What’s Known

Leishmania tropica and Leishmania major cause cutaneous leishmaniasis in Iran and many other countries. Metabolomics is a powerful tool to identify the metabolite profile and differentially expressed metabolites and can help gain further information on the metabolic pathways between species, the presentation of potential drug targets, and host-pathogen interactions. No data exist on comparative metabolomics between the Iranian clinical isolates of Leishmania tropica and Leishmania major.

What’s New

Our data showed markedly different metabolic patterns and their biochemical pathways for Leishmania tropica and Leishmania major. Betaine metabolism, ammonia recycling, and the metabolism of methionine, asparagine, and aspartate were the most important metabolomic pathways between the two species. Significant metabolites confer greater insights into novel potential drug targets and how pathogens adapt to their hosts.

Introduction

Leishmaniasis is a parasitic and vector-borne disease created by various Leishmania species (L. spp). Cutaneous leishmaniasis (CL) is regarded as a major public health concern in the Middle East, including Iran. ¹ The Leishmania parasite has different two stages including infective metacyclic promastigote forms inside the sand fly midgut, which is transmitted during the blood feeding, by the bite of phlebotomine sand flies into a vertebrate (mammalian) host. In the vertebrate hosts, metacyclic promastigotes are rapidly swallowed by macrophages and converted to amastigotes. ^{2 , 3} CL is the most common form of leishmaniasis; it is endemic in more than half of the provinces of Iran. ^{1 , 4} Leishmania tropica (L. tropica) and Leishmania major (L. major) cause CL in Iran; they represent the two main forms of CL: anthroponotic (dry) CL and zoonotic (wet) CL, respectively. ¹ The recent years have witnessed a rise in the number of CL cases in different parts of Iran. ¹

“Omics” approaches, including proteomics and metabolomics, have gained much attention in the recent years. ^{3 , 5} Metabolomics is an instance of such modern technologies and is capable of detecting and quantifying metabolites in specific samples. Thus, it provides a global picture in time of the metabolic changes that occur during the course of a disease. ³ Metabolomics enjoys better dynamism than both genomics and proteomics, which enables to detect metabolic changes associated with different physiological sates in a shorter time frame. ⁶ There are multiple changes in biochemical and metabolite pathways in the different stages of L. spp, ³ including L. tropica and L. major, which are responsible for the creation of the two different clinical forms of cutaneous ulcer. A thorough understanding of these differences in the life cycle of the parasite and the identification of the metabolites related to these biochemical dissimilarities that play an important role not only in the prevention and treatment of leishmaniasis but also in the prediction of new potential drugs against it.

For all the metabolomics-based studies performed on L. spp to assess their drug resistance mechanisms, metabolomics, and stage-specific metabolic profiling, ^{3 , 7} there is a dearth of data on comparative metabolomics between the Iranian clinical field isolates of the parasites. We, accordingly, performed the present study to identify differentially expressed metabolites between the Iranian isolates of L. major and L. tropica via the analytical platform of proton nuclear magnetic resonance (¹H-NMR).

Materials and Methods

Patients and Parasite Species Identification

Iranian clinical field isolates were obtained from the cutaneous lesions of seven patients infected with L. major and nine patients infected with L. tropica who referred to laboratories in Gonbad, Golestan Province, and Bam, Kerman Province, for parasitological diagnoses between 2017 and 2018. All the subjects were new cases, and had not been on medication. The study protocol was approved by the Ethics Committee of Shahid Beheshti University of Medical Sciences (Code No: IR.SBMU.REC.1398.086), and written informed consent was obtained from all the participants.

Samples (smears) were prepared through sampling the border of the skin lesions of each case with clinically suspected CL. The samples were then stained with Giemsa stain and examined for the amastigote stage by light microscope (ZEISS, Germany). Positive skin scrapings were aseptically inoculated into tubes containing the Novy-MacNeal-Nicolle medium (NNN). The isolates were identified via the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) technique, in which the internal transcribed-spacer-1 (ITS1) region of the parasites’ ribosomal-RNA gene was amplified, followed by the resulting amplicons digested by the HaeIII enzyme (Fermentas, Leon-Rot, Germany). PCR was conducted through the use of a forward primer (5’-CTGGATCATTTTCCGATG-3’), and a reverse primer (5’-TGATACCACTTATCGCACTT-3’). After the use of the restriction enzyme, the banding patterns of the isolates were obtained in comparison with the molecular profiles of the World Health Organization (WHO) reference strains of L. tropica (MHOM/IR/02/MHOM/R), and L. major (MRHO/IR/75/ER).

Preparation of Metacyclic Promastigotes

The promastigotes were initially grown on the NNN medium and for mass culture. The parasites were prepared in the RPMI 1640 medium (Gibco, Germany), supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Gibco, Germany), and Pen-Strep (100 U/mL of penicillin and 100 μg/mL of streptomycin; Gibco, Germany) at 24 to 25 °C. The promastigotes were cultured with repeated medium refreshment every three to five days until the number of parasites reached approximately 2×10⁷ cells/mL by Neubauer chamber counting (Marienfeld, Germany). The culture was incubated without adding the fresh medium for 10 (range=8–12) days, and then the metacyclic promastigotes (the stationary phase) were evaluated using both the morphometric analysis and the agglutination assay by lectin. ^{8 , 9} The cell body size and flagellum lengths for 300 parasites in each day were measured, and the percentage of metacyclic forms was calculated. (The promastigotes whose flagellum/body length ratio was ≥2 were considered metacyclic forms, as described by da Silva and colleagues. ⁸ ) To ensure the collection of purified metacyclic promastigotes, we also performed the agglutination assay with peanut agglutinin (PNA) (Sigma, CA, USA). ⁹ Briefly, we added PNA to phosphate-buffered saline (PBS)-washed metacyclic promastigotes to a final concentration of 30 µg/mL for 2×10⁷ cells/mL. After 30 minutes at room temperature, the separation of PNA- cells (metacyclic promastigotes) from PNA+ cells (procyclic promastigotes) was performed by centrifugation at 200 g for five minutes. The non-agglutinated metacyclic promastigotes were then harvested from the supernatants. Stationary phase L. major and L. tropica promastigotes were washed at least three successive times in sterile PBS (pH 7.4) to remove the culture medium compounds, and collected by centrifugation at 3500 g at 4 °C for 20 minutes. The practical conditions and processes/steps for both parasite species examined were thoroughly identical.

Cell Extraction and Proton Nuclear Magnetic Resonance (1H-NMR) Spectroscopy

Cell disruption and metabolite extraction were performed according to Gupta and others, ¹⁰ with slight modifications. Child 1.8 M perchloric acid, Merck, Germany, (800 μL) was added to the cell suspension, and then each sample was vortexed and sonicated for five minutes at 4 °C. The lysed cells were centrifuged at 12000g for 10 minutes at 4 °C, and the pH of each supernatant was adjusted to 6.8 with potassium hydroxide (Merck, Germany). The supernatant was kept on ice for one hour to allow the precipitation of potassium perchlorate and centrifuged again as above. The lysate supernatant was transferred into new Eppendorf tubes (Sigma Aldrich, Germany). For ¹H-NMR spectroscopy, D2O (20%) with trimethylsilyl propionate (TSP, 0.25 mM) (Sigma, CA, USA), as the NMR chemical shift reference (as an internal standard), was added to each sample, and mixed by vortexing. The samples were spun down at 12000g for 15 minutes at 4 °C, and then 550 μL of the supernatant was transferred into 5-mm NMR tubes (Sigma Aldrich, Germany). All the experiments were performed using a Bruker AVANCE 400 MHz (Bruker, Germany), equipped with a 5-mm probe at 298 K. The Carr-Purcell Meiboom-Gill (CPMG) spin-echo pulse sequencing was used to remove macromolecule signals. The water impact on the baseline was minimized by irradiating the water frequency during the relaxation delay of two seconds and a mixing time of 0.1 seconds. ¹H-NMR spectra were acquired in 150 scans per sample, at 298 K, with an acquisition time of 3.0 seconds and a spectral width of 8389.26 Hz.

Data Processing and Statistical Analysis

The ¹H-NMR spectra were processed using ProMatab in the MATLAB (version 7.8.0.347) environment, and necessary corrections were made on the spectra. The spectra were referenced to the TSP standard at 0.0 ppm. Chemical shifts between 0.2 and 10 ppm were normalized and subjected to spectral binning in 0.01 ppm. The spectral region affected by the water-related signal was removed in the range of 4.54 to 5.0 ppm. The 2D data matrix obtained from ¹H-NMR was 16 at 813, comprising the number of the studied samples (nine samples of L. tropica and seven samples of L. major) and bins, respectively.

Multivariate statistical analyses, including the unsupervised principal component analysis (PCA) and the supervised orthogonal projections to latent structures (OPLS) discriminant analysis, were applied to identify the most significant relevant metabolites between L. tropica and L. major. PCA was applied to detect similarities or trends in different samples, as well as to reveal the outliers and relationships that existed between the observations. The OPLS discriminant analysis was employed to maximize the covariance between the measured data and to compare the two classes of samples. The PCA and OPLS diagrams were plotted in the R software (Version 3.3.2). In each comparison, the variable influence in projection (VIP) was used to find the most significant discriminating metabolites. Metabolites with a VIP of greater than 1 and a P value of less than 0.05 were marked as significant variables.

Metabolite Identification and Pathway Analysis

The metabolites were identified using metabolite validated databases such as the Biological Magnetic Resonance Data Bank (BMRB)11 and the Human Metabolome Database (HMDB).12 Finally, the significantly changed metabolites were used to determine the most important pathway differences between the two L. spp. The pathway enrichment analysis was performed using the MetaboAnalyst server.13 MetaboAnalyst (www.metaboanalyst.ca) is a web server for metabolomic data analysis and interpretation.

Results

Diagnosis of Parasite Species

The PCR products of both positive specimens and standard strains with approximately 350 bp were digested by the HaeIII enzyme. The reference strain on agarose gel exhibited two bands (135 bp and 215 bp) that corresponded to L. major and two bands (57bp and 185bp) corresponded to L. tropica (figure 1).

Figure 1. Results of the restriction fragment length polymorphism patterns of ITS1-rDNA amplicons digestion with the HaeIII (BsuRI) enzyme for the Leishmania isolates are illustrated. Lane 1 and 2: Leishmania tropica sample; Lane 3 and 4: Leishmania major sample; Lane 5: standard strain of Leishmania major (MHOM/IR/75/ER); Lane 6: standard strain of Leishmania tropica (MHOM/IR/02/MHOM/R); Lane 7: marker 50 bp

Discrimination between L. major and L. tropica Using Multivariate Analysis

Multivariate analyses were performed on the result matrix to find metabolites, that mostly discriminated L. major from L. tropica. The values of the first two components in the PCA with the highest role in separating the two groups (L. major and L. tropica) in in vitro conditions were 92.9% (PC1) and 4% (PC2) (figure 2). The score plot based on the PCA analysis showed that the two groups were well-separated based on the first two dimensions of the analysis. Additionally, all the data were within the 95% confidence interval, and no outlier data were observed. The OPLS discriminant analysis is a supervised method for finding a combination based on existing variables with the most variance between the groups. The OPLS discriminant analysis separated L. major and L. tropica clusters, which is visualized in score plots in figure 3.

Figure 2. Scatter score plot of the principal component analysis displays the discrimination between the Iranian isolates of Leishmania major (○) and Leishmania tropica (∆). Principal component 1 (Dim1) and principal component 2 (Dim2) explain 92.9% and 4.0% of the total variance, respectively.

Figure 3.Leishmania tropica

The OPLS model is validated by R² (coefficient of determination) and Q² (cross-validated R²), which was performed in the present study via the seven-fold cross-validation measure. All the models had Q² values of greater than 0.4 with the intercept of less than zero, which demonstrated that the models were validated. The R²X, R²Y, Q²Y, and RMSE values of the OPLS model were 0.983, 0.595, 0.566, and 0.355, respectively (figure 3), which demonstrated the model’s good predictive ability.

Metabolite Identification of Two Leishmania Species and Pathway Analysis

The differentially expressed metabolites were determined among the metacyclic promastigotes of the Iranian isolates of L. major and L. tropica, and the spectral bins of the highest importance according to VIP values were selected. Twenty-four significantly differentially expressed metabolites were identified among the metacyclic promastigotes of the L. major and L. tropica samples (P≤0.0152). The details of the metabolite alterations that reached statistical significance are indicated in table 1.

The metabolite set enrichment analysis of biochemical pathways was performed using the MetaboAnalyst database. In this analysis, the significantly differentially expressed metabolites between L. tropica and L. major were measured relative to the predefined biochemical pathway sets in the database, which were involved in specific biological pathways, as is shown in figure 4. According to the result, the significantly altered biochemical pathways with a P value of less than 0.05 were eight metabolic pathways. Most of the altered metabolic pathways in the metacyclic promastigotes of the Iranian isolates of L. major and L. tropica are presented in table 2.

Figure 4. Metabolite set enrichment analysis results of differentially expressed metabolites are shown between the Iranian isolates of Leishmania tropica and Leishmania major.

Discussion

In this study, the metabolites detected by the ¹H-NMR spectroscopy of the Iranian clinical isolates of L. major and L. tropica showed a considerable overlap; however, overall, 24 metabolites were significantly expressed in abundance at least with a P value of less than 0.05 between the cell extracts of the two species. The discriminating metabolites in L. tropica compared with L. major included the increment of creatinine, galactose, tryptophan, L-serine, hippurate, pantothenate, betaine, creatine, mannose, aspartate, methionine, homocitrulline, and acetylcarnitine along with the decline of 3-hydroxybutyrate, phosphoserine, L-proline, kynurenine, lactate, choline, histidine, asparagine, phenylalanine, tyrosine, and leucine. A comparative metabolomic analysis between the cell extracts of the promastigotes of L. donovani, L. major, and L. Mexicana by Westrop and others ¹⁴ revealed 64 different metabolites. They reported many interesting differences between the metabolites of the three L. spp, especially tryptophan, asparagine, arginine, proline, glycine, and serine.

We found many interesting differences between the metabolism of the two L. spp, particularly in the area of amino acid metabolism, which is somewhat in accordance with several other studies. ^{3 , 14 , 15}

Our comparative analysis revealed 13 out of 24 metabolites with significantly increased differential expressions in L. tropica and the remaining metabolites in L. major. Among them, the top four differential metabolites were methionine, aspartate, betaine, and acetylcarnitine, which were increased in L. tropica by 3.45-fold, 3.48-fold, 2.87-fold, and 2.85-fold compared with L. major, respectively. On the other hand, asparagine, 3-hydroxybutyrate, L-proline, leucine, kynurenine, and phosphoserine were increased remarkably in L. major in comparison with L. tropica. Consistent with these results, alterations in some metabolites have been previously recorded on the promastigotes and /or amastigotes of L. donovani, L. major, and L. Mexicana. ^{3 , 10 , 14} The results concerning amino acids also propound speculative hypotheses on how the species differ and could explain the distinct disease phenotypes resulting from the infections of humans with the two different species, if they occur also with the amastigote forms that are responsible for human diseases. ¹⁴ Leishmania spp are auxotrophic for L-leucine, and it can be converted through 4-methyl-2-oxopentanoate and hydroxymethylglutaryl coenzyme A into acetyl-CoA, and then metabolized through the tricarboxylic acid cycle. ¹⁶ Tryptophan Metabolism via the Kynurenine Pathway is leading to synthesis of nicotinamide adenine dinucleotide (NAD+); it also regulates the biological processes including immune cell response and inflammation. ¹⁷

As indicated in table 2, we detected eight metabolic pathways with a P value of less than 0.05: ammonia recycling; betaine metabolism; methionine metabolism; glycine, serine, and threonine metabolism; arginine and proline metabolism; aspartate metabolism; beta-alanine metabolism; and phenylalanine and tyrosine metabolism. Based on the results of the previous studies, valine, leucine, and isoleucine biosynthesis, pyruvate metabolism, glutathione metabolism, fatty acid biosynthesis, and sulfur metabolism are the most important pathways changed between the procyclic and metacyclic phases of L. major. ^{3 , 7}

In the present study, the ammonia recycling biochemical pathway analysis had the most important pathway involved in the biological pathways (P=0.007): L-aspartic acid, L-histidine, L-serine, and L-asparagine were the amino acids involved in this pathway. Amiri and others ⁵ and Arjmand and others ⁷ disclosed that the major alterations in metabolic pathways in the metacyclogenesis process of L. major were citraconic acid, isopropylmalic acid, L-leucine related to valine, leucine, and isoleucine biosynthesis pathways, respectively. One interesting hypothesis for these differences in metabolic pathways could be due to the fact that our findings pertain to comparisons between two species (L. tropica and L. major), while their results are related to one species (only the standard strain of L. major). Asparagine/glutamine related pathways in Leishmania play a key role in cellular homeostasis maintenance. ¹⁸ According to another study, aspartate uptake was essential in the tricarboxylic acid cycle in L. Mexicana. ¹⁹ Previous research supports the notion that the L-asparagine synthetase is a survival factor in L. infantum and can serve as a potential drug target in Leishmania. ²⁰ As discussed above, similar roles may have been played by aspartate and asparagine in L. tropica and L. major.

The other significant biochemical pathways in this study were betaine and methionine metabolism; betaine, choline, L-methionine, and L-serine were involved in these pathways (table 2 and figure 4). Betaine and methionine metabolites were increased by 2.87-fold and 3.45-fold, respectively, and choline was decreased more in L. tropica than L. major. A similar study on L. donovani life stages was performed by Gupta and others, ¹⁰ using the ¹H-NMR method. They detected significantly altered metabolites in the promastigote stage, including betaine and hydroxybutyrate; nonetheless, succinate and valine metabolites were present in the amastigote/ axenic amastigote stage. Choline and its derivatives are lipoprotein and membrane lipids components in eukaryote cells. Betaine serves as a substrate in the betaine-homocysteine–methionine synthesis pathway. ²¹

We observed that methionine was markedly upregulated in L. tropica than L. major. This metabolite is also one of the amino acids involved in the betaine metabolism pathway, which is a sulfur-containing proteinogenic amino acid. L-methionine contributes to polyamine biosynthesis. ²²

As seen in figure 4 and table 2, the results of the pathway enrichment analysis showed that glycine, serine, and threonine metabolism, arginine and proline metabolism, beta-alanine metabolism, and phenylalanine and tyrosine metabolism constituted the other biochemical pathways with significant differences in metabolic pathways, particularly in amino acids. This observation is relatively consistent with reports from other studies. ^{3 , 14} For example, Westrop and others ¹⁴ using the LC-MS method detected significant differences in metabolic pathways in amino acids, especially tryptophan, aspartate, arginine, proline, glycine, serine, and threonine compared with the global metabolome of the studied L. spp.

In the present study, phenylalanine and tyrosine were decreased more in L. tropica than L. major. These two amino acids were also involved in significant pathways. Lamour and others ¹⁵ found that these amino acids were differentially expressed in activated and non-activated macrophages affected by L. major.

In this study, proline was one of the other metabolites whose amount differed between the two species (L. tropica<L. major). Leishmania parasites in the insect stage of (promastigotes) use proline as an important carbon and energy source. A recent study by Westrop and others ¹⁴ showed that proline and asparagine were decreased more in L. major promastigotes than L. infantum promastigotes but were increased in comparison with L. Mexicana promastigotes. Tryptophan in L. tropica had a higher concentration than in L. major in this study. Furthermore, Westrop and others compared the metabolites of three L. spp via the LC-MS method and demonstrated that tryptophan had a considerably lower level in L. major than in L. donovani and L. Mexicana. Besides, its fundamental role in protein synthesis, tryptophan is an essential amino acid that is the precursor of many physiologically significant metabolites produced during the process of its degradation. It has been reported that 95% of the overall tryptophan degradation takes place in the kynurenine pathway. ²³ According to this content, one of the methods against the pathogen by host cells is the evacuation of the parasite from tryptophan via the kynurenine pathway. ²⁴

A main limitation of the current investigation is that we evaluated the metabolic profile of promastigotes, while the metabolic profiling of amastigotes would have yielded more robust data on the species-specificity of survival and pathogenicity.

Conclusion

The metabolites of each species have a distinct metabolic profile and involve different numbers of amino acids and their catabolism. Significantly different metabolites may explain the distinct phenotypes and clinical patterns of CL resulting from the infection of humans with the two different L. spp (L. major and L. tropica), if they occur also with the amastigote forms, which are responsible for human diseases. Our findings could serve as a framework in future studies, aimed at exploring the host-parasite interaction and species-specific pathogenicity and can also help to present new potential drug targets.

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