Activity of chlorpromazine on nfa1 and Mp2CL5 genes of Naegleria fowleri trophozoites

doi:10.4236/health.2011.33032

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Vol.3, No.3, 166-171 (2011) Health

doi:10.4236/health.2011.33032

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.

ABSTRACT

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

1. INTRODUCTION

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-

tion.

It has been reported that nfa1 and Mp2CL5 genes

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167

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. METERIALS AND METHODS

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-

tion.

2.3. Effect of Single Drugs on N. Fowleri

Growth

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

168

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. RESULTS

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)

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 (×

1,000).

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

169

(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.

4. DISCUSSIONS

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

170

1 2 3 4 5 6 7

(a)

1 2 3 4 5 6 7

(b)

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.

5. CONCLUSIONS

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

6. ACKNOWLEDGMENTS

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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