From the Departments of Medicine,
§ Molecular Microbiology, and
Biochemistry and
Molecular Biophysics, Washington University School of Medicine, St.
Louis, Missouri 63110 and the ¶ Department of Microbiology,
Southern Illinois University, Carbondale, Illinois 62901
Received for publication, February 12, 2001, and in revised form, March 21, 2001
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ABSTRACT |
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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.
The anaerobic intestinal protozoan parasite Entamoeba
histolytica converts pyruvate to ethanol in its fermentation
pathway (1). The last two steps of this pathway are the conversion of
acetyl-CoA to acetaldehyde followed by the reduction of acetaldehyde to
ethanol (1). 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) (2-5). There are at least
two enzymes with ALDH activity, the EhADH2 enzyme and a
NADP-dependent ALDH, EhALDH1 (2, 5, 6). Given the presence
of multiple ADH and ALDH enzymes in E. histolytica, an important question is whether any of these enzymes are essential for
E. 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 (2, 7-12). 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 (8). The EhADH2 enzyme utilizes NAD and Fe2+ as co-factors and does not
demonstrate homology with the zinc-dependent ADH
enzymes (13). 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 (2). The
prototype member of the family is the Escherichia coli ADHE
enzyme, which is required for the anaerobic growth of E. coli (8). 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 (5, 14, 15).
Episomal expression of the amebic EhADH2 gene in
E. coli can complement an ADHE mutation, providing a system
for rapid evaluation of the activity of EhADH2 mutants and for testing
inhibitors of EhADH2 (16).
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.
Escherichia coli Strains--
Strains DH5 EhADH2 Antisense Constructs--
To construct the antisense
insert for EhADH2, a PCR product was generated from nucleotides
The EhADH2 antisense RNA under an inducible expression system was
generated by PCR from Assays of EhADH2 Expression and E. histolytica Growth--
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. histolytica
trophozoites 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 in
E. 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 (16). Scanning
densitometry of autoradiographs was performed using a DuoScan
scanner from Agfa (Ridgeway Park, NJ).
Assessment of ADH and ALDH Activity in E. histolytica Transfected
with EhADH2 Antisense Constructs--
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 mM
Tris-HCl, pH 6.5, 2 µM leupeptin, 5 mM
N-ethylmaleimide, 2 mM phenylmethylsulfonyl
fluoride, 2 mM benzamidine, and 5 mM
E-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 mM
dithiothreitol, 5 mM MgSO4, 0.1 mM
Fe (NH4)2(SO4)2,
0.2 mM NADH, 0.5 mM acetaldehyde, and 0.1 Tris-HCl buffer, pH 6.5 (16). The ALDH activity was performed in
the same buffer, replacing the acetaldehyde with 0.2 mM
acetyl-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 (16). Km values were calculated using
Lineweaver-Burk plots.
Construction of the pEhADH2-(1-532) and pEhADH2-(417-870)
Expression Vectors--
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 (16). 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- and
BamHI-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 (2).
The fragment containing EhADH2-(417-870) was then ligated into
NheI- and BamHI-digested pET23a vector creating plasmid pEhADH2-(417-870).
Site-directed and Deletion Mutagenesis of EhADH2--
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
(19).
Expression of Truncated and Mutant Versions of
EhADH2--
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 (16). For functional studies, SHH31
( Co-expression of EhADH2(H754R) and EhADH2 in
SHH31--
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 ( Sizing Exclusion Analysis--
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.
Spirosome Detection by Electron Microscopy--
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 (15).
Episomal Expression of an Antisense RNA to the EhADH2 Gene Inhibits
E. histolytica Growth--
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) (17). 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. histolytica
trophozoites 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.
3A, both sets of transgenic
E. 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.
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. histolytica
trophozoites. The pEhHYG-tetR-O-EhADH2 plasmid was used to transform
E. 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. 3B, 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).
The ADH Activity of EhADH2 Resides in the C-terminal Half of
the Enzyme--
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
(2). 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 on
E. 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 (Table
III). 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 (Table
IV). 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) (16). Episomal expression of EhADH2-(417-870) could not rescue the ability of E. coli SHH31 to grow under
anaerobic conditions (Fig. 6).
The ALDH Activity of EhADH2 Requires Residues in the C-terminal
Half of EhADH2--
The N-terminal ALDH domain of EhADH2 has
positional identities (32%) with the CoA- and
NADP-dependent succinate semialdehyde dehydrogenase of
Clostridium kluyveri and retains a number of residues known
to be required for ALDH activity (20-22). To determine whether the
N-terminal region of EhADH2 encoded a functional ALDH enzyme, we
expressed nucleotides 1-1566 of EhADH2 in E. coli BL21 (DE3) and E. coli SHH31 ( Conserved Histidines in the ADH Domain of EhADH2 Are Required for
Both ADH and ALDH Activity--
EhADH2 possesses a histidine at
position 754 that is similar in location to a histidine implicated in
the catalytic mechanism of the Z. mobilis ADH(II)
enzyme (23). Replacement of His754 with either Gln or Arg
completely eliminated both the ADH and ALDH activity of recombinant
EhADH2 (Table III). In gel filtration analysis, EhADH2(H754R) was found
in the 200-kDa and >2000-kDa fraction, and spirosomes were found in
the >2000-kDa fraction (Fig. 5). Episomal expression of the mutant
EhADH2(H754R) enzyme could not restore the ability of E. coli SHH31 to grow under anaerobic conditions (Fig. 6). Another
set of highly conserved histidine residues,
GMDH730SMAH734KVGAAFHLPH744G,
is found in the putative iron-binding domain of the EhADH2 enzyme. To
determine whether these residues are required for EhADH2 activity, we
generated mutant EhADH2 enzymes with selected histidines replaced by
arginine: EhADH2(H730R), EhADH2(H734R), EhADH2(H744R). In addition, we
made EhADH2(H88R) because this histidine is not predicted to be within
the active sites of either the ADH or ALDH domains. Lysates from
E. coli expressing EhADH2(H730R) and EhADH2(H744R) lacked both ADH and ALDH activity, whereas EhADH2(H734R) possessed reduced ADH activity. EhADH2(H88R) was identical to wild type EhADH2 in
both ADH and ALDH activity (Table III). Among the mutant EhADH2
enzymes, only EhADH2(H88R) could rescue the anaerobic growth of
E. coli SHH31 Spirosome-forming EhADH2(H754R) Functions as a Dominant Negative
Mutant--
It has been proposed that EhADH2 and the E. coli adhE enzyme must form dimers or multimers (spirosomes) for
activity (2, 5, 14); this suggests that the episomal expression of a
nonfunctional EhADH2 enzyme capable of forming spirosomes could serve
as a dominant negative mutant by pairing and oligomerizing with the
native enzyme. We found that the nonfunctional mutant EhADH2(H754R)
mutant was capable of forming spirosomes (Fig. 5) but did not possess
EhADH2 activity. We co-transformed E. coli SHH31 with EhADH2
on plasmid pET3a/EhADH2 ampR and pEhADH2(H754R)
kanR. E. coli expressing both antibiotic
resistance plasmids were obtained by selection with kanamycin and
ampicillin under aerobic conditions, and spirosome formation was
assessed. As shown in Fig. 5, spirosomes could be detected in partially
purified lysates obtained from E. coli SHH31 transformed
with either pEhADH2 or pEhADH2(H754R) or with both plasmids. No
spirosomes were seen in E. coli co-transformed with both
parent plasmids (pET3a and pET29b). Subsequently, the double transformants were screened for the ability to grow under anaerobic conditions. E. coli expressing both EhADH2 and EhADH2(H754R)
failed to grow under anaerobic conditions in either liquid (Fig.
7) or solid media (data not shown).
Curing of SHH31 doubly transformed with pEhADH2 and pEhADH2(H754R) of
the pEhADH2(H754R) plasmid by removing kanamycin selection restored
the ability of E. coli SHH31 to grow under anaerobic
conditions (Fig. 7).
The glycolytic pathway for the anaerobic protozoan parasite
E. histolytica was first described by Reeves (1, 24) more than two decades ago. A number of the E. histolytica
metabolic enzymes appear to be derived from ancestral prokaryotic
enzymes and are significantly different from eukaryotic homologues (7). The EhADH2 enzyme (also referred to as EhADHE) is a 97-kDa NAD- and
Fe2+-dependent bifunctional enzyme with both
ADH and ALDH activities (2,5). This enzyme does not have a homologue in
man. Its proposed function in E. histolytica is to catalyze
the final two steps in the amebic glycolytic pathway, which are the
conversion of acetyl-CoA to acetaldehyde and the reduction of
acetaldehyde to ethanol. However, E. histolytica possesses
other ADH and ALDH enzymes that could potentially serve a similar
function. There are two structurally distinct
NADP-dependent ADH molecules, EhADH1 and EhADH3 (3, 4).
There is also a NADP-dependent ALDH, EhALDH1 (6). Despite
the presence of these other enzymes, there is experimental evidence
that the EhADH2 enzyme is required for both the conversion of
acetyl-CoA to acetaldehyde and acetaldehyde to ethanol in E. histolytica. The conversion of acetaldehyde to ethanol is NADH-
rather than NADPH-dependent (1). The EhADH1 enzyme, which
is NADP-dependent, shows a marked preference for branched
chain alcohols, whereas EhADH2 prefers ethanol as a substrate (2,3). In
addition, the EhALDH1 enzyme does not utilize acetyl Co-A as a
substrate, suggesting that EhADH2 may be solely responsible for the
conversion of acetyl-CoA to acetaldehyde (6).
To test directly whether EhADH2 is necessary for E. histolytica growth and survival, we specifically decreased the
expression of EhADH2 through the episomal expression of an antisense
RNA to the EhADH2 gene. Targeted disruption of amebic genes
has not yet been achieved; however, episomal expression of antisense
RNA has been used specifically to inhibit the expression of E. histolytica genes. This approach does not result in complete
inhibition of protein expression, but it has reduced the levels of the
target protein by 70-90% (17). In our hands, constitutive expression of an antisense RNA to EhADH2 reduced NAD-dependent ADH
activity by 60-70%. This was accompanied by a marked decrease in
amebic growth and viability, compared with either amebae carrying the vector lacking the antisense insert or amebae expressing an antisense construct to the amebic cysteine proteinase gene. Similar results were
seen using an inducible expression system in which E. histolytica growth was significantly inhibited with induction of
the antisense RNA. These results are very different from the data seen
with antisense inhibition of expression of the E. histolytica cysteine proteinase 5 gene, the E. histolytica amebapore A gene, or the light chain of the galactose
binding lectin, where expression of antisense RNA did not significantly
inhibit trophozoite growth or survival (17, 25, 26). These data suggest
that EhADH2 is responsible for most, if not all, of the
NAD-dependent ADH and ALDH activity in E. histolytica and that the expression of EhADH2 is necessary for
growth and survival of amebic trophozoites under anaerobic conditions.
A potential problem with the use of antisense RNA is whether the
inhibition of protein expression is limited to the targeted molecule.
We found no changes in the levels of amebic cysteine proteinase
activity or expression of the amebic serine-rich protein in amebae
carrying the EhADH2 antisense construct but cannot absolutely exclude
the possibility that the expression of other proteins could have been affected.
Enzymes homologous to EhADH2 have been found in Gram-positive and
Gram-negative bacteria and in the intestinal protozoan parasite G. lamblia (7, 8-12, 20). Despite the potential clinical importance of this new family of enzymes, little is known about their
structure and catalytic mechanisms. Based on homology with separate ADH
and ALDH enzymes, members of this family appear to be fusion enzymes,
with a discrete C-terminal ADH domain and N-terminal ALDH domain (8).
The ADH domain of EhADH2 displays limited homology with members of the
small family of class III microbial ADH enzymes, the prototype of which
is the Fe2+-dependent ADH(II) enzyme from the
anaerobic bacterium Z. mobilis (2, 27, 28). We found that
the C-terminal 453 amino acids of EhADH2 contained all of the residues
necessary for NAD-dependent ADH activity, consistent with
the fusion enzyme hypothesis. These data are similar to those found in
studies of the E. coli AdhE enzyme, in which ADH activity
could be localized to a peptide containing ~300 amino acids in the
C-terminal region (14). Based on its migration under gel filtration,
the mutant EhADH2-(417-870) enzyme appeared to form tetramers rather
than the dimers or spirosomes seen with the full-length EhADH2 enzyme.
Interestingly, other class III iron-dependent ADH enzymes,
including ADHII of Z. mobilis and Bacillus
stearothermophilus RS93, also form homotetramers (27, 29). There
were some significant differences in Km values for
the mutant EhADH2-(417-870) enzyme compared with the full-length
recombinant EhADH2, and the mutant enzyme, unlike full-length EhADH2
could utilize the branched chain alcohol isopropanol as a substrate.
These data indicate that removal of the N-terminal half of the enzyme
results in conformational changes that alter the substrate binding
pocket in the EhADH2-(417-870) mutant enzyme. However, acetyl Co-A was
not a substrate for the ADH domain mutant, consistent with the primary
structure of EhADH2 in which residues that could be involved in acetyl
Co-A binding are found within the N-terminal half of EhADH2 (2).
Alignment of the N-terminal sequences of members of the ADHE family,
including EhADH2, with consensus ALDH sequences shows the conservation
of a number of key residues in the catalytic site2 (2, 22). These include
a potential catalytic cysteine at Cys252, a conserved
glutamate involved in proton binding at position 350, and a conserved
asparagine at position 121 that may be involved in hydrogen bonding to
the nicotinamide ring of NAD (21, 22). The EhADH2 enzyme also possesses
a classic GXGXXG NAD binding fingerprint,
but it is located at the junction of the putative ALDH and ADH domains
(2, 30). A second motif that resembles an NAD-binding domain (GVGAG) is
present in the ALDH region, and these residues could play a role in
binding the adenine residue of CoA. Thus, the ALDH domain of E. histolytica EhADH2 appears to contain the key catalytic residues
necessary for ALDH activity and, possibly, appropriate motifs for NADH
and acetyl-CoA binding. Despite these homologies, we found that a
truncated protein containing the N-terminal 522 amino acids of EhADH2
did not possess either ADH or ALDH activity. One possible explanation
for our findings is that the lack of the iron-binding domain within the
EhADH2-(1-522) mutant renders it inactive. However, we also found that
the deletion of only 17 amino acids in the C terminus of the EhADH2
(which left the putative iron-binding domain intact) abrogated both ADH and ALDH activity. This mutant enzyme failed to form dimers or spirosomes, suggesting that residues in the C terminus may be necessary
for EhADH2 dimer and spirosome formation and thus indirectly for both
ADH and ALDH activity.
By comparing the primary structure of the C-terminal domain of EhADH2
with the ADHII of Z. mobilis, we were able to identify EhADH2 residues that could play a role in the ADH activity of the
enzyme. The ADH domain of members of the ADHE family and the Z. mobilis ADH(II) enzyme contains the sequence
GXXHXXAHXXGXXXXXPHG, which
is the consensus sequence proposed by Bairoch (31) for an iron binding
domain (28). It has been suggested that the imidazole rings of
the histidines coordinate the Fe2+ ion (31). Direct
evidence for the role of iron in enzymatic activity comes from Kessler
et al. (14), who showed that the ADH, ALDH, and pyruvate
formate-lyase activities of the E. coli adhE enzyme were
stimulated by iron. However, in a recent study of the
Giardia EhADH2 homologue, Sanchez (9) reported that its ADH
and ALDH activities were not dependent on iron or other metal ions for
activity. We were able to demonstrate that the three conserved
histidines located in the putative iron-binding domain of the EhADH2
enzyme are required for both its ADH and ALDH activity. This is
consistent with the finding that both the ADH and ALDH activity of the
EhADH2 enzyme require the presence of iron (2).
A histidine residue serves as a proton donor at the active site of
zinc-dependent ADH molecules. Studies of the
iron-dependent enzyme ADH(II) of Z. mobilis have
shown that the histidine in position 277 (YNLPH277GV), which is 10 amino acids removed from
the terminal histidine within the putative iron-binding domain, appears
to serve a similar function in Z. mobilis ADH(II) (23). The
EhADH2 enzyme has a histidine at position 754 that is within the
peptide VLLPH754VI; this histidine is also 10 residues
removed from the terminal histidine within the putative iron-binding
domain (2). We found that the conservative replacement in EhADH2 of
histidine 754 eliminates EhADH2 enzymatic activity. These data suggest
that the catalytic mechanism of the ADH domain of EhADH2 may be similar
to other members of the class III microbial enzymes. Although the
inactivation of the ADH activity of EhADH2 by replacement of
His754 was not unexpected, the finding that the
site-directed mutagenesis of a single histidine in the putative active
site of the ADH domain also completely eliminates ALDH enzymatic
activity is surprising. Thus, our current findings suggest that the
ALDH activity of EhADH2 is dependent on both a structurally intact C
terminus and a functional ADH domain. Further studies will be necessary
to determine whether this is because the ALDH activity of EhADH2
requires residues in the C terminus for substrate binding, catalysis,
co-factor binding, or to maintain its appropriate conformation.
Studies of EhADH2 and the E. coli AdhE enzyme indicate that
the minimal functional unit of the enzyme is a dimer and that both
enzymes are capable of assembling dimers into helical structures called
spirosomes, containing 20 or more dimers (2, 5, 8, 14). As
noted above, we found that the recombinant EhADH2 molecule could form
spirosomes, as did the mutant EhADH2(H754R), which lacks both ADH and
ALDH activity. Expression of EhADH2 in the adhE deletion
E. coli strain SHH31 rescues its ability to grow in minimal
media under anaerobic conditions. We found that co-expression of EhADH2 and EhADH2(H754R) in SHH31 led to spirosome production but
did not rescue the ability of SHH31 to grow under anaerobic conditions.
These data suggest that EhADH2(H754R) may serve as a dominant negative
mutant by forming nonfunctional dimers with the EhADH2 enzyme. This
could represent a potential approach for inhibiting the native E. histolytica enzyme as well. An alternative explanation for our
findings would be a recombination event between the pEhADH2 and
pEhADH2(H754R) plasmids resulting in loss of the functional EhADH2
enzyme gene in doubly transformed SHH31. However, curing SHH31 of only
the pEhADH2(H754R) plasmid restored the ability of SHH31 to grow under
anaerobic conditions, indicating that an intact pEhADH2 plasmid was
still present. Unfortunately, our inability to successfully express a
mutant EhADH2 with an epitope tag that was capable of forming
spirosomes3 precluded the
demonstration of heterodimer formation in these experiments.
In summary, the EhADH2 enzyme is required for E. histolytica
growth and survival. Structural studies suggest that the C-terminal ADH
domain of the enzyme functions as a separate entity, with a catalytic
mechanism that may be similar to that seen in other class III microbial
enzymes. However, the second component of the EhADH2, the N-terminal
ALDH region, appears to be dependent upon an intact C terminus and
intact ADH activity for ALDH enzyme activity. Further studies will be
necessary to determine whether this dependence reflects
conformational requirements or the evolutionary migration of substrate
binding, co-factor binding, or catalytic sites from the N-terminal ALDH
domain to the C terminus of EhADH2.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, BL21 (DE3), and
SHH31 (
adhE fadR met tyrT) were used for
expression of recombinant proteins (16). 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 GasPakTM system, Becton
Dickinson, Sparks, MD) with anaerobic system envelopes (BBL GasPak
Plus) (16). Indicator strips were used to confirm anaerobic conditions.
50 to
+1312 of the EhADH2 gene in the antisense orientation
using oligonucleotide primers 5'-AATTGCGGCCGCCTAACCCGTTTTTGGATCAG (forward primer) and 5'-ATAGATCTCCTCCATATGATCCACATCCAAG incorporating NotI and BglII restriction sites, respectively.
This fragment was then inserted into plasmid pSA8 (17) 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 (17).
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.
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) (18) using the
KpnI 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 (18). Hygromycin selection (10 µg/ml) was initiated
48 h after transfection.
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Fig. 1.
Schematic diagram of EhADH2 and EhADH2 mutant
proteins. The locations of the putative ADH and ALDH domains, the
NAD-binding motif, iron-binding domain, and hydrophobic tail are
indicated. The histidines replaced by site-directed mutagenesis are
also marked. The composition of the three main deletion mutants,
EhADH2-(1-532), EhADH2-(417-870), and EhADH2-(1-853), is
also shown.
adhE) was transformed separately with plasmids carrying
the altered version of EhADH2. Expression of the mutant proteins by
SHH31 (
adhE) 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 (16). Growth was measured
spectrophotometrically by absorbance at 600 nm or by counting colonies
on agar.
adhE) 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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Constitutive expression of an antisense RNA to EhADH2 significantly
reduces NAD-dependent ADH and ALDH activities in E. histolytica trophozoites
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Fig. 2.
Lysates from E. histolytica
trophozoites transfected with pZZ1 (antisense RNA to
Ehadh2) contain a reduced amount of EhADH2.
SDS-PAGE-separated lysates from 106 E. histolytica HM1:IMSS trophozoites transfected with pZZ1
(lane 1) or wild-type HM1:IMSS (lane 2) were
subjected to immunoblotting with anti-EhADH2 antiserum. The intensity
of the bands at 97 kDa (EhADH2) is compared.
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Fig. 3.
Antisense inhibition of EhADH2 synthesis
reduces E. histolytica growth. Panel A,
separate starting cultures of 5 × 103 trophozoites
carrying either pSA8 or pZZ1 or the control plasmid pNeo-Act were grown
under G418 selection and counted at 48 and 72 h (n = 6 for each group). There was a statistically significant
(p < 0.001) difference in the number of
pZZ1-transfected trophozoites at 72 h. Panel B, 48-h
cultures of E. histolytica trophozoites carrying
pEhHYG-tetR-O-CAT or pEhHYG-tetR-O-EhADH2 were treated with
tetracycline and their growth monitored over 24 and 48 h.
The addition of tetracycline significantly inhibited growth
(p < 0.001) in the pEhHYG-tetR-O-EhADH2-transfected
amebae at 48 h.
Induced expression of an antisense RNA to EhADH2 significantly reduces
NAD-dependent ADH and ALDH activities in E. histolytica
trophozoites
NAD-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 insert
Km values for different substrates for EhADH2417-870
compared with the wild type EhADH2 enzyme
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Fig. 4.
Size analysis of purified fractions of the
full-length EhADH2 and the truncated EhADH2-(1-532) (ALDH) and
EhADH2-(417-870) (ADH) peptides. The ordinate
represents protein concentration in at
A280 nm. The EhADH2 enzyme was detected
at >2000 kDa as well as at 200 kDa. The ADH domain protein migrates at
~200 kDa, whereas the ALDH domain migrates at 66 kDa, consistent with
a monomer. The arrows show the fraction where the indicated
molecular mass standards elute.
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Fig. 5.
Spirosome formation in partially purified
lysates from bacteria expressing either EhADH2 or EhADH2(H754R) or both
EhADH2 and EhADH2(H754R). Electron micrographs show spirosomes
(arrows) in these fractions. Lysates from bacteria carrying
both vectors without inserts did not show spirosome formation
(pET3a/pET29b).
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Fig. 6.
Rescue of anaerobic growth of E. coli SHH31 ( adhE) by
wild type EhADH2 and mutant EhADH2 enzymes. E. coli
SHH31 (
adhE) transformed with plasmids encoding
full-length EhADH2, EhADH2-(1-532) (ALDH),
EhADH2-(417-870) (ADH), EhADH2-(1-853)
(1-853), EhADH2(H88R) (H88R), EhADH2(H730R)
(H730R), EhADH2(H734R) (H734R), EhADH2(H744R)
(H744R), EhADH2(H754R) (H754R), or EhADH2(H754Q)
(H754Q) was grown in liquid medium under anaerobic
conditions, and A600 nm was measured at 0, 24, 48, and 72 h after inoculation. Only full-length EhADH2 and the
EhADH2(H88R) mutant could restore growth under anaerobic
conditions.
adhE) generating a
truncated protein (EhADH2-(1-522)) containing the entire ALDH domain
plus a potential NAD binding site (Fig. 1). Expression of the
EhADH21-522 protein (predicted molecular mass, 58 kDa) was
confirmed by immunoblotting using polyclonal antiserum to the
full-length EhADH2 protein (data not shown). Assays on E. coli SHH31 lysates and purified EhADH2-(1-522) protein showed no
ALDH or ADH activity (Table III). In gel filtration, EhADH2-(1-522)
migrated in a fraction consistent with a molecular mass of ~66 kDa
and was not detected in the >2000-kDa fraction or the 200-kDa fraction
(Fig. 4), and electron microscope analysis of the >2000-kDa
fraction did not show spirosomes (data not shown). The episomal
expression of EhADH2-(1-522) could not rescue the ability of E. coli SHH31 (
adhE) to grow under anaerobic conditions (Fig. 6). To determine whether the lack of ALDH activity of
EhADH2-(1-522) was due to a lack of additional C-terminal residues
including the putative iron-binding domain, we generated a deletion
mutant lacking only the C-terminal 17 amino acids of EhADH2
(EhADH2-(1-853)) (Fig. 1). This mutant enzyme lacked both ADH and ALDH
activity (Table III), and under gel filtration it migrated primarily in the >2000-kDa fraction (data not shown). However, electron microscopic analysis of that fraction failed to reveal spirosomes (data not shown).
Episomal expression of EhADH2-(1-853) could not rescue the ability of
E. coli SHH31 to grow under anaerobic conditions (Fig.
6).
adhE (Fig. 6).
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Fig. 7.
Co-expression of EhADH2(H754R) blocks the
ability of wild type EhADH2 to rescue the anaerobic growth of E. coli SHH31 ( adhE).
Anaerobic growth of SHH31 transformed with plasmids expressing either
EhADH2 or EhADH2(H754R) (H754R), both EhADH2 and
EhADH2(H754R) (H754R/EhADH2), or both plasmids without
inserts (pET3a/pET29b) is shown. E. coli SHH31
(
adhE) transformed with both EhADH2 and EhADH2(H754R) was
subsequently grown without kanamycin selection to cure it of the
pEhADH2(H754R) plasmid (H754R(cured)/EhADH2). Only E. coli SHH31 (
adhE), transformed with a plasmid
expressing EhADH2, or the doubly transfected E. coli SHH31
(
adhE), cured of the plasmid expressing EhADH2(H754R),
could grow under anaerobic conditions.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Linda Kurz and John Hempel for many helpful discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AI37977, AI30084, and DK52574 and U. S. Department of Energy Contract DE-FG02-88ER13941. Presented in part at the XIII Seminar on Amebiasis, Mexico City, 1997.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** Burroughs Wellcome Scholar in Molecular Parasitology. To whom correspondence should be addressed. Tel.: 314-362-107; Fax: 314-362-3525; E-mail: sstanley@im.wustl.edu.
Published, JBC Papers in Press, March 26, 2001, DOI 10.1074/jbc.M101349200
2 J. Hempel (University of Pittsburgh), personal communication.
3 A. Espinosa, L. Yan, Z. Zhang, L. Foster, D. Clark, E. Li, and S. L. Stanley, Jr., unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: ADH, alcohol dehydrogenase; ALDH, acetaldehyde dehydrogenase; EhADH2, Entamoeba histolytica alcohol dehydrogenase 2; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.
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