Visceral leishmaniasis (VL) is a potentially fatal protozoan infection caused by Leishmania donovani
on the Indian subcontinent and L. infantum
in the Mediterranean region. In the absence of an effective vaccine, treatment of VL relies largely upon chemotherapy. Reduced efficacy and increasing relapses against available antileishmanial agents require exploring more druggable targets. Internalization of parasites and their proliferation within the host cell are of primary concern to understand the key players involved in establishing infection and parasite persistence [1
]. The genome of Leishmania
shows constitutive expression in both flagellar and aflagellar forms. Most often, mRNA abundance does not correlate with the expression level of proteins in Leishmania
]. Transcriptomic studies have provided a plethora of information regarding parasitic factors associated with drug resistance, parasite fitness, and the adaptive changes during different developmental stages of Leishmania
]. However, mRNA-based studies do not provide information regarding translational and post-translational modifications, which are required to understand the gene function and host pathogenesis [4
]. Therefore, it becomes essential to explore the Leishmania
proteome, which will provide a detailed understanding of gene functions and cellular reprogramming. Regulation of protein expression at developmental stages in Leishmania
is evident at post-transcriptional and post-translational levels [5
]. Recent developments in proteomic studies have opened the window for understanding disease mechanisms, identifying biomarkers for diagnosis, and identifying several vital candidates for vaccine development [6
Being an obligate intracellular parasite, Leishmania
exploits several strategies to survive and replicate within the host cell. Internalization of the parasite into the host exhibits a classical receptor-mediated process that initiates phagocytosis [8
]. The host cell invasion process requires contact with the host cell plasma membrane, and the interacting proteins have been targeted in many infections [9
]. Major surface molecules from both the parasite as well as the host interact with each other during the process of internalization. Upon phagocytosis, to resist degradation within the host cell to establish infection, the parasites envelope themselves within the parasitophorous vacuole, commonly known as Leishmania
parasitophorous vacuoles (LPVs), derived from the host endocytic pathway [10
]. Molecules from the endocytic and secretory pathways are localized and expressed on LPVs [12
]. The previous study has highlighted that parasite-derived proteins are present throughout the host cell; however, their molecular contributions are poorly described to the complex host-pathogen interactions [13
The interaction of cell membrane proteins between both the host and the pathogen is a prerequisite for the initial encounter and further penetration of the parasite to the host cell [14
]. Membrane proteins have an indispensable role in host-pathogen interaction and regulatory pathways that operate within parasites and the host cells. Membrane proteins predominantly represent almost half of the potential drug targets [5
A study on the Leishmania
secretome highlighted several potential virulence-related proteins, and proteins associated with signal transduction, parasite survival, immuno-suppression, transport processes and antioxidant defense mechanisms [15
]. Identifying such proteins involved at the host-pathogen interface will help to understand the virulence factors involved in host invasion. Such virulence factors responsible for parasite establishment may serve as new drug targets. The present study aims to identify Leishmania
promastigote membrane proteins interacting with host macrophages using 2-dimensional gel electrophoresis (2-DE) and mass spectrometry. Such proteins may be exploited further as novel targets for chemotherapeutic intervention.
In the present study, we identified a number of interacting membrane proteins involved in the Leishmania-macrophage interaction. Based on their biological relevance, the identified proteins belonged to different pathways/categories, including antioxidant defense (peroxidoxin), electron transport chain of mitochondria (cytchrome c oxidase), cytoskeleton, and cell motility (β-actin, SMP-1, and FILIP1), as well as a protein involved in cell proliferation and signal transduction (activated C kinase).
Study of the host-pathogen interaction is critical for understanding the disease pathogenesis. The Leishmania
-macrophage interaction provides an excellent model for studying the proteins involved during the establishment of a successful infection, parasite survival, and persistence within the host [1
]. Several genomic and proteomic studies have shed light on stage-specific regulation during parasite development and on membrane proteins of the parasite and host during cross-talk [16
]. Proteomic studies have identified cytosolic, membrane, and secretory proteins involved in the host-parasite interaction, parasite survival, virulence, and signaling pathways, and proteins with diagnostic and vaccine potential [4
]. Parasites circumvent host-induced oxidative and nitrosative stresses, either by manipulating their surface proteins or by modulating the host-gene expression and epigenetic modification [22
In this study, we identified peroxidoxin from the parasite origin, which is a critical thiol-specific antioxidant. This protein, ubiquitous and over-expressed in the drug-resistant parasite, plays a crucial role against endogenous and host-derived oxidative and nitrosative stress [24
]. Another parasite membrane protein interacting with the host macrophage was identified as SMP-1, which constitutes a major flagellar membrane protein and guides flagellar movement, as evident in the SMP-1 deleted mutant Leishmania
]. Among interacting membrane proteins identified in the present study, cytochrome c oxidase, an enzyme complex located in an inner mitochondrial membrane required to meet the energy demand of the parasite to combat hostile mammalian environments/niches, was found to be important during the host-parasite interaction.
Kinases are the key molecules involved in phosphorylation of specific amino acids that play a role in different signaling cascades. They are involved in growth and proliferation and serve as important drug-target molecules [18
]. We identified activated C kinase from the L. donovani
membrane interacting with host macrophages. Previous proteomic studies have highlighted its role in parasite viability, progression of infection, and persistence of the parasite within the host macrophage [28
]. Additionally, Leishmania
-activated C kinase facilitates the expression of virulence-associated proteins in the mammalian host, enhancing parasite fitness during the invasion to host macrophages, and helps parasite replication in the hostile mammalian environment [29
Our current finding of Leishmania
-activated C kinase protein interacting with the macrophage membrane protein, strongly backed by previous studies, indicated this as an important molecule to explore as a potential target for chemotherapeutic intervention. Withaferin A (WA), an inhibitor of activated C kinase and other enzymes of Leishmania
, significantly reduced the proliferation of L. donovani
to macrophages, highlighting its role in the host-parasite interaction [31
-activated C kinase (LACK) facilitates the cytochrome c oxidase subunit expression to promote the fitness of L. major
. Cytochrome c oxidase (COX) activity is favored by LACK, as evident in a study where it was shown that COX activity and oxygen consumption are reduced in LACK-deficient L. major
]. Besides the parasite membrane protein, we also identified interacting membrane proteins of macrophage origin, namely β-actin and FILIP-1. Actins are conserved proteins and responsible for the integrity of the host cytoskeleton which is a requisite for parasite infection to the host [33
]. Previous investigations reported that actin destabilization in the host macrophage reduced the binding of the Leishmania
promastigote to the host, suggesting its importance during the attachment of the parasite to the host plasma membrane [33
]. FILIP-1, which interacts with Filamin A, is an actin-binding protein required for cell motility. The role of Filamin A has been well studied in cancer progression and other microbial pathogenesis; however, in Leishmania
infection biology, its role remains to be investigated [34
In conclusion, we identified proteins involved in the host-parasite interaction that supposedly have a vital role during Leishmania pathogenesis and may serve as potential chemotherapeutic intervention targets.
4. Materials and Methods
4.1. Parasite Culture
AG83 (MHOM/IN/83/AG83) and L. donovani
K133 (MHOM/IN/2000/K133) parasites were cultured at 24 °C in M199 medium with 25 mM HEPES (pH7.4) supplemented with 10% heat-inactivated foetal bovine serum, 100 IU and 100 µg/mL each of penicillin G and streptomycin, respectively. Promastigotes were grown for six days to get the stationary phase parasites. Both these isolates are sensitive to various antileishmanial drugs [35
]. LdK133, a patient-derived isolate, was used for proteomic analysis to identify the interacting membrane proteins. The validation experiments to characterize the role of LACK were conducted with both LdAG83 and LdK133.
4.2. Macrophage Culture
Human mononuclear cell line THP-1 and murine macrophage adherent cell line RAW 264.7 were maintained in RPMI 1640 medium, supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO2
in flat-bottom tissue-culture flasks. THP-1 cells were differentiated into macrophages by incubating them with 50 ng/mL phorbol-12-myristate-13-acetate (PMA) for 24 h. Cells were washed with 1× phosphate buffer saline (PBS) to remove non-adherent cells and PMA. Fresh medium was added, and adherent macrophages were removed after 24 h by gentle scraping [37
4.3. Isolation and Quantification of Membrane Proteins
Plasma membrane proteins were isolated from Leishmania
promastigotes and the THP-1 macrophage following standard protocol [21
]. Briefly, parasite cells (1010
cells) and macrophages (3 × 106
) were washed thrice with ice cold 1× PBS buffer by centrifuging at 3000× g
/10 min/4 °C. Cells were resuspended in ice-cold homogenization buffer (10 mM Tris pH 7.4, 1 mM EDTA, and 250 mM sucrose) containing a protease inhibitor cocktail (Sigma Aldrich). The suspension was lysed by sonication (25 W, 10 × 30 s) and incubated on ice for 1 h. Crude lysate was centrifuged at 15,000× g
/30 min/4 °C, and supernatant was collected. Membrane proteins were pelleted down by ultracentrifugation of supernatant at 100,000× g
/1 h/4 °C, and supernatant was collected as soluble fraction proteins. Membrane proteins were resuspended in buffer (40 mM Tris pH 7.4, 4% CHAPS, 2% CHAPSO). Proteins were quantified by the Bradford method.
4.4. Verifying Integrity and Purity of Membrane Proteins
The integrity of soluble and membrane proteins isolated was verified by SDS-PAGE. Further, the purity of membrane protein was confirmed by Western blot using an anti-sodium potassium ATPase antibody, which is a plasma membrane marker protein [39
]. Proteins were separated by SDS-PAGE on 12% acrylamide gels prior to transfer onto the nitrocellulose membrane. Blot was developed using the Membrane Fraction WB Cocktail (Cat No. ab140365abcam), which contains 5 monoclonal Abs each targeting proteins located in different compartments of the cell. After primary and secondary antibodies’ incubations, the blot was developed using the ECL kit (Amersham GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s protocol. The presence of the plasma membrane protein is shown by the anti-sodium potassium ATPase corresponding band (~112 kDa).
4.5. Evaluation of Protein Interaction by ELISA
First, we investigated the interaction of parasite membrane proteins with the macrophage membrane by enzyme-linked immunosorbent assay (ELISA Sigma-Aldrich, Saint Louis, MI, USA), as described elsewhere with modifications [38
]. Briefly, the plate was coated with the THP-1 membrane protein (10 ng/well) in 0.1 M bicarbonate buffer (pH 9.0) overnight at 4 °C. The plate was blocked with 3% BSA overnight at 4 °C and incubated with the parasite membrane protein (10 ng/well) for 2 h. The plate was incubated for 2 h with the primary antibody (VL patient serum 1/100 dilution), followed by the addition of the secondary antibody (horseradish peroxidase conjugated anti-human IgG, 1/5000 dilution) and incubated for 2 h. After a subsequent wash at each step with PBST, an orthophenylenediamine (OPD) substrate with hydrogen peroxide was added, and the optical density (OD) of each well was measured at 492 nm. Wells coated with THP-1 lysate without the interaction of the parasite membrane protein was taken as a control. Wells coated with the parasite membrane protein were considered as a positive control.
4.6. Identification of Membrane Proteins Involved in Host-Parasite Interaction
Membrane proteins isolated from the parasite/macrophage were labeled with N-hydroxysuccinimidyl ester-derivatives of the cyanine dye (Cy5 NHS-Ester kit from Amersham, GE Healthcare, Buckinghamshire, UK). Labeling of 50 µg of membrane protein was done according to the manufacturer’s instructions. Briefly, the pH of the membrane protein was adjusted to 8.5 with 50 mM NaOH, and then Cy 5 dye (400 pico mole) was added, and kept on ice for 30 min, and then 1 µL lysine (10 mM) was added to stop the reaction. The Cy5-labeled parasite membrane protein was incubated with THP-1 macrophage for 1 h. Similarly, Cy5-labeled macrophage membrane proteins were incubated with intact parasites for 1 h.
4.7. Two-Dimensional Gel Electrophoresis (2 DE)
Iso-Electric Focusing (IEF)
2-DE of the membrane protein isolated was performed following the standard procedure [21
] with modifications. Briefly, a total of 200 µg protein dissolved in the rehydration buffer (50 mM DTT, 2% of IPG-buffer, 0.002% bromophenol blue) was used for passive rehydration of the IPG-strip, pH range 3–10, 13 cm (GE Healthcare) overnight. IEF was performed on the Protean i12 isoelectric focusing (IEF) system (Bio-Rad, Hercules, CA, USA) at 20 °C for separation of proteins in the first dimension. The IEF run comprised seven steps including 250 V for 1 h, 500 V for 1 h, 1000 V for 1 h, 2000 V for 2 h, 4000 V for 2 h, 6000 V for 2 h, and 8000 V for 2.5 h, with a maximum current of a 50 µA/IPG strip. The proteins were further separated in the 2nd dimension using SDS-PAGE.
The strip was sequentially equilibrated for 30 min each in an equilibration buffer (6 M urea, 0.375 M Tris–HCl, pH 8.8, 2% SDS, 20% glycerol) supplemented with 2% (w/v) DTT and then in an equilibration buffer with 2.5% (w/v) iodoacetamide. The strip was carefully loaded on the 12.5% PAGE gel, and air bubbles were removed, and sealed with 1% agarose gel. Gel was run in a SDS-electrophoresis buffer (25 mM Tris base, 192 mM glycine, 0.1% SDS) at 15 mA for 30 min and then at 30 mA until the bromophenol blue front reached the bottom of the gel. The gel was stained with Colloidal Coomassie Brilliant Blue (CBB) and scanned using the Typhoon trio using a Cy 5 setting of 670 BP 30 with red laser 633 (which transmits light between 655 nm and 685 nm and has a transmission peak centered at 670 nm). The reproducibility of the 2-D pattern was considered final when two consecutive runs produced an identical pattern with the same membrane protein fraction. The ten most intense protein spots from the parasite membrane fraction and 6 protein spots from the macrophage membrane fraction were selected for protein identification.
4.9. In-Gel Digestion
The in-gel digestion of protein spots of interest and purification of peptides from the gel was carried out using standard procedures described elsewhere with modifications [17
]. Briefly, 2-DE protein spots (n = 10 from parasite and n = 6 from macrophage) were excised by the pipette tip, washed with HPLC grade water, and destained by a 50% acetonitrile/50 mM NH4
solution. The gel was then dehydrated with 100% acetonitrile and then rehydrated and digested with Trypsin (0.25 µg/sample) overnight at 37 °C. Peptides were extracted from the gel using 0.4% formic acid in 3% ACN twice, once using 0.4% formic acid in 50% ACN and once using 100% ACN. The extracted peptides were dried using speed vac and stored at −80 ℃ until the MALDI-MS analysis.
4.10. Protein Identification by MALDI-TOF/TOF
The peptide extract was reconstituted in the 10µl of 60% acetonitrile and 0.1% TFA. The peptides were mixed with the α-cyano-4-hydroxycinnamic acid matrix in a ration of 1:1 and spotted on the MALDI plate. MALDI-MS data were acquired automatically over a mass range of 0.8–3.5 kDa within a reflector ion mode at a fixed laser intensity for 1500 shots/spectrum on the 4800 MALDI-TOF/TOF Analyzer (Applied Biosystems) with the 4000 Series Explorer v3.5 software. Instrument settings were optimized to achieve optimal sensitivity at a collision energy of 1keV. The single collision condition was achieved by using air as the collision gas. The 10 most abundant MS peaks were selected for MS/MS in each MS spectrum, employing an acquisition method that excluded ions with S/N below 50. Only the strongest precursor was selected, and identical peaks detected in adjacent spots were filtered out. A MS/MS operating mode of 1 kV was used with a relative precursor mass window fixed at 250 (full-width half mass), keeping metastable suppression enabled. A total of 1250 shots with 50 shots per sub-spectrum were used for the MS/MS acquisition of selected precursors at a fixed laser intensity. The acquisition was stopped when a minimum of one hundred S/N on greater than seven peaks within the spectrum was reached after the minimum thousand shots was acquired.
To identify the peptide, peptide masses obtained from the mass spectrometric analysis were searched in the MASCOT search engine with the NCBI nr database for the BPK282A1 strain of the L. donovani and Homo sapiens reference sequence for the identification of proteins. The detected protein threshold was a fixed confidence score of 99.9%.
The search parameters used were as follows: (a) trypsin was set as a proteolytic enzyme allowing up to one missed cleavage; (b) a precursor peptide mass error tolerance of 20 ppm was selected; (c) a fragment mass error tolerance of 0.1 Da was selected; (d) the oxidation of methionine, deamidation of asparagine and glutamine, and acetylation of protein N-termini were set as variable modifications, whereas the carbamidomethylation of cysteine was set as a fixed modification.
4.11. Sensitivity of Leishmania Promastigote towards Withaferin A
The sensitivity of the Leishmania
promastigote to withaferin A (WA) was investigated by the standard resazurin assay, described elsewhere [35
]. Briefly, late log phase promastigotes were plated into a 96-well culture plate (105
promastigotes/well), and exposed to increasing concentrations of WA (0.0625 µM to 10 µM). Post-72-h incubation at 25 °C, 50 μL resazurin (0.0125 % (w/v
in PBS)) were added, and plates were incubated for a further 24 h. Viability of cell was measured fluorometrically (λex 550 nm; λem 590 nm). The results were interpreted as percentage reduction in the parasite viability compared to untreated control wells. Fifty percent inhibitory concentration (IC50
) was calculated by sigmoidal regression analysis using Microcal Origin 6.0 software (https://microcal-origin.software.informer.com/6.0/
, accessed on 13 May 2021). The experiments were repeated at least twice in quadruplicates.
4.12. Cytotoxicity of WA on THP-1 Macrophages
Cytotoxicity of WA on THP-1was assessed using the mitochondrial-respiration-dependent 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) reduction method described elsewhere [40
] with minor modification. Briefly, THP-1 cells were seeded into 96 well tissue culture plate at a confluence of 30,000 cells per well and incubated with 50 ng/mL of PMA for 48 h at 37 °C and 5% CO2.
After differentiation THP-1 cells were treated with different concentration of WA (0.0625 µM to 10 µM) for 48 h. Cells were washed with PBS and incubated with 1 mg/mL MTT in PBS for 2 h at 37 °C in 5% CO2
, followed by DMSO treatment. Absorbance of each well was then read at 540 nm using a microplate reader (Tecan 200). The optical density of formazan formed in control cells (without treatment with WA) was taken as 100% viability.
4.13. Proliferation of L. donovani Parasite within Host Macrophage in Presence and Absence of Activated C kinase Inhibitor (Withaferin A)
Proliferation of the L. donovani
parasite was investigated by infecting THP-1 and RAW 264.7 murine macrophages with L. donovani
promastigote using standard protocols with modifications [37
]. These differentiated macrophages were infected with parasites at a 1/10 macrophage/parasite ratio in a humidified atmosphere at 37 °C/5% CO2
overnight with or without WA (0.1 µM and 1 µM). After 24 h, slides were air-dried, fixed in absolute methanol for 5 min, and stained with Diff–Quik solutions. To calculate the number of amastigotes/cell, 100 macrophages were examined under oil immersion light a microscopy at 1000× magnification.