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 Dehydrogenase and Acetaldehyde Dehydrogenase Activity
2001; Elsevier BV; Volume: 276; Issue: 23 Linguagem: Inglês
10.1074/jbc.m101349200
ISSN1083-351X
AutoresAvelina Espinosa, Yan Le, Zhi Zhang, Lynne Foster, David P. Clark, Ellen Li, Samuel L. Stanley,
Tópico(s)Parasitic Infections and Diagnostics
ResumoThe 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 forE. 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 inEscherichia 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. 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 forE. 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 inEscherichia 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. alcohol dehydrogenase acetaldehyde dehydrogenase Entamoeba histolytica alcohol dehydrogenase 2 polyacrylamide gel electrophoresis polymerase chain reaction The anaerobic intestinal protozoan parasite Entamoeba histolytica converts pyruvate to ethanol in its fermentation pathway (1Lo H.-S. Reeves R.E. Biochem. J. 1978; 171: 225-230Crossref PubMed Scopus (40) Google Scholar). The last two steps of this pathway are the conversion of acetyl-CoA to acetaldehyde followed by the reduction of acetaldehyde to ethanol (1Lo H.-S. Reeves R.E. Biochem. J. 1978; 171: 225-230Crossref PubMed Scopus (40) Google Scholar). E. histolytica possesses at least three enzymes with alcohol dehydrogenase (ADH)1 activity: a NADP-dependent ADH (EhADH1); a 97-kDa NAD(+)-dependent and Fe2+-dependent bifunctional enzyme with both ADH and acetaldehyde dehydrogenase (ALDH) activities (EhADH2, also known as EhADHE); and a 43-kDa NADP-dependent ADH with some sequence homology to class III microbial alcohol dehydrogenases (EhADH3) (2Yang W. Li E. Kairong T. Stanley Jr., S.L. Mol. Biochem. Parasitol. 1994; 64: 253-260Crossref PubMed Scopus (79) Google Scholar, 3Kumar A. Shen P.-S. Descoteaux S. Pohl J. Bailey G. Samuelson J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10188-10192Crossref PubMed Scopus (55) Google Scholar, 4Rodriguez M.A. Baez-Camargo M. Delgadillo D.M. Orozco E. Biochim. Biophys. Acta. 1996; 1306: 23-26Crossref PubMed Scopus (19) Google Scholar, 5Bruchhaus I. Tannich E. Biochem. J. 1994; 303: 743-748Crossref PubMed Scopus (64) Google Scholar). There are at least two enzymes with ALDH activity, the EhADH2 enzyme and a NADP-dependent ALDH, EhALDH1 (2Yang W. Li E. Kairong T. Stanley Jr., S.L. Mol. Biochem. Parasitol. 1994; 64: 253-260Crossref PubMed Scopus (79) Google Scholar, 5Bruchhaus I. Tannich E. Biochem. J. 1994; 303: 743-748Crossref PubMed Scopus (64) Google Scholar, 6Zhang W.-W. Shen P.-S. Descoteaux S. Samuelson J. Mol. Biochem. Parasitol. 1994; 63: 157-161Crossref PubMed Scopus (10) Google Scholar). Given the presence of multiple ADH and ALDH enzymes in E. histolytica, an important question is whether any of these enzymes are essential forE. histolytica growth and survival and thus potential targets for anti-amebic therapy. The EhADH2 enzyme is part of a newly described family of multifunctional enzymes found in Gram-negative and Gram-positive bacteria and the intestinal protozoan parasite Giardia lamblia (2Yang W. Li E. Kairong T. Stanley Jr., S.L. Mol. Biochem. Parasitol. 1994; 64: 253-260Crossref PubMed Scopus (79) Google Scholar, 7Rosenthal B. Mai Z.M. Caplivski D. Ghosh S. De la Vega H. Graf T. Samuelson J. J. Bacteriol. 1997; 179: 3736-3745Crossref PubMed Scopus (94) Google Scholar, 8Goodlove P.E. Cunningham P.R. Parker J. Clark D.P. Gene. 1989; 85: 209-214Crossref PubMed Scopus (90) Google Scholar, 9Sanchez L.B. Arch Biochem. Biophys. 1998; 354: 57-64Crossref PubMed Scopus (55) Google Scholar, 10Fischer R.J. Helms J. Durre P. J. Bacteriol. 1993; 175: 6959-6969Crossref PubMed Google Scholar, 11Nair R.V. Bennett G.N. Papoutsakis E.T. J. Bacteriol. 1994; 176: 871-885Crossref PubMed Google Scholar, 12Arnau J. Jorgensen F. Madsen S.M. Vrang A. Israelsen H. J. Bacteriol. 1998; 180: 3049-3055Crossref PubMed Google Scholar). EhADH2 and other members of the family appear to be composed of separate C-terminal ADH and N-terminal ALDH domains linked together to create a fusion enzyme (8Goodlove P.E. Cunningham P.R. Parker J. Clark D.P. Gene. 1989; 85: 209-214Crossref PubMed Scopus (90) Google Scholar). The EhADH2 enzyme utilizes NAD and Fe2+ as co-factors and does not demonstrate homology with the zinc-dependent ADH enzymes (13Danielsson O. Atrian S. Luque T. Hjelmqvist L. Gonzàlez-Duarte R. Jörnvall H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4980-4984Crossref PubMed Scopus (124) Google Scholar). Regions of EhADH2 that could be involved in iron binding and NAD binding have been identified, but a requirement for specific residues in enzymatic activity has not been demonstrated (2Yang W. Li E. Kairong T. Stanley Jr., S.L. Mol. Biochem. Parasitol. 1994; 64: 253-260Crossref PubMed Scopus (79) Google Scholar). The prototype member of the family is the Escherichia coli ADHE enzyme, which is required for the anaerobic growth of E. coli (8Goodlove P.E. Cunningham P.R. Parker J. Clark D.P. Gene. 1989; 85: 209-214Crossref PubMed Scopus (90) Google Scholar). Both the ADHE enzyme and the native EhADH2 enzyme form protomers that assemble into large helical structures called spirosomes, which are visible by electron microscopy (5Bruchhaus I. Tannich E. Biochem. J. 1994; 303: 743-748Crossref PubMed Scopus (64) Google Scholar, 14Kessler D. Leibrecht I. Knappe J. FEBS Lett. 1991; 281: 59-63Crossref PubMed Scopus (120) Google Scholar, 15Matayoshi S. Oda H. Sarwar G. J. Gen. Microbiol. 1989; 135: 525-529PubMed Google Scholar). Episomal expression of the amebic EhADH2 gene inE. coli can complement an ADHE mutation, providing a system for rapid evaluation of the activity of EhADH2 mutants and for testing inhibitors of EhADH2 (16Yong T.-S. Li E. Clark D. Stanley Jr., S.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6464-6469Crossref PubMed Scopus (16) Google Scholar). Here, we describe studies designed to better understand the structure and function of the EhADH2 enzyme. We have used the constitutive and inducible episomal expression of antisense RNA to EhADH2 in amebae specifically to inhibit EhADH2 expression and assess whether EhADH2 is required for amebic growth and survival. We have analyzed whether EhADH2 is functionally a fusion enzyme with autonomous ADH and ALDH domains by expressing truncated proteins corresponding to the putative ADH and ALDH regions of EhADH2. Finally, we have used site-directed mutagenesis and deletion analyses to identify residues of EhADH2 that are required for both ADH and ALDH activity and a region of the enzyme that may play a role in EhADH2 dimerization and polymerization. Strains DH5α, BL21 (DE3), and SHH31 (ΔadhE fadR met tyrT) were used for expression of recombinant proteins (16Yong T.-S. Li E. Clark D. Stanley Jr., S.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6464-6469Crossref PubMed Scopus (16) Google Scholar). Cultures were grown in LB broth medium with agitation at 37 °C, and 1.5% Bacto agar (Difco, Sparks, MD) was added for solid media. For anaerobic growth, the transformed strains were grown on solid (1.5% Bacto agar, Difco) or liquid M9 media in anaerobic jars (BBL GasPak™ system, Becton Dickinson, Sparks, MD) with anaerobic system envelopes (BBL GasPak Plus) (16Yong T.-S. Li E. Clark D. Stanley Jr., S.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6464-6469Crossref PubMed Scopus (16) Google Scholar). Indicator strips were used to confirm anaerobic conditions. To construct the antisense insert for EhADH2, a PCR product was generated from nucleotides −50 to +1312 of the EhADH2 gene in the antisense orientation using oligonucleotide primers 5′-AATTGCGGCCGCCTAACCCGTTTTTGGATCAG (forward primer) and 5′-ATAGATCTCCTCCATATGATCCACATCCAAG incorporatingNotI and BglII restriction sites, respectively. This fragment was then inserted into plasmid pSA8 (17Ankri S. Stolarsky T. Mirelman D. Mol. Microbiol. 1998; 28: 777-785Crossref PubMed Scopus (104) Google Scholar) replacing the antisense ehcp5 insert to create pZZ1. 1 × 107 E. histolytica HM1:1MSS trophozoites in log phase growth were transfected with 100 ng of plasmid pZZ1, pSA8 (antisense construct against E. histolytica cysteine proteinase 5), or the parent pNeo-Act plasmid without an insert (17Ankri S. Stolarsky T. Mirelman D. Mol. Microbiol. 1998; 28: 777-785Crossref PubMed Scopus (104) Google Scholar). Selection was performed by adding 10 μg/ml G418 to TYI-S33 media beginning 48 h after transfection and gradually increasing G418 concentration to 50 μg/ml over 2 weeks' time. The EhADH2 antisense RNA under an inducible expression system was generated by PCR from −50 to +1312 of the EhADH2 sequence as template and synthetic oligonucleotide primers 5′-GAGGATCCTAACCCGTTTTTGGATCAG (forward primer) and 5′-GAGGTACCTCCATATGATCCACATCCAAG incorporating BamHI and KpnI restriction sites, respectively. The PCR product was ligated into pEhHYG-tetR-O-CAT (graciously provided by Egbert Tannich, Bernhard Nocht Institute, Hamburg, Germany) (18Hamann L. Buss H. Tannich E. Mol. Biochem. Parasitol. 1997; 84: 83-91Crossref PubMed Scopus (70) Google Scholar) using theKpnI and BamHI restriction sites to obtain plasmid pEhHYG-tetR-O-EhADH2 (Fig. 2). 1 × 107 E. histolytica trophozoites were transfected with 100 ng of plasmid pEhHYG-tetR-O-EhADH2 or pEhHYG-tetR-O-CAT as described previously (18Hamann L. Buss H. Tannich E. Mol. Biochem. Parasitol. 1997; 84: 83-91Crossref PubMed Scopus (70) Google Scholar). Hygromycin selection (10 μg/ml) was initiated 48 h after transfection. To assay E. histolytica trophozoite growth during expression of the antisense RNA to EhADH2, starting cultures of 5 × 103 trophozoite transfected with pZZ1, pSA8, or pNeo-Act were grown under 50 μg/ml G418 selection and counted at 48 and 72 h. Results were averaged from three replicate tubes and three separate experiments. For studies of the inducible expression of the EhADH2 antisense RNA, separate cultures of 5 × 103 E. histolytica pEhHYG-tetR-O-EhADH2 (anti-EhADH2) or pEhHYG-tetR-O-CAT (p-CAT) transfected trophozoites were grown for 48 h and treated with 1 μg/ml tetracycline. After tetracycline induction, cultures were grown for 48 h. E. histolyticatrophozoites were harvested by chilling culture tubes on ice for 10 min and centrifuging them at 430 × g for 5 min. The resulting pellets were resuspended in 1 ml of BYI-S-33 medium and the trophozoites counted with a hemocytometer. EhADH2 expression inE. histolytica trophozoites transfected with pZZ, pSA8, pNeo-Act, pEhHYG-tetR-O-EhADH2, or pEhHYG-tetR-O-CAT was assessed by immunoblotting using a 1:10000 dilution of rabbit antiserum raised to a recombinant 6His-EhADH2 fusion protein on SDS-PAGE-separated lysates obtained from 1 × 106 trophozoites (16Yong T.-S. Li E. Clark D. Stanley Jr., S.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6464-6469Crossref PubMed Scopus (16) Google Scholar). Scanning densitometry of autoradiographs was performed using a DuoScan scanner from Agfa (Ridgeway Park, NJ). Approximately 1 × 108 ameba trophozoites from each of the transfectants described above (pZZ, pEhHYG-tetR-O-EhADH2, pSA8, and pEhHYG-tetR-O-CAT), grown under conditions to maximize antisense RNA expression, were harvested by chilling on ice for 10 min and centrifuged at 430 × g at 4 °C for 5 min. The pellet was resuspended in 800 μl of lysis buffer B (20 mmTris-HCl, pH 6.5, 2 μm leupeptin, 5 mm N-ethylmaleimide, 2 mm phenylmethylsulfonyl fluoride, 2 mm benzamidine, and 5 mmE-64), freeze-thawed five times in ethanol/CO2, and centrifuged at 150,000 × g at 4 °C for 40 min. The supernatant was kept on ice and the activity assays performed immediately. Protein concentrations were calculated using the Bradford method. ADH activity of the amebic lysates (or bacterial lysates from subsequent experiments) was determined by measuring the decrease in absorbance at 340 nm following the oxidation of NADH to NAD. The standard assay was performed in the presence of 6 mmdithiothreitol, 5 mm MgSO4, 0.1 mmFe (NH4)2(SO4)2, 0.2 mm NADH, 0.5 mm acetaldehyde, and 0.1 Tris-HCl buffer, pH 6.5 (16Yong T.-S. Li E. Clark D. Stanley Jr., S.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6464-6469Crossref PubMed Scopus (16) Google Scholar). The ALDH activity was performed in the same buffer, replacing the acetaldehyde with 0.2 mmacetyl-CoA. One unit of enzyme activity was defined as that which consumes 1 μmol of NADH or NAD+/min. Values are expressed as the means of at least three independent experiments. For the determination of Km values for the EhADH2 and EhADH2-(417–870) proteins, 5 μg of purified recombinant protein was used in each assay. Km determinations for alcohol substrates with EhADH2 and EhADH2-(417–870) were made with the spectrophotometric assay in the reverse reaction, using 1 mm NAD+ rather than NADH as previously described (16Yong T.-S. Li E. Clark D. Stanley Jr., S.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6464-6469Crossref PubMed Scopus (16) Google Scholar). Km values were calculated using Lineweaver-Burk plots. The T7 promoter-based vector pET23a (Novagen, Madison, WI) was used for E. coli expression of recombinant proteins comprising the N-terminal domain (EhADH2-(1–532)) and the C-terminal domain (EhADH2-(417–870)) of EhADH2 (Fig.1). Nucleotides encoding EhADH2-(1–532) were obtained by PCR from the pET3a/EhADH2 construct (16Yong T.-S. Li E. Clark D. Stanley Jr., S.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6464-6469Crossref PubMed Scopus (16) Google Scholar). The sequences flanking the nucleotides encoding EhADH2-(1–532) were modified by the incorporation of a stop codon and a BamHI site at position 1598 of the EhADH2 DNA sequence. The fragment containing EhADH2-(1–532) was then ligated into a NdeI- andBamHI-digested pet23a vector to generate plasmid pEhADH2-(1–532). Nucleotides encoding EhADH2-(417–870) were also obtained by PCR from the pET3a/EhADH2 construct. The sequences flanking regions encoding EhADH2-(417–870) were modified by the incorporation of a BamHI site next to the termination codon TAA at position 2612 at the 3′ end of EhADH2 and a NheI site with an initiating codon at position 1251 of the EhADH2 DNA sequence (2Yang W. Li E. Kairong T. Stanley Jr., S.L. Mol. Biochem. Parasitol. 1994; 64: 253-260Crossref PubMed Scopus (79) Google Scholar). The fragment containing EhADH2-(417–870) was then ligated intoNheI- and BamHI-digested pET23a vector creating plasmid pEhADH2-(417–870). The recombinant pET3a/EhADH2 vector was used to generate site-directed and deletion mutants using the QuickChangeTM site-directed mutagenesis kit (Stratagene, La Jolla, CA). In separate constructs, the histidines at position 88, 730, 734, or 744 of recombinant EhADH2 were replaced by arginine, and the histidine at position 754 was replaced by arginine or glutamine, generating mutants EhADH2(H88R), EhADH2(H730R), EhADH2(H734R), EhADH2(H744R), EhADH2(H754R), and EhADH2(H754Q). All changes were confirmed by sequence analysis of each plasmid. These replacements are marked in Fig. 1. To generate a C-terminal deletion, the hydrophobic stretch of 17 amino acids at the end of EhADH2 was deleted using a modified PCR reaction (19Wang W. Malcolm B.A. BioTechniques. 1999; 26: 680-682Crossref PubMed Scopus (488) Google Scholar). Plasmids carrying the N- and C-terminal domains of EhADH2 and plasmids carrying mutant EhADH2 were expressed separately in BL21 (DE3) by standard procedures. For each strain, a single colony was grown overnight in 1-liter cultures under aerobic conditions. Cells were collected by centrifugation at 1,500 × g for 30 min and resuspended in 20 mm Tris-HCl (pH 6.5). Samples were disrupted by sonication and sedimented by centrifugation at 13,000 × g for 30 min at 4 °C. The supernatant was filtered with a 0.22-micron pore unit, and protein concentration was determined by the Bradford assay (Bio-Rad). Each sample (5 μg) was analyzed by SDS-PAGE analysis to detect expression of the altered EhADH2 protein. Western blot analysis was performed using a 1:10,000 dilution of rabbit antiserum raised to a recombinant 6His-EhADH2 fusion protein as described previously (16Yong T.-S. Li E. Clark D. Stanley Jr., S.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6464-6469Crossref PubMed Scopus (16) Google Scholar). For functional studies, SHH31 (ΔadhE) was transformed separately with plasmids carrying the altered version of EhADH2. Expression of the mutant proteins by SHH31 (ΔadhE) under aerobic conditions was confirmed by SDS-PAGE and immunoblot analyses. Bacterial lysates from each transformed strain were tested for ADH and ALDH activities as described above. To test for the ability of a mutant EhADH2 protein to complement SHH31 for anaerobic growth, individual colonies from the transformed strain were grown in 14 ml of M9 liquid or on M9 agar for 24, 48, and 72 h under anaerobic conditions (16Yong T.-S. Li E. Clark D. Stanley Jr., S.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6464-6469Crossref PubMed Scopus (16) Google Scholar). Growth was measured spectrophotometrically by absorbance at 600 nm or by counting colonies on agar. The sequence encoding EhADH2(H754R) was inserted in plasmid pET29b, which contains a selectable marker for kanamycin resistance (Novagen). The pEhADH2 plasmid contains an ampicillin selection marker. Strain SHH31 (ΔadhE) was co-transformed by electroporation with 2 μg (each) of pEhADH2 (recombinant wild type) and pEhADH2(H754R). Isolated colonies were selected from clones grown in the presence of both kanamycin and ampicillin. Single colonies were grown overnight in 1 liter of LB broth medium under double antibiotic selection. Plasmids pEhADH2 (positive control), pEhADH2(H754R), pET3a, and pET29b (negative controls) were expressed separately. Bacterial lysates were obtained and tested for expression of recombinant EhADH2 proteins, anaerobic growth, and ADH/ALDH activities as described above. Retention of pEhADH2 within SHH31 was shown by reselection of the doubly transfected SHH31 with ampicillin alone, followed by assessment of growth under anaerobic conditions. Sepharose CL-6B (Sigma) was used to size engineered EhADH2 proteins. A single colony of each transformed strain was grown overnight under aerobic conditions. Bacterial cells were pelleted by centrifugation, resuspended in lysis buffer, disrupted by French press, and sedimented by centrifugation as described above. Filtered samples were separated over a Sepharose CL-6B column equilibrated with 20 mm Tris-HCl (pH 6.5). Fractions were collected and measured spectrophotometrically at 280 nm. Each protein was sized by comparison with molecular weight standards (Sigma). EhADH2 proteins in samples were identified by immunoblotting of SDS-PAGE separated fractions. For studies of spirosome formation by EhADH2 and its altered derivatives, 100 ng of protein from cell lysates were absorbed to a 200-mesh nickel Formvar- and carbon-coated grids, negatively stained with 0.05% uranyl acetate, and photographed using a Zeiss 902 electron microscope (15Matayoshi S. Oda H. Sarwar G. J. Gen. Microbiol. 1989; 135: 525-529PubMed Google Scholar). To analyze the role of EhADH2 enzymatic activity in the growth and survival of E. histolytica, we transformed amebae with the plasmid pZZ1, constitutively expressing an antisense EhADH2 transcript. Amebic lysates derived from pZZ1-transfected trophozoites had 30% of the ADH activity of lysates derived from equivalent numbers of wild type HM1:IMSS trophozoites or trophozoites transformed with plasmid pSA8 (which contains an antisense transcript to the E. histolytica cysteine proteinase 5 gene) (Table I) (17Ankri S. Stolarsky T. Mirelman D. Mol. Microbiol. 1998; 28: 777-785Crossref PubMed Scopus (104) Google Scholar). Using immunoblotting with antiserum raised against EhADH2, we found that EhADH2 was present in lower amounts (83% reduction by scanning densitometry of the autoradiograph bands) in SDS-PAGE-separated lysates from pZZ1-transfected E. histolytica compared with lysates from pSA8-transfected E. histolytica (Fig.2). In contrast, expression of the serine-rich E. histolytica protein (SREHP) (measured by immunoblotting with anti-SREHP monoclonal antibody) did not differ between pZZ1 and wild type trophozoites (data not shown). To look at the effects of reduced EhADH2 synthesis, we measured the growth of pZZ1 transfected E. histolytica trophozoites by inoculating a culture tube with 5 × 103 E. histolyticatrophozoites and then counting viable trophozoites 48 and 72 h after inoculation. We used pSA8-transfected trophozoites as a control for both G418 selection and possible nonspecific effects of antisense expression on amebic growth. As shown in Fig.3 A, both sets of transgenicE. histolytica trophozoites under antibiotic selection with G418 have reduced growth compared with wild type trophozoites. However, amebic trophozoites transfected with the pZZ1 plasmid show significantly reduced growth compared with trophozoites transfected with pSA8.Table IConstitutive expression of an antisense RNA to EhADH2 significantly reduces NAD-dependent ADH and ALDH activities in E. histolytica trophozoitesPlasmidAntisense toADH activityALDH activitymilliunits/mgpSA8E. histolytica cysteine proteinase 5 gene (Ehcp5)185 ± 1578 ± 13pZZ1Ehadh256 ± 1123 ± 9Lysates were obtained from 106 amebae transfected with either pSA8 (expressing an antisense RNA for Ehcp5) or pZZ1 (expressing the antisense RNA for Ehadh2). Results are expressed as the mean ± standard error of the mean from three separate experiments. Open table in a new tab Lysates were obtained from 106 amebae transfected with either pSA8 (expressing an antisense RNA for Ehcp5) or pZZ1 (expressing the antisense RNA for Ehadh2). Results are expressed as the mean ± standard error of the mean from three separate experiments. We confirmed that the reduced growth seen in these trophozoites was caused by expression of the antisense EhADH2 RNA by inducing expression of the antisense RNA in log phase E. histolyticatrophozoites. The pEhHYG-tetR-O-EhADH2 plasmid was used to transformE. histolytica HM1:IMSS trophozoites, while HM1:IMSS trophozoites transformed with the pEhHYG-tetR-O-CAT construct served as controls. Transformants were selected for growth in 10 μg/ml hygromycin, and both sets of transformants showed similar growth curves under continuous hygromycin selection (data not shown). We then grew equivalent starting cultures of trophozoites carrying pEhHYG-tetR-O-EhADH2 and trophozoites carrying pEhHYG-tetR-O-CAT in the added presence of 1 μg/ml tetracycline, and the number of trophozoites was counted at 24 and 48 h following the addition of tetracycline. As shown in Fig. 3 B, the addition of tetracycline caused a significant inhibition of the growth of pEhHYG-tetR-O-EhADH2-transformed amebae at 48 h but did not alter the growth of pEhHYG-tetR-O-CAT-transformed trophozoites. To establish that this difference in growth was due to inhibition of EhADH2 synthesis, we measured ADH and ALDH activity in the lysates of 106 pEhHYG-tetR-O-EhADH2-transfected or pEhHYG-tetR-O-CAT-transfected trophozoites that had been cultured for 48 h in the presence of tetracycline. There was a 70% reduction in ADH activity and a 66% reduction in ALDH activity in pEhHYG-tetR-O-EhADH2-transfected trophozoites compared with pEhHYG-tetR-O-CAT-transfected trophozoites at 48 h after tetracycline addition (Table II).Table IIInduced expression of an antisense RNA to EhADH2 significantly reduces NAD-dependent ADH and ALDH activities in E. histolytica trophozoitesPlasmidADH activitiesALDH activitiesTetracycline+−+−milliunits/mgpEhHYG-tetR-O-CAT210 ± 21203 ± 18113 ± 13117 ± 19pEhHYG-tetR-O-EhADH283 ± 12191 ± 2337 ± 8122 ± 22ADH and ALDH activities were measured in the lysates from 106amebae transfected with either the vector alone (pEhHYG-tetR-O-CAT) or with the plasmid encoding the Ehadh2 antisense sequence (pEhHYG-tetR-O-EhADH2). For values after tetracycline induction,E. histolytica trophozoites were isolated 48 h after growth in 1 μg/ml tetracycline. Open table in a new tab ADH and ALDH activities were measured in the lysates from 106amebae transfected with either the vector alone (pEhHYG-tetR-O-CAT) or with the plasmid encoding the Ehadh2 antisense sequence (pEhHYG-tetR-O-EhADH2). For values after tetracycline induction,E. histolytica trophozoites were isolated 48 h after growth in 1 μg/ml tetracycline. The C-terminal 400 amino acids of EhADH2 show significant homology with class III microbial ADH from organisms such as Clostridium acetobutylicum and Zymomonas mobilis (2Yang W. Li E. Kairong T. Stanley Jr., S.L. Mol. Biochem. Parasitol. 1994; 64: 253-260Crossref PubMed Scopus (79) Google Scholar). If the EhADH2 enzyme is truly a fusion enzyme, with distinct ADH and ALDH enzymes linked to form a single peptide, then expression of the individual domains should produce separate functional ADH and ALDH enzymes. To test this hypothesis, we first expressed nucleotides 1251–2612 of EhADH2 in E. coli BL21 and SHH31 to generate a truncated protein containing the entire ADH domain (EhADH2-(417–870)). Expression of EhADH2-(417–870) was confirmed by immunoblotting using polyclonal antiserum to the full-length EhADH2 protein (data not shown). Assays of ADH activity onE. coli SHH31 lysates and on purified EhADH-(417–870) demonstrated that the truncated protein retained ADH activity similar in magnitude to that seen with the full-length EhADH2 enzyme (TableIII). EhADH2-(417–870) did not possess ALDH activity (Table III). The Km values for EhADH2-(417–870) and EhADH2 for the substrates ethanol, acetaldehyde, acetyl-CoA, propanol, and isopropanol were determined (TableIV). The Km values for EhADH2-(417–870) were lower than those obtained for EhADH2 for all substrates tested, and EhADH2-(417–870) was able to utilize isopropanol as a substrate. Gel filtration analysis revealed that EhADH2-(417–870) (predicted molecular mass, 49 kDa) migrated in a fraction consistent with a molecular mass of ∼200 kDa (Fig.4). Electron microscopic analysis of either the >2000-kDa fraction or the 200-kDa fraction did not reveal spirosome formation (data not shown). For comparison, the full-length EhADH2 enzyme (97.4 kDa) is found in the > 2000-kDa fraction (Fig. 4), where it forms spirosomes (Fig.5), or at 200 kDa consistent with dimer formation (Fig. 4) (16Yong T.-S. Li E. Clark D. Stanley Jr., S.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6464-6469Crossref PubMed Scopus (16) Google Scholar). Episomal expression of EhADH2-(417–870) could not rescue the ability of E. coli SHH31 to grow under anaerobic conditions (Fig. 6).Table IIINAD-dependent ADH and ALDH dehydrogenase activities measured from partially purified bacterial lysates of E. coli SHH31 expressing either wild type recombinant EhADH2 enzyme, one of the indicated EhADH2 mutants, or plasmid without an insertEhADH2 versionADH activityALDH activitymilliunits/mg(None) plasmid pET3aNDNDEhADH2 wild type500 ± 43220 ± 32EhADH2-(417–870)808 ± 313NDEhADH2-(1–532)NDNDEhADH2-(1–853)NDND(None) pET3a and pET39bNDNDEhADH2(H88R)423 ± 110198 ± 72EhADH2(H730R)NDNDEhADH2(H734R)149 ± 24NDEhADH2(H744R)NDNDEhADH2(H754R)NDNDEhADH2(H754Q)NDNDEhADH2 and EhADH2(H754R)NDNDEhADH2 (after curing EhADH2(H754R))537 ± 54350 ± 117ND, no activity (<1 milliunit/mg) detected). Open table in a new tab Table IVKm values for different substrates for EhADH2417–870compared with the wild type EhADH2 enzymeReactionsKm for EhADH2Km for EhADH2417–870mmAcetaldehyde + NADH0.210.0830.320.028Acetyl-CoA + NADH0.028ND0.1NDEthanol + NAD+500.0790.20.062l-Propanol + NAD+340.0810.190.073Isopropanol + NAD+ND0.077ND0.06525 μg of purified enzyme was used for each assay. ND, no activit
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