Evaluation of Organelle Changes in Promastigotes of Unresponsive Leishmania Tropica to Meglumine Antimoniate in Comparison with Sensitive and Standard Isolates by Electron Microscopy.

BACKGROUND: The control of leishmaniasis faces serious challenges because of resistance to the first-line antimonial drugs. We aimed to evaluate the differences in organelle changes of cultivated promastigotes obtained from skin lesions of sensitive and unresponsive isolates to meglumine antimoniate (Glucantime) by electron microscopy.
MATERIAL AND METHODS: This study was done in Bam city, southeastern Iran, in which the incidence of disease has sharply increased since the earthquake in 2003. The samples were taken from 66 patients who were referred to the cutaneous leishmaniasis (CL) treatment center in Bam. A questionnaire was completed for each individual, recording their demographic characteristics and CL status. The scraping smears provided from the edge of active lesions with sterile blades were fixed with methanol, stained by Giemsa, and examined under a compound light microscope for amastigote form simultaneously. To prepare the specimens for transmission electron imaging, promastigotes were centrifuged and resuspened.
RESULTS: Transmission electron microscopic study of the cultivated promastigotes revealed that there were alterations in the organelles and structures of sensitive isolates compared with unresponsive and standard ones. Organelles and structures such as mitochondria, kinetoplast, microtubules, cytoplasmic vacuoles, plasma membrane and vesicles were studied. The alterations such as disintegration of kinetoplast into thin filaments and condensation of kinetoplast DNA core, changes in size, number and location of vesicles and microtubules were observed. We noted intense cytoplasmic vacuolization, and considerable swelling of mitochondria.
CONCLUSION: The significance and relevance of these changes might help understand drug resistance patterns and help localize the best target site for inactivating the organism.

What was known?

Organelle changes in sensitive isolates of Leishmania tropica induced by meglumine antimoniate (Glucantime) are prominent.

Introduction

Leishmaniasis is a vector borne disease, affecting 98 countries and territories around the world which is transmitted by the bite of the female sandfly phlebotomine[1] (WHO, 2010). This disease causes a considerable morbidity and mortality in tropical and sub-tropical countries. Leishmaniasis presents with four major clinical forms: Visceral, cutaneous, diffuse cutaneous and mucocutaneous leishmaniasis. Cutaneous leishmaniasis (CL) is an important public health problem worldwide. 90% of the cases occur in seven countries including Iran, Afghanistan, Peru, Saudi Arabia, Syria, Algeria, and Brazil[2] (Desjeux, 2004). It has been estimated that each year 1-1.5 million new cases of CL and 500,000 cases of visceral leishmaniasis occur. Overall, the prevalence is 12 million and the population at risk is 350 million[2] (Desjeux, 2004). The epidemiology of CL has been significantly changed, mainly because of ecological factors, population displacements and individual risk factors[3] (Desjeux, 2001). Vector and reservoir control methods are difficult due to the diverse ecology of many species of sandfly vectors and reservoirs. The standard drugs, pentavalent antimonials, have been used for decades with severe side-effects. Moreover, prolonged use and resistant forms have been frequently reported[4] (Hadigi et al., 2006).

In Iran two epidemiological forms of CL are present: Zoonotic CL (ZCL) caused by L. major and anthroponotic CL (ACL) caused by Leishmania tropica. The latter species are prevalent in Kerman province[5] (Sherifi et al., 2012 and Nadim et al., 1995) and have developed resistance to antimonials[46] (Hadigi et al., 2006; Pour et al., 2010). In the present study, we characterized the ultrastructure alteration of different cell organelles in Glucantime unresponsive L. tropica isolates compared with standard strains or sensitive isolates. The evaluation of resistant isolates is important because it permits the development of diagnostic tools for early recognition of unresponsiveness, thus avoiding inefficient and often toxic chemotherapy. To the best of our knowledge, there is no previous study that has been done on electron microscopic changes in unresponsive and sensitive isolates of L. tropica in clinical settings.

Material and Methods

Sampling and study area

This study is experimental and was done in the city of Bam during 2008-2012 in Kerman province, south-eastern Iran. Bam is a well-known focus for ACL, where Vaccine trials of single and multiple doses of L. major against L. tropica were conducted (Noazi et al., 2008, 2009).[78] A devastating earthquake had struck Bam in 2003 destroying nearly 90% of medical and social infrastructures. Ever since, the mean annual incidence of ACL has significantly increased in this region (Sharifi et al., 2011).[9] The samples were taken from 66 patients who were referred to the CL treatment center in Bam. A questionnaire was completed for each individual, recording their demographic characteristics and CL status including age, sex, area of residence, number, type and location of CL lesions, and history of the disease. Consent of the patient was obtained and all patients received proper medication free of charge.

Smear preparation and culture

The scraping smears provided from the edge of active lesions with sterile blades were fixed with methanol, stained by Giemsa, and examined under a compound light microscope for amastigote form simultaneously. Skin scrapings were inoculated for mass production of promastigotes into NNN biphasic medium and incubated at 24±1°C for 7 days. Promastigotes were then sub-cultured in RPMI 1640 medium (Gibco) containing 15% of heat-inactivated fetal calf serum (Gibco) with streptomycin (200 mg/ml), and penicillin (200 units/ml) (Gibco), and incubated at 24±1°C. The cultures were checked every 7 days for promastigote growth for up to 28 days (Sharifi et al., 1999).[10] Diagnosis was confirmed by smear and culture media, while identification of species was carried out by nested-PCR, as it was previously performed elsewhere (unpublished data) based on national guidelines. Resistant case was referred to those CL-infected patients who did not heal after receiving two full courses of treatment by Glucantime. On the other hand, a sensitive case was an infected individual who often responded to a full course of Glucantime. The L. tropica standard strain (MHOM/IR/02/Mash2) was provided by the Center for Research and Training in Skin Diseases and Leprosy, Tehran university of Medical Sciences, Tehran, Iran.

Low-Voltage Scanning Transmission Electron Microscopic Study

To prepare the specimens for transmission electron imaging, promastigotes were fixated with 3% cold glutaraldehyde (Merck Chemical Co.) for at least half an hour. The samples were centrifuged and resuspended in 0.1 M sodium cacodylate buffer (pH 7.2). Then, it was again centrifuged, the supernatant was discarded, and resuspended in 3% agar 50°C. Finally, the promastigotes were centrifuged and stored in the refrigerator. The gel was cut and the sedimentation zone was separated. The promastigotes were further fixed in 1% osmium tetroxide for 2 hours, washed with buffer, and dehydrated through series of ethanol concentrations. The samples were embedded in Agar-100 resin and baked at 65°C for 24 hours. Using an ultramicrotome, the blocks were cut into 1 μm sections, then stained by toluidine blue and were examined under a light microscope to confirm the preparation success (Oryan and Mehrabani, 2007).[11] The resin block preparation was done at Shiraz Medical School, Shiraz, Iran. The rest of the electron microscopic sample preparation and imaging were done at Janelia Research Campus, Howard Hughes Medical Institute (HHMI). The imaging at HHMI was performed with a Scanning Transmission Electron Microscope (STEM) detector. This detector was developed utilizing a Carl Zeiss, Ultra 55 Zeiss Schottky Field Emission SEM system, resulting a low voltage (20–30 kV, which is a few times smaller than the voltage used in conventional TEM) high throughput STEM (LVSTEM). The detector is based on a scintillator and photo-multiplier tube technology and the detector's optics’ geometry gets the highest signal to noise ratio in an image (Bolorizadeh, 2009).[12] Furthermore, the column design of the SEM, having no cross over point and having a capability of upgrading to high current modes (tens of nA), made it an ideal candidate for this study.

Electron microscopic studies prior to imaging were prepared with 40 nm Pioloform substrates on 0.5 mm TEM slot grids. Then, 5 nm Carbon was thermally evaporated onto the top surface (the side facing of primary electron beam) of the TEM grids, to eliminate any charging during the electron beam imaging. To make the grids more hydrophilic, the grids were glow discharged (using Nitrogen DC Plasma) prior to laying down the sections on the grid slots. Immediately after the grid substrate preparation, the resin blocks were cut into 40 nm thick sections using an ultramicrotome and laid on the prepared TEM slot grids. These sections were post-stained with 1% osmium tetroxide at room temperature for 30 minutes. All the images in this paper were taken with a 30 keV, 16 nA electron beam at 10 MHz bandwidth (0.1 μsec per pixel) without any image noise reduction and alteration.

Results

Our ultrastructural findings in unresponsive promastigote revealed that the cytoplasmic mitochondrion was mostly circular [Figure 1a]. The cytoplasmic microtubules were seen in the middle of the promastigote [Figure 1a]. The presence of large vacuole close to flagellum and its apparatus were seen. Small vacuole inside the flagellar pocket [Figure 1b] was also observed. The nucleus was bigger than normal and located close to the posterior end of the kinetoplast. The kinetoplast, which is enlarged in Figure 1c, was not degenerated as in the sensitive strain The plasma membrane was smooth with normal thickness [Figure 1a and b].

Figure 1

Ultrathin sections in unresponsive strain of promastigote forms of Leishmania tropica. (a) No change in mitochondrial structure was observed. Microtubules were close to the center compared with standard strains. (b) Nucleus became swollen, nuclear chromatin fragmented and dispensed instead of rimming around nuclear membrane. Small vacuoles were in flagellar pocket compared with the sensitive strain. Kinetoplast was the same as standard. Large vacuole was close to flagellum and nucleus. (c) Enlarged view of Mitochondrion-kinetoplast complex. N, Nucleus; K, Kinetoplast; V, Vesicle; M, Mitochondrion; F, Flagellum; MI, Microtubule; PM, Plasma membrane; V, Vacuole; FP, Flagellar pocket. Horizontal bars are 3 μm while the vertical bar is 1μm

The ultrastructural finding in sensitive promastigote revealed the cytoplasmic mitochondrion that was swollen, elongated and located at a short distance from the plasma membrane of the promastigote [Figure 2a]. Intense vacuolization of flagellar pocket were seen [Figure 2b]. The plasma membrane was smooth with normal thickness [Figure 2a-c]. The nucleus became elongated and the nuclear chromatin was fragmented and dispensed instead of rimming around the nuclear membrane. Its location was at a further distance from the kinetoplast at the posterior end of the organism as compared with the standard or unresponsive isolates as compared with the standard [Figure 3] or unresponsive isolates [Figure 1].

Figure 2

Ultrathin sections of sensitive strain of promastigote forms of Leishmania tropica revealed different ultrastructural alteration induced by treatment with meglumine antimoniate (Glucantime). (a) Nucleus became elongated; nuclear chromatin became fragmented and dispensed instead of rimming around nuclear membrane, mitochondrial swelling, loss of its matrix and cristae. (b) Kinetoplast DNA core was condensed, vacuolization of flagellar pocket. (c) Intense vacuolization (V) of cytoplasm. Intense vesiculation (v) were seen. (d) Enlarged view of the kinetoplast. (e) Enlarged view of the vacuole and vesicles. Bars are 3 μm

Figure 3

Electron microscopy of standard sample showing: (a) Few vesicles scattered throughout the cytoplasm. (b) normal mitochondria, (c) mitochondrion with normal matrix and cristae

The mitochondrion-kinetoplast's complex loss, its matrix and cristae, condensation of DNA core and disintegration into a complex of thin filaments [Figure 2b and d]. Meglumine treatment induced intense vesicles which could be seen around and under the nucleus, mostly at the posterior end of the promastigote [Figure 2c]. Intense vacuolization of promastigote was observed in sensitive patients [Figure 2c and e]. The microtubules were not seen in sensitive strain. The vacuoles which were not seen in the standard stain were visible close to the kinetoplast and flagellum and at the posterior end of the promastigote [Figure 2c].

The ultrastructural findings of standard sample revealed few vesicles which were located throughout the cytoplasm [Figure 3a] and also at the posterior end of the promastigote stage [Figure 3b]. The nucleus had a normal size and was located at a short distance posterior to the kinetoplast. Nuclear chromatin was rimming around the nuclear membrane. Mitochondrion matrix and cristae were normal [Figure 3b and c]. The plasma membrane was rough [Figure 3a]. The cytoplasmic mitochondria were circular and located throughout the promastigote [Figure 3a and b]. The cytoplasmic microtubules were located at a short distance from the cytoplasmic membrane [Figure 3a]. The results discussed for unresponsive, sensitive, and standard promastigotes are given in Table 1.

Table 1

Organelle changes in different forms of L. tropica promastigotes

Discussion

This electron microscopic study was done to evaluate the effect of drugs on L. tropica parasite due to the development of drug resistance. We demonstrated the effect of Glucantime on various L. tropica promastigote organelles to provide a better understanding of drug-resistance. The results indicated that meglumine acted mainly on the mitochondrial physiology of this species which was distributed in branches close to the promastigote membrane and in the region rich in DNA called kinetoplast (de Souza, 2009).[13] Our ultrastructural observation of the mitochondria and kinetoplast showed disintegration into a complex of thin filaments, condensation of DNA core, loss of its matrix and cristae. Similar findings were observed for L. amazonesis with different drugs (Rodrigues et al., 2005, 2007, Mitochondria are essential organelles in energy metabolism, oxidative phosphorylation, and respiration. They also have a vital role in the survival of the organism and are an exceptionally chemotherapeutic target (Job et al., 2012). Another deleterious effect was also observed on the microtubules, they disappeared in sensitive promastigote. The microtubules play important roles in intracellular transport, morphogenesis, and maintenance of cellular shape, and are involved in mitosis (Bell, 1998).[16] The extensive increase in vacuole size and number in sensitive strains indicated the cessation of the cell cycle which is consistent with previous findings by de Macedo and his colleagues (2011).[17] Other ultrastructural alteration was structural changes in the nucleus including nuclear chromatin which was fragmented and dispensed instead of rimming around the nuclear membrane and became elongated. In a study that was done by de Macedo and his colleagues (2011),[17] they observed many changes in mitochondria, nucleus, vesicles and mitochondrion-kinetoplast complexes in L. amazonensis treated with Amiodarone drugs.

A study by Rodrigues and colleagues (2008)[14] showed the effect of drugs on the organelles of L. amazonensis as alteration of mitochondrion such as swelling, loss of cristae and matrix content. They also reported the alteration in kinetoplast and its condensation of DNA core. They also observed a large vacuole close to the flagellar pocket and small ones inside it.

We also observed intense vacuolization of promastigotes, marked swelling of cytoplasmic mitochondrion and its elongation. There were also intense vacuolization in flagellar pocket. All of these alterations in sensitive promastigotes are characteristics of cell death: Necrosis and apoptosis. Similar findings were observed by de Macedo and his colleagues (2011)[17] who showed the effect of Amio drugs on the organelles of L. amazonensis.

It seems that resistant forms get more amastigote like structure and more atrophic but viable organelles close to each other for better hemostasis of the parasites. Kinetoplasts are the most important damageable organelles.

With respect to ultrastructural findings in L. tropica, so far, no study has been done on L. tropica isolates unresponsive to Glucantime. To the best of our knowledge, this study is the only investigation performed on ultrastructure of unresponsive and sensitive isolates.

Conclusion

The significance and relevance of these changes might help understand drug resistance patterns and help localize the best target site for inactivating the organism. Further, ultrastructural studies from skin biopsies of different forms of CL are needed to evaluate in vivo changes of amastigotes.

What is new?

Kinetoplasts are the most important damageable organelles

Microtubules play important roles in surveillance of Leishmania.