-beta-galactopyranoside as the sole carbon source.     

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.

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.     

Cloning, overexpression, purification, and characterization of O-acetylserine sulfhydrylase-B from Escherichia coli

O-Acetylserine sulfhydrylase-B (OASS-B, EC 2.5.1.47) is one of the two isozymes produced by Escherichia coli that catalyze the synthesis of L-cysteine from O-acetyl-L-serine and sulfide. The cysM gene encoding OASS-B was cloned and the enzyme was overexpressed in E. coli using pUC19 with a lacUV5 promoter. The enzyme was purified to homogeneity, as evidenced by SDS-PAGE. Approximately 300 mg of purified OASS-B was obtained from 1600 mL of culture broth with a purification yield of 60% or higher. The purified OASS-B was characterized and its properties compared with OASS-A. OASS-B did not form a complex with E. coli serine acetyltransferase (SAT, EC 2.3.1.30) and showed a wide range of substrate specificity in nonproteinaceous amino acid synthesis.     

Interaction of serine acetyltransferase with O-acetylserine sulfhydrylase active site: evidence from fluorescence spectroscopy

Serine acetyltransferase is a key enzyme in the sulfur assimilation pathway of bacteria and plants, and is known to form a bienzyme complex with O-acetylserine sulfhydrylase, the last enzyme in the cysteine biosynthetic pathway. The biological function of the complex and the mechanism of reciprocal regulation of the constituent enzymes are still poorly understood. In this work the effect of complex formation on the O-acetylserine sulfhydrylase active site has been investigated exploiting the fluorescence properties of pyridoxal 5'-phosphate, which are sensitive to the cofactor microenvironment and to conformational changes within the protein matrix. The results indicate that both serine acetyltransferase and its C-terminal decapeptide bind to the alpha-carboxyl subsite of O-acetylserine sulfhydrylase, triggering a transition from an open to a closed conformation. This finding suggests that serine acetyltransferase can inhibit O-acetylserine sulfhydrylase catalytic activity with a double mechanism, the competition with O-acetylserine for binding to the enzyme active site and the stabilization of a closed conformation that is less accessible to the natural substrate.     

Molecular and biochemical characterization of D-phosphoglycerate dehydrogenase from Entamoeba histolytica. A unique enteric protozoan parasite that possesses both phosphorylated and nonphosphorylated serine metabolic pathways.

A putative phosphoglycerate dehydrogenase (PGDH), which catalyzes the oxidation of d-phosphoglycerate to 3-phosphohydroxypyruvate in the so-called phosphorylated serine metabolic pathway, from the enteric protozoan parasite Entamoeba histolytica was characterized. The E. histolytica PGDH gene (EhPGDH) encodes a protein of 299 amino acids with a calculated molecular mass of 33.5 kDa and an isoelectric point of 8.11. EhPGDH showed high homology to PGDH from bacteroides and another enteric protozoan ciliate, Entodinium caudatum. EhPGDH lacks both the carboxyl-terminal serine binding domain and the 13-14 amino acid regions containing the conserved Trp139 (of Escherichia coli PGDH) in the nucleotide binding domain shown to be crucial for tetramerization, which are present in other organisms including higher eukaryotes. EhPGDH catalyzed reduction of phosphohydroxypyruvate to phosphoglycerate utilizing NADH and, less efficiently, NADPH; EhPGDH did not utilize 2-oxoglutarate. Kinetic parameters of EhPGDH were similar to those of mammalian PGDH, for example the preference of NADH cofactor, substrate specificities and salt-reversible substrate inhibition. In contrast to PGDH from bacteria, plants and mammals, the EhPGDH protein is present as a homodimer as demonstrated by gel filtration chromatography. The E. histolytica lysate contained PGDH activity of 26 nmol NADH utilized per min per mg of lysate protein in the reverse direction, which consisted 0.2-0.4% of a total soluble protein. Altogether, this parasite represents a unique unicellular protist that possesses both phosphorylated and nonphosphorylated serine metabolic pathways, reinforcing the biological importance of serine metabolism in this organism. Amino acid sequence comparison and phylogenetic analysis of various PGDH sequences showed that E. histolytica forms a highly supported monophyletic group with another enteric protozoa, cilliate E. caudatum, and bacteroides.     

Expression of a bacterial serine acetyltransferase in transgenic potato plants leads to increased levels of cysteine and glutathione

The coding sequence of the wild-type, cys-sensitive, cysE gene from Escherichia coli, which encodes an enzyme of the cysteine biosynthetic pathway, namely serine acetyltransferase (SAT, EC 2.3.1. 30), was introduced into the genome of potato plants under the control of the cauliflower mosaic virus 35S promoter. In order to target the protein into the chloroplast, cysE was translationally fused to the 5'-signal sequence of rbcS from Arabidopsis thaliana. Transgenic plants showed a high accumulation of the cysE mRNA. The chloroplastic localisation of the E. coli SAT protein was demonstrated by determination of enzymatic activities in enriched organelle fractions. Crude leaf extracts of these plants exhibited up to 20-fold higher SAT activity than those prepared from wild-type plants. The transgenic potato plants expressing the E. coli gene showed not only increased levels of enzyme activity but also exhibited elevated levels of cysteine and glutathione in leaves. Both were up to twofold higher than in control plants. However, the thiol content in tubers of transgenic lines was unaffected. The alterations observed in leaf tissue had no effect on the expression of O-acetylserine(thiol)-lyase, the enzyme which converts O-acetylserine, the product of SAT, to cysteine. Only a minor effect on its enzymatic activity was observed. In conclusion, the results presented here demonstrate the importance of SAT in plant cysteine biosynthesis and show that production of cysteine and related sulfur-containing compounds can be enhanced by metabolic engineering.     

Role of CysE in production of an extracellular signaling molecule in Providencia stuartii and Escherichia coli: loss of CysE enhances biofilm formation in Escherichia coli

A mini-Tn5Cm insertion has been identified that significantly reduced the amount of an extracellular activating signal for a lacZ fusion (cma37::lacZ) in Providencia stuartii. The transposon insertion was located immediately upstream of an open reading frame encoding a putative CysE ortholog. The CysE enzyme, serine acetyltransferase, catalyzes the conversion of serine to O-acetyl-L-serine (OAS). This activating signal was also produced by Escherichia coli, and production was abolished in a strain containing a null allele of cysE. Products of the CysE enzyme (OAS, N-acetyl-L-serine [NAS], O-acetyl-L-threonine, and N-acetyl-L-threonine) were individually tested for the ability to activate cma37::lacZ. Only OAS was capable of activating the cma37::lacZ fusion. The ability of OAS to activate the cma37::lacZ fusion was abolished by pretreatment at pH 8.5, which converts OAS to NAS. However, the activity of the native signal in conditioned medium was not decreased by treatment at pH 8.5. In contrast, conditioned medium prepared from cells grown at pH 8.5 exhibited a 4- to 10-fold-higher activity, relative to pH 6.0. Additional genes regulated by the CysE-dependent signal and OAS were identified in P. stuartii and E. coli. The response to the extracellular signal in E. coli was dependent on CysB, a positive activator that requires NAS as a coactivator. In E. coli, a cysE mutant formed biofilms at an accelerated rate compared to the wild type, suggesting a physiological role for this extracellular signal.     

Molecular and biochemical characterisation of a serine acetyltransferase of onion, Allium cepa (L.)

We have previously cloned a cDNA, designated SAT1, corresponding to a gene coding for a serine acetyltransferase (SAT) from onion (Allium cepa L.). The SAT1 locus was mapped to chromosome 7 of onion using a single-stranded conformation polymorphism (SSCP) in the 3' UTR of the gene. Northern analysis has demonstrated that expression of the SAT1 gene is induced in leaf tissue in response to low S-supply. Phylogenetic analysis has placed SAT1 in a strongly supported group (100% bootstrap) that comprises sequences that have been characterised biochemically, including Allium tuberosum, Spinacea oleracea, Glycine max, Citrullus vulgaris, and SAT5 (AT5g56760) of Arabidopsis thaliana. This group can be divided further with the SAT1 of A. cepa sequence grouping strongly with the A. tuberosum sequence. Translation of SAT1 from onion generates a protein of 289 amino acids with a calculated molecular mass of 30,573 Da and pI of 6.52. The conserved G277 and H282 residues that have been identified as critical for L-cysteine inhibition are observed at G272 and H277. SAT1 has been cloned into the pGEX plasmid, expressed in E. coli and SAT activity of the recombinant enzyme has been measured as acetyl-CoA hydrolysis detected at 232 nm. A Km of 0.72 mM was determined for l-serine as substrate, a Km of 92 microM was calculated with acetyl-CoA as substrate, and an inhibition curve for L-cysteine generated an IC50 value of 3.1 microM. Antibodies raised against the recombinant SAT1 protein recognised a protein of ca. 33 kDa in whole leaf onion extracts. These properties of the SAT1 enzyme from onion are compared with other SAT enzymes characterised from closely related species.     

Benzotriazoles and indazoles are scaffolds with biological activity against Entamoeba histolytica

Parasitic infections caused by Entamoeba histolytica are still major threats against public health, especially in developing countries. Although current therapies exist, the problems associated with parasite resistance and negative side effects make it imperative to search for new therapeutic agents. A systematic scaffold analysis reported herein of a public database containing 474 antiamoebic compounds reveals that benzimidazole is the most active scaffold reported thus far. To gain insights into the antiamoebic activity of novel compounds, the authors report herein the biological activity of 12 compounds, including benzotriazole and indazole derivatives, scaffolds not previously tested against E. histolytica. Compounds with the benzotriazole and indazole scaffolds showed low micromolar activity (IC(50) = 0.304 and 0.339 μM) and are more active than metronidazole, which is the drug of choice used for the treatment of amebiosis. The novel compounds have similar properties to approved drugs. Compounds with novel scaffolds represent promising starting points of an optimization program against E. histolytica.     

Entamoeba histolytica: construction and applications of subgenomic databases

Knowledge about the influence of environmental stress such as the action of chemotherapeutic agents on gene expression in Entamoeba histolytica is limited. We plan to use oligonucleotide microarray hybridization to approach these questions. As the basis for our array, sequence data from the genome project carried out by the Institute for Genomic Research (TIGR) and the Sanger Institute were used to annotate parts of the parasite genome. Three subgenomic databases containing enzymes, cytoskeleton genes, and stress genes were compiled with the help of the ExPASy proteomics website and the BLAST servers at the two genome project sites. The known sequences from reference species, mostly human and Escherichia coli, were searched against TIGR and Sanger E. histolytica sequence contigs and the homologs were copied into a Microsoft Access database. In a similar way, two additional databases of cytoskeletal genes and stress genes were generated. Metabolic pathways could be assembled from our enzyme database, but sometimes they were incomplete as is the case for the sterol biosynthesis pathway. The raw databases contained a significant number of duplicate entries which were merged to obtain curated non-redundant databases. This procedure revealed that some E. histolytica genes may have several putative functions. Representative examples such as the case of the delta-aminolevulinate synthase/serine palmitoyltransferase are discussed.     

Effect of cysteine desulfhydrase gene disruption on L-cysteine overproduction in Escherichia coli

In Escherichia coli, the enzyme called cysteine desulfhydrase (CD), which is responsible for L-cysteine degradation, was investigated by native-PAGE and CD activity staining of crude cell extracts. Analyses with gene-disrupted mutants showed that CD activity resulted from two enzymes: tryptophanase (TNase) encoded by tnaA and cystathionine beta-lyase (CBL) encoded by metC. It was also found that TNase synthesis was induced by the presence of L-cysteine. The tnaA and metC mutants transformed with the plasmid containing the gene for feedback-insensitive serine acetyltransferase exhibited higher L-cysteine productivity than the wild-type strain carrying the same plasmid. These results indicated that TNase and CBL did act on L-cysteine degradation in E. coli cells.     

Antimicrobial properties of allicin from garlic

Allicin, one of the active principles of freshly crushed garlic homogenates, has a variety of antimicrobial activities. Allicin in its pure form was found to exhibit i) antibacterial activity against a wide range of Gram-negative and Gram-positive bacteria, including multidrug-resistant enterotoxicogenic strains of Escherichia coli; ii) antifungal activity, particularly against Candida albicans; iii) antiparasitic activity, including some major human intestinal protozoan parasites such as Entamoeba histolytica and Giardia lamblia; and iv) antiviral activity. The main antimicrobial effect of allicin is due to its chemical reaction with thiol groups of various enzymes, e.g. alcohol dehydrogenase, thioredoxin reductase, and RNA polymerase, which can affect essential metabolism of cysteine proteinase activity involved in the virulence of E. histolytica.     

The bifunctional Entamoeba histolytica alcohol dehydrogenase 2 (EhADH2) protein is necessary for amebic growth and survival and requires an intact C-terminal domain for both alcohol dahydrogenase and acetaldehyde dehydrogenase activity

The intestinal protozoan pathogen Entamoeba histolytica lacks mitochondria and derives energy from the fermentation of glucose to ethanol with pyruvate, acetyl enzyme Co-A, and acetaldehyde as intermediates. A key enzyme in this pathway may be the 97-kDa bifunctional E. histolytica alcohol dehydrogenase 2 (EhADH2), which possesses both alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase activity (ALDH). EhADH2 appears to be a fusion protein, with separate N-terminal ALDH and C-terminal ADH domains. Here, we demonstrate that EhADH2 expression is required for E. histolytica growth and survival. We find that a mutant EhADH2 enzyme containing the C-terminal 453 amino acids of EhADH2 has ADH activity but lacks ALDH activity. However, a mutant consisting of the N-terminal half of EhADH2 possessed no ADH or ALDH activity. Alteration of a single histidine to arginine in the putative active site of the ADH domain eliminates both ADH and ALDH activity, and this mutant EhADH2 can serve as a dominant negative, eliminating both ADH and ALDH activity when co-expressed with wild-type EhADH2 in Escherichia coli. These data indicate that EhADH2 enzyme is required for E. histolytica growth and survival and that the C-terminal ADH domain of the enzyme functions as a separate entity. However, ALDH activity requires residues in both the N- and C-terminal halves of the molecule.     

Molecular cloning and characterization of a protein farnesyltransferase from the enteric protozoan parasite Entamoeba histolytica

Genes encoding alpha- and beta-subunits of a putative protein farnesyltransferase (FT) from the enteric protozoan parasite Entamoeba histolytica were obtained and their biochemical properties were characterized. Deduced amino acid sequences of the alpha- and beta-subunit of E. histolytica FT (EhFT) were 298- and 375-residues long with a molecular mass of 35.6 and 42.6 kDa, and a pI of 5.43 and 5.65, respectively. They showed 24% to 36% identity to and shared common signature domains and repeats with those from other organisms. Recombinant alpha- and beta-subunits, co-expressed in Escherichia coli, formed a heterodimer and showed activity to transfer farnesyl using farnesylpyrophosphate as a donor to human H-Ras possessing a C-terminal CVLS, but not a mutant H-Ras possessing CVLL. Among a number of small GTPases that belong to the Ras superfamily from this parasite, we identified EhRas4, which possesses CVVA at the C terminus, as a sole farnesyl acceptor for EhFT. This is in contrast to mammalian FT, which utilizes a variety of small GTPases that possess a C-terminal CaaX motif, where X is serine, methionine, glutamine, cysteine, or alanine. EhFT also showed remarkable resistance against a variety of known inhibitors of mammalian FT. These results suggest that remarkable biochemical differences in binding to substrates and inhibitors exist between amebic and mammalian FTs, which highlights this enzyme as a novel target for the development of new chemotherapeutics against amebiasis.     

In vitro validation of acetyltransferase activity of GlmU as an antibacterial target in Haemophilus influenzae

GlmU is a bifunctional enzyme that is essential for bacterial growth, converting D-glucosamine 1-phosphate into UDP-GlcNAc via acetylation and subsequent uridyl transfer. A biochemical screen of AstraZeneca's compound library using GlmU of Escherichia coli identified novel sulfonamide inhibitors of the acetyltransferase reaction. Steady-state kinetics, ligand-observe NMR, isothermal titration calorimetry, and x-ray crystallography showed that the inhibitors were competitive with acetyl-CoA substrate. Iterative chemistry efforts improved biochemical potency against gram-negative isozymes 300-fold and afforded antimicrobial activity against a strain of Haemophilus influenzae lacking its major efflux pump. Inhibition of precursor incorporation into bacterial macromolecules was consistent with the antimicrobial activity being caused by disruption of peptidoglycan and fatty acid biosyntheses. Isolation and characterization of two different resistant mutant strains identified the GlmU acetyltransferase domain as the molecular target. These data, along with x-ray co-crystal structures, confirmed the binding mode of the inhibitors and explained their relative lack of potency against gram-positive GlmU isozymes. This is the first example of antimicrobial compounds mediating their growth inhibitory effects specifically via GlmU.     

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.