Multid rug Resistance
in Entamoeba histolytica
E. Orozco, D.G. Perez, M.C. Gomez and P. Ayala
Drug rewstance rn protozoan parasites
has been ernerglng rn the post decade as
on obstacle to [her control. Amoebiasrs.
caused by Entamoeba h~stolytlca 1s a
woddwrde drsease that provokes hrgh races
of rnohrdrty and rnortalrty. Reports of failed
drug rreatrnent and dlferences rn drug
suscep~brlrtres among E. h~stolyt~ca
probably herald the development of drug
resistance rn th~s paraate. In th~s revrew.
Esther Orozco and co-worken surnrnanze
recent progress on the eluodaoon of physio-
logrcal and molecular evrdence of rnultrdrug
resistance ~n thrs parasrte.
Entarnoeba hrstolytica is the protozoan
responsible for human amoebiasis. It in-
fects 10% of the world population, caus-
ing 50 million cases of dysentery or liver
abscesses and lulling. 100000 humans per
yeart. As with oth& parasitic infections.
amoeblasis is a cor$equence of poverty
and bad sanitary conditions. Unfortu-
nately, with the exception of malaria?.
no vacclnes against protozoan parasites
are in sight, and poor countries cannot
afford to finance large preventive medi-
cine programs. Therefore, amoebiasis is
controlled primarily' by drug treatment
of symptomatic individuals using metro-
nidazole, emetine'and chloroquine, al-
though other drugs such as diloxanide
and iodoquinol are also ~ s e d ~ . ~ .
ences in drug susceptlbilrty have been re-
ported in E. h~srolyoca strain~~,~
are case reports of failed drug-treat-
ments7a, suggestmg that drug resistme
could be occurnng. In other parasites.
such as Plasmodrum, the emergence of
drug resistance has been a major ob-
stacle to its control9. The development
of drug resistance by amoeba in infected
patients would represent an additional
burden in health care cost and in the
planning of public health policies in the
countries affected by this infection.
The development of drug-resistant
parasite cell lines in vivo has been central
to the understanding of the mechanisms
underlying drug resistance in protozoa
To study the physiological and molecular
basis of drug resistance in E hrstolyoca.
Orozco et a1.IO generated emetine-
resistant mutants by mutagenesis with
ethyl methanesulfonate of the clone A.
Parasrtology Today, vol. I I, no 12. 1995
strain HM I:IMSS (Fig. I). Emetine has
been a major drug for clinical treatment
of human amoebiasis. It is an alkaloid
that ~nhibits eukaryotic protein synthesis
by bloclung translocation of the peptidyl-
tRNA from acceptor to donor site on
that drug resistance in clone C2 may
be based upon overexpression of the
P-glycopr~tetn-like pump. P-glycoprotein
Should be the product of rndr genes de-
scribed first In transformed mammalian
Cloning and Expression
of rndr Genes
Physiology of Drug Resistance
in E. histolytica
The mutagenized trophozoites were
selected by their ability to grow in I0 p M
emetine. Two different phenotypes were
exhibited by the emetine-resistant mu-
tants: (I) clones C9, which were resist-
ant to 35 p~ emetine and sensitive to
other drugs; and (2) clones C2, which
were resistant up to 90 p~ emetine and
showed cross resistance to unrelated
hydrophobic drugs, such as colch~cine.
diloxanide and iodsquino110.'3. While
clones C9 are apparently altered in the
protern synthesis mach~nery, clones C2
are altered in drug transport (Fig. I ).
Phenotype of clones C2 suggested
the possibility of the presence of a multi-
drug resistance (MDR) mechanism in E
histolytica, resembling the one first de-
scribed in mammalian transformed cellsl~.
An MDR phenotype has also been found
in protozoan parasites including Plas-
rnodrurn, Giardia and Le~shrnanra sppy.15,1~.
It is based upon the overexpression of a
175 kDa P-glycoprotein, with two ATP-
binding sites that provide the energy for
its function. P-glycoprotein operates a s
an energy-dependent drug efflux pump.
decreasing intracellular drug depotsI7.
MDR in mammalian transformed cells
and in parasites can be reversed by cal-
cium-channel blockers. such a s verapamil.
These compounds inhibit P-glycoprotein-
mediated drug efflux, increasing sensi-
tivrty to the drug18.
The physioloa of MDR was studied
in a selected clone C2 using l[H]emetine
and 3[H]colchicine. Drug uptake experi-
ments showed that emetine-resistant
trophozoites accumulated less drug than
did the sens~tive ones19. Similar results
were obtained when 3[H]colchicine was
used instead of emetine (Fig. 2). In the
presence of verapamil, drug accumu-
lation was increased in the mutant C2.
to a level close to that of the sensitive
clone A. The verapamil effect was the
revecion of drug resistance in mutant
trophozoite~'~. These findings suggested
O 1995. Elsewer jc~ence Lrd
To confirm the presence of a bona
fide MDR phenotype in E. hrstolyt~co.
rndr-like genes were cloned and se-
q:enced. The conserved amino acid se-
qu&ces of the ATP-binding sites in the
mammalian P-glycoprotein were used
to synthesize sense and antisense
oligonucleotides. Nucleotide sequences
were based on the codon usage of E.
hrstolytica20. The oligonucleotides were
used In a polymerase chain reaction
(PCR) wlth E. hraolytrco DNA a s tem-
plate. Amplified fragments (Ehrndrl and
Ehrndr4) showed 46-5 1 % identity in
their predicted ammo acid sequence to
HM1 : IMSS
Fig. I. Origin of drug-resistant clones o f
Entomoebo hkto/y-uco. Swain HM I : IMSS
was cloned in semi-solid agar to obtain
the clone A. The clone A was mutagen-
ized with ethyl methansulfonate (EMS).
Mutagenized vophozoices were selec.
ted by their ability co grow in emetine
(Emt). Two differenc types of emetine-
resistant clones were obtained: C9 anc
C2. Only C2 showed multidrug resisr
ance (MDR) phenotype. 5, sensitive; R
o j $ $ & o Sensitive
Fig. 2. Physiology of multidrug resistance (MDR) in Entornoebo hiscolpco, showing a
model of the MDR phenomenon in which drugs are accumulated in sensitive tropho-
zoites (a). while in resistant trophozoites (b) drugs are pumped out. (c) Trophozoites
of clone A (sen~itive) (circles) and clone C2 (resistant) (triangles) were incubated
with 1.5 mn cafd colchicine and 2.5 pCi ml-' tritiated colchicine with (closed) or
without (open) I 0 p n venpamil at 37%
the human mdrl (Ref. 2 1 ). Fragments
were used as probes to select mdr genes
from a genomic library constwed
uslng DNA from the mutant clone C2
SIX different mdr genes have been iso-
lated from E, h~stolytica: four complete
genes (EhPgpl. EhPgp2. EhPgp5 and
EhPgp6) and bvo pseudogenes (EhPgp3
and EhPgp4) with stop codons in the
coding sequence22 (P. Ayala PhD Thesis.
CINVESTAV IPN. Mexico, 1994). They
show 38-41% identity to mammalian
rndr amino acid predicted sequences, and
22-27% and 1 1-
modium and Leishmania, respectively. The
hydropathy plot of EhPgp genes drawn
using the algorithm of Kyte and Doolittle
were nearly superimposable onto each
other and onto the hydrophaty plot of
human mdrl. It is a fair assumption then
that the predicted E. histolytica P-glyco-
1 8% to those of Plas-
Table I. Differential expression of EhPgpl. EhPgp2 and EhPgp5 in clones of Entornoebo
RNA from clones A and C2 was hybridized wich specific probes for each EhPgp genes. Acun probe
was used as a control.
b Emeune concentndon (pn) chat war used to grow trophozoites before RNA exuaction.
proteins have the same transmembrane
structure a s the mammalian P-glyco-
proteins22 (P. Ayala, op. cit.).
DNA from both the mutant clone Q
and the sensitive clone A, digested with
several different endonucleases were
hybridized against Ehmdrl and Ehmdr4
PCR fragments in Southern blot experi-
ments. Muitiple similar bands in both
clones corroborated the presence of a
multigene mdr family in E. histoly~co. The
number of mdr genomic coples was not
increased in the drug-resiqant clone C2,
and no reanangements of mdr genes
DNA probes specific for each of the
three E. h~stoty~ca
mdr genes (EhPgp I,
EhPgp2. EhPgp5) were obtained by PCR
using sense and antisense oligonucleo-
tides from the linker region bebeen the
two ATP-binding sites. Southern experi-
'hknts using the specific probes showed
that there is a unique copy of EhPgpI
and EhPgp2. However, a specific probe
for EhPgp5 recognized PNO bands in the
total E. histolyt~ca DNA (P. Ayala, op.
cit.) (data not shown), suggesting that
there is more than one, unique copy of
EhPgp5, or that there is a crossreact~on
with another EhPgp gene.
Northern blot expenments showed
the presence of a 4.5-5.0 kb transcript.
exclusively in the drug-resistant tropho-
zoites (clone C2). The transcript was
augmented when trophozoites were
grown in the presence of emetinell.
EhPgp / gene is transcribed constitutively
in clone C2, independently of the drug
concentration in the medium. Interest-
ingly. EhPgp5 IS transcribed only in
mutant trophozoites growlng in the
presence of a high drug concentration
( 2 0 0 ~ ~ ) , while EhPgp2 RNA was not
detected in the mutant nor in the wild-
type trophozo~tes (P. Ayala, op. cit.)
The MDR phenotype in E. h~stolytica
is a consequence of an increased P-glyco-
protein activrty, regulated at the trans-
criptional level. Comprehension of the
regulatory mechanisms underlying the
differential expression of EhPgp genes is
cumntly under study. We have recently
initiated the characterization of the
EhPgp I and EhPgp5 upstream regions.
In EhPgp I a TATA box-like sequence is
not present in the position proposed
for other TATA boxes In E. histolyt'ca
gene+. However. a A T A A T se-
quence is located at -61 bp upstream
from the ATG start codon. In EhPgp5.
a putative TATA box (TATAAA) is
located at -26 bp upstream, similar in
sequence and position to the putatwe
TATA boxes reported for other E. histo-
lytica genes (Fig. 3). In addition, the
Paras~rology Todoy, vol. 1 I, no. 12, 1 995
Fig. 3. The 5' end flanking sequences of several structural genes of Entamoeba histohca.
The putative TATA-box consensus sequences a s well a s the conserved motifs ATTCA
or ATCA at the start of transcription are printed in bold capital letters. Nucleotides
that do not match the consensus sequences are underlined.
Table 2. Comparison of multidrug resistance in mammals and in Entamoeba histolyt~ca
Mutants resistant to more than one drug
Vera~amil reversal of drug resistance
Lower drug accumulGon into cytoplasm
Resistance based up% energy-dependent efflux
of the drugs
Resistance correlates'with P-glycoprotein
(170 kDa) in the plasma membrane
Multiple drug resistance gene encodes for
Expression of 4.5 kb rndr mRNA
Increasing drug resistance correlates with:
Increased expression of rndr gene
Amplification of rndr gene
Multidrug resistance phenotype is conferred
when the rndr gen'4is uansfected into
650bp upstream sequences of both
genes have 53-66% positional identity
with promoter regions of several -&-
tyostel~um drscordeum genes (data not
shown). Transfection experiments cur-
rently in progress will determine if TATA
box position and other putative con-
sensus sequences in the upstream re-
gion are ~nvolved in the control of the
differential expression of EhPgp 1 and
The MDR phenotype in E. hisrolpco
shares several features with the MDR
phenotype described in mammalian
transformed cells: (I ) Trophozoites are
resistant to structurally unrelated drugs;
(2) resistance is reversed by calcium-
channel blockers, such as verapamil;
(3) accumulation of the drug in drug-
resistant cells is lower than it is in the
wild-type cell; (4) several genes with
40-4 1 % homology to rndr human genes
Para~tology Today. vol. 1 I. no. I 2. 1 995
were identified in E. hrstolyt'co tropho-
zoi+.es; and (5) at least two of these
genes are transcribed in drug-resistant
trophozo~tes (Table 2). Transfection ex-
periments will definitively test whether
or not the E. hrstolytica EhPgpl and
EhPgp5 genes are involved in the MDR
phenotype. However. the available data
suggest that there is a strong similarity
among transformed mammalian cells.
E. histolpco and other parasites in the
MDR phenotype. Several common fea-
tures between mammalian transformed
cells and E hrstolpco have been re-
ported24. Both types of cell are invasive
and metastatic. They show a higher ex-
pression of proteolytic enzymes and
other proteins and genes such as onco-
genes, present genomic reanangements25
and are able to present MDR pheno-
type by overexpression of rndr genes.
Although drug resistance in E. histolyt'co
is not yet a serious problem, in this re-
view we have shown the evidence that
MDR is an evolutionarily conserved
mechanism that could be present in the
natural population of this parasite.
We thank &ma Gharabeh for critical read~ng
of th~s manuscript The authors acknowl-
edge John Samuelson for his valuable com-
ments and collaboration on the study of
mukidrug resistance In E h~stolpco. This work
was supported by an International Research
Scholar Award from the Howard Hughes
Medical lnstrtute and by the International
Agency for Atomic Energy.
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1.1. (1 990) Amebros~s:
Cancer Suppl. 70.
Esther Orozco. Gu~ilermo Perez. Consuelo
Gomez and Parnua Ayala are at the Depar-
tomento de Patologia Expenmental. Programa
de Biomedicina Molecular. Centn, de Invesu-
gac~dn y de Gtudios Avanzados del lnstituto
Politecnico Nac~onal (UNVESTAV IPN). A P
14-740, Mexico 07000 DF. Tel: +525 747
7000x5650. Fax: +525 747 7108, mail: