ABSTRACT

Objective:

The relationship between drug resistance and the expression of hexokinase (HK) has been indicated in leishmaniasis. According to the prolonged treatment period in cutaneous leishmaniasis (CL) patients co-infected with Crithidia in Iran, this study aims to investigate the expression of HK in the proteome of Leishmania major and Crithidia using a proteomic approach.

Methods:

A total of 205 samples were removed from the lesions of patients in Fars province, Iran, for the characterization of L. major and Crithidia using polymerase chain reaction (PCR). After protein extraction, two-dimensional gel electrophoresis was employed for protein separation. Several spots were isolated for HK determination in the proteomes of L. major and Crithidia using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF/TOF MS).

Results:

The PCR results showed 5 positive cases for Crithidia and 96 positive cases for L. major. MALDI TOF/TOF MS indicated HK as a common protein in the proteome of L. major and Crithidia. HK was up-regulated in the Crithidia proteome in comparison with the L. major proteome.

Conclusion:

Since a relationship between HK expression and drug resistance has been indicated in leishmaniasis, the overexpression of HK in Crithidia might be related to the increased duration of the treatment period in CL patients co-infected with Crithidia.

Keywords: Leishmania major, Crithidia, proteomics, hexokinase, drug resistance

INTRODUCTION

Leishmaniasis is one of the endemic diseases in the tropical and subtropical regions of the world which is caused by protozoan parasites of the genus Leishmania. According to the World Health Organization data, endemic leishmaniasis has been reported in 98 countries on five continents. Leishmania major is considered one of the main agents of cutaneous leishmaniasis (CL) in the world. Iran is an endemic region for CL and it is estimated that the annual incidence of CL is 20,000-60,000 in this country (1-4). Crithidia species (flagellate protozoa) belongs to the order of lower Trypanosomatida. Different orders of insects including Diptera, Hemiptera, and Hymenoptera are considered as possible vectors for these flagellated protozoa (5). In comparison to the Leishmania genus (heterogeneous parasites), Crithidia spp. are considered single-host parasites (5).

Recently, the co-infection of L. major and Crithidia has been reported in CL patients in Iran (4). Clinical investigations have indicated the increased duration of the treatment period in CL patients co-infected with Crithidia (4). According to the obtained gene sequencing data, the sequence of the internal transcribed spacer 1 (ITS1) gene in L. major has shown a 10% similarity to Crithidia fasciculata (6-8). The presence of similar genes in L. major and Crithidia genomes suggests the expression of common functional proteins involved in the pathogenicity and treatment of these parasites (6).

In comparison with the genomic data, proteomic data reveal more details regarding the structure and the function of the expressed proteins in the pathogens and diseases. The different aspects of Leishmania parasites have been investigated using proteomic studies in recent years; however, there is no information regarding the proteome of the Crithidia spp. (9-11). The use of proteomics concerning the involved proteins in drug resistance in leishmaniasis provides further information in the treatment of leishmaniasis in the future.

The relationship between drug resistance and the expression of the hexokinase (HK) has been indicated in leishmaniasis. According to the prolonged the treatment period in CL patients co-infected with Crithidia in Iran, this study was conducted to investigate the possible expression of the HK in the proteome of Leishmania major and Crithidia using a proteomic approach and two-dimensional gel electrophoresis (2-DE).

METHODS

Ethical Approval

All human specimens were obtained from the CL patients with the approval of the ethical committee of Shiraz University of Medical Sciences, Shiraz, Iran (IR.SUMS.REC.1395.S1).

Samples Collecting

Totally, in this study, 205 patients were selected. In the first step, the important criterion for patient selection was the observation of cutaneous lesion in individuals. All patients had one or more lesions on their hand, leg, and face. Two hundred five samples were taken from the lesions of the patients from Marvdasht and Kharameh cities, Fas province, Iran. Other significant clinical manifestations were not seen. After sample collecting, all samples were checked for L. major and Crithidia using microscopic, cultivation, and polymerase chain reaction (PCR) methods. The ages of the patients ranged from 6 months to 70 years old.

Samples Cultivation

The obtained samples were transferred to the Novy-MacNeal-Nicolle (NNN) media in sterile conditions. After the growth of the promastigote in NNN, the promastigotes were transferred to RPMI-1640 (Shelmax Company, China) supplemented with 10% (v/v) heat-inactivated fetal calf serum, 100 µg/mL streptomycin and 100 U/mL penicillin at 25 °C and mass-cultivated for obtaining the logarithmic growth phase of the parasites.

Characterization of the L. major and Crithidia

Genus identification

The cultivated promastigotes were characterized by PCR for finding and confirming the presence of Leishmania and Crithidia spp. The positive controls of L. major (MHOM/TM/1973/5ASKH) and Crithidia spp. were provided by the Department of Parasitology and Mycology, Shiraz University of Medical Sciences, Shiraz, Iran (4).

DNA extraction

In the first step, the DNA of the cultivated promastigotes (Leishmania and Crithidia) was extracted using a commercial kit (QIAGEN 28106, USA).

PCR

Specific primers were designed using Kinetoplastid Genomics Resource (TriTrypDB) (http://tritrypdp.org/tritrypdp) and based on the genomic sequence of the Glyceraldehyde-3-phosphate dehydrogenase in L. major and Crithidia. Used primers (Forward: 5’ATGGTCAAAGTGGGCATTAACGG3’ and Reverse: 5’TCCATGTGCGAGGACAACGTGCT3’) were able to characterize the Leishmania and Crithidia spp. (4,12). The amplification program was set to start denaturation at 94 °C for 3 min; followed by 10 cycles each at 95 °C for 30 sec, 62 °C for 30 sec, and 72 °C for 45 sec and a final extension stage at 72 °C for 5 min (4,12).

Agarose-gel electrophoresis

5 µL of loading buffer (3X) (Ampliqon Company, Cat. No. A608304) was added to 10 µL of the final PCR products. Then, PCR products were run to electrophoresis in 1.5% agarose gel. After running, the gels were visualized under ultraviole light with ethidium bromide.

Protein extraction

The cultivated promastigotes of the L. major and Crithidia were centrifuged at 3.000×g at 4 °C for 20 min. The obtained pellets were washed 3 times with PBS (pH: 7.2-7.4), each time at 3.000×g in 4 °C for 10 min. For protein extraction, 10 cc of the extraction buffer (acetone solution containing 10% TCA) was added to the obtained pellets and were kept for 1 h at-20 °C for protein precipitation. In the next step, the tubes were centrifuged at 17.500×g in 4 °C for 15 min. Then, the obtained pellets were dissolved in an acetone solution containing dithiothreitol (DTT) for 1 h at-20 °C for more protein precipitation (13). After centrifugation at 12.000×g at 4 °C, the precipitated proteins were dissolved in a lysis buffer (9.5 M urea, 2 M thiourea, 2% CHAPS, 8% immobilized pH gradient (IPG) buffer (pH 3-10) (GE Healthcare, Uppsala, Sweden) and stored at -70 °C. The protein concentration was measured by using the Bradford method.

2-DE

For each experiment, 250 µgr of extracted protein was developed in the first dimension on the IPG strips (18 cm, pH=3-10, GE healthcare) and the rehydration was done overnight at 50 V. Rehydration solution contained 8 M urea, 2% CHAPS, 50 mM DTT, and 0.5% IPG buffer. After rehydration, isoelectric focusing (IEF) (first dimension) was performed at 50-55.000 Vh using the protean IEF cell (Bio-Rad). Then the strips were equilibrated for 15 min in equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, and 2% SDS) containing 65 mM DTT which was followed by a 15 min incubation in equilibration buffer containing 135 mM iodoacetamide. In the second dimension, the equilibrated strips were sealed to the top of the 12% SDS-PAGE using a twin gel apparatus (Sci Plus/15 mA/gel) until the tracking dye reached the bottom of the gel (14). All experiments were performed in triplicate for confirming the results.

Gel staining, imaging, and image analysis

After fixation of the 2-DE gels with fixation buffer (5% acetic acid, 30% ethanol), the gels were stained with silver staining method (silver stain solution: 12.5 mL of 1 N silver nitrate solution per liter, development solution: 30 g anhydrous potassium carbonate, 250 µL of 37% formaldehyde and 125 µL of 10% thiosulfate solution per liter) (15). After washing, the gels were scanned using GS-800 calibrated densitometer (Bio-Rad) and were analyzed using the progenesis same spot software (version 4.1). Matching and analysis were performed with both automatic and manual methods.

In-gel digestion of protein samples, MS, and database search

In-gel digestion of protein spots and MALDI TOF/TOF MS analysis were done by the metabolomics and proteomics lab technology facility, Department of Biology, University of York. MALDI TOF/TOF MS was performed on a Bruker Autoflex Mass Spectrometer. The punched gels were placed into the wells of a ZipPlate (Montage In-Gel-DigestZP Kit, Millipore). The proteins in the punched gels were destained, digested with trypsin (Promega), extracted, purified on a C18 reverse phase matrix, and eluted in 8 µL of 60% acetonitrile, 0.1% trifluoroacetic acid (TFA). MALDI TOF/TOF MS was done on a BrukerAutoflex Mass Spectrometer (BrukerDaltonics, Bremen). Measurements were done in the reflection mode, using Ion source 1 voltage: 19 kV; ion source 2 voltages: 16.5 kV; reflector voltage 20 kV; lens voltage 8 kV; 40 ns pulse time; 120 ns pulse extraction time; and matrix suppression <500 Da. Using the Xtof analysis software package, version 5.1.5 (BrukerDaltonics), all spectra were analyzed. Using a mix of peptides (Sigma-Aldrich), the mass spectra we calibrated (9,16). Obtained masses generated by MALDI TOF/TOF MS were searched in the MASCOT program. MASCOT scores more than 62 were significant (p<0.05) for obtained proteins.

Statistical Analysis

Statistical analysis was not applicable in this study.

RESULTS

PCR

For identification of the parasites’ genus, PCR was performed on the cultivated samples (promastigotes). PCR amplification and electrophoresis results for L. major and Crithidia have been shown in Figure 1, 2, respectively. In Figure 1, positive control and protein ladder have been indicated as Figure 1 a, b, respectively. Samples that produced a 760 bp band were positive for L. major (Figure 1 c, d,) (e: negative control, f: positive sample with a weak band). In Figure 2, samples that illustrated a band in 858 bp were positive for Crithidia spp. Protein ladder (Figure 2a), positive control (Figure 2 b), positive samples for Crithidia spp. (Figure 2 c, d, e,), and negative control (Figure 2 f) have been represented in Figure 2. Generally, the PCR results showed 5 positive cases of Crithidia spp. and 96 positive cases of CL (L. major).

Figure 1

Figure 2

2-DE and MS

As shown in Figure 3, different spots were matched in this study. The sharp and repeatable spots in different 2-DE experiments were selected for MS analysis (Figure 3). In peptide mass fingerprinting, unknown proteins (peptide profile obtaining from MS data) are compared with theoretical peptide libraries provided from sequences in the different protein databases (17). Accordingly, the results of the MALDI TOF/TOF MS indicated the HK (spot 48) as a common and repeatable protein in the proteome of L. major and Crithidia (Table 1). The score of the obtained protein was significant (>62). The up-regulation of the HK in Crithidia proteome (Figure 4 a) in comparison with L. major proteome (Figure 4 b) has been shown in obtained graph from progenesis same spot software (Figure 4 c). The putative role of the identified protein (HK) was investigated using L. major and Crithidia spp. genome project database (www.genedb.org).

Figure 3

Table 1

Figure 4

DISCUSSION

Co-infection of the Crithidia spp. and Leishmania parasites in CL patients has received attention in recent years in Iran (4). It seems that molecular techniques should be used more frequently in the diagnosis of co-infection of Leishmania and Crithidia spp. in research and clinical fields in the future.

Protein profiling of the microorganisms can be changed due to the routine subculture in vitro conditions. Since sample preparation is an important stage in proteomic studies, for increasing the accuracy of the obtained data, L. major and Crithidia were taken from the CL patients and it was a remarkable issue in our study.

Although the information regarding HK is rare in Crithidia spp., genomic data have revealed the presence of two types of HK (HK1, HK2) on chromosome 21 in Leishmania parasites. The obtained results of our proteomic study confirmed the expression of HK in both proteomes of L. major and Crithidia. The report of the expression of the HK in the proteome of the Crithidia by Alcolea et al. (11) in 2014 confirms our results.

The proteomic studies have elucidated the critical functions of portions involving mitochondrion and metabolic pathways in the pathogenicity, diagnosis, treatment, and vaccination of protozoan parasites (18). HK, as an identified protein in the proteome profile of the Crithidia spp. and L. major belongs to the glycolysis pathway. Glycolysis is one of the important pathways in the metabolism of Leishmania parasites and the associated-glycolysis enzymes are located in glycosome organelle (19).

A study in 1999 indicated the relationship of drug resistance in Leishmania parasite and the expression of the HK gene. Their results showed that the up-regulation of the HK gene increases the drug resistance of the Leishmania parasites (20). The inhibitory effect of anti-leishmanial drugs against HK highlights the important role of HK as a therapeutic target and a possible protein in drug resistance in Leishmania parasites (21,22). According to our results, the up-regulation of the HK in Crithidia proteome in comparison to the L. major proteome might be related to the increased duration of the treatment period with an unknown mechanism in CL patients co-infected with Crithidia.

Evidence shows that the identification of diverse HK-zymodemes leads to distinguishing the different strains of L. major (23). Indication of the HK as a common protein in the proteome of L. major and Crithidia might be suggested for the detection of Leishmania and Crithidia spp. according to the HK-zymodemes profiles. Therefore, the results of the isoenzyme and zymography studies regarding HK might be used in the detection of the co-infection of L. major and Crithidia spp. in CL patients.

CONCLUSION

The report of Crithidia spp. in CL patients in Iran suggests more attention be given to the presence of this co-infection in other parts of the world. This study suggested the expression of the HK in Crithidia spp. as a possible factor in the increased duration of the treatment period in CL patients; however, further investigation such as using HK-inhibitors can reveal more information in this regard.

ACKNOWLEDGEMENTS

This article was extracted from Mohammadreza Karimazar’s Ph.D. thesis which was supported by Shiraz University of Medical Sciences (grant no: 95-7660).

* Ethics

Ethics Committee Approval: All human specimens were obtained from the CL patients with the approval of the Ethical Committee of Shiraz University of Medical Sciences, Shiraz, Iran (IR.SUMS.REC.1395.S1).

Informed Consent: Informed consent was taken from all present patients in this study.

Peer-review: Internally peer-reviewed.

* Authorship Contributions

Concept: M.R.T., M.H.M., Design: M.R.T., M.H.M., Data Collection or Processing: M.K., Q.A., S.R., Analysis or Interpretation: M.K., Q.A., S.R., Literature Search: M.K., S.R., Writing: M.K., Q.A., S.R., M.R.T., M.H.M.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: This study was supported by Shiraz University of Medical Sciences (grant no: 95-7660).

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Expression of Hexokinase in the Proteome Profile of Leishmania major and Crithidia