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Nov 13, 2012 - Isoenzyme and ultrastructural characterization of Leishmania tropica axenic amastigotes and promastigotes. Gholam Reza Hatam & Somayeh ...
Isoenzyme and ultrastructural characterization of Leishmania tropica axenic amastigotes and promastigotes Gholam Reza Hatam, Somayeh Bahrami, S. Mostafa Razavi & Ahmad Oryan

Parasitology Research Founded as Zeitschrift für Parasitenkunde ISSN 0932-0113 Volume 112 Number 2 Parasitol Res (2013) 112:643-648 DOI 10.1007/s00436-012-3179-0

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Author's personal copy Parasitol Res (2013) 112:643–648 DOI 10.1007/s00436-012-3179-0

ORIGINAL PAPER

Isoenzyme and ultrastructural characterization of Leishmania tropica axenic amastigotes and promastigotes Gholam Reza Hatam & Somayeh Bahrami & S. Mostafa Razavi & Ahmad Oryan

Received: 5 July 2012 / Accepted: 28 October 2012 / Published online: 13 November 2012 # Springer-Verlag Berlin Heidelberg 2012

Abstract Leishmania tropica is one of the main etiological agents of cutaneous leishmaniasis in Iran. For ultrastructural and isoenzyme study, axenic amastigotes were cultured in a brain–heart infusion medium containing 20 % fetal calf serum, pH4.5, and incubated at 37 °C in 5 % CO2. Different stages of L. tropica revealed the same isoenzyme profiles after comparing four enzyme systems including phosphoglucomutase, 6-phosphogluconate dehydrogenase, malate dehydrogenase, and nucleoside hydrolase II. Different isoenzyme patterns for glucose-phosphate isomerase, glucose6-phosphate dehydrogenase, nucleoside hydrolase I, and malic enzyme enzymic systems were seen; thus, these isoenzyme systems among the eight systems studied were more efficient in characterizing L. tropica amastigotes. The structure of the axenic amastigotes was essentially similar to that of the promastigotes except for some important characteristics G. R. Hatam (*) Basic Sciences in Infectious Diseases Research Center, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran e-mail: [email protected] G. R. Hatam e-mail: [email protected] S. Bahrami : S. M. Razavi Department of Parasitology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran S. Bahrami Department of Parasitology, Faculty of Veterinary Medicine, Shahid Chamran University, Ahvaz, Iran A. Oryan Department of Pathology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran

including the flagellum, flagellar pocket, paraxial rod, and the subpellicular microtubules.

Introduction Leishmania are a group of protozoan parasites which cause a wide range of human diseases from localized self-healing cutaneous lesions to fatal visceral infections (Debrabant et al. 2004). Because of their significant impact on global public health in developing countries, they are listed by the World Health Organization as the etiologic agents of one of the six major tropical diseases (Ghedin et al. 1997). The Leishmania have a digenetic life cycle comprising an extracellular promastigote stage in the insect vector and an intracellular stage in mammalian mononuclear phagocytes (Ghosh et al. 2003). The flagellated promastigotes are released at the site of the sand fly bite and ingested by mammalian macrophages, whereupon they lose their flagellae and transform into amastigotes. The intracellular amastigotes are able to eventually cause clinical manifestations of leishmaniasis (Gantt et al. 2001). Leishmania tropica is one of the causative agents of cutaneous leishmaniasis (CL) which is more resistant to treatment than other species causing CL in the Old World. In addition, it is also occasionally the causative agent of viscerotropic leishmaniasis (VTL) in endemic regions and has been isolated from the bone marrow and spleens of patients with VTL and visceral leishmaniasis (VL) and from dogs with VL (Lemrani et al. 2002). L. tropica may also cause leishmaniasis recidivans that is resistant to treatment, with new lesions appearing at the edge of scar tissue. The geographical distribution of this parasite extends from India throughout central Asia, the Middle East, and Southeast Europe into North and equatorial Africa (Ashford 2000; Jacobson et al. 2003). Epidemics due to L. tropica with

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extensive morbidity have been previously reported in Iran (Sharifi et al. 2011; Zahraei-Ramazani et al. 2007). It has been suggested that rats (Rattus rattus) and rock hyraxes may be the reservoir hosts of this parasite (Nasereddin et al. 2010). The promastigotes and amastigotes of Leishmania species clearly differ both morphologically and on the basis of their bioenergetics, including utilization of enzymes at different stages of the parasite, fatty acids, the enzymes of fatty acid oxidation, and the glycolytic enzyme pathways and glycosomes (Callahan et al. 1997; Bahrami et al. 2011). A better understanding of Leishmania characteristics will hopefully lead to the design of a more effective control program for this zoonotic disease which is one of the major goals of the World Health Organization. The current investigation describes the ultrastructure and isoenzyme patterns of L. tropica in three in vitro phases: logarithmic as procyclic promastigotes, the stationary as metacyclic promastigotes, and the axenic amastigotes as intracellular stages.

Materials and methods

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stained with toluidine blue, and used for checking the desired samples with a light microscope. Ultrathin sections were cut from selected areas, mounted on copper grids, and double stained with uranyl acetate and lead citrate. The sections were examined using a transmission electron microscope (TEM) (Philips CM10; Eindhoven, the Netherlands). Preparation of lysates Ten samples of each stage including 100 ml medium containing 1×107 parasite/ml were prepared. The logarithmic and stationary phases of the organisms were obtained 3 and 10 days after cultivation, respectively. The culture tubes were centrifuged at 2,000×g for 20 min at 4 °C, the supernatant was discarded, and the pelleted organisms were washed three times by resuspension and recentrifugation in cold PBS. An equal volume of hypotonic aqueous solution of enzyme stabilizers (1 mM amino-n-caproic acid, 1 mM dithiotheritol, and 1 mM EDTA, Sigma) was then added and mixed thoroughly. Freeze/thaw cycles were performed five times. The extract was centrifuged at 18,000×g for 30 min at 4 °C, and the supernatants were stored at −70 °C.

Culture of the parasite Enzyme electrophoresis The Leishmania strain used in this study was L. tropica (MHOM/AF/88/KK27). The logarithmic and the stationary promastigotes were grown at 26 °C in brain–heart infusion (BHI) medium plus 10 % heat-inactivated fetal calf serum (FCS), pH7.0, and 1 % of penicillin (50 U/ml) streptomycin (50 μg/ml) solution (Sigma, St. Louis, MO, USA). The logarithmic and stationary phases of organisms were obtained 3 and 10 days after cultivation, respectively. The methods used for in vitro transformation of metacyclic promastigotes to axenic amastigotes were done according to our previous investigation (Bahrami et al. 2011). Therefore, amastigote-like forms were obtained in BHI medium supplemented with 20 % FCS at pH4.5 after 48 h of incubation at 37 °C in the presence of 5 % CO2. To evaluate the morphology of the transformed parasites, the axenic amastigotes were harvested from the culture, centrifuged at 400×g with 50 mM phosphate-buffered saline (PBS), applied to microscope slides, fixed and stained with Giemsa, and examined with an ordinary light microscope (Olympus Optical, NY, model BX60, Tokyo, Japan) at ×1,000. Transmission electron microscopy The promastigotes and axenic amastigotes were fixed in 3 % glutaraldehyde in 0.2 M cacodylate buffer at 4 °C. The samples were postfixed in 1 % osmium tetroxide and dehydrated through graded ethanol and embedded in agar 100 resin. Semithin sections were cut by an ultramicrotome,

Analysis was performed by discontinuous polyacrylamide gel electrophoresis using a 3 % stacking gel and 7.5 % separating gel. The stacking buffer was composed of Tris/HCl (pH06.7), resolving buffer of Tris/HCl (pH08.9), and the tank buffer of Tris/HCl (pH08.3), which ran under a constant current of 2 mA/well. Each stain was tested for eight enzymatic systems, namely malate dehydrogenase (MDH) E.C. 1.1.1.37, phosphoglucomutase (PGM) E.2.7.51, glucose-phosphate isomerase (GPI) E.C.5.3.1.9, nucleoside hydrolase I (NH1) E.C.3.2.2.1, nucleoside hydrolase II (NH2) E.C.3.2.2.1, malic enzyme (ME) E.C.1.1.1.40, 6-phosphogluconate dehydrogenase (6PGD) E.C. 1.1.1.44, and glucose-6-phosphate dehydrogenase (G6PD) E.C. 1.1.1.49. The electrophoretic band developing conditions were as described earlier (Hatam et al. 1999 and 2005).

Results In the Giemsa-stained dry smears, the structure of the axenic amastigote of L. tropica as seen with the light microscope consists of a nucleus and usually a rod-like kinetoplast within a homogeneous mass of cytoplasm, while the extracellular or culture form, in addition to these structures, had an anterior flagellum. The intracellular form was usually round or ovoid measuring 4–5 μm (Fig. 1). Electron microscopy was used to analyze the structural relationships

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Fig. 1 Light micrograph of the L. tropica axenic amastigotes obtained from the BHI medium with pH4.5 at 37 °C in the presence of 5 % CO2

among the promastigotes and the cultured amastigotes. The promastigotes and amastigotes at the ultrastructural level were in general agreement with the vast amount of morphological data available (Pan and Pan 1986; Balanco et al. 1998). Axenic amastigotes differed from the promastigotes in some aspects. The flagellum of promastigotes contained a paraxial rod originating at the axosome level within the flagellar pocket, whereas the flagellum of amastigotes lacks this structure. The flagellar pocket of promastigotes was usually small, whereas amastigotes had a distended reservoir. Subpellicular microtubules of promastigotes terminated at the posterior end, whereas those of amastigotes ended subterminally. Figures 2, 3, 4, and 5 show some of these differences. The logarithmic and stationary promastigotes as well as the axenic amastigotes of L. tropica were tested for eight enzymes using the acrylamide electrophoresis method. All the stages had the same allomorphs (bands of the enzyme activity by electrophoresis) in MDH, NH2, 6PGD, and PGM electrophoresis, but they were not similar in GPI, G6PD, NH1, and ME. The results are shown diagrammatically in Fig. 6. The electrophoretic profiles obtained with soluble extracts of the logarithmic and stationary promastigotes and axenic amastigotes for the eight enzymatic systems are shown in Fig. 6.

Discussion Establishment of the amastigote axenic culture system has substantially advanced the understanding of parasitic protozoa, and in many cases, they can advantageously replace the use of laboratory animals. In vitro propagation permits observation of the parasites under nearly physiological conditions and facilitates screening for antiparasitic compounds (Maser et al. 2002).

Fig. 2 Transmission electron micrograph of L. tropica promastigotes showing different organelles. Abbreviations: a axoneme, er endoplasmic reticulum, f flagellum, g Golgi apparatus, m mitochondrion, n nucleus, s subpellicular microtubules. Scale bar015.3 μm

In present study, in the first step, we prepared the optimum conditions for obtaining large amounts of axenic amastigotes (Bahrami et al. 2011). The axenic parasites have been shown to be similar to the intracellular amastigotes by several criteria, including morphology, virulence, and protein profile (Bates 1993; Pan et al. 1993; Gupta et al. 2001; Teixeira et al. 2002). Albeit it should be considered that axenic amastigotes cannot mimic intracellular amastigotes in absolute terms because of the varied environments, overall, they do closely resemble intracellular amastigotes and are different from promastigotes (Gupta et al. 2001). Due to the small dimensions of the parasite and the limited resolution of the light microscope, relatively little information is available on the fundamental organization of L. tropica. With the greater resolving power of the TEM, several workers have attempted to study its ultrastructure (Zakai et al. 1999). Although the fine structure of Leishmania has been previously studied, few investigations were aimed specifically at differentiating between the ultrastructure of promastigotes and amastigote forms of L. tropica (Pan and Pan, 1986; Balanco et al. 1998). In the present study, the ultrastructure of axenic amastigotes and promastigotes of L. tropica was studied by TEM. The structure of

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Fig. 3 Transmission electron micrograph of L. tropica promastigotes. Note the pointed posterior end, the long flagellum, and terminally subpellicular microtubules. Abbreviations: c carbohydrate droplet, f flagellum, k kinetoplast, l lipid droplet, mi mitochondrion, n nucleus, s subpellicular microtubules. Scale bar024.0 μm

Fig. 4 Ultramicrograph of L. tropica, an amastigote obtained from BHI medium with pH4.5 at 37 °C in the presence of 5 % CO2. Note the greatly dilated flagellar pocket and short flagellum it encloses. The cytoplasm contains electron-dense structures which vary in size. Abbreviations: ed electron-dense structures, er endoplasmic reticulum, fp flagellar pocket, k kinetoplast, mi mitochondrion, n nucleus, pl phagolysosome. Scale bar015.3 μm

the axenic amastigotes was essentially similar to that of the promastigotes except for some important characteristics. For example, in the promastigotes, the flagellum arising at the base of the flagellar pocket and extending free beyond the reservoir contains a paraxial rod which originates at the level of the basal body. The paraxial rod, which consists of a network of microfilaments that runs along the axonemal microtubules of the flagella, has the appearance of a crosshatch paracrystalline structure, but in the amastigotes, the short flagellum that is usually contained within the flagellar pocket lacks a paraxial rod (Figs. 2, 3, 4, and 5). Absence of the paraflagellar rod in the axenic amastigotes of Leishmania braziliensis has been confirmed by western blots using the monoclonal antibody developed against the Trypanosoma cruzi epimastigote paraflagellar structure (Balanco et al. 1998). Hyams (1982) indicated that since only the promastigotes are motile due to the active motion of a long flagellum, the paraxial rod along the axoneme may be important for the vigorous flagellar motility of the organisms. In addition, he noted that the paraxial rod in Euglena spp. had an ATPase activity distinct from that of dynein in the axoneme, thus making it possibly associated with flagellar beating.

The flagellar pocket was usually not distended in the promastigotes but greatly distended in the amastigotes. The plasmalemma was underlined by subpellicular microtubules of 15–25-nm diameter. The subpellicular microtubules end subterminally and terminally in amastigotes and promastigotes, respectively. Pan and Pan (1986) showed that the subterminal subpellicular microtubules in the amastigotes frequently caused depression at the posterior end due to fixation and dehydration procedures. Identification of the Leishmania species is important not only from the epidemiological point of view but has also relevance to clinical illness and management. In many studies, isoenzyme electrophoresis techniques have been used for species identification and also differentiation of various strains of the same species. For example, using this approach, L. tropica has been identified as the main causative agent of the lupoid form of cutaneous leishmaniasis in Iran (Hatam et al. 1999). In Mexico, 93.3 % of the isolates causing localized cutaneous leishmaniasis have been identified by isoenzyme markers as Leishmania mexicana and 6.7 % as L. braziliensis. Kreutzer and Christensen (1980) have characterized different Leishmania species by analyzing 14 isoenzymes,

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using cellulose acetate electrophoresis, and found distinct isoenzyme patterns for ASAT, G6PDH, and 6PGDH among the

different Leishmania species. Shamsuzzamana et al. (2000) examined 11 isoenzymes of which 6GPDH, ASAT, GPI, and PGM showed similar migration bands in isolates from kalaazar patients which were consistent with reference strains of Leishmania donovani (DD8). Hatam et al. (1999) found MDH, NH1, and GPI as the efficient enzymes in characterizing L. tropica, Leishmania major, and Leishmania infantum. Another purpose of the present study was to compare the electromorphs of different stages of the same strain. Several developmental forms exist in the Leishmania life cycle. These forms represent an adaptation to the changing environmental conditions encountered by the parasites within their hosts. Normally, in the insect midgut, the actively dividing, immature, procyclic promastigotes differentiate into nondividing metacyclic forms, which migrate to the thoracic midgut and proboscis. These latter forms have been shown to be the infective stage of the parasite. The virulent metacyclic promastigotes of Leishmania are introduced into the host tissues by the bite of an infected phlebotomine sand fly and are subsequently phagocytosed by the tissue macrophages, where differentiation into the amastigote stage takes place (Barak et al. 2005). These cytodifferentiations involve many changes which are crucial for the pathogenecity and survival of the parasites within the host (Racoosin and Beverley 1997). Different stages of L. tropica revealed the same isoenzyme profiles for the four enzyme systems including PGM, 6PGD, NH2, and MDH. However, different isoenzyme patterns in the GPI, G6PD, NH1, and ME electrophoresis were seen. The enzymes GPI, G6PD, ME, and NH1 were found to be more efficient in characterizing L. tropica amastigotes.

Fig. 6 Photographs of the enzyme profiles for different stages of L. tropica. a GPI pattern; lane 1, L. major logarithmic promastigotes; lane 2, L. tropica axenic amastigotes; lane 3, L. tropica stationary promastigotes; lane 4, L. tropica logarithmic promastigotes. b G6PD pattern; lane 1, L. tropica logarithmic promastigotes; lane 2, L. tropica stationary promastigotes; lane 3, L. tropica axenic amastigotes; lane 4, L.

major logarithmic promastigotes. c NH1 pattern; lane 1, L. tropica axenic amastigotes; lane 2, L. tropica stationary promastigotes; lane 3, L. tropica logarithmic promastigotes; lane 4, L. major logarithmic promastigotes. d Lane 1, L. tropica axenic amastigotes; lane 2, L. tropica stationary promastigotes; lane 3, L. tropica logarithmic promastigotes; lane 4, L. major logarithmic promastigotes

Fig. 5 Ultramicrograph of an axenically cultured amastigote of L. tropica. The flagellum is completely enclosed within the flagellar pocket. Abbreviations: fp flagellar pocket, k kinetoplast, mi mitochondrion, n nucleus, pl phagolysosome. Scale bar017.8 μm

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In comparison, the logarithmic promastigotes, stationary phase, revealed different allomorphs in G6PD, NH1, and ME isoenzyme electrophoresis (Fig. 6). To the best of our knowledge, this is the first study of isoenzymic comparison of various stages of L. tropica. In conclusion, due to the difference in isoenzyme patterns between different stages of the parasite's life cycle, in studies based on characterization and identification of the causative species and the zymodem phenotypes, samples should be prepared from the same stage. Acknowledgments This manuscript was extracted from a part of the Ph.D. thesis of Somayeh Bahrami which was financially supported by the Office of the Vice Chancellor for Research of the Shiraz University of Medical Sciences and the Veterinary School, Shiraz University authorities (grant no. 2698)

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