J. Agrobiotech. Vol 2, 2011, p. 67-71. ©Universiti Sultan Zainal Abidin ISSN 1985 5133
Fatimah H. et al. Surface Morphological Changes of Pathogenic Acanthamoeba spp. Treated with Mahanimbine.
Short Communication Surface Morphological Changes of Pathogenic Acanthamoeba spp. Treated with Mahanimbine Fatimah H. 1,5 , * Nakisah M. A. 1,2 , A. M. Ali 3 and S. Aspollah 1
4
Department of Biological Sciences, Faculty of Science and Technology, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu Darul Iman, MALAYSIA.
2Institute
of Oceanography, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu Darul Iman, MALAYSIA. *
[email protected] 3 Department of Biotechnology, Faculty of Agriculture and Biotechnology, Universiti Sultan Zainal Abidin, Kota Campus, Jalan Sultan Mahmud, 20400 Kuala Terengganu, Terengganu Darul Iman, MALAYSIA. 4 Department of Chemistry, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor Darul Ehsan, MALAYSIA. 5Faculty of Applied Science, Universiti Teknologi MARA, Kuala Terengganu , 21080 Kuala Terengganu, Terengganu Darul Iman, MALAYSIA. ABSTRACT Acanthamoeba spp. are the most common free-living amoebae (FLA) found abundantly in the environment. These opportunistic organisms are causative agents of Acanthamoeba keratitis, a painful eye infection that could result in loss of vision and is commonly associated with users of contact lenses. Two species of Acanthamoeba, namely Acanthamoeba sp. (SW isolate) and A. polyphaga (CCAP 1501/3A) were chosen in order to study the mechanism of action of a plant compound, mahanimbine, against these amoebae. Scanning electron microscopy (SEM) was used to analyze the cellular morphological changes that occurred to the amoebae after treatment with this pure compound at their IC50 concentration. The IC50 value for Acanthamoeba sp. (SW isolate) and A. polyphaga (CCAP 1501/3A) were 7.2 µg/mL and 1.00 µg/mL, respectively. The treatment was carried out for 72 hr at 30 °C and involved culturing the amoebae on cover slips in six-well culture plates, followed by normal processing methods of the amoebae for SEM. The evidences of damage on the trophozoites of amoebae were their rounded forms with cystic appearances and reduction in size. The number of acanthapodia decreased as compared to numerous spine-like acanthapodia as observed on untreated trophozoites. The research findings in the current study revealed that mahanimbine can cause cellular morphological changes especially to the cell shape and number of acanthapodia on the amoeba trophozoites. Keywords: Acanthamoeba, scanning electron microscopy (SEM), plant compounds, Acanthamoeba keratitis, mahanimbine ABSTRAK Acanthamoeba spp. adalah ameba hidup bebas yang terdapat dengan banyak sekali di persekitaran. Organisma bersifat mengambil peluang ini adalah agen penyebab keratitis Acanthamoeba, sejenis jangkitan mata yang boleh menyebabkan kehilangan penglihatan dan biasanya berlaku kepada pengguna kanta lekap. Dua spesis Acanthamoeba, dinamakan sebagai Acanthamoeba sp. (isolat SW) serta
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A. polyphaga (CCAP 1501/3A), telah dipilih untuk kajian mekanisma tindakan satu sebatian tumbuhan iaitu mahanimbin terhadap kedua-dua spesis ameba ini. Mikroskop imbasan elektron (SEM) telah digunakan untuk menganalisis perubahan morfologi sel yang terdapat pada ameba setelah dirawat dengan mahanimbin pada kepekatan IC50 untuk setiap spesis. Nilai IC50 mahanimbin yang diperolehi untuk Acanthamoeba sp. (isolat SW) dan A. polyphaga (CCAP 1501/3A) ialah 7.2 µg/mL dan 1.00 µg/mL, secara berurutan. Rawatan telah dijalankan selama 72 jam pada suhu 30 °C yang melibatkan pengkulturan ameba di atas sisip kaca di dalam piring kultur enam lubang, diikuti dengan kaedah pemprosesan biasa ke atas ameba untuk SEM. Bukti-bukti kerosakan pada sel yang dapat dilihat ialah trofozoit-trofozoit ameba berbentuk bulat dan seperti sista. Saiz trofozoittrofozoit ini juga berkurangan serta mengecut. Bilangan akantapodia juga berkurangan berbanding bilangan akantapodia seperti duri pada trofozoit yang tidak dirawat. Hasil kajian ini menunjukkan mahanimbin boleh menyebabkan perubahan morfologi terutamanya pada bentuk sel dan bilangan akantapodia pada trofozoit-trofozoit ameba. Kata kunci: Acanthamoeba, mikroskopi imbasan elektron (SEM), sebatian tumbuhan, keratitis Acanthamoeba, mahanimbin
INTRODUCTION Diagnosis and management of Acanthamoeba keratitis, infection of the human cornea, remains a difficult problem even though this eye disease has been recognized since 1973 in the USA (Jones et al., 1975). The cases became more apparent worldwide due to the increase in wearing of contact lenses in the 1990s (Illingworth et al., 1995). Acanthamoeba keratitis is frequently reported to occur mostly in users of all types of contact lenses (Illingworth and Cook, 1998). Several cases of keratitis associated with Acanthamoeba have been reported in Malaysia and the first case reported involved a patient with a long history of using contact lenses (Kamel and Norazah, 1995). According to Page (1988), Acanthamoeba is the most common amoeba to be found in soil and water samples. In addition, Acanthamoeba spp. have been known to be resistant to many antibacterial, antifungal and antiviral compounds (Kamel and Norazah, 1995). Mahanimbine, a pure compound isolated from a local plant Murraya koenigii, was tested against two different species of Acanthamoeba (namely Acanthamoeba sp., an environmental isolate from Setiu Wetland, Terengganu, Malaysia; and Acanthamoeba polyphaga, CCAP 1501/3A). In our previous study, the susceptibility of Acanthamoeba against this compound was reported and observed under light microscopy. The results obtained indicated different species of Acanthamoeba exhibited different responses towards the compound. The concentrations of mahanimbine to inhibit the growth of the amoeba trophozoites are very low. Alterations at the cellular morphological level of Acanthamoeba spp. after treatment with mahanimbine indicated that the compound has the potential to be used in the chemotherapy of Acanthamoeba keratitis. Besides, the compounds from the same plant have been reported to have antibacterial and antifungal activities (Rahman and Gray, 2005), as well as antileukemic properties (Roy et al., 2003) which indicates the potential importance of this plant for medicinal use.
MATERIALS AND METHODS Trophozoites of Acanthamoeba were cultured on cover slips in six-well plates for 72 hr in a 30 °C incubator containing culture medium and the compound at their IC50 concentration. The concentrations of mahanimbine used were 7.2 µg/mL for Acanthamoeba sp. (SW isolate), and 2.0 µg/mL for A. polyphaga (CCAP 1501/3A). For fixation, the culture media was replaced with warm 2.5% (v/v) gluteraldehyde in phosphate buffered saline (PBS) for 1 h at 30 °C. The cover slips were washed three times with PBS followed by post-fixation in 1% osmium tetraoxide at room
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temperature for 1 hour. After post-fixation, the cover slips were rinsed with PBS buffer twice and continued with dehydration in a graded series of ethanol to 100%. The specimens on the cover slips were rapidly transferred to a critical-point drying device with liquid carbon dioxide by using Baltec 030 Apparatus. After critical point drying, the cover slips were attached to a 13 mm aluminium stub ducting paint and coated with gold with a JEOL JFC-1600 auto fine coater, ion-sputtering device and examined under scanning electron microscope (JEOL JSM- 6360LA, Analytical SEM). RESULTS Observations under SEM of untreated Acanthamoeba, showed that acanthapodia that arise from the surface of untreated Acanthamoeba trophozoites were mainly for cellular movement since both isolates of Acanthamoeba were grown axenically or in the absence of external live food organisms (Figures 1a and 2a). Acanthamoeba treated with mahanimbine however, showed significant changes after 72 hr treatment when analysed under SEM (Figures 1b and 2b). Collectively, the trophozoites became reduced in size, with cystic appearance, sunken food cups, loss of acanthapodial structure and wrinkled cell surfaces. These observations provide convincing evidences that the compound tested in this study generated alterations of the Acanthamoeba spp’s external morphology. a
b fc
Fig. 1.
Scanning electron micrograph of Acanthamoeba sp. (SW isolate): (a) Untreated trophozoite demonstrates a healthy cell shape indicated by numerous acanthapodia (arrows), (b) Mahanimbine- treated trophozoites with cystic appearance, sunken food cups (fc) and fewer number of acanthapodia (arrows) a
Fig. 2.
fc
b
Scanning electron micrograph showing trophozoites of A. polyphaga (CCAP 1501/3A): (a) untreated trophozoites display normal cell shape with numerous acanthapodia on the cell surface (arrows), (b) Mahanimbine treated-trophozoite shows cystic appearance of cell with shortened and fewer number of acanthapodia (arrows)
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DISCUSSION Many chemical agents and plant extracts were tested in vitro for the susceptibility study of different species, strains and isolates of Acanthamoeba (Schuster and Visvesvara, 2002). However, the mode of action of the therapeutic agents against the amoeba species is poorly known even though many experiments have been done on trophozoites and cysts in cell cytotoxicity studies. Dudley et al. (2005) suggested that in amoeba pathogenesis, Acanthamoeba must be present in the trophozoite stage, as cysts do not bind or infect the corneal epithelial cells. Therefore, this study had focused on the effects of plant compounds towards the infective form of Acanthamoeba spp. Structural alteration of the affected trophozoites, reflected by the membrane and the outer structure of Acanthamoeba, was observed by SEM. Mahanimbine was found to stimulate alteration on tested Acanthamoeba. After 72 hour incubation time, Acanthamoeba exposed to the compound revealed irregularity in shape, became rounded (encystment) and exhibited cell shrinkage; at this stage, the trophozoites are probably metabolically inactive and will be differentiated later into double-walled cysts (ecto- and endo-cyst). In the presence of the compound, the cell environment is not conducive for the amoeba to stay in the vegetative or active form. The number of food cups was reduced, collapsed and shrunk after exposure to the compound. As a result, the uptake of nutrients from the environment was disturbed. Untreated trophozoites showed numerous acanthapodia on the cell surface indicating the amoebae lived in a normal condition. Acanthopodia in treated cells were less and shorter compared to untreated trophozoites, and this affects the cell attachment to the surface. To infect the host cells, cell attachment is essential to occur. Untreated trophozoites of two species of Acanthamoeba showed normal shape with intact plasma membrane, and spine-like acanthapodia were normal. In the amoeba pathogenesis, vegetative cells/trophozoites of Acanthamoeba multiply in the host and establish an infection. A specific stimulus normally induced them to encyst before leaving for a new host. In this study, mahanimbine induced the trophozoites to become less active and begin to encyst. This is the first execution step of the amoeba while facing the unfavorable conditions. In this condition, the amoebae will not be able to infect the host cells. In this study, SEM was used to observe details on the cell membrane of amoebae after treatment with mahanimbine. The results obtained so far are sufficient to interpret the effects of this compound on the amoebae. Most results obtained in this study are very much dependent on the proper techniques of cell processing for SEM. In order to be able to get better quality images of SEM for carrying out the cytotoxic effect study of the compounds on the amoebae, techniques for processing single cell organisms should be further improved and explored. CONCLUSION These present SEM observations indicated that mahanimbine affects the structure of trophozoites in Acanthamoeba spp. The alterations of the cell structure observed were mainly on the cell membrane, food cups, acanthapodia as well as the formation of cyst-like trophozoites. Formation of the cell membrane’s blebbing was also seen on treated trophozoites. The results of this study can differentiate between species susceptibility against mahanimbine compound, as indicated by different changes in their morphology (size and acanthapodia) after treatment with the compound. ACKNOWLEDGEMENTS This work was funded by IRPA RM8 and Fundamental Research Grant Scheme (FRGS), and SEM facilities were provided by the Institute of Oceanography (INOS), Universiti Malaysia Terengganu (UMT), Terengganu Darul Iman, Malaysia.
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J. Agrobiotech. Vol 2, 2011, p. 86-95. ©Universiti Sultan Zainal Abidin ISSN 1985 5133
Hailmi M. S. et al. Plant-Pathogen Interaction between Exserohilum monoceras with Oryza sativa and Echinochloa crus-galli.
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