Vol.3, No.3, 166-171 (2011) Health
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
Activity of chlorpromazine on nfa1 and Mp2CL5 genes
of Naegleria fowleri trophozoites
Supathra Tiewcharoen1, Jundee Rabablert2*, Dusit Worawirunwong3,
Titima Pratumsrikajorn2, Saksun limsangurai2, Virach Junnu1,
1Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand;
2Department of Biology, Faculty of Science, Silpakorn University, Nakorn Phathom, Thailand;
*Corresponding Author: jundee@su.ac.th
3National Institute of Health, Department of Medical Sciences, Nonthaburi, Thailand.
Received 14 January 2011; received 15 February 2011; accepted 28 February 2011.
Amoeba treatment of patients suffering from
primary amoebic meningoencephalitis caused
by Naegleria fowleri has not been successful.
Damaged morphology and effect on genes of N.
fowleri as the result of its initial interaction with
drug may provide clue to the success of treat-
ment. In this study, we investigated the activity
of chlorpromazine compared with amphotericin
B and voriconazole against N.fowleri Khon Kaen
strain using cell based assay and molecular
techniques. Scanning electron and light micro-
graph showed the drug interaction of treated
amoebae with 0.098 ug/ml chlorpromazine was
faster than 0.002 ug/ml amphotericin B and 12.5
ug/ml of voriconazole. The morphological char-
acteristics of treated amoebae with Gomori’s
trichrome stain correlated to the scanning elec-
tron microscope study. The effect of drugs to
nfa1 and Mp2CL5 genes of treated amoebae
found that at 120 min post exposure, chlorpro-
mazine, voriconazole inhibited both genes ex-
cept amphotericin B. Most of drug inhibited nfa1
except fluconazole. The results evaluated that
chlorpromazine was higher potency and rapidly
activity than amphotericin B and voriconazole
against N. fowleri trophozoites.
Keywords: Naegleria fowleri; Chlorpromazine;
Scanning Micrograph; Gene Expression
Naegleria fowleri, amoebo-flagellate free-living, is
the causative agent of primary amebic meningoencepha-
litis (PAM) in humans, a fatal disease of central nervous
system that affects mainly children and young adu lts [1].
The sensitivity of drugs against N. fowleri has been stu d-
ied since 1982 [2]. To date, there is no available drug for
PAM [3]. The activity o f drugs to morphological charac-
teristic of the Naegleria trophozoites provided an over
view understanding of the potential activity. Stains have
been used for classify microorganisms; acid- fast stain,
fluorochrome stain, gram stain Gormori’s trichrome stain
is rapid and cost-effective information for preliminary
diagnosis of infectious diseases. This technique is simple
procedure that produce well-stained smear of intestinal
protozoa, human cell, yeast cells, and artifact material [4].
The descriptive study on surface membrane of the amoe-
bae, scanning electron microscope (SEM) has been es-
tablished on free living amoeba [5]. SEM study on che-
motherapeutic agents against N. fowleri revealed the
obvious cell destruction. Previous study showed the mi-
crographs of N. fowleri HB-1 treated for different period
of time with purify amoebacin A12-B. The results dem-
onstrated the characteristic of cell damaged; abnormal
pseudopodia and the cell rounded up at 10 min then the
amoebae cell membrane ruptured with the release of
cytoplasmic material after 30 min of inoculation [6]. Cy-
topathology of pathogenic and nonpathogenic Naegleria
species; N. australiensis, N. fowleri, N. gruberi, and N.
lovaniensis for cultured rat neuroblastoma cells B-103
and the pathogenicity for B-103 mice were reported. All
four species of Naegleria exhibited surface extensions
termed food cups by electron microscope including ei-
ther N. australiensis or N. lovaniensis with B-103 cells
established the cytopathology involved lysis of the B-103
target cells [7]. SEM has also been studied on cytopatho-
genesis of N. fowleri in humam neuroblastoma SK-N - MC
cells [8]. The drug activity correlated between morpho-
logical characteristic and gene expression of trophic
amoebae should be explained the detail of drug interac-
It has been reported that nfa1 and Mp2CL5 genes
S. T iewcharoen et al. / Health 3 (2011) 166-171
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
were use for distinguish pathogenic N. fowleri in water
and soil samples [9,10] which both of them; lo calized to
the pseudopodia apparatus [11] and a unique membrane
respectively [12,13]. In additio n the function of nfa1 and
Mp2CL5 genes were responsible for ingestion of cells
during feeding and cells proliferation [14]. Many inves-
tigators reported the effect of drugs against N. fowleri
using in vitro and in vivo [15,16]. However, no report
has been studied the effect of drugs against morphology
and virulent genes of N. fowleri. In present study, we
focus on series morphological damage as well as effect
of gene expression of treated amoebae. The purpose of
this studies was to explore the effect of chlorpromazine
and voriconazole which were compared with ampho-
tericin B against N. fowleri in both morphogenesis and
gene expression. This study should be either initially
demonstrated on a series destruction of trophic amoebae
or inhibited pathogenic gene regulation.
2.1. Amoebae Cultivation
Cultivatio n of free liv ing N. fowleri (Khon-Kaen strain)
trophozoites was cultured in 75—cm2 tissue culture flask
containing Nelson’s medium supplemented with 5% heat
inactivated fetal calf serum (FCS) without antibiotics at
37˚C. The trophozoite pellet was harvested and kept at
4˚C for 10 min, then centr ifuged at 5,00 0 × g fo r 10 min.
The pellets were resuspended in 5 ml fresh culture me-
dium and the number of viable cells in each preparation
was determined by using 0.4% trypan blue exclusion
method [17].
2.2. Pharmacological Agents
Powders of amphotericin B (Advanced Remedies Pvt,
India), artesunate (Gulin Pharmaceutical, Guangxi, China),
azithromycin (Ben Venue Laboratories, Ohio, USA) , and
voriconazole, (Vfend (R); Roerig/Pfizer Inc., NewYork,
NY, USA) were dissolved in distilled water, as manu-
facture’s instruction. Chlorpromazine (aqueous form,
Government Pharmaceutical Organization, Bangkok,
Thailand) and fluconazole (aqueous form Siam Bheasach,
Bangkok, Thailand) were kept as manufacture’s instruc-
2.3. Effect of Single Drugs on N. Fowleri
The trophozoites (5 × 106 cells/ml) were suspended in
Nelson’s mediu m in the presence or the absence of drugs
at 10% of IC50, duplicates [13]. The samples were then
incubated at 37˚C for use in this experiment. Ampho-
tericin B 0.002 ug/ml, voriconazole 12.56 ug/ml, flu-
conazole 3.19 ug/ml, azithromycin 0.67 ug/ml, artesu-
nate 0.39 ug/ml, and chlorpromazine 0.098 ug/ml against
amoebae trophozoites were performed in 1.5 ml centri-
fuges tubes (Corning, USA) at 37. All experiments
were repeated three times. At the indicated times, tro-
phozoites were harvested and centrifuged at 5 000 × g
for 10 min. The pellet was washed twice with 0.9%
normal saline to stop reaction and centrifuged at 5 000 g
for 10 min. The number of total cells was counted using
Trypan blue exclusion method and observed under light
microscope. The morphology changing of cells was
stained with Gormori’s trichrome and detected under
light microscope as well as the ultrastructure surface
membrane was investigated by scanning electron micro-
scope. The remaining cell pellets were frozen at –80
for detection of gene expression. The effect of drugs in
our experiment was derived from a previous report on
these agents on N. fowleri.
2.4. Morphological Study
Untreated and treated trophozoites were smeared,
fixed and stained with Gormori’s trichrome staining, as
previously described [18]. Morphological changes of the
amoeba were observed under light microscope at 1, 3, 5
h after exposure. Untreated and treated trophozoites were
also observed by scanning electron microscope. Briefly,
the cells were pre-warmed 2.5% glutaraldehyde in 0.1 M
PBS, pH 7.3 at 4 for 3 h and post fixed with 1% os-
mium tetroxide (OsO4) in 0.1 M PBS for 2 hr. After that,
the specimens were dehydrated in a graded series of
ethanol, critical point dried, mounted on to the metal
stubs and coated with gold/palladium. Finally the speci-
mens were ex amined and photograph ed under SEM (Hi-
tachi S – 510) at an accelerating voltage of 25 kV. [8]
2.5. Reverse Transcriptase Polymerase
Chain Reaction (RT-PCR)
Total RNA of the untreated and treated cell pellets at
indicated times were extracted by Tri Reagent No TR
118, as the manufacture described. Total RNA of the un-
treated and treated cell pellets were evaluated by RT-PCR
using primers of nfa1 [12] and Mp2CL5 [13] were fol-
lowed as previously described. One µg of total RNA fro m
each individual sample was used as a template to synthe-
size first strand cDNA using Maxime RT PreMix Kit
(iNtRON Biotechnology, Korea), following manufac-
turer ’s instruction. Briefly, template RNA and RNase-fre e
water were added into the MaximeRT PreMix tubes
(Random primer) to a total volume of 20 µl, duplicate.
The cDNA reaction was performed at 45˚C for 60 min
and at 95˚C for 5 min, respectively. The amplification
was carried out in a 20 µl total reaction volume by using
S. T iewcharoen et al. / Health 3 (2011) 166-171
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
Maxime PCR PreMix (iNtRON Biotechnology) with 50
pmol of forward and reverse primers and 20 µl of tem-
plate. The thermal profile of PCR was 94˚C for 5 min,
followed by 35 cycles of 94˚C for 30 s, 55˚C 148 for 30
s, and 72˚C for 45 s and a final extension cycle at 72˚C
for 5 min using Thermal cycler (DNA Engine® & Peltier
Thermal Cyclers, PCT-200, Bio-Rad, USA). Agarose gel
electrophoresis PCR products were subjected to 1.5%
agarose Trisborate-EDTA gel electrophoresis at 100 V
for 30 min. Gels were stained with ethidium bromide and
visualization on a UV Tran illuminator at 260 nm.
2.6. Statistical Analysis
Results were expressed as the mean ± standard devia-
tion error from three independent experiments carried
out in tripli cate. Student’s t-test and the P-values of <0.05
was used to determine the level of significance.
3.1. Light Microscopic Study
Morphological changes of N. fowleri amoeba in the
absence or presence of amphotericin B, voriconazole,
fluconazole, artesunate, azithromicin, and chlorpromaz-
ine were observed. Untreated trophozoites was stained
with Gomori’s trichrome staining and observed under
light microscope for 5 h post-treatment. The results re-
vealed that viable trophozoites contained homogeneous
green cytoplasm. Large nucleolus was located in central
of nucleus and surrounded with completed nuclear mem-
brane (Figure 1(a)). Most treated amoebae were clumped
with course and fine hyperpigment granules. Tropho-
zoites with 0.002 ug/ml amphotericin B showed cracking
of surface membrane (Figure 1(b)) whereas trophozoites
with 12.56 ug/ml voriconazole had numerous small dense
granules inside cytoplasm but not observed nuclear mem-
brane (Figure 1(c)). Amoebae trophozoites with 0.098
ug/ml chlorpromazine showed increasing number of the
vacuoles and developing sinus from fusion of food vacu-
ole (Figure 1(d)). The other drugs showed slightly de-
structive morphology of trophozoites at indicated times.
3.2. Scanning Electron Microscope Study
SEM micrograph of trophozoites demonstrated wrin-
kle, sucker-like apparatus and binary fission (Figure
2(a)). Treated amoebae with amphotericin B showed sub-
sequently decreasing of surface membrane, sucker–like
apparatus, and bi nary fission duri n g 1- 3 h p ost treatment.
Amphotericin B destroyed surface membrane and de-
creased a number of sucker apparatus, but increased wrin-
kle edema at 3 h post-treatment (Figures 2(b)-(d)). Chlor-
promazine rapidly increased bleb formation at 1 h (Fig-
(a) (b)
(c) (d)
Figure 1. Light micrographs of N. fowleri Khon Kaen tropho-
zoites with Gomori’s trichrome stain in Nelson‘s media at 1 h
showed binary fission and the cytoplasm was homogeneous
green content. The vesicular nucleus showed normal charac-
teristics (arrow) (a). The treated amoebae with 0.002 ug/ml
amphotericin B showed cracking of surface membrane while
vesicular nucleus was normal at 1 h (arrow) (b). At 3 h, vori-
conazole (12.57 ug/ml)-treated amoeba, the cytoplasm was
small dense granule inside and the nuclear membrane disap-
peared (c). At 0.098 ug/ml chlorpromazine, the small tropho-
zoites was found with the appearance of dominant large size
food vacuoles and the wall of a few food vacuoles were broken
and merged into sinus (arrow) at 1h (d). bf-binary fission, fv-
food vacuole, sm-surface membrane, vn-vesicular nucleus (×
ure 2(e)), and rough wrinkle (Figure 2(f)). However,
chlorpromazine did not inhibit binary fission of the
amoebae at 5 h post treatment. (Figure 2(g)). Slightly
different morphology of N. fowleri trophozoites after
treatment with fluconazole, voriconazole, artesunate and
azithromicin compared with untreated trophozoites was
observed at indicated times (data not shown). It was in-
dicating that patterns of drug panel against N. fowleri
were demonstrated by scanning electron microscope.
3.3. Effects of Drugs on Naegleria
Trophozoites at Gene Levels
Generally, the expression of nfa1 (360 bp) and Mp2CL5
genes (110 bp) of trophozoites were observed during
proliferation growth. The activities of amphotericin B,
voriconazole, fluconazole, artesunate, azithromycin, or
chlorpromazine at 1/10 IC 50 against nfa1 and Mp2CL5
genes of trophozoites were observed at 15, 30, 45, 60
and 120 min, respectively, using RT-PCR . During 15-60
min, we found inhibition of nfa1 genes after treatment
with amphotericin B, voriconazole, fluconazole, azithro-
S. T iewcharoen et al. / Health 3 (2011) 166-171
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
(a) (b) (c)
(d) (e) (f) (g)
Figure 2. Scanning electron micrograph of N. fowleri trophozoite in the absence (a) or presence of amphotericin B (0.002 ug/ml)
(b)-(d) or chlorpromazine at 0.098 ug/ml (e-g). Untreated trophozoites showed normal characteristics; equal binary fission and sucker
apparatus (arrow) (a). Amphotericin B-treated amoebae showed sucker apparatus at 1 h (b). Treated amoebae showed the destructive
surface membrane started at 3 h; disappearance of sucker apparatus and edematous wrinkles surface (c). The series damage continued
until wrinkles surface was not found (d). Chlorpromazine -treated amoebae showed the surface membrane was rapidly damage and
the bleb formations were found (arrow) at 1 h (e). The destruction of amoebae cell was continued; sucker and pseudopodia formation
were disappeared including the surface membrane showed edematous change at 3h (arrow) (f). The unequal binary fission of the
amoeba was seen at 5 h (arrow) (g). Bars represent 10 μm: b-bleb formation, bf-binary fission, sa-sucker apparatus, sm-surface mem-
brane, vn-vesicular nucleus.
mycin, artesunate, and chlorpromazine (data not shown).
Furthermore, all drugs, except fluconazole inhibited nfa1
genes at 120 min (Figure 3(a)). During 15-30 min, all
drugs inhibited mp2c15 gene expression of trophozoites
(data not shown). From 30 min to 120 min, voriconazole,
fluconazole, and chlorpromazine also inhibited Mp2CL5
gene expression (Figure 3(b)), there by amphotericin B,
azithromycin, and artesunate did not inhibit Mp2CL5
gene. It was indicating that the activities of drugs against
N. fowleri trophozoites were time-dependent manner.
PAM is a severe infection of human that typically
leads to death during 1 to 2 weeks from the onset of
symptoms [19]. Amphotericin B is only drug of choice
for PAM treatment [1]. However, it was a very toxic
antibiotic and it caused renal toxicity, electrolyte distur-
bances, hematopoietic effects, and other organ damage,
as well as chills, fever, nausea, vomiting, and headache
[20]. In our study, we investigated the patterns of drugs
against the trophozoites using light microscope and
scanning electron microscope. Furthermore, we studied
the activities of drugs against viru lent nfa1 and Mp 2CL5
gene expression. This is the first report which demon-
strated the characterization activity of drugs against
amoeba trophozoites in both cellular and molecular lev-
els. In the present study, light micrographs of N. fowleri
Khon Kaen trophozoites showed binary fission and
showed normal characteristics of the vesicular nucleus.
SEM micrograph of trophozoites demonstrated wrinkle,
sucker-like apparatus and binary fission. Morphological
characteristic of N. fowleri in this study was consistent
with previous reports [8,21]. Morphology changing of
trophozoites after drug treatment was revealed. Among
these drugs, we found that the activity of chlorpromazine
was higher potency than amphotericin B and voricona-
zole due to increasing number of the vacuoles, bleb for-
mation and rough wrinkle at 1 h. As previously de-
scribed, our study correlated to the mechanism of chlor-
promazine that acted on lipophilic action which effected
on the plasma membrane and reflected sensitivity of
amoeba calcium regulatory protein. Many researches
revealed that nfa1 and Mp2CL5 genes were found only
pathogenic N. fowleri [10,11]. Nfa1 protein expressed
from nfa1 11 gene was located in pseudopodia and
around food vacuole [22]. Nfa1 protein was specifically
localized food cups which are involved in phagocytic
activity [11]. In this study, we found that most of drugs
inhibited nfa1 gene which represented that the amoebae
should be loss of phagocytic activity Reve iller et al., de-
monstrated that the expression of native Mp2CL5 protein
in N. flowleri appeared to be growth phase regul at ed [12].
The increased expression of the protein in the stationary
phase of growth when the cells are experiencing nutrient
S. T iewcharoen et al. / Health 3 (2011) 166-171
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
1 2 3 4 5 6 7
1 2 3 4 5 6 7
Figure 3. The nfa1 (a) and Mp2CL5 (b) genes of N. fowleri
trophozoites expressed in the absence or presence of drugs at
120 min post-treatment. Untreated N. fowleri (lane 1). N. fowl-
eri treated with amphotericin B (lane 2), voriconazole (lane 3),
fluconazole (lane 4), azithromycin (lane 5), artesunate (lane 6),
and chlorpromazine (lane 7), respectively.
deprivation is consistent with our hypothesis that the
protein may be involved in nutrient or environment
sensing. In our study, we found that chlorpromazine and
voriconazole inhibited Mp2CL5 gene, suggesting that
activity of both drugs invo lved losing of cell recogn ition ,
sensing of environment and growth of amoeba. However,
previously our report showed that at MIC100 ampho-
tericin B and chlorpromazine inhibited trophic amoeba
in 3 days but voriconazole did not [15]. Therefore,
chlorpromazine should be proposed for treatment p ri mary
amoebic meningoencephalitis.
Our studies presented the correlation activity of drugs
at ultrastructure and gene expressions of N. fowleri tro-
phozoites. Chlorpromazine was rapidly damage surface
membrane due to bleb formation occurred at 1 hour
whereas the activity of amphotericin B was slow re-
sponse at the same time in contrast it showed completely
destruction during 5 hours. However both drugs could
inhibit nfa1 and Mp2CL5 gene of N. fowleri
We are thank Associate Professor Dr. Darawan Wana-
chiwanawin, head of Department of Parasitology, Fac-
ulty of Medicine, Siriraj hospital, Mah idol University for
providing the research facility. We also thank to Dr.
Pathom awanpanyalert Director, National Institute of
Health, Department of Medical Sciences. This work was
supported by with Grant Number 017(II)50 from Faculty
of Medicine, Siriraj Hospital, Mahidol University Foun-
dation in 2007-2009 and partial fund from Grant Num-
ber RGP 2551/01 from Department of Biology, Faculty
of Science, Silpakorn University at Sanamchan Palace,
Nakhon Pathom, Thailand.
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